U.S. patent number 6,022,853 [Application Number 08/278,730] was granted by the patent office on 2000-02-08 for morphogen-enriched dietary composition.
This patent grant is currently assigned to Creative BioMolecules, Inc.. Invention is credited to Charles M. Cohen, Thangavel Kuberasampath, Hermann Oppermann, Roy H. L. Pang, David C. Rueger.
United States Patent |
6,022,853 |
Kuberasampath , et
al. |
February 8, 2000 |
Morphogen-enriched dietary composition
Abstract
Disclosed are methods and compositions useful in dietary
applications and capable of enhancing tissue morphogenesis,
including tissue development and viability in a mammal,
particularly a human. The methods and compositions include a
morphogen which, when provided to an individual as a food
formulation or supplement, is capable of enhancing tissue
development and viability in the individual.
Inventors: |
Kuberasampath; Thangavel
(Medway, MA), Cohen; Charles M. (Medway, MA), Rueger;
David C. (Hopkinton, MA), Oppermann; Hermann (Medway,
MA), Pang; Roy H. L. (Etna, NH) |
Assignee: |
Creative BioMolecules, Inc.
(Boston, MA)
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Family
ID: |
27560274 |
Appl.
No.: |
08/278,730 |
Filed: |
July 20, 1994 |
Related U.S. Patent Documents
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946235 |
Sep 16, 1992 |
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922813 |
Jul 31, 1992 |
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923780 |
Jul 31, 1992 |
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938336 |
Aug 28, 1992 |
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938337 |
Aug 28, 1992 |
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752764 |
Aug 30, 1991 |
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922813 |
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752764 |
Aug 30, 1991 |
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667274 |
Mar 11, 1991 |
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923780 |
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752857 |
Aug 30, 1991 |
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667274 |
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938336 |
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753059 |
Aug 30, 1991 |
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667274 |
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938337 |
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753059 |
Aug 30, 1991 |
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667274 |
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Current U.S.
Class: |
424/464; 424/439;
514/8.8 |
Current CPC
Class: |
A61L
27/227 (20130101); A61L 27/24 (20130101); C07K
14/51 (20130101); C07K 16/22 (20130101); A23L
33/18 (20160801); A61K 38/00 (20130101); G01N
2500/10 (20130101) |
Current International
Class: |
A01N
1/02 (20060101); A23L 1/305 (20060101); A61L
27/22 (20060101); A61L 27/00 (20060101); A61L
27/24 (20060101); A61K 6/00 (20060101); C07K
14/435 (20060101); C07K 14/51 (20060101); C07K
16/22 (20060101); C07K 16/18 (20060101); A61K
38/00 (20060101); A61K 038/18 () |
Field of
Search: |
;514/12 ;424/439,464
;426/657,800,801 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0148155 |
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Jul 1985 |
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EP |
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0 295 009 |
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0 313 515 |
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Apr 1989 |
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EP |
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0416578A2 |
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Mar 1991 |
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EP |
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88/00205 |
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Jan 1988 |
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WO |
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89/09787 |
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Oct 1989 |
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WO |
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89/09788 |
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Oct 1989 |
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WO |
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89/10409 |
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Nov 1989 |
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WO |
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90/03733 |
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Apr 1990 |
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WO |
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Primary Examiner: Kemmerer; Elizabeth
Attorney, Agent or Firm: Elriff; Ivor R. Morency; Michael
Mintz, Levin, Cohn, Ferris, Glovsky and Popeo, P.C.
Parent Case Text
CROSS REFERENCE TO RELAYED APPLICATIONS
This application is a continuation of Ser. No. 07/946,235, filed
Sep. 16, 1992, now abandoned, which is continuation-in-part of (1)
U.S. Ser. No. 07/922,813, filed Jul. 31, 1992, now abandoned which
is a continuation-in-part of U.S. Ser. No. 07/752,764, filed Aug.
30, 1991, now abandoned, which in turn is a continuation in part of
U.S. Ser. No. 07/667,274, filed Mar. 11, 1991, now abandoned; (2)
U.S. Ser. No. 07/923,780, filed Jul. 31, 1992, now abandoned which
is a continuation-in-part of U.S. Ser. No. 07/752,857, filed Aug.
30, 1991, now abandoned which in turn is a continuation-in-part of
U.S. Ser. No. 07/667,274 filed Mar. 11, 1991, now abandoned; (3)
U.S. Ser. No. 07/938,336 and U.S. Ser. No. 07/938,337, both, filed
Aug. 28, 1992, both now abandoned and both continuations-in-part of
U.S. Ser. No. 07/753,059, filed Aug. 30, 1991, now abandoned which
in turn is a continuation-in-part of U.S. Ser. No. 07/667,274,
filed Mar. 11, 1991, now abandoned; and (4) U.S. Ser. No.
07/752,764, filed Aug. 30, 1991, now abandoned which is a
continuation-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11,
1991, now abandoned. The disclosures of these applications are
incorporated herein by reference. The disclosure of U.S. Ser. No.
07/946,238, filed Sep. 16, 1992, now abandoned, and currently
pending as U.S. Ser. No. 08/445,468, filed May 22, 1995, now U.S.
Pat. No. 5,849,686, is also incorporated herein by reference.
Claims
What is claimed is:
1. A composition comprising:
(a) a nutrient, trace mineral, or vitamin required for normal
metabolism in mammalian tissue; and
(b) a morphogen capable of being absorbed from the digestive system
into the bloodstream, wherein it retains morphogenic activity, said
morphogen comprising an acid-stable, protease-resistant dimeric
protein that
(i) induces tissue-specific morphogenesis of at least mammalian
bone tissue; and
(ii) comprises a pair of folded polypeptides, each having an amino
acid sequence comprising a conserved cysteine skeleton of
approximately 102 amino acids, wherein cysteines occupy relative
positions 1, 30, 34, 66, 67, 99, and 101, and wherein at least 70%
of amino acids in said skeleton are selected independently from
amino acids occupying corresponding positions within the C-terminal
seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No.
5, and conservative substitution variants of said amino acids,
said morphogen being dispersed in said composition in a dosage
sufficient to induce tissue-specific morphogenesis and to protect
mammalian tissue from tissue damage or reduced tissue function due
to metabolic stress or dysfunction.
2. A composition comprising:
(a) a nutrient, trace mineral, or vitamin required for normal
metabolism in mammalian tissue; and
(b) a morphogen capable of being absorbed from the digestive system
into the bloodstream, wherein it retains morphogenic activity, said
morphogen comprising an acid-stable, protease-resistant dimeric
protein that
(i) induces tissue-specific morphogenesis of at least mammalian
bone tissue; and
(ii) comprises a pair of folded polypeptides, each having an amino
acid sequence comprising a conserved cysteine skeleton of
approximately 102 amino acids, wherein cysteines occupy relative
positions 1, 30, 34, 66, 67, 99, and 101, an wherein at least 70%
of amino acids in said skeleton are selected independently from
amino acids occupying corresponding positions within the C-terminal
seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No.
5, and conservative substitution variants said amino acids,
said morphogen being dispersed in said composition in a dosage
sufficient to induce tissue-specific morphogenesis and to restore
the function of senescent or quiescent mammalian tissue or
mammalian tissue afflicted with tissue damage or reduced tissue
function due to metabolic stress or dysfunction.
3. The composition of claim 1 or 2, wherein said morphogen is
associated with a controlled release component, adapted such that
the morphogen is released in a controlled manner in the
gastrointestinal tract subsequent to passage through the
stomach.
4. The composition of claim 1 or 2 adapted for enteral
administration.
5. The composition of claim 1 or 2, formulated as a solid.
6. The composition of claim 5, wherein said solid is a tablet,
troche or lozenge.
7. The composition of claim 1 or 2 formulated as a liquid.
8. The composition of claim 7, wherein said liquid is a beverage or
a syrup.
9. The composition of claim 1 or 2, wherein said morphogen is
associated with a morphogen-solubilizing molecule.
10. The composition of claim 9, wherein said molecule is casein or
a derivative, salt or analog thereof.
11. The composition of claim 9, wherein said morphogen-solubilizing
molecule comprises one or more prodomains selected from the group
consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A,
GDF-1, BMP2A, BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, BMP6, and
conservative substitution variants of any of the foregoing which
retain morphogenic activity.
12. The composition of claim 1 or 2, wherein at least 80% of amino
acids within said skeleton are selected independently from amino
acids occupying corresponding positions within the C-terminal seven
cysteine domain of human OP-1, residues 38-139 of Seq. ID No. 5,
and conservative substitution variants of said amino acids.
13. The composition of claim 1 or 2, wherein said skeleton has
greater than 60% amino acid sequence identity with the C-terminal
seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No.
5.
14. The composition of claim 13, wherein said skeleton has greater
than 65% amino acid sequence identity with the C-terminal seven
cysteine domain of human OP-1, residues 38-139 of Seq. ID No.
5.
15. The composition of claim 14, wherein said morphogen comprises
an acid-stable, protease-resistant dimeric protein comprising a
pair of folded polypeptides, each comprising a C-terminal seven
cysteine domain of human OP-1, residues 43-139 of Seq. ID No.
5.
16. The composition of claim 1 or 2, wherein said nutrient is
selected from the group consisting of proteins, amino acids,
carbohydrates, lipids, fatty acids, sugars, nucleosides, and
nucleotides.
17. The composition of claims 1 or 2, wherein said mineral is
selected from the group consisting of calcium, phosphorus, sodium,
potassium, chloride, magnesium, iron, zinc, copper, manganese, and
iodine.
18. The composition of claim 1 or 2, wherein said vitamin is
selected from the group consisting of vitamin A, vitamin D3,
vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12,
pantothenic acid, vitamin E, vitamin K1, folic acid and biotin.
19. The composition of claim 1 or 2, wherein said morphogen is
present in its pro form.
20. The composition of claim 19, wherein said morphogen is an
acid-stable, protease-resistant dimeric protein comprising a pair
of folded polypeptides, each comprising a sequence of human pro
OP-1, residues 30-431 of Seq. ID No. 17.
21. A composition comprising:
(a) a nutrient, trace mineral, or vitamin required for normal
metabolism in mammalian tissue; and
(b) a morphogen capable of being absorbed from the digestive system
into the bloodstream, wherein it retains morphogenic activity, said
morphogen comprising an acid-stable, protease-resistant dimeric
protein that
(i) induces tissue-specific morphogenesis of at least mammalian
bone tissue; and
(ii) comprises a pair of folded polypeptides, each comprising an
amino acid sequence defined by Generic Sequence 6, Seq. ID No. 31,
said morphogen being dispersed in said composition at a dosage
sufficient to induces tissue-specific morphogenesis and to protect
mammalian tissue at risk of incurring tissue damage or reduced
tissue function, or for treating mammalian tissue afflicted with
tissue damage or reduced tissue function due to metabolic stress or
dysfunction.
22. The composition of claim 21, wherein said morphogen comprises
an acid-stable, protease-resistant dimeric protein comprising a
pair of folded polypeptides, each comprising an amino acid sequence
defined by OPX, Seq. ID No. 29.
Description
FIELD OF THE INVENTION
This invention relates generally to the field of dietary
compositions and supplements.
BACKGROUND OF THE INVENTION
The present invention relates to compositions useful as mammalian
dietary compositions and supplements. In particular, the invention
relates to food additives and dietary supplements capable of
enhancing tissue morphogenesis and development, particularly in
individuals at risk for normal tissue development and viability.
Examples of such individuals include infants, particularly
prematurely-born ("preterm") and low birth weight infants, and
juveniles; aged individuals; and individuals experiencing altered
metabolic function and/or suffering from metabolic dysfunctions and
other disorders that threaten organ or tissue function or
viability, such as can result from malnutrition or starvation,
autoimmune diseases, organ cirrhosis and other tissue necrotizing
dysfunctions, or disorders associated with aging cells (cell
senescence.)
Mammalian infants are nourished by mother's milk until such time as
they can digest food solids. Infant formulas now exist for humans
and other mammals which can supplant or supplement mother's milk.
The formulas may be milk based (e.g., cow milk) or non-milk-based
(e.g., soy). Particularly at risk are prematurely born infants
whose tissues and organs are at an earlier stage of development,
and whose nutritional requirements may differ from those of full
term infants. Formula development is an ongoing endeavor to more
accurately mimic the beneficial aspects of mother's milk.
Nevertheless, despite the efforts of many researchers, infant
formulas still differ in a number of significant ways from human
milk. In part this is due because human milk has many substances,
such as immunoglobulins, free amino acids, polyamines, nucleotides
and polyunsaturated fatty acids not present, for example, in cow's
milk. In addition, while infant formulas try to mimic the protein
quantity found in human milk, the foreign proteins typically are
present in the formula as hydrolysates to avoid rejection or
reaction by the infant's digestive system. The proteins are present
primarily as amino acid sources rather than as functional proteins
as might normally be transmitted by the nursing mother to the
infant. In addition, human milk may contain unidentified growth and
differentiation factors that are important for overall tissue and
skeletal development.
Another group of individuals with potentially unique nutritional
requirements are individuals undergoing metabolic changes which may
result from periods of intense growth or stress, including, for
example, pregnant women and drowning victims. Other sources of
stress to the body may result from malnutrition or starvation, or
from metabolic disorders that affect organ viability, such as
autoimmune disease and organ cirrhosis. Aged individuals, and
postmenopausal women also have altered or slower metabolic
function. All of these individuals are at risk for tissue damage or
loss of tissue function due to altered metabolic function.
Reduced or lost tissue function due to malnutrition also is found
in many patients admitted to hospitals (protein energy
malnutrition, "PEM"). Proper nutritional support for such patients,
while not a primary mode of treatment is, nevertheless, an
important factor for therapy and recovery. It is, therefore
important to administer a nutritionally balanced diet given orally,
enterally or parenterally, adequate to the needs of the patient.
This is especially true for those patients where conventional
feeding is contraindicated (e.g., in dehydrated or
gastroenterological patients) or is insufficient (e.g., in
hypercatabolic patients). The enteral or oral mode of
administration of foods typically is preferable to parenteral modes
because of the lower morbidity, trophic effect upon the intestinal
mucosa, reduced dependency on instrumentation and lower costs.
It is an object of this invention to provide dietary compositions
and supplements for enhancing tissue morphogenesis, including
tissue growth, development, maintenance and viability in a mammal,
particularly a human. Another object of the invention is to provide
an infant formula capable of enhancing tissue development in an
infant or juvenile. Still another object is to provide an infant
formula that more closely mimics a nursing mother's milk. Another
object of the invention is to provide dietary supplements for
individuals at risk for normal tissue development, growth,
maintenance and viability, including premature infants, aged
individuals and individuals with altered metabolic function and/or
suffering from disorders or metabolic dysfunctions which threaten
organ viability and function. These and other objects and features
of the invention will be apparent from the description, drawings,
and claims which follow.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods useful in
dietary applications and capable of enhancing tissue morphogenesis,
including tissue growth, development, maintenance and viability in
a mammal, particularly a human. The dietary compositions and
supplements of this invention comprise a morphogenic protein
("morphogen"), as described herein, which, when provided to an
individual as a food formulation or supplement, is capable of
enhancing tissue development, growth, maintenance and/or viability
in the individual. The compositions and processes provided herein
are suitable for both infants and adults, and as part of clinical
nutrition.
As used herein, "enhancing tissue viability" is understood to mean
protecting tissue from lost or reduced tissue function due to cell
damage or cell senescence, including inducing cells to maintain
their differentiated phenotype, inducing regeneration of damaged
tissue, and/or inhibiting additional damage thereto.
"Morphogenically effective concentration" is understood to mean a
concentration sufficient to enhance tissue development and tissue
viability in an individual at risk for tissue damage and/or reduced
or lost tissue function due to insufficient nutritional
considerations, tissue damage associated therewith, and/or
incomplete tissue development, regardless of etiology. The ability
of morphogens to repair, regenerate and protect various disparate
tissues, including but not limited to, tissues of the
gastrointestinal tract, including the oral mucosa, liver tissue,
dentin tissue, periodontal tissue, nerve tissue, bone tissue, and
any tissue at risk of damage due to immune response-mediated tissue
destruction, including ischemia-reperfusion related tissue damage
are disposed in copending U.S. Ser. No. 07/945,286 filed Sep. 15,
1992, (currently pending as U.S. Ser. No. 08/461,397), U.S. Ser.
No. 07/946,238, filed Sep. 16, 1992 (currently pending as U.S. Ser.
No. 08/445,468), U.S. Ser. No. 07/752,764, filed Aug. 30, 1991
(currently pending as U.S. Ser. No. 08/404,113), U.S. Ser. No.
07/945,285, filed Sep. 15, 1992 (currently pending as U.S. Ser. No.
08/155,343), U.S. Ser. No. 07/922,813, filed Jul. 31, 1992
(currently pending as U.S. Ser. No. 08/260,675), U.S. Ser. No.
07/923,780, filed Jul. 31, 1992 (currently pending as U.S. Ser. No.
08/432,883), and U.S. Ser. No. 07/938,336, filed Aug. 2, 1992
(currently pending as U.S. Ser. No. 08/445,467), respectively, the
disclosures of which are incorporated herein above by reference.
"Morphogen-solubilizing molecule" is understood to mean a molecule
capable of maintaining a morphogen in soluble form in
physiologically buffered solutions. "Food formulation" is
understood to mean a dietary composition normally ingested by an
individual to satisfy the body's fundamental nutritional
requirements; "dietary supplement" is understood to mean
supplemental compositions ingested by an individual in addition to
the food formulations ingested to satisfy the fundamental
nutritional requirements. Multivitamin and iron tablets are
examples of common dietary supplements. "Dietary composition" is
understood to include both food formulations and dietary
supplements. As used herein, the term "infant formula" is
understood to refer to the well established infant compositions as
defined by the American Academy of Pediatrics (AAP) and the AAP
Committee on Nutrition ((1985) Pediatrics 75: 976, the European
Society of Pediatric Gastroenterology and Nutrition (ESPGAN) and
the ESPGAN Committee on Nutrition ((1987) Acta Paed Scan Suppl:
330), including recent updates published by these committees on
infant formula nutritional guidelines.
The dietary composition or supplement preferably is administered
orally, and may be provided in liquid form or as a powder to be
dissolved in a beverage. Alternatively, the dietary supplement may
be provided as a solid, e.g., in a capsular, tablet, troche or
lozenge form; or, the supplement may be provided as an aerosol, for
oral or nasal administration. Where oral administration is not
possible or desirable, other administration routes are envisioned.
For example, for some premature infants, or for intubated patients,
parenteral administration may be required, e.g., via an enteral
feeding tube.
The morphogen may be provided alone or in association with one or
more suitable excipients or carriers, and/or in combination with
other beneficial molecules such as vitamins, minerals, lipids,
fiber sources and the like. The dietary supplements also may
include pharmaceutically acceptable inert materials for use as
binders or stabilizers, including magnesium stearate or calcium
carbonate. The morphogen may be formulated together with one or
more normal food ingredients, e.g., as part of a food formulation.
Alternatively or, in addition, the morphogen may be provided as a
dietary supplement in, for example, tablet or syrup form.
The mature form of the morphogen, or active truncated forms thereof
which may be formulated in the composition, further may be provided
in association with a morphogen precursor "pro" domain, which is
known to enhance the solubility of the protein in physiologically
buffered solutions. Other useful molecules known to enhance protein
solubility include casein, including derivatives, salts and analogs
thereof, as well as other milk components, and various serum and
milk serum proteins. Additional useful molecules which may be
associated with the morphogen include tissue targeting molecules
capable of directing the morphogen to a desired target tissue.
Tissue targeting molecules envisioned to be useful in the treatment
protocols of this invention include antibodies, antibody fragments
or other binding proteins which interact specifically with surface
molecules on the target tissue cells.
Still another useful tissue targeting molecule may be part or all
of a morphogen precursor "pro" domain. Morphogens may be
synthesized in one tissue and secreted and transported to another
tissue. For example, while the protein has been shown to be active
in bone tissue, the primary source of OP-1 synthesis appears to be
the tissue of the urogenic system (e.g., renal and bladder tissue),
with secondary expression levels occurring in the brain, heart,
lungs and gastrointestinal tract (GI tract, see below.) Moreover,
the protein has been identified in serum, saliva and various milk
forms. In addition, the secreted form of the protein comprises the
mature dimer in association with the pro domain of the intact
morphogen sequence. Accordingly, the associated morphogen pro
domains may act to target specific morphogens to different tissues
in vivo. As described below, morphogen species comprising the pro
domain may be obtained from the culture medium of
morphogen-secreting mammalian cells. Alternatively, a
tissue-targeting species may be formulated by complexing the mature
dimer (or an active fragment thereof) with part or all of a pro
domain.
Associated tissue targeting or solubility-enhancing molecules also
may be covalently linked to the morphogen using standard chemical
means.
In one preferred embodiment, the morphogen comprises part of an
infant formula. The infant formula may be milk-based or
nonmilk-based, e.g., soy-based. A typical ready-to-feed
morphogen-enriched formulation for infants, when diluted to feeding
concentrations, comprises, in addition to the morphogen added to
the formula, from about 1-5% by weight fat, from about 0.01 to
about 0.5% by weight immunoglobulins as appropriate, from about
4-10% by weight carbohydrate in a quantity substantially to mimic
the carbohydrate content of human mother's milk, from about 0.5 to
4% by weight protein in a quantity substantially to mimic the
protein content of human mother's milk, optional vitamins and
minerals as required, a total solids content of from about 8 to 17%
by weight, and the remainder water.
In another preferred embodiment, the dietary composition is
formulated for individuals at risk for reduced or lost tissue
function, such as postmenopausal women, elderly individuals,
undernourished or malnourished individuals, dehydrated individuals,
drowning victims, individuals suffering from metabolic disorders
including an endocrine imbalance, gastrointestinal disorders, or
immune-compromised individuals. Undernourished or malnourished
individuals include those suffering from a lack of food
(starvation) and/or eating disorders (e.g., anorexia nervosa),
and/or suffering from a maladsorption syndrome (e.g., individuals
afflicted with digestive or intestinal fistulas, shortened bowel,
or hypercatabolism.) Individuals receiving a medical therapy,
including radiotherapy, chemotherapy or a surgical procedure also
are at risk for reduced or lost tissue function as a result of a
therapy-related malabsorption-malnutrition dysfunction. In another
embodiment, the dietary supplement is formulated for individuals
undergoing periods of increased growth or stress, such as infants
and juveniles, or pregnant or lactating women. In another
embodiment, the dietary supplement is formulated for individuals at
risk for reduced or lost organ function as results from tissue
cirrhosis or an autoimmune disease.
Morphogen-enriched nutritional products, particularly clinical
nutrition products for use in hospital or other clinical settings,
in addition to comprising a morphogen preferably are based on the
utilization of diverse other protein sources (casein, sodium and
calcium caseinate, isolated soy protein, protein hydrolyzates
and/or crystalline amino acids) mixtures of vegetable and animal
fats, carbohydrates (basically glucose polymers), vitamins and
minerals to meet, at least, the dietary intakes recommended for
healthy individuals (see, for example, Committee on Dietary
Allowances, Food and Nutrition Board, Nat Acad Sci, 9th Ed,
1980).
Among the morphogens useful in this invention are proteins
originally identified as osteogenic proteins, such as the OP-1,
OP-2 and CBMP2 proteins, as well as amino acid sequence-related
proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1
(from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.),
GDF-1 (from mouse, see Lee (1991) PNAS 88: 4250-4254), all of which
are presented in Table II and Seq. ID Nos.5-14), and the recently
identified 60A protein (from Drosophila, Seq. ID No. 24, see
Wharton et al. (1991) PNAS 88: 9214-9218.) The members of this
family, which include members of the TGF-.beta. super-family of
proteins, share substantial amino acid sequence homology in their
C-terminal regions. The proteins are translated as a precursor,
having an N-terminal signal peptide sequence, typically less than
about 30 residues, followed by a "pro" domain that is cleaved to
yield the mature sequence. The "pro" form of the protein includes
the pro domain and the mature domain, and forms a soluble species
that appears to be the primary form secreted from cultured
mammalian cells. The signal peptide is cleaved rapidly upon
translation, at a cleavage site that can be predicted in a given
sequence using the method of Von Heijne ((1986) Nucleic Acids
Research 14: 4683-4691.) Table I, below, describes the various
morphogens identified to date, including their nomenclature as used
herein, their Seq. ID references, and publication sources for the
amino acid sequences for the full length proteins not included in
the Seq. Listing. The disclosure of these publications is
incorporated herein by reference.
TABLE I ______________________________________ "OP-1" Refers
generically to the group of morphogenically active proteins
expressed from part or all of a DNA sequence encoding OP-1 protein,
including allelic and species variants thereof, e.g., human OP-1
("hOP-1", Seq. ID No. 5, mature protein amino acid sequence), or
mouse OP-1 ("mOP-1", Seq. ID No. 6, mature protein amino acid
sequence.) The conserved seven cysteine skeleton is defined by
residues 38 to 139 of Seq. ID Nos. 5 and 6. The cDNA sequences and
the amino acids encoding the full length proteins are provided in
Seq. Id Nos. 16 and 17 (hOP1 and Seq. ID Nos. 18 and 19 (mOP1.) The
mature proteins are defined by residues 293-431 (hOP1) and 292-430
(mOP1). The "pro" regions of the proteins, cleaved to yield the
mature, morphogenically active proteins are defined essentially by
residues 30-292 (hOP1) and residues 30-291 (mOP1). "OP-2" refers
generically to the group of active proteins expressed from part or
all of a DNA sequence encoding OP-2 protein, including allelic and
species variants thereof, e.g., human OP-2 ("hOP-2", Seq. ID No. 7,
mature protein amino acid sequence) or mouse OP-2 ("mOP-2", Seq. ID
No. 8, mature protein amino acid sequence). The conserved seven
cysteine skeleton is defined by residues 38 to 139 of Seq. ID Nos.
7 and 8. The cDNA sequences and the amino acids encoding the full
length proteins are provided in Seq. ID Nos. 20 and 21 (hOP2) and
Seq. ID Nos. 22 and 23 (mOP2.) The mature proteins are defined
essentially by residues 264-402 (hOP2) and 261-399 (mOP2). The
"pro" regions of the proteins, cleaved to yield the mature,
morphogenically active proteins likely are defined essentially by
residues 18-263 (hOP2) and residues 18-260 (mOP2). (Another
cleavage site also occurs 21 residues upstream for both OP-2
proteins.) "CBMP2" refers generically to the morphogenically active
proteins expressed from a DNA sequence encoding the CBMP2 proteins,
including allelic and species variants thereof, e.g., human CBMP2A
("CBMP2A(fx)", Seq ID No. 9) or human CBMP2B DNA ("CBMP2B(fx)",
Seq. ID No. 10). The amino acid sequence for the full length
proteins, referred to in the literature as BMP2A and BMP2B, or BMP2
and BMP4, appear in Wozney, et al. (1988) Science 242:1528- 1534.
The pro domain for BMP2 (BMP2A) likely includes residues 25-248 or
25-282; the mature protein, residues 249-396 or 283-396. The pro
domain for BMP4 (BMP2B) likely includes residues 25-256 or 25-292;
the mature protein, residues 257-408 or 293-408. "DPP(fx)" refers
to protein sequences encoded by the Drosophila DPP gene and
defining the conserved seven cysteine skeleton (Seq. ID No. 11).
The amino acid sequence for the full length protein appears in
Padgett, et al (1987) Nature 325: 81-84. The pro domain likely
extends from the signal peptide cleavage site to residue 456; the
mature protein likely is defined by residues 457-588. "Vgl(fx)"
refers to protein sequences encoded by the Xenopus Vgl gene and
defining the conserved seven cysteine skeleton (Seq. ID No. 12).
The amino acid sequence for the full length protein appears in
Weeks (1987) Cell 51: 861-867. The prodomain likely extends from
the signal peptide cleavage site to residue 246; the mature protein
likely is defined by residues 247-360. "Vgr-1(fx)" refers to
protein sequences encoded by the murine Vgr-1 gene and defining the
conserved seven cysteine skeleton (Seq. ID No. 13). The amino acid
sequence for the full length protein appears in Lyons, et al,
(1989) PNAS 86: 4554-4558. The prodomain likely extends from the
signal peptide cleavage site to residue 299; the mature protein
likely is defined by residues 300-438. "GDF-1(fx)" refers to
protein sequences encoded by the human GDF-1 gene and defining the
conserved seven cysteine skeleton (Seq. ID No. 14). The cDNA and
encoded amino sequence for the full length protein is provided in
Seq. ID. No. 32. The prodomain likely extends from the signal
peptide cleavage site to residue 214; the mature protein likely is
defined by residues 215-372. "60A" refers generically to the
morphogenically active proteins expressed from part or all of a DNA
sequence (from the Drosophila 60A gene) encoding the 60A proteins
(see Seq. ID No. 24 wherein the cDNA and encoded amino acid
sequence for the full length protein is provided). "60A(fx)" refers
to the protein sequences defining the conserved seven cysteine
skeleton (residues 354 to 455 of Seq. ID No. 24.) The prodomain
likely extends from the signal peptide cleavage site to residue
324; the mature protein likely is defined by residues 325-455.
"BMP3(fx)" refers to protein sequences encoded by the human BMP3
gene and defining the conserved. seven cysteine skeleton (Seq. ID
No. 26). The amino acid sequence for the full length protein
appears in Wozney et al. (1988) Science 242: 1528-1534. The pro
domain likely extends from the signal peptide cleavage site to
residue 290; the mature protein likely is defined by residues
291-472. "BMP5(fx)" refers to protein sequences encoded by the
human BMP5 gene and defining the conserved seven cysteine skeleton
(Seq. ID No. 27). The amino acid sequence for the full length
protein appears in Celeste, et al. (1991) PNAS 87: 9843-9847. The
pro domain likely extends from the signal peptide cleavage site to
residue 316; the mature protein likely is defined by residues
317-454. "BMP6(fx)" refers to protein sequences encoded by the
human BMP6 gene and defining the conserved seven cysteine skeleton
(Seq. ID No. 28). The amino acid sequence for the full length
protein appears in Celeste, et al. (1990) PNAS 87: 9843-5847. The
pro domain likely includes extends from the signal peptide cleavage
site to residue 374; the mature sequence likely includes residues
375-513. ______________________________________
The OP-2 proteins have an additional cysteine residue in this
region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in addition
to the conserved cysteine skeleton in common with the other
proteins in this family. The GDF-1 protein has a four amino acid
insert within the conserved skeleton (residues 44-47 of Seq. ID No.
14) but this insert likely does not interfere with the relationship
of the cysteines in the folded structure. In addition, the CBMP2
proteins are missing one amino acid residue within the cysteine
skeleton.
The morphogens are inactive when reduced, but are active as
oxidized homodimers and when oxidized in combination with other
morphogens of this invention. Thus, as defined herein, a morphogen
is a dimeric protein comprising a pair of polypeptide chains,
wherein each polypeptide chain comprises at least the C-terminal
six cysteine skeleton defined by residues 43-139 of Seq. ID No. 5,
including functionally equivalent arrangements of these cysteines
(e.g., amino acid insertions or deletions which alter the linear
arrangement of the cysteines in the sequence but not their
relationship in the folded structure), such that, when the
polypeptide chains are folded, the dimeric protein species
comprising the pair of polypeptide chains has the appropriate
three-dimensional structure, including the appropriate intra- or
inter-chain disulfide bonds such that the protein is capable of
acting as a morphogen as defined herein. Specifically, the
morphogens generally are capable of all of the following biological
functions in a morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the differentiation
of progenitor cells; stimulating the proliferation of
differentiated cells; and supporting the growth and maintenance of
differentiated cells, including the "redifferentiation" of
transformed cells. In addition, it is also anticipated that these
morphogens are capable of inducing redifferentiation of committed
cells under appropriate environmental conditions.
In one preferred aspect, the morphogens of this invention comprise
one of two species of generic amino acid sequences: Generic
Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2);
where each Xaa indicates one of the 20 naturally-occurring
L-isomer, .alpha.-amino acids or a derivative thereof. Generic
Sequence 1 comprises the conserved six cysteine skeleton and
Generic Sequence 2 comprises the conserved six cysteine skeleton
plus the additional cysteine identified in OP-2 (see residue 36,
Seq. ID No. 2). In another preferred aspect, these sequences
further comprise the following additional sequence at their
N-terminus:
______________________________________ Cys Xaa Xaa Xaa Xaa (Seq. ID
No. 15) 1 5 ______________________________________
Preferred amino acid sequences within the foregoing generic
sequences include: Generic Sequence 3 (Seq. ID No. 3), Generic
Sequence 4 (Seq. ID No. 4), Generic Sequence 5 (Seq. ID No. 30) and
Generic Sequence 6 (Seq. ID No. 31), listed below. These Generic
Sequences accommodate the homologies shared among the various
preferred members of this morphogen family identified in Table II,
as well as the amino acid sequence variation among them.
Specifically, Generic Sequences 3 and 4 are composite amino acid
sequences of the following proteins presented in Table II and
identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5
and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and
mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9),
CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11),
Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No.
13), and GDF-1 (from mouse, Seq. ID No. 14.) The generic sequences
include both the amino acid identity shared by the sequences in
Table II, as well as alternative residues for the variable
positions within the sequence. Note that these generic sequences
allow for an additional cysteine at position 41 or 46 in Generic
Sequences 3 or 4, respectively, providing an appropriate cysteine
skeleton where inter- or intramolecular disulfide bonds can form,
and contain certain critical amino acids which influence the
tertiary structure of the proteins.
______________________________________ Generic Sequence 3
______________________________________ Leu Tyr Val Xaa Phe 1 5 Xaa
Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 10 Xaa Ala Pro Xaa Gly Xaa Xaa Ala
15 20 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Pro Xaa Xaa Xaa Xaa
Xaa 35 Xaa Xaa Xaa Asn His Ala Xaa Xaa 40 45 Xaa Xaa Leu Xaa Xaa
Xaa Xaa Xaa 50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 55 60 Cys Xaa Pro
Xaa Xaa Xaa Xaa Xaa 65 Xaa Xaa Xaa Leu Xaa Xaa Xaa 70 75 Xaa Xaa
Xaa Xaa Val Xaa Leu Xaa 80 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 85 90
Xaa Cys Gly Cys Xaa 95 ______________________________________
wherein each Xaa is independently selected from a group of one or
more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg,
Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or
Val); Xaa at res.11=(Gln, Leu, Asp, His or Asn); Xaa at
res.12=(Asp, Arg or Asn); Xaa at res.14=(Ile or Val); Xaa at
res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or
Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp,
Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at
res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or
Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe, Leu
or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp, Ala
or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at
res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or
Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly);
Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at
res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at
res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at
res.50=(His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala or Val); Xaa at
res.53=(Asn, Lys, Ala or Glu); Xaa at res.54=(Pro or Ser); Xaa at
res.55=(Glu, Asp, Asn, or Gly); Xaa at res.56=(Thr, Ala, Val, Lys,
Asp, Tyr, Ser or Ala); Xaa at res.57=(Val, Ala or Ile); Xaa at
res.58=(Pro or Asp); Xaa at res.59=(Lys or Leu); Xaa at res.60=(Pro
or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr or Ala);
Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or
Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro or
Ser); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala);
Xaa at res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at
res.75=(Phe, Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at
res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn or
Tyr); Xaa at res.79=(Ser, Asn, Asp or Glu); Xaa at res.80=(Asn, Thr
or Lys); Xaa at res.82=(Ile or Val); Xaa at res.84=(Lys or Arg);
Xaa at res.85=(Lys, Asn, Gln or His); Xaa at res.86=(Tyr or His);
Xaa at res.87=(Arg, Gin or Glu); Xaa at res.88=(Asn, Glu or Asp);
Xaa at res.90=(Val, Thr or Ala); Xaa at res.92=(Arg, Lys, Val, Asp
or Glu); Xaa at res.93=(Ala, Gly or Glu); and Xaa at res.97=(His or
Arg);
______________________________________ Generic Sequence 4
______________________________________ Cys Xaa Xaa Xaa Xaa Leu Tyr
Val Xaa Phe 1 5 10 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 15 Xaa Ala
Pro Xaa Gly Xaa Xaa Ala 20 25 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 30 35
Xaa Pro Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Asn His Ala Xaa Xaa 45
50 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys 60 65 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Leu Xaa
Xaa Xaa 75 80 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 85 Xaa Xaa Xaa Xaa
Met Xaa Val Xaa 90 95 Xaa Cys Gly Cys Xaa 100
______________________________________
wherein each Xaa is independently selected from a group of one or
more specified amino acids as defined by the following: "Res."
means "residue" and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or
Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly,
Arg or Pro); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg,
Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or
Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa at
res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at
res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or
Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp,
Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at
res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or
Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or
Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or
Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at
res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or
Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or
Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa
at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at
res.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at
res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met,
Asn, Ala or Val); Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at
res.59=(Pro or Ser); Xaa at res.60=(Glu, Asp, or Gly); Xaa at
res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at
res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at
res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala
or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg
or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser
or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr
or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met);
Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at
res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at
res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or
Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val);
Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His);
Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln or Glu); Xaa at
res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr or Ala); Xaa at
res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly or
Glu); and Xaa at res.102=(His or Arg).
Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence
6 (Seq. ID No. 31) accommodate the homologies shared among all the
morphogen protein family members identified in Table II.
Specifically, Generic Sequences 5 and 6 are composite amino acid
sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse
OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq.
ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID
No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus,
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1
(from mouse, Seq. ID No. 14), human BMP3 (Seq. ID No. 26), human
BMP5 (Seq. ID No. 27), human BMP6 (Seq. ID No. 28) and 60(A) (from
Drosophila, Seq. ID Nos. 24-25). The generic sequences include both
the amino acid identity shared by these sequences in the C-terminal
domain, defined by the six and seven cysteine skeletons (Generic
Sequences 5 and 6, respectively), as well as alternative residues
for the variable positions within the sequence. As for Generic
Sequences 3 and 4, Generic Sequences 5 and 6 allow for an
additional cysteine at position 41 (Generic Sequence 5) or position
46 (Generic Sequence 6), providing an appropriate cysteine skeleton
where inter- or intramolecular disulfide bonds can form, and
containing certain critical amino acids which influence the
tertiary structure of the proteins.
______________________________________ Generic Sequence 5
______________________________________ Leu Xaa Xaa Xaa Phe 1 5 Xaa
Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 10 Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala
15 20 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Pro Xaa Xaa Xaa Xaa
Xaa 35 Xaa Xaa Xaa Asn His Ala Xaa Xaa 40 45 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 55 60 Cys Xaa Pro
Xaa Xaa Xaa Xaa Xaa 65 Xaa Xaa Xaa Leu Xaa Xaa Xaa 70 75 Xaa Xaa
Xaa Xaa Val Xaa Leu Xaa 80 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 85 90
Xaa Cys Xaa Cys Xaa 95 ______________________________________
wherein each Xaa is independently selected from a group of one or
more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile);
Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or
Ala); Xaa at res.7=(Asp, Glu or Lys); Xaa at res.8=(Leu, Val or
Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at
res.12=(Asp, Arg, Asn or Glu); Xaa at res.14=(Ile or Val); Xaa at
res.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at
res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or
Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp,
Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at
res.26=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.28=(Glu, Lys,
Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln or Asn); Xaa at
res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at
res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu,
Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at
res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa
at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser);
Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu or Ile);
Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at
res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at
res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val or Leu); Xaa at
res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or
Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val or Lys); Xaa at
res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at
res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at
res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro or Ala); Xaa at
res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at
res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa
at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser);
Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or
Pro); Xaa at res.72=(Val, Met or Ile); Xaa at res.74=(Tyr or Phe);
Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or
Leu); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser,
Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys);
Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val or Asn);
Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His or
Val); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln, Glu or
Pro); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr, Ala
or Ile); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at
res.93=(Ala, Gly, Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa
at res.97=(His or Arg).
______________________________________ Generic Sequence 6
______________________________________ Cys Xaa Xaa Xaa Xaa Leu Xaa
Xaa Xaa Phe 1 5 10 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 15 Xaa Xaa
Pro Xaa Xaa Xaa Xaa Ala 20 25 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 30 35
Xaa Pro Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Asn His Ala Xaa Xaa 45
50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys 60 65 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Leu Xaa
Xaa Xaa 75 80 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 85 Xaa Xaa Xaa Xaa
Met Xaa Val Xaa 90 95 Xaa Cys Xaa Cys Xaa 100
______________________________________
wherein each Xaa is independently selected from a group of one or
more specified amino acids as defined by the following: "Res."
means "residue" and Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa at
res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); Xaa at
res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at
res.7=(Tyr or Lys); Xaa at res.8=(Val or Ile); Xaa at res.9=(Ser,
Asp or Glu); Xaa at res.11=(Arg, Gln, Ser, Lys or Ala); Xaa at
res.12=(Asp, Glu, or Lys); Xaa at res.13=(Leu, Val or Ile); Xaa at
res.16=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.17=(Asp, Arg,
Asn or Glu); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or
Val); Xaa at res.21=(Ala or Ser); Xaa at res.23=(Glu, Gln, Leu,
Lys, Pro or Arg); Xaa at res.24=(Gly or Ser); Xaa at res.25=(Tyr or
Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Gln, Leu, or Gly);
Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp,
Gln or Ser); Xaa at res.33=Glu, Lys, Asp, Gln or Ala); Xaa at
res.35=(Ala, Ser, Pro, Gln or Asn); Xaa at res.36=(Phe, Leu or
Tyr); Xaa at res.38=(Leu, Val or Met); Xaa at res.39=(Asn, Asp,
Ala, Thr or Pro); Xaa at res.40=(Ser, Asp, Glu, Leu, Ala or Lys);
Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe,
Gly or Leu); Xaa at res.43=(Asn, Ser or Lys); Xaa at res.44=(Ala,
Ser, Gly or Pro); Xaa at res.45=(Thr, Leu or Ser); Xaa at
res.49=(Ile, Val or Thr); Xaa at res.50=(Val, Leu or Ile); Xaa at
res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at
res.53=(Leu or Ile); Xaa at res.54=(Val or Met); Xaa at
res.55=(His, Asn or Arg); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.57=(Ile, Met, Asn, Ala, Val or Leu); Xaa at
res.58=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.59=(Pro, Ser or
Val); Xaa at res.60=(Glu, Asp, Gly, Val or Lys); Xaa at
res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at
res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at
res.64=(Lys, Leu or Glu); Xaa at res.65=(Pro or Ala); Xaa at
res.68=(Ala or Val); Xaa at res.70=(Thr, Ala or Glu); Xaa at
res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa
at res.73=(Asn, Ser, Asp or Gly); Xaa at res.74=(Ala, Pro or Ser);
Xaa at res.75=(Ile, Thr, Val or Leu); Xaa at res.76=(Ser, Ala or
Pro); Xaa at res.77=(Val, Met or Ile); Xaa at res.79=(Tyr or Phe);
Xaa at res.80=(Phe, Tyr, Leu or His); Xaa at res.81=(Asp, Asn or
Leu); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser,
Gln, Asn, Tyr or Asp); Xaa at res.84=(Ser, Asn, Asp, Glu or Lys);
Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile, Val or Asn);
Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln, His or
Val); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln, Glu or
Pro); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr, Ala
or Ile); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at
res.98=(Ala, Gly, Glu or Ser); Xaa at res.100=(Gly or Ala); and Xaa
at res.102=(His or Arg).
Particularly useful sequences for use as morphogens in this
invention include the C-terminal domains, e.g., the C-terminal
96-102 amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A,
CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as
well as proteins comprising the C-terminal domains of 60A, BMP3,
BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at
least the conserved six or seven cysteine skeleton. In addition,
biosynthetic constructs designed from the generic sequences, such
as COP-1, 3-5, 7, 16, disclosed in U.S. Pat. No. 5,011,691, also
are useful. Other sequences include the inhibins/activin proteins
(see, for example, U.S. Pat. Nos. 4,968,590 and 5,011,691).
Accordingly, other useful sequences are those sharing at least 70%
amino acid sequence homology or "similarity", and preferably 80%
homology or similarity with any of the sequences above. These are
anticipated to include allelic and species variants and mutants,
and biosynthetic muteins, as well as novel members of this
morphogenic family of proteins. Particularly envisioned in the
family of related proteins are those proteins exhibiting
morphogenic activity and wherein the amino acid changes from the
preferred sequences include conservative changes, e.g., those as
defined by Dayoff et al., Atlas of Protein Sequence and Structure;
vol. 5, Suppl. 3, pp. 345-362, (M. O. Dayoff, ed., Nat'l BioMed.
Research Fdn., Washington, D.C. 1979). As used herein, potentially
useful sequences are aligned with a known morphogen sequence using
the method of Needleman et al. ((1970) J.Mol.Biol. 48: 443-453) and
identities calculated by the Align program (DNAstar, Inc.).
"Homology" or "similarity" as used herein includes allowed
conservative changes as defined by Dayoff et al.
The currently most preferred protein sequences useful as morphogens
in this invention include those having greater than 60% identity,
preferably greater than 65% identity, with the amino acid sequence
defining the conserved six cysteine skeleton of hOP1 (e.g.,
residues 43-139 of Seq. ID No. 5). These most preferred sequences
include both allelic and species variants of the OP-1 and OP-2
proteins, including the Drosophila 60A protein. Accordingly, in
another preferred aspect of the invention, useful morphogens
include active proteins comprising species of polypeptide chains
having the generic amino acid sequence herein referred to as "OPX",
which accommodates the homologies between the various identified
species of OP1 and OP2 (Seq. ID No. 29).
The morphogens useful in the methods, composition and devices of
this invention include proteins comprising any of the polypeptide
chains described above, whether isolated from naturally-occurring
sources, or produced by recombinant DNA or other synthetic
techniques, and includes allelic and species variants of these
proteins, naturally-occurring or biosynthetic mutants thereof, as
well as various truncated and fusion constructs. Deletion or
addition mutants also are envisioned to be active, including those
which may alter the conserved C-terminal cysteine skeleton,
provided that the alteration does not functionally disrupt the
relationship of these cysteines in the folded structure.
Accordingly, such active forms are considered the equivalent of the
specifically described constructs disclosed herein. The proteins
may include forms having varying glycosylation patterns, varying
N-termini, a family of related proteins having regions of amino
acid sequence homology, and active truncated or mutated forms of
native or biosynthetic proteins, produced by expression of
recombinant DNA in host cells.
The morphogenic proteins can be expressed from intact or truncated
cDNA or from synthetic DNAs in procaryotic or eucaryotic host
cells, and purified, cleaved, refolded, and dimerized to form
morphogenically active compositions. Currently preferred host cells
include E. coli or mammalian cells, such as CHO, COS or BSC cells.
A detailed description of the morphogens useful in the methods,
compositions and devices of this invention is disclosed in
copending U.S. patent application Ser. No. 752,764, filed Aug. 30,
1991, and Ser. No. 667,274, filed Mar. 11, 1991, the disclosure of
which are incorporated herein by reference.
Thus, in view of this disclosure, skilled genetic engineers can
isolate genes from cDNA or genomic libraries of various different
species which encode appropriate amino acid sequences, or construct
DNAs from oligonucleotides, and then can express them in various
types of host cells, including both procaryotes and eucaryotes, to
produce large quantities of active proteins useful as dietary
compositions for enhancing tissue morphogenesis, including
enhancing tissue development and tissue viability in a variety of
mammals, including humans.
The foregoing and other objects, features and advantages of the
present invention will be made more apparent from the following
detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects and features of this invention, as
well as the invention itself, may be more fully understood from the
following description, when read together with the accompanying
drawings, in which:
FIG. 1A and B shows relative amounts of protein present in mammary
gland extract eluate fractions of a C-18 reverse phase
chromatography column (A), and the corresponding results of a
Western Blot (B);
FIG. 2A and B shows relative amounts of protein present in bovine
colostrum eluate fractions from purification scheme A of a C-18
reverse phase chromatography column (A), and the corresponding
results of a Western blot under reduced (1) and oxidized (2)
conditions (B);
FIG. 3A and B shows relative amounts of protein present in bovine
colostrum eluate fractions from purification scheme B of a C-18
reverse phase chromatography column (A), and the corresponding
results of a Western Blot under reduced conditions (B);
FIG. 4A and B shows relative amounts of protein present in bovine
57 day milk eluate fractions of a C-18 reverse phase chromatography
column (A), and the corresponding results of a Western Blot under
reduced (1) and oxidized (2) conditions (B);
FIG. 5 shows Western Blot analysis of bovine colostrum using OP-1
and BMP2-specific antibodies;
FIG. 6A and B show results of in vivo and in vitro activity assays,
respectively, for the corresponding fractions shown in FIG. 1;
FIG. 7 is a photomicrograph of an immunoblot showing the presence
of hOP-1 in serum; and
FIG. 8A is a dose response curve for the induction of the 180 kDa
and 140 kDa N-CAM isoforms in morphogen-treated NG108-15 cells;
FIG. 8B is a photomicrograph of a Western blot of whole cell
extracts from morphogen-treated NG108-15 cells with an
N-CAM-specific antibody; and
FIG. 9 (A and B) are photographs showing the effect of
morphogen-specific antibody on mouse development (9B) compared to
untreated, control mice (9A).
DETAILED DESCRIPTION OF THE INVENTION
It now has been discovered that the proteins described herein are
found in nursing mother's milk and are useful as components of a
dietary composition for enhancing tissue morphogenesis in a mammal,
particularly in an individual at risk for normal tissue development
and viability. As described herein, these proteins ("morphogens")
are capable of enhancing tissue development in growing mammals,
stimulating CAM expression and maintaining the normal tissue
function in adult tissue.
Provided below are detailed descriptions of suitable morphogens
useful in the compositions and methods of this invention, as well
as methods for their administration and application, and numerous,
nonlimiting examples which demonstrate the suitability of the
morphogens described herein as active components of a dietary
composition for a mammal; and 2) provide assays with which to test
candidate morphogens for their efficacy. Specifically, examples are
provided which (1) demonstrate the presence of endogenous morphogen
in milk and human serum (Examples 1 and 2), (2) demonstrate the
ability of morphogens to induce CAM expression in a mammal (Example
3), (3) demonstrate the ability of morphogens to enhance tissue
development in developing embryos (Example 4) and juveniles
(Example 5); (4) demonstrate the ability of morphogens to reduce an
osteoporotic condition in a mammal (Example 6); (5) demonstrate the
presence of morphogens in developing tissues and adult stomach and
gut tissue, demonstrate the ability of parenterally provided
morphogen to localize to stomach tissue, and describe protocols for
identifying morphogen-synthesizing tissue (Example 7) and (6)
describe protocols for obtaining morphogen-specific antibodies and
measuring morphogens in solution (Example 8).
I. Useful Morphogens
As defined herein a protein is morphogenic if it is capable of
inducing the developmental cascade of cellular and molecular events
that culminate in the formation of new, organ-specific tissue and
comprises at least the conserved C-terminal six cysteine skeleton
or its functional equivalent (see supra). Specifically, the
morphogens generally are capable of all of the following biological
functions in a morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the differentiation
of progenitor cells; stimulating the proliferation of
differentiated cells; and supporting the growth and maintenance of
differentiated cells, including the "redifferentiation" of
transformed cells. Details of how the morphogens useful in the
method of this invention first were identified, as well as a
description on how to make, use and test them for morphogenic
activity are disclosed in U.S. Ser. No. 667,274 abandoned in favor
of U.S. Ser. No. 752,764 (currently pending as U.S. Ser. No.
08/404,113), the disclosures of which
A candidate morphogen or morphogen composition can be evaluated for
in vivo morphogenic utility generally according to the procedures
set forth in U.S. Ser. No. 08/404,113. The proteins and
compositions may be injected or surgically implanted in a mammal,
following any of a number of procedures well known in the art. For
example, surgical implant bioassays may be performed essentially
following the procedure of Sampath et al. (1983) PNAS 80:
6591-6595.
Histological sectioning and staining is preferred to determine the
extent of morphogenesis in vivo, particularly in tissue repair
procedures. Excised implants are fixed in Bouins Solution, embedded
in paraffin, and cut into 6-8 .mu.m sections. Staining with
toluidine blue or hemotoxylin/eosin demonstrates clearly the
ultimate development of the new tissue. Twelve day implants are
usually sufficient to determine whether the implants contain newly
induced tissue.
Successful implants exhibit a controlled progression through the
stages of induced tissue development allowing one to identify and
follow the tissue-specific events that occur. For example, in
endochondral bone formation the stages include:
(1) leukocytes on day one;
(2) mesenchymal cell migration and proliferation on days two and
three;
(3) chondrocyte appearance on days five and six;
(4) cartilage matrix formation on day seven;
(5) cartilage calcification on day eight;
(6) vascular invasion, appearance of osteoblasts, and formation of
new bone on days nine and ten;
(7) appearance of osteoblastic and bone remodeling and dissolution
of the implanted matrix on days twelve to eighteen; and
(8) hematopoietic bone marrow differentiation in the ossicle on day
twenty-one.
In addition to histological evaluation, biological markers may be
used as a marker for tissue morphogenesis. Useful markers include
tissue-specific enzymes whose activity may be assayed (e.g.,
spectrophotometrically) after homogenization of the implant. These
assays may be useful for quantitation and for obtaining an estimate
of tissue formation quickly after the implants are removed from the
animal. For example, alkaline phosphatase activity may be used as a
marker for osteogenesis.
Incorporation of systemically provided morphogens may be followed
using tagged morphogens (e.g., radioactively labelled) and
determining their localization in new tissue, and/or by monitoring
their disappearance from the circulatory system using a standard
pulse-chase labeling protocol. The morphogen also may be provided
with a tissue-specific molecular tag, whose uptake may be monitored
and correlated with the concentration of morphogen provided. As an
example, ovary removal in female rats results in reduced bone
alkaline phosphatase activity, rendering the rats predisposed to
osteoporosis. If the female rats now are provided with a morphogen,
e.g., OP-1, a reduction in the systemic concentration of calcium
(CA.sup.2+) is seen, which correlates with the presence of the
provided morphogen and can be shown to correspond to increased
alkaline phosphatase activity.
The morphogen to be assayed according to the above-described
exemplary procedures can be purified from naturally-sourced
material, or can be recombinantly produced from procaryotic or
eucaryotic host cells, into which genetic material encoding a
morphogen, e.g., genetic material bearing one of the nucleic acid
sequences disclosed herein, has been introduced. Alternatively, the
above-described exemplary procedures can be used to determine
whether a novel protein suspected of being a morphogen indeed has
morphogenic activity.
Particularly useful proteins include those which comprise the
naturally derived sequences disclosed in Table II. Other useful
sequences include biosynthetic constructs such as those disclosed
in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated
herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and
COP-16).
Accordingly, the morphogens useful in the methods and compositions
of this invention also may be described by morphogenically active
proteins having amino acid sequences sharing 70% or, preferably,
80% homology (similarity) with any of the sequences described
above, where "homology" is as defined herein above.
The morphogens useful in the method of this invention also can be
described by any of the 6 generic sequences described herein
(Generic Sequences 1, 2, 3, 4, 5 and 6). Generic sequences 1 and 2
also may include, at their N-terminus, the sequence
______________________________________ Cys Xaa Xaa Xaa Xaa (Seq. ID
No. 15) 1 5 ______________________________________
Table II, set forth below, compares the amino acid sequences of the
active regions of native proteins that have been id entified as
morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17),
mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2
(Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), CBMP2B
(Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP (from Drosophila, Seq.
ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse,
Seq. ID No. 13), GDF-1 (from mouse, Seq. ID Nos. 14, 32 and 33),
60A protein (from Drosophila, Seq. ID Nos. 24 and 25), BMP5 (Seq.
ID No. 27) and BMP6 (Seq. ID No. 28). The sequences are aligned
essentially following the method of Needleman et al. (1970) J. Mol.
Biol., 48: 443-453, calculated using the Align Program (DNAstar,
Inc.) In the table, three dots indicates that the amino acid in
that position is the same as the amino acid in hOP-1. Three dashes
indicates that no amino acid is present in that position, and are
included for purposes of illustrating homologies. For example,
amino acid residue 60 of CBMP-2A and CBMP-2B is "missing". Of
course, both these amino acid sequences in this region comprise
Asn-Ser (residues 58, 59), with CBMP-2A then comprising Lys and
Ile, whereas CBMP-2B comprises Ser and Ile.
TABLE II
__________________________________________________________________________
hOP-1 Cys Lys Lys His Glu Leu Tyr Val mOP-1 . . . . . . . . . . . .
. . . . . . . . . . . . hOP-2 . . . Arg Arg . . . . . . . . . . . .
. . . mOP-2 . . . Arg Arg . . . . . . . . . . . . . . . DPP . . .
Arg Arg . . . Ser . . . . . . . . . Vgl . . . . . . Lys Arg His . .
. . . . . . . Vgr-1 . . . . . . . . . . . . Gly . . . . . . . . .
CBMP-2A . . . . . . Arg . . . Pro . . . . . . . . . CBMP-2B . . .
Arg Arg . . . Ser . . . . . . . . . BHP3 . . . Ala Arg Arg Tyr . .
. Lys . . . GDF-1 . . . Arg Ala Arg Arg . . . . . . . . . 60A . . .
Gln Met Glu Thr . . . . . . . . . BHP5 . . . . . . . . . . . . . .
. . . . . . . . . . BMP6 . . . Arg . . . . . . . . . . . . . . . .
. . 1 5 hOP-1 Ser Phe Arg Asp Leu Gly Trp Gln Asp mOP-1 . . . . . .
. . . . . . . . . . . . . . . . . . . . . hOP-2 . . . . . . Gln . .
. . . . . . . . . . Leu . . . mOP-2 Ser . . . . . . . . . . . . . .
. . . . Leu . . . DPP Asp . . . Ser . . . Val . . . . . . Asp . . .
Vgl Glu . . . Lys . . . Val . . . . . . . . . Asn Vgr-1 . . . . . .
Gln . . . Val . . . . . . . . . . . . CBMP-2A Asp . . . Ser . . .
Val . . . . . . Asn . . . CBMP-2B Asp . . . Ser . . . Val . . . . .
. Asn . . . BHP3 Asp . . . Ala . . . Ile . . . . . . Ser Glu GDF-1
. . . . . . . . . Glu Val . . . . . . His Arg 60A Asp . . . Lys . .
. . . . . . . . . . His . . . BHP5 . . . . . . . . . . . . . . . .
. . . . . . . . . . . BHP6 . . . . . . Gln . . . . . . . . . . . .
. . . . . . 10 15 hOP-1 Trp Ile Ile Ala Pro Glu Gly Tyr Ala mOP-1 .
. . . . . . . . . . . . . . . . . . . . . . . . . . hOP-2 . . . Val
. . . . . . . . . Gln . . . . . . Ser mOP-2 . . . Val . . . . . . .
. . Gln . . . . . . Ser DPP . . . . . . Val . . . . . . Leu . . . .
. . Asp Vgl . . . Val . . . . . . . . . Gln . . . . . . Met Vgr-1 .
. . . . . . . . . . . . . . Lys . . . . . . . . . CBMP-2A . . . . .
. Val . . . . . . Pro . . . . . . His CBMP-2B
. . . . . . Val . . . . . . Pro . . . . . . Gln BHP3 . . . . . . .
. . Ser . . . Lys Ser Phe Asp GDF-1 . . . Val . . . . . . . . . Arg
. . . Phe Leu 60A . . . . . . . . . . . . . . . . . . . . . . . .
Gly BHP5 . . . . . . . . . . . . . . . . . . . . . . . . . . . BHP6
. . . . . . . . . . . . . . . Lys . . . . . . . . . 20 25 hOP-1 Ala
Tyr Tyr Cys Glu Gly Glu Cys Ala mOP-1 . . . . . . . . . . . . . . .
. . . . . . . . . . . . hOP-2 . . . . . . . . . . . . . . . . . . .
. . . . . Ser mOP-2 . . . . . . . . . . . . . . . . . . . . . . . .
. . . DPP . . . . . . . . . . . . His . . . Lys . . . Pro Vgl . . .
Asn . . . . . . Tyr . . . . . . . . . Pro Vgr-1 . . . Asn . . . . .
. Asp . . . . . . . . . Ser CBMP-2A . . . Phe . . . . . . His . . .
Glu . . . Pro CBMP-2B . . . Phe . . . . . . His . . . Asp . . . Pro
BMP3 . . . . . . . . . . . . Ser . . . Ala . . . Gln GDF-1 . . .
Asn . . . . . . Gln . . . Gln . . . . . . 60A . . . Phe . . . . . .
Ser . . . . . . . . . Asn BMP5 . . . Phe . . . . . . Asp . . . . .
. . . . Ser BMP6 . . . Asn . . . . . . Asp . . . . . . . . . Ser 30
35 hOP-1 Phe Pro Leu Asn Ser Tyr Met Asn Ala mOP-1 . . . . . . . .
. . . . . . . . . . . . . . . . . . . hOP-2 . . . . . . . . . Asp .
. . Cys . . . . . . . . . mOP-2 . . . . . . . . . Asp . . . Cys . .
. . . . . . . DPP . . . . . . . . . Ala Asp His Phe . . . Ser Vgl
Tyr . . . . . . Thr Glu Ile Leu . . . Gly Vgr-1 . . . . . . . . . .
. . Ala His . . . . . . . . . CBMP-2A . . . . . . . . . Ala Asp His
Leu . . . Ser CBMP-2B . . . . . . . . . Ala Asp His Leu . . . Ser
GDF-1 Leu . . . Val Ala Leu Ser Gly Ser** . . . BMP3 . . . . . .
Met Pro Lys Ser Leu Lys Pro 60A . . . . . . . . . . . . Ala His . .
. . . . . . . BMP5 . . . . . . . . . . . . Ala His Met . . . . . .
BMP6 . . . . . . . . . . . . Ala His Met . . . . . . 40 hOP-1 Thr
Asn His Ala Ile Val Gln Thr Leu mOP-1
. . . . . . . . . . . . . . . . . . . . . . . . . . . hOP-2 . . . .
. . . . . . . . . . . Leu . . . Ser . . . mOP-2 . . . . . . . . . .
. . . . . Leu . . . Ser . . . DPP . . . . . . . . . . . . Val . . .
. . . . . . . . . Vgl Ser . . . . . . . . . . . . Leu . . . . . . .
. . Vgr-1 . . . . . . . . . . . . . . . . . . . . . . . . . . .
CBMP-2A . . . . . . . . . . . . . . . . . . . . . . . . . . .
CBMP-2B . . . . . . . . . . . . . . . . . . . . . . . . . . . BHP3
Ser . . . . . . . . . Thr Ile . . . Ser Ile GDF-1 Leu . . . . . . .
. . Val Leu Arg Ala . . . 60A . . . . . . . . . . . . . . . . . . .
. . . . . . . . BHP5 . . . . . . . . . . . . . . . . . . . . . . .
. . . . BHP6 . . . . . . . . . . . . . . . . . . . . . . . . . . .
45 50 hOP-1 Val His Phe Ile Asn Pro Glu Thr Val mOP-1 . . . . . . .
. . . . . . . . . . . Asp . . . . . . hOP-2 . . . His Leu Met Lys .
. . Asn Ala . . . mOP-2 . . . His Leu Met Lys . . . Asp Val . . .
DPP . . . Asn Asn Asn . . . . . . Gly Lys . . . Vgl . . . . . . Ser
. . . Glu . . . . . . Asp Ile Vgr-1 . . . . . . Val Met . . . . . .
. . . Tyr . . . CBMP-2A . . . Asn Ser Val . . . Ser -- Lys Ile
CBMP-2B . . . Asn Ser Val . . . Ser -- Ser Ile BHP3 . . . Arg Ala**
Gly Val Val Pro Gly Ile GDF-1 Met . . . Ala Ala Ala . . . Gly Ala
Ala 60A . . . . . . Leu Leu Glu . . . Lys Lys . . . BHP5 . . . . .
. Leu Met Phe . . . Asp His . . . BHP6 . . . . . . Leu Met . . . .
. . . . . Tyr . . . 55 60 hOP-1 Pro Lys Pro Cys Cys Ala Pro Thr Gln
mOP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . hOP-2 .
. . . . . Ala . . . . . . . . . . . . . . . Lys mOP-2 . . . . . .
Ala . . . . . . . . . . . . . . . Lys DPP . . . . . . Ala . . . . .
. Val . . . . . . . . . Vgl . . . Leu . . . . . . . . . Val . . . .
. . Lys Vgr-1 . . . . . . . . . . . . . . . . . . . . . . . . Lys
CBMP-2A . . . . . . Ala . . . . . . Val . . . . . . Glu CBMP-2B . .
. . . . Ala . . . . . . Val . . . . . . Glu BMP3 . . .
Glu . . . . . . . . . Val . . . Glu Lys GDF-1 Asp Leu . . . . . . .
. . Val . . . Ala Arg 60A . . . . . . . . . . . . . . . . . . . . .
. . . Arg BHP5 . . . . . . . . . . . . . . . . . . . . . . . . Lys
BMP6 . . . . . . . . . . . . . . . . . . . . . . . . Lys 65 70
hOP-1 Leu Asn Ala Ile Ser Val Leu Tyr Phe mOP-1 . . . . . . . . . .
. . . . . . . . . . . . . . . . . hOP-2 . . . Ser . . . Thr . . . .
. . . . . . . . Tyr mOP-2 . . . Ser . . . Thr . . . . . . . . . . .
. Tyr Vgl Met Ser Pro . . . . . . Met . . . Phe Tyr Vgr-1 Val . . .
. . . . . . . . . . . . . . . . . . . . . DPP . . . Asp Ser Val Ala
Met . . . . . . Leu CBMP-2A . . . Ser . . . . . . . . . Met . . . .
. . Leu CBMP-2B . . . Ser . . . . . . . . . Met . . . . . . Leu
BMP3 Met Ser Ser Leu . . . Ile . . . Phe Tyr GDF-1 . . . Ser Pro .
. . . . . . . . . . . Phe . . . 60A . . . Gly . . . Leu Pro . . . .
. . . . . His BMP5 . . . . . . . . . . . . . . . . . . . . . . . .
. . . BMP6 . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
80 hOP-1 Asp Asp Ser Ser Asn Val Ile Leu Lys mOP-1 . . . . . . . .
. . . . . . . . . . . . . . . . . . . hOP-2 . . . Ser . . . Asn . .
. . . . . . . . . . Arg mOP-2 . . . Ser . . . Asn . . . . . . . . .
. . . Arg DPP Asn . . . Gln . . . Thr . . . Val . . . . . . Vgl . .
. Asn Asn Asp . . . . . . Val . . . Arg Vgr-1 . . . . . . Asn . . .
. . . . . . . . . . . . . . . CBMP-2A . . . Glu Asn Glu Lys . . .
Val . . . . . . CBMP-2B . . . Glu Tyr Asp Lys . . . Val . . . . . .
BHP3 . . . Glu Asn Lys . . . . . . Val . . . . . . GDF-1 . . . Asn
. . . Asp . . . . . . Val . . . Arg 60A Leu Asn Asp Glu . . . . . .
Asn . . . . . . BMP5 . . . . . . . . . . . . . . . . . . . . . . .
. . . . BHP6 . . . . . . Asn . . . . . . . . . . . . . . . . . . 85
hOP-1 Lys Tyr Arg Asn Met Val Val Arg mOP-1 . . . . . . . . . . . .
. . .
. . . . . . . . . hOP-2 . . . His . . . . . . . . . . . . . . . Lys
mOP-2 . . . His . . . . . . . . . . . . . . . Lys DPP Asn . . . Gln
Glu . . . Thr . . . Val Vgl His . . . Glu . . . . . . Ala . . . Asp
Vgr-1 . . . . . . . . . . . . . . . . . . . . . . . . CBMP-2A Asn .
. . Gln Asp . . . . . . . . . Glu CBMP-2B Asn . . . Gln Glu . . . .
. . . . . Glu BHP3 Val . . . Pro . . . . . . Thr . . . Glu GDF-1
Gln . . . Glu Asp . . . . . . . . . Asp 60A . . . . . . . . . . . .
. . . Ile . . . Lys BHP5 . . . . . . . . . . . . . . . . . . . . .
. . . BMP6 . . . . . . . . . Trp . . . . . . . . . . . . 90 95
hOP-1 Ala Cys Gly Cys His mOP-1 . . . . . . . . . . . . . . . hOP-2
. . . . . . . . . . . . . . . mOP-2 . . . . . . . . . . . . . . .
DPP Gly . . . . . . . . . Arg Vgl Glu . . . . . . . . . Arg Vgr-1 .
. . . . . . . . . . . . . . CBMP-2A Gly . . . . . . . . . Arg
CBMP-2B Gly . . . . . . . . . Arg BHP3 Ser . . . Ala . . . Arg
GDF-1 Glu . . . . . . . . . Arg 60A Ser . . . . . . . . . . . .
BMP5 Ser . . . . . . . . . . . . BMP6 . . . . . . . . . . . . . . .
100
__________________________________________________________________________
**Between residues 56 and 57 of BMP3 is a Val residue; between
residues 4 and 44 of GDF1 lies the amino acid sequence
GlyGly-Pro-Pro.
As is apparent from the foregoing amino acid sequence comparisons,
significant amino acid changes can be made within the generic
sequences while retaining the morphogenic activity. For example,
while the GDF-1 protein sequence depicted in Table II shares only
about 50% amino acid identity with the hOP1 sequence described
therein, the GDF-1 sequence shares greater than 70% amino acid
sequence homology (or "similarity") with the hOP1 sequence, where
"homology" or "similarity" includes allowed conservative amino acid
changes within the sequence as defined by Dayoff, et al., Atlas of
Protein Sequence and Structure vol.5, supp.3, pp.345-362, (M. O.
Dayoff, ed., Nat'l BioMed. Res. Fd'n, Washington D.C. 1979.)
The currently most preferred protein sequences useful as morphogens
in this invention include those having greater than 60% identity,
preferably greater than 65% identity, with the amino acid sequence
defining the conserved six cysteine skeleton of hOP1 (e.g.,
residues 43-139 of Seq. ID No. 5). These most preferred sequences
include both allelic and species variants of the OP-1 and OP-2
proteins, including the Drosophila 60A protein. Accordingly, in
still another preferred aspect, the invention includes morphogens
comprising species of polypeptide chains having the generic amino
acid sequence referred to herein as "OPX", which defines the seven
cysteine skeleton and accommodates the identities between the
various identified mouse and human OP1 and OP2 proteins. OPX is
presented in Seq. ID No. 29. As described therein, each Xaa at a
given position independently is selected from the residues
occurring at the corresponding position in the C-terminal sequence
of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or Seq. ID
Nos. 16-23).
II. Formulations and Methods for Administering Therapeutic
Agents
A. General Considerations
The morphogens may be provided to an individual by any suitable
means, most preferably orally, or, alternatively, parenterally.
Where the morphogen is to be provided parenterally, such as
intravenously or by enteral feeding tube, the morphogen preferably
comprises part of an aqueous solution. The solution is
physiologically acceptable so that in addition to delivery of the
desired morphogen to the patient, the solution does not otherwise
adversely affect the patient's electrolyte and volume balance. The
aqueous medium for the morphogen thus may comprise normal
physiologic saline (0.85% NaCl, 0.15 M), pH 7-7.4. The aqueous
solution containing the morphogen can be made, for example, by
dissolving the protein in 50% ethanol containing acetonitrile in
0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent
solvents. One volume of the resultant solution then is added, for
example, to ten volumes of phosphate buffered saline (PBS), which
further may include 0.1-0.2% human serum albumin (HSA). The
resultant solution preferably is vortexed extensively. If desired,
a given morphogen may be made more soluble by association with a
suitable molecule. For example, the pro form of the morphogenic
protein comprises a species that is soluble in physiologically
buffered solutions. In fact, the endogenous protein is thought to
be transported (e.g., secreted and circulated) in this form. This
soluble form of the protein may be obtained from the culture medium
of morphogen-secreting mammalian cells. Alternatively, a soluble
species may be formulated by complexing the mature dimer (or an
active fragment thereof) with part or all of a pro domain. Another
molecule capable of enhancing solubility and particularly useful
for oral administrations, is casein, including salts, derivatives
and analogs thereof. For example, addition of 0.2% casein increases
solubility of the mature active form of OP-1 in physiologically
buffered solutions by 80%. Other components found in milk and/or
various serum proteins also may be useful.
Useful solutions for parenteral administration may be prepared by
any of the methods well known in the pharmaceutical art, described,
for example, in Remington's Pharmaceutical Sciences (Gennaro, A.,
ed.), Mack Pub., 1990. Formulations may include, for example,
polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, hydrogenated naphthalenes, and the like. Biocompatible,
preferably bioresorbable, polymers, including, for example,
hyaluronic acid, collagen, polybutyrate, tricalcium phosphate,
lactide and lactide/glycolide copolymers, may be useful excipients
to control the release of the morphogen in vivo.
As described above, the dietary supplements comprising the
morphogens described herein preferably are provided orally. Oral
administration of proteins as therapeutics generally is not
practiced as most proteins are readily degraded by digestive
enzymes and acids in the mammalian digestive system before they can
be absorbed into the bloodstream. However, the morphogens described
herein typically are acid stable and protease-resistant (see, for
example, U.S. Pat. No. 4,968,590.) In addition, at least one
morphogen, OP-1, has been identified in mammary gland extract,
colostrum and 57-day milk (see Example 1, below). Moreover, the
OP-1 purified from mammary gland extract is morphogenically active.
Specifically, this protein induces endochondral bone formation in
mammals when implanted subcutaneously in association with a
suitable matrix material, using a standard in vivo bone assay, such
as is disclosed in U.S. Pat. No. 4,968,590. Moreover, the morphogen
also is detected in the bloodstream (see Example 2, below). These
findings indicate that oral and parenteral administration are
viable means for administering morphogens to an individual. In
addition, while the mature forms of certain morphogens described
herein typically are sparingly soluble, the morphogen form found in
milk (and mammary gland extract and colostrum) is readily soluble,
probably by association of the mature, morphogenically active form
with part or all of the pro domain of the intact sequence and/or by
association with one or more milk components. Accordingly, the
compounds provided herein also may be associated with molecules
capable of enhancing their solubility in vitro or in vivo.
The dietary compositions for oral administration may be formulated
as a liquid, for example, as part of an aqueous medium as described
above for parenteral administration, and which further may contain
flavoring and coloring agents. The formulation also may be combined
with a beverage or may be provided in a syrup. The dietary
composition also may be provided as an aerosol for oral or nasal
administration. Formulations for inhalation administration contain
as excipients, for example, lactose, or may be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or oily solutions for administration
in the form of nasal drops, or as a gel to be applied intranasally.
Alternatively, the dietary composition may be provided as a solid,
for example as a tablet, capsule or lozenge. As for parenteral
administration, formulations for oral administration also may
include molecules to enhance a controlled release of the morphogen
in vivo.
As will be appreciated by those skilled in the art, the
concentration of the compounds described in a given dietary
supplement composition will vary depending upon a number of
factors, including the dosage number to be administered, the
chemical characteristics (e.g., hydrophobicity) of the compounds
employed, and the route of administration. The preferred dosage to
be administered also is likely to depend on such variables as the
type and extent of tissue development enhancement desired, the type
and extent of any tissue damage present to be repaired, the overall
health status of the particular individual, the relative biological
efficacy of the compound selected, the formulation of the compound
excipients, and its route of administration. In general terms, the
compounds of this invention may be provided in a formulation
containing about 0.001 to 10% w/v of morphogen to formulation.
Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body
weight per day; a preferred dose range is from about 0.1 .mu.g/kg
to 100 mg/kg of body weight per day. Optimally, the morphogen
dosage given is between 0.1-100 .mu.g of protein per kilogram
weight of the individual. No obvious morphogen induced pathological
lesions are induced when mature morphogen (e.g., OP-1, 20 .mu.g) is
administered daily to normal growing rats for 21 consecutive days.
Moreover, 10 .mu.g systemic injections of morphogen (e.g., OP-1)
injected daily for 10 days into normal newborn mice does not
produce any gross abnormalities.
In administering morphogens parenterally in the methods of the
present invention, preferably a large volume loading dose is used
at the start of the treatment. The treatment then is continued with
a maintenance dose. In all cases administration dosages then can be
monitored by measuring at intervals the levels of the morphogen in
the blood.
B. Considerations for Infant and Other Formulas
1. Infant Formulas
In all cases the morphogens of this invention preferably are added
to an infant formula that complies with the nutritional guidelines
provided by the AAP and ESPGAN. Basic ingredients for infant
formulas include cow's milk, protein, whey proteins, casein and its
salts (i.e. calcium caseinate). Soy protein isolates may be
substituted for milk-derived proteins, and preferably are used in
the products made for infants with lactose intolerance and/or cow's
protein intolerance. Protein hydrolyzates (i.e. casein and
lactalbumin hydrolyzates) with low molecular weight, also may be
used for these products.
The proportions of the diverse component nutrients preferably are
similar to those of human milk. Thus, the ratio of whey proteins to
casein preferably varies from 60:40 to 70:30 in infant formulas
based on milk. The mixture of fats employed is made up of edible
fats to provide an essential fatty acid profile. Lactose preferably
is used as the carbohydrate source for at-term newborns infants,
and dextrinmaltose preferably is employed in products used for the
treatment of lactose intolerance and malabsorption syndromes in
infancy.
Infant formulas according to the invention also preferably contain
minerals (including calcium, phosphorus, sodium, potassium,
chloride, magnesium, iron, zinc, copper, manganese and iodine) and
vitamins (including vitamin A, vitamin D3, vitamin C, vitamin B1,
vitamin B2, vitamin B6, vitamin B12, pantothenic acid, vitamin E,
vitamin K1, folic acid, biotin) adequate for the infants'
requirements. Also, in the products whose source of proteins is
derived from soy or protein isolates or hydrolyzates, carnitine
preferably is included to satisfy the nutritional requirements for
this compound in infants with malabsorptive syndromes.
A typical ready-to-feed morphogen-enriched formulation for infants,
when diluted to feeding concentrations, preferably comprises in
addition to the added morphogen, from about 1-5% by weight fat,
from about 0.01 to about 0.5% by weight immunoglobulins as
appropriate, from about 4-10% by weight carbohydrate in a quantity
substantially to mimic the carbohydrate content of human mother's
milk, from about 0.5 to 4% by weight protein in a quantity
substantially to mimic the protein content of human mother's milk,
optional vitamins and minerals as required, a total solids content
of from about 8 to 17% by weight, and the remainder water.
A typical protein source for use in infant formula is
electrodialyzed whey or electrodialyzed skim milk or milk whey,
although other protein sources are also available and may be used.
Preferred sugars include food grade substances such as glucose,
dextrose, sucrose, or edible lactose. The following vitamins and
minerals may also be incorporated in the infant formula: calcium,
phosphorus, potassium, sodium, chloride, magnesium, manganese,
iron, copper, zinc, selenium, iodine, and vitamins A, E, D, and B
complex. These micronutrients are added in the form of commonly
accepted nutritional compounds in amounts equivalent to those
present in human milk on a per calories basis.
The infant formula according to the present invention also
preferably is sterilized and subsequently used on a ready-to-feed
basis, or can be stored as a concentrate. The concentrate can be
prepared using standard procedures known in the art, and the
formula can be reconstituted by rehydrating the concentrate. The
infant formula preferably is a stable liquid and has a suitable
shelf life. A more detailed description of infant formula
considerations, including preferred formulations for newborn,
preterm and low birth-weight infants, lactose-intolerant infants,
may be found, for example, in U.S. Pat. No. 5,066,500 to Gil et
al., the disclosure of which is incorporated herein by
reference.
2. Other Nutritional Products
The morphogen-enriched dietary products for balanced nutrition
(e.g., dietary food formulations) according to the present
invention, preferably have, in addition to added morphogen, a
composition of nutrients adequate to the specific requirements of
not only healthy human in need of a balanced nutritional product,
but also those individuals at risk for lost or reduced tissue
function due malnutrition-maladsorption disorder, and/or altered
metabolism. Individuals particularly affected by an altered
metabolic function include postmenopausal women or aged
individuals, hypercatabolic individuals, and individuals undergoing
periods of rapid growth or physical stress, such as developing
juveniles, and pregnant, lactating and nursing mothers. Other
individuals at risk are those suffering from malnutrition, induced,
for example, by starvation and/or an eating disorder, and
individuals affected with energy-protein malnutrition and in
hypercatablic states derived from traumatic, septic, surgical
processes and other clinically-derived malabsorption syndromes.
Morphogen-enriched nutritional products according to the present
invention preferably also provide mineral elements which include
trace elements and vitamins in adequate proportions to satisfy the
specific requirements of normal healthy individuals as well as
individuals at risk, such as those suffering
malabsorption-malnutrition processes and in a hypercatabolic state.
The nutritional products also preferably are enriched with amino
acids sources, vitamins, nucleosides and/or nucleotides in similar
amounts to those present in ordinary foods.
As described above for infant formulas, liquid products may be
formulated ready for consumption or as concentrates to be diluted
before use. Preferably, liquid dietary compositions have pH values
generally ranging from about 6.0 to about 8.0, most preferably
6.8-7.5.
Useful dietary compositions and considerations for their
formulation are well described in the medical and nutritional arts.
Useful compositions for clinical nutrition, also are described in
detail in U.S. Pat. No. 5,066,500.
III. Examples
Example 1
Determination of the Presence of Morphogen in Milk
Morphogenically active OP-1 was demonstrated to be present in
mammary gland extract, colostrum, and milk, as described below. The
discovery that the morphogen naturally is present in milk, together
with the known observation that mature, active OP-1 is acid-stable
and protease-resistant, indicate that oral administration is a
useful route for therapeutic administration of morphogen to a
mammal. Oral administration typically is the preferred mode of
delivery for extended or prophylactic therapies. In addition, the
identification of morphogen in all milk forms, including colostrum,
indicates that the protein plays a significant role in tissue
development, including skeletal development of juveniles.
Rat mammary gland extract and bovine colostrum and 57 day milk were
subjected to purification procedures designed to partially purify
OP-1. The partially purified product then was examined for the
presence of OP-1 by Western blot analysis using OP-1-specific
antisera, and tested for in vivo and in vitro activity.
1.1 Purification
The purification protocol for all three "milk" forms (e.g., mammary
gland extract, colostrum and 57-day milk), involved three
chromatography steps: (1) cation-exchange chromatography
(S-Sepharose and followed by Phenyl-Sepharose chromatography); (2)
Copper-Immobilized Metal Affinity chromatography (Cu++-IMAC); and
finally, (3) C-18 reverse phase chromatography. Fractions were
sampled at each step for the presence of OP-1. Fraction samples for
testing were dialyzed versus water/0.1% TFA, then against 30%
acetonitrile/0.1% TFA for analysis on SDS-polyacrylamide gels and
immunoblots, using standard methodologies well described in the
art. Unless otherwise stated, the primary antibody used for the
immunoblots was made against full length OP-1 produced in E.coli
using standard recombinant DNA and antibody production techniques
(see, for example, Example 8, below for a general description for
producing morphogen-specific antibodies.) Fractions found to
contain the morphogen then were applied to the next column step or
used in the immunoreactivity or activity assays described
below.
Essentially the same protocol was followed for all three milk
sources, except that two alternative cation-exchange methodologies
were employed for colostrum purification, described in detail
below. Unless otherwise indicated, all chemicals referenced are
standard, commercially available reagents, readily available from a
number of sources, including Sigma Chemical, Co., St. Louis;
Calbiochem, Corp., San Diego and Aldrich Chemical Co.,
Milwaukee.
step 1. Cation-Exchange Chromatography
The S-Sepharose purification step was performed as follows. 200 ml
of cation exchanger (S-Sepharose, Sigma Chemical Corp.) were
equilibrated with equilibration buffer (6M urea, 20 mM MES, 70 mM
NaCl, pH 6.5). The supernatant from the centrifuged extract was
diluted to final concentration of 6M urea, 20 mM MES, 70 mM NaCl,
pH 6.5. After loading, the column was washed to baseline using
equilibration buffer, and the bound components were eluted stepwise
from the column with 6M urea, 20 mM MES, 100 mM and 500 mM NaCl, pH
6.5. The more tightly bound components then were eluted with 4M
guanidine, 20 mM sodium phosphate, pH 7.0.
The Phenyl-Sepharose purification step was performed as follows. 15
ml of Phenyl-Sepharose CL-4B (Sigma) were equilibrated with 6M
urea, 20 mM HEPES, 1M ammonium sulfate, 300 mM NaCl, pH 7.0. The
500 mM NaCl eluate from the S-Sepharose step was diluted with 6M
urea, 20 mM HEPES, 3M ammonium sulfate, 300 mM NaCl, pH 7.0, to a
final concentration of 1M ammonium sulfate, pH 7.0. After loading,
the column was washed to baseline with equilibration buffer. The
column was eluted with 6M urea, 20 mM HEPES, 0.6M ammonium sulfate,
300 mM NaCl, pH 7.0, and then with 4M guanidine, 20 mM sodium
phosphate, pH 7.0.
Two alternative cation-exchange chromatography schemes (A and B)
were exploited in the purification of OP-1 from colostrum, as
follows. For both schemes, 200 ml of S-Sepharose (Sigma) was poured
into a 5.times.10 cm Bio-Rad econocolumn (Bio-Rad, Inc.
Cambridge.)
Scheme A: The colostrum, which had been diluted to 6M urea, 20 mM
sodium phosphate, pH 7.0, was loaded onto a column equilibrated
with 6M urea, 20 mM sodium phosphate, 50 mM NaCl, pH 7.0. Elution
was stepwise, with 6M urea, 20 mM sodium phosphate, 100 mM and then
500 mM NaCl, pH 7.0; and the final wash was with 4M guanidine, 20
mM sodium phosphate, pH 7.0. The Phenyl-Sepharose column was run as
described above, except that sodium phosphate was used as the
running buffer instead of HEPES. The Phenyl-Sepharose bound
fraction (0.0M ammonium sulfate eluate) from scheme A then was
dialyzed into 6M urea, 20 mM Hepes, 500 mM NaCl, pH 7.0, before it
was applied to the Cu++-IMAC column, which was run as described
below.
Scheme B: The alternative S-Sepharose purification was performed as
follows. Ethanol-precipitated protein was loaded onto an
S-Sepharose column equilibrated in 6M urea, 20 mM MES, 50 mM NaCl,
pH 6.5. Elution was stepwise with 6M urea, 20 mM MES, 100 mM NaCl
and then 500 mM NaCl, and finally 4M guanidine, 20 mM sodium
phosphate, pH 7.0. The Phenyl-Sepharose column was run as described
above, with the 0.0M ammonium sulfate eluate then applied to a
Cu++-IMAC column.
step 2. Cu++IMAC Chromatography
The Cu++IMAC purification step was performed as follows. 10 ml of
Pharmacia Fast Flow Chelating Resin were charged with 0.2M cupric
sulfate, and equilibrated with 6M urea, 20 mM HEPES, 0.5M NaCl, pH
7.0. After loading, the column was washed to baseline with
equilibration buffer. Elution from the column was stepwise, using
equilibration buffer containing 1 mM, 5 mM, or 10 mM imidazole. The
column then was stripped with equilibration buffer containing 10 mM
EDTA. The 10 mM imidazole elution was dialyzed against water/0.1%
TFA, then against 30% acetonitirile/0.1% TFA.
step 3. Reverse Phase Chromatography
The C-18 reverse phase chromatography purification step was
performed as follows. An HPLC C-18 semi-prep column was used for
the final purification step. The gradient used was 30-50%
acetonitrile/0.1% TFA over 60 minutes at 3 ml/minutes. After the
sample was loaded, the column was washed to baseline with 30%
acetonitrile/0.1% TFA before the gradient is started. Fractions
collected were 3 ml in size. Chromatograms were read at 214 nm.
(a) OP-1 from Rat Mammary Gland Extract
Mammary glands were obtained from 2 female Long Evan rats (Charles
River Labs, Wilmington, Mass.) one week post-partum. The excised
glands were mildly homogenized in 6M urea, 20 mM
methylethansulfonate (MES), 0.5M NaCl, pH 6.5 using a polytron
homogenizer. The suspension then was centrifuged for 20 minutes at
8,000 RPM, and the supernatant removed for further
purification.
Following S-Sepharose chromatography, fractions containing 6M urea,
20 mM MES containing 500 mM NaCl, also appeared to contain OP-1 as
determined by SDS and immunoblot, and were applied to the
Phenyl-Sepharose column. The eluate from the 6M urea, 20 mM HEPES,
300 mM NaCl, pH 7.0 elution step from this column were found to
contain OP-1. This eluate then was applied to a Cu++-IMAC column.
Eluate fractions found to contain OP-1 were then applied to the
C-18 column and chromatographed as described.
FIG. 1(A) shows the chromatogram and 1(B) the corresponding Western
blot for fractions from the C-18 reverse phase chromatography step
run under reducing conditions. Lane S of the Western blot is a
standard, containing reduced, purified, recombinantly-produced
OP-1. The arrows show molecular weight markers corresponding to 17,
27, and 39 Kd. The reduced monomer run at approximately 16-18 Kd;
the oxidized homodimer at approximately 36 Kd. Lanes 13-30
represent the corresponding fractions of the C-18 reverse phase
column as numbered in FIG. 1(A). As can be seen in FIG. 1(B),
mammary extract OP-1 elutes primarily in fractions 21-25 from this
final chromatography step.
(b) OP-1 from Bovine Colostrum
Colostrum is the first milk to be produced by the mother following
birth. Approximately 5 gallons of bovine colostrum were obtained
from a local dairy farm and delipidated by centrifugation (8000 rpm
for approximately 10 min. at 4.degree. C.). The supernatant then
was filtered through cheese cloth. The filtered supernatant was
stored in 500 ml aliquots at 70.degree. C.
50 ml of colostrum were diluted with 100 ml of 9M urea, 30 mM
sodium phosphate, pH 7.0. Alternately, 50 ml of colostrum was added
to 50 ml of 8M guanidine-HCl, 50 mM Tris, pH 7.2 and precipitated
with 40%, then 85% ice cold ethanol. The pellet was washed with 90%
cold ethanol and lyophilized overnight. The lyophilized pellet was
resuspended in 6M urea, 20 mM MES, 500 mM NaCl, pH 6.5, stirred
overnight at 4.degree. C., and centrifuged at 9,000 RPM for 10
minutes to clarify the suspension before loading onto the column as
described in schemes A and B, above.
Following S-Sepharose chromatography by scheme A, both the 100 mM
and the 500 mM eluate fractions were found to contain OP-1, with
the 100 mM fraction containing relatively more morphogen. This
fraction then was loaded onto the Phenyl-Sepharose column following
dilution with an equal volume of 6M urea, 20 mM sodium phosphate,
2M ammonium sulfate, and 300 mM NaCl.
Following S-Sepharose chromatography by scheme B, the 500 mM NaCl
eluate was found to contain OP-1 and was loaded onto a
Phenyl-Sepharose column as described above, following dilution with
6M urea, 40 mM HEPES, 2M ammonium sulfate, pH 7.0.
Following Cu++IMAC chromatography OP-1 was identified in the 5 mM
and 10 mM imidazole eluates for both purification schemes, and was
dialyzed for further purification on the C-18 column.
Both purification schemes produce purified OP-1, as determined by
immunoblot. FIG. 2 shows the chromatogram (A) and corresponding
Western blot (B) for results of purification scheme A (FIG. 2B-1,
reduced and FIG. 2B-2, oxidized); and FIG. 3 shows the chromatogram
(A) and Western blot (B, reduced) for C-18-purified protein from
scheme B. As for FIG. 1B, lane S in FIGS. 2B and 3B is a standard,
containing purified, recombinantly produced OP-1; 17, 27 and 39 are
molecular weight markers, and lane numbers correspond to fraction
numbers in the corresponding chromatograms. OP-1 purified by scheme
A appears predominantly in fractions 18-27, and OP-1 purified by
scheme B appears predominantly in fractions 18-25.
OP-1 from 57-day milk
Milk was obtained from the same cow from which the colostrum came,
57 days after the birth of the calf. The milk was delipidated by
centrifugation at 10,000 RPM for 15 minutes, and the milk was
poured off and away from the fat layer.
100 ml of milk then were diluted with 200 ml of 9M urea, 30 mM MES,
pH 6.5 and loaded onto a 200 ml S-Sepharose column which had been
equilibrated with 6M urea, 20 mM MES, 50 mM NaCl, pH 6.5. Elution
was with 6M urea, 20 mM MES, 100 mM and 500 mM NaCl, and 4M
guanidine, 20 mM sodium phosphate, pH 7.0. The 500 mM elution was
put over a Phenyl-Sepharose column after being diluted with an
equal volume of 6M urea, 20 mM MES, 2M ammonium sulfate, pH
7.0.
The Phenyl-Sepharose column then was run as described above. The
Phenyl-Sepharose-bound sample was eluted and applied to a Cu++IMAC
column, prepared and run as described above. The 10 mM imidazole
eluate was found to contain OP-1 and was dialyzed for further
purification on the C-18 column.
The C-18 reverse phase chromatography column and gradient were
performed as described above. The results are presented in FIG. 4A
(chromatogram) and 4B (immunoblot, 10B-1, oxidized; 4B-2, reduced.)
As above, lane S is a standard, containing purified, recombinantly
produced OP-1; 17, 27, and 39 are molecular weight markers, and the
lane numbers correspond to the fractions numbers in FIG. 4A. OP-1
purified from 57-day milk appears predominantly in fractions
18-26.
1.2 OP-1 Characterization by immunoreactivity
OP-1 purified from the different milk sources as described above
also were characterized by Western blotting using antibodies raised
against OP-1 and BMP2. Antibodies were prepared using standard
immunology protocols well known in the art, and as described in
Example 8, below using full-length E. coli-produced OP-1 and BMP2
as the immunogens.
As shown in FIG. 5 OP-1 purified from colostrum reacts with the
anti-OP-1 antibody, but not with anti-BMP2 antibody. In FIG. 5A and
B, lane 1 contains reduced, purified, recombinantly-produced OP-1;
lane 2 contains C-18 purified bovine colostrum, and lane 3 contains
reduced COP-16, a biosynthetic construct having morphogenic
activity and an amino acid sequence modeled on the proteins
described herein, but having highest amino acid sequence homology
with BMP2 (see U.S. Pat. No. 5,011,691 for the COP-16 amino acid
sequence.) In FIG. 5A the gel was probed with anti-OP-1 antibody;
in FIG. 5B, the gel was probed with anti-BMP2 antibody. As can be
seen in the figure, anti-OP-1 antibody hybridizes with protein in
lanes 1 and 2, but not 3; while anti-BMP2 antibody hybridizes with
lane 3 only.
C-18 purified mammary gland extract and 57-day milk also were shown
to react with anti-OP-1 antibodies, including antibody raised
against the full length E. coli OP-1, full length
mammalian-produced OP-1, and the OP-1 Ser-17-Cys peptide (e.g., the
OP-1 N-terminal 17 amino acids).
1.3 OP-1 Characterization by Activity
The morphogenic activity of OP-1 purified from mammary gland
extract was evaluated in vivo as follows. 33% of each OP-1
immunoreactive fraction of C-18-purified mammary gland extract was
lyophilized and resuspended in 220 .mu.l of 50% acetonitrile/0.1%
TFA. After vortexing, 25 mg of collagen matrix was added. The
samples were lyophilized overnight, and implanted in Long Evans
rats (Charles River Laboratories, Wilmington, Mass., 28-35 days
old). Each fraction was implanted in duplicate. For details of the
collagen matrix implantation procedure, see, for example, U.S. Pat.
No. 4,968,590, hereby incorporated by reference. After 12 days, the
implants were removed and evaluated for new bone formation by
histological observation.
The results are presented in FIG. 6A, where "% activity" refers to
the % of bone formation/total area covered by bone in the histology
sample. In the figure, solid bars represent implants using mammary
extract-derived OP-1, where the fraction numbers correspond to the
related fractions eluted from the C-18 reverse phase column (see
FIG. 1B), and the hatched bar represents implants using
recombinantly produced OP-1 (600 ng). The results demonstrate that
the peak bone forming activity of C-18-purified mammary gland
extract corresponds with the immunoreactive fraction peaks of FIG.
1B (compare FIG. 6A and 1B.)
Similarly, the morphogenic activity of OP-1 purified from mammary
gland extract was evaluated in vitro by measuring alkaline
phosphatase activity in vitro using the following assay. Test
samples were prepared using 15-20% of individual immunoreactive
fractions from the C-18 run which were precipitated and resuspended
in a smaller volume of 50% acetonitrile/0.1% TFA. Alkaline
phosphatase activity was tested using ROS 17/2.8 cells (Rat
Osteosarcoma, e.g., obtained, for example, from Dr. Robert J.
Majeska, Mt. Sinai Medical Center, New York, N.Y., in a standard
alkaline phosphatase activity assay (see, for example, U.S. Pat.
No. 4,968,590). The results, presented in FIG. 6B, indicate that
the immunoreactive fractions obtained from C-18-purified mammary
gland extract correspond with alkaline phosphatase activity in
vitro (compare FIG. 6B and FIG. 1B.) In FIG. 6B solid bars
represent assays performed with mammary gland-purified OP-1, where
the fraction numbers correspond to the related fractions eluted
from the C-18 reverse phase column (see FIG. 1B), the hatched bar
represents the assay performed with purified,
recombinantly-produced OP-1 (100 ng/ml), and the cross-hatched bar
represents background. As for FIG. 6A, alkaline phosphatase
activity corresponds with immunoreactivity of the C-18-purified
extract (compare FIG. 6B and 1B.)
Example 2.
Morphogen Identification in Human Serum
OP-1 was detected in human serum using the following assay. A
monoclonal antibody raised against mammalian, recombinantly
produced OP-1 using standard immunology techniques well described
in the art and described generally in Example 8, was immobilized by
passing the antibody over an activated agarose gel (e.g.,
Affi-Gel.TM., from Bio-Rad Laboratories, Richmond, Calif., prepared
following manufacturer's instructions), and used to purify OP-1
from serum. Human serum then was passed over the column and eluted
with 3M K-thiocyanate. K-thiocyanante fractions then were dialyzed
in 6M urea, 20 mM PO.sub.4, pH 7.0, applied to a C8 HPLC column,
and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient.
Mature, recombinantly produced OP-1 homodimers elute between 20-22
minutes. Fractions then were collected and tested for the presence
of OP-1 by standard immunoblot. FIG. 7 is an immunoblot showing
OP-1 in human sera under reducing and oxidized conditions. In the
figure, lanes 1 and 4 are OP-1 standards, run under oxidized (lane
1) and reduced (lane 4) conditions. Lane 5 shows molecular weight
markers at 17, 27 and 39 kDa. Lanes 2 and 3 are human sera OP-1,
run under oxidized (lane 2) and reduced (lane 3) conditions.
Example 3.
Morphogen-Induced CAM Expression
The morphogens described herein induce CAM expression as part of
their induction of morphogenesis. CAMs are morphoregulatory
molecules identified in all tissues as an essential step in tissue
development. N-CAMs, which comprise at least 3 isoforms (N-CAM-180,
N-CAM-140 and N-CAM-120, where "180", "140" and "120" indicate the
apparent molecular weights of the isoforms as measured by
polyacrylamide gel electrophoresis) are expressed at least
transiently in developing tissues, and permanently in nerve tissue.
Both the N-CAM-180 and N-CAM-140 isoforms are expressed in both
developing and adult tissue. The N-CAM-120 isoform is found only in
adult tissue. Another neural CAM is L1.
CAMs are implicated in normal tissue development; N-CAMs are
implicated in appropriate neural development, including appropriate
neurulation, neuronal migration, fasciculation, and synaptogenesis.
Inhibition of N-CAM production, as by complexing the molecule with
an N-CAM-specific antibody, inhibits retina organization, including
retinal axon migration, and axon regeneration in the peripheral
nervous system, as well as axon synapses with target muscle cells.
CAMs also have been postulated as part of a morphoregulatory
pathway whose activity is induced by a to date unidentified
molecule (See, for example, Edelman, G. M. (1986) Ann. Rev. Cell
Biol. 2: 81-116). Without being limited to any given theory, the
morphogens described herein may act as the inducer of this
pathway.
The morphogens described herein can stimulate CAM production. As
described below, the morphogens stimulate L1 and N-CAM production,
including all three isoforms of the N-CAM molecule, in nerve
tissue. A detailed description of this protocol is described in
copending U.S. Ser. No. 922,813 the disclosure of which is
incorporated hereinabove by reference.
In this example NG108-15 cells were cultured for 4 days in the
presence of increasing concentrations of OP-1 and standard Western
blots performed on whole cells extracts. The NG108-15 cell line is
a hybrid cell line (neuroblastoma x glioma, American Type Culture
Collection, Rockville, Md.). N-CAM isoforms were detected with an
antibody which crossreacts with all three isoforms, mAb H28.123,
obtained from Sigma Chemical Co., St. Louis, the different isoforms
being distinguishable by their different mobilities on an
electrophoresis gel. Control NG108-15 cells (untreated) express
both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa, as
determined by western blot analyses using up to 100 .mu.g of
protein. As shown in FIG. 8, treatment of NG108-15 cells with OP-1
resulted in a dose-dependent increase in the expression of the 180
kDa and 140 kDa isoforms, as well as the induction of the 120 kDa
isoform. FIG. 8B is a Western blot of OP1-treated NG108-15 cell
extracts, probed with mAb H28.123, showing the induction of all
three isoforms. FIG. 8A is a dose response curve of N-CAM-180 and
N-CAM-140 induction as a function of morphogen concentration.
N-CAM-120 is not shown in the graph as it could not be quantitated
in control cells. However, as is clearly evident from the Western
blot in FIG. 8A, N-CAM-120 is induced in response to morphogen
treatment. The induction of the 120 isoform also indicates that
morphogen-induced redifferentiation of transformed cells stimulates
not only redifferentiation of these cells from a transformed
phenotype, but also differentiation to a phenotype corresponding to
a developed cell. The differential induction of N-CAM 180 and 140
isoforms seen may be because constitutive expression of the 140
isoform is close to maximum. In addition, the increase in N-CAM
expression corresponded in a dose-dependent manner with the
morphogen induction of multicellular aggregates.
In addition, the cell aggregation effects of OP-1 on NG108-15 cells
can be inhibited with anti-N-CAM antibodies or antisense N-CAM
oligonucleotides. Antisense oligonucleotides can be made
synthetically on a nucleotide synthesizer, using standard means
known in the art. Preferably, phosphorothioate oligonucleotides
("S-oligos") are prepared, to enhance transport of the nucleotides
across cell membranes. Concentrations of both N-CAM antibodies and
N-CAM antisense oligonucleotides sufficient to inhibit N-CAM
induction also inhibited formation of multilayered cell aggregates.
Specifically, incubation of morphogen-treated NG108-115 cells with
0.3-3 .mu.M N-CAM antisense S-oligos, 5-500 .mu.M unmodified N-CAM
antisense oligos, or 10 .mu.g/ml mAb H28.123 significantly inhibits
cell aggregation. It is likely that morphogen treatment also
stimulates other CAMs, as inhibition is not complete.
The experiments also have been performed with soluble morphogen
(e.g., mature OP-1 associated with its pro domain) which also
specifically induced CAM expression.
Example 4.
Effect of Morphogen Neutralization on Embryogenesis
As described in Example 7, below, at least one morphogen, OP2, is
found principally in early developing embryos (8-day embryos). As
described below, morphogen neutralization with morphogen-specific
antibodies inhibits embryogenesis.
Morphogen inhibition in developing embryos inhibits tissue and
organ development. Specifically, 9-day mouse embryo cells, cultured
in vitro under standard culturing conditions, were incubated in the
presence and absence of an OP-1-specific monoclonal antibody
prepared using recombinantly produced, purified mature OP-1 as the
immunogen. The antibody was prepared using standard antibody
production means well known in the art and essentially as described
for Example 9, below. After two days, the effect of the antibody on
the developing embryo was evaluated by histology using standard
histology procedures well known in the art. As determined by
histological examination, the OP-1-specific antibody specifically
inhibits eye lobe formation in the developing embryo. In
particular, the diencephalon outgrowth does not develop. In
addition, the heart is malformed. Moreover, in separate
immunolocalization studies on embryo sections with labelled OP-1
specific antibody, the OP-1-specific antibody localizes to neural
epithelia.
Similarly, morphogen activity may be demonstrated in fetal
development in the mouse model using the following assay. Single
lip injections comprising 10-100 .mu.g/injection of
morphogen-specific antibody are administered to pregnant female
mice during each day of the gestation period and tissue development
(e.g., bone development) in treated and control new mice evaluated
by standard histomorphometric analysis at birth.
Finally, stimulation of endogenous morphogen antibody production in
egg-laying hens interferes with shell formation in the developing
eggs.
All of these data demonstrate that inhibition of morphogen activity
significantly interferes with tissue development during
embryogenesis.
Example 5.
Effect of Morphogen Neutralization on Juvenile Tissue
Development
The effect of the morphogens described herein on tissue development
in developing mammals also may be demonstrated using neutralizing
antibodies specific for particular morphogens and assessing the
effect of these antibodies on tissue development as described
below. Specifically, anti-morphogen monoclonal and/or polyclonal
antibodies may be prepared using standard methodologies including,
for example, the protocol provided in Example 8, below, and
provided to juveniles to inhibit the activity of endogenous
morphogens.
Generally, purified antibodies are provided regularly to new born
mice, e.g., 10-100 .mu.g/injection/day for 10-15 days. At 10 or 21
days, the mice are sacrificed and the effect of morphogen on bone
development assessed by body weight, gross visual examination and
histology. In this example, anti-OP-1 antibodies were used in 10
.mu.g injections/day for 14 days, and the mice were sacrificed at
21 days. As is dramatically demonstrated in FIG. 9, mice treated
with OP-1 specific antibody show consistent and significant stunted
growth, including reduced body length and body weight, (9B) as
compared with untreated mice (9A). Histological examination showed
reduced bone growth as evidenced by reduced bone size in the
treated mice.
In a variation on this protocol, single lip injections also may be
provided to older juveniles and adult mice (e.g., 10-100 .mu.g)
over a prolonged time (e.g., 10-15 days) to evaluate the effect or
morphogen neutralization on bone growth and bone integrity and to
evaluate the onset of osteoporosis.
Example 6.
Morphogen Treatment of Osteoporosis
6.1 Effect of Morphogen on Trabecular Bone in Ovariectomized (OVX)
Rats
Aged individuals, and particularly postmenopausal women are
particularly at risk for osteoporosis. Provided below is an animal
osteoporosis model demonstrating the ability of morphogens to
substantially inhibit and/or reduce the tissue damage effects
associated with osteoporosis, wherein osteoporosis is induced by
ovary removal in rats. Bone growth is evaluated in these animals by
measuring serum alkaline phosphatase and osteocalcin levels in
treated and untreated rats.
Forty Long-Evans rats (Charles River Laboratories, Wilmington)
weighing about 200 g each are ovariectomized (OVX) using standard
surgical procedures, and ten rats are sham-operated. The
ovariectomization of the rats produces an osteoporotic condition
within the rats as a result of decreased estrogen production. Food
and water are provided ad libitum. Eight days after ovariectomy,
the rats, prepared as described above, were divided into five
groups: (A), 10 sham-operated rats; (B), 10 ovariectomized rats
receiving 1 ml of phosphate-buffered saline (PBS) i.v. in the tail
vein; (C) 10 ovariectomized rats receiving about 1 mg of
17.beta.E.sub.2 (17-.beta.-estradiol E.sub.2) by intravenous
injection through the tail vein; (D) 9 ovariectomized rats
receiving daily injections of approximately 2 .mu.g of morphogen by
tail vein for 22 days; and (E) 9 ovariectomized rats receiving
daily injections of approximately 20 .mu.g of morphogen by tail
vein for 22 days. In this example, OP-1 was the morphogen
tested.
On the 15th and 21st day of the study, each rat was injected with 5
mg of tetracycline, and on day 22, the rats were sacrificed. The
body weights, uterine weights, serum alkaline phosphate levels,
serum calcium levels and serum osteocalcin levels then were
determined for each rat. The results are shown in Tables III and
IV.
TABLE III ______________________________________ Body Weights,
Uterine Weights and Alkaline Phosphatase Body Weights Uterine
Weights Alk. Phosphatase Group (g) (g) (U/L)
______________________________________ A-SHAM 250.90 .+-. 17.04
0.4192 .+-. 0.10 43.25 .+-. 6.11 B-OVX + PBS 273.40 .+-. 16.81
0.1650 .+-. 0.04 56.22 .+-. 6.21 C-OVX + E2 241.66 .+-. 21.54
0.3081 .+-. 0.03 62.66 .+-. 4.11 D-OVX + OP-1 266.67 .+-. 10.43
0.1416 .+-. 0.03 58.09 .+-. 12.97 (2 .mu.g) E-OVX + OP-1 272.40
.+-. 20.48 0.1481 .+-. 0.05 66.24 .+-. 15.74 (20 .mu.g)
______________________________________
TABLE IV ______________________________________ Serum Calcium and
Serum Osteocalcin Levels Serum Calcium Serum Osteocalcin Group
(ng/dl) (ng/ml) ______________________________________ A-SHAM 8.82
.+-. 1.65 64.66 .+-. 14.77 B-OVX + PBS 8.95 .+-. 1.25 69.01 .+-.
10.20 C-OVX + E2 9.20 .+-. 1.39 67.13 .+-. 17.33 D-OVX + OP-1 8.77
.+-. 0.95 148.50 .+-. 84.11 (2 .mu.g) E-OVX + OP-1 8.67 .+-. 1.94
182.42 .+-. 52.11 (20 .mu.g)
______________________________________
The results presented in Table III and IV show that intravenous
injection of morphogen into ovariectomized rats produces a
significant increase in serum alkaline phosphatase and serum
osteocalcin levels and demonstrates that systemic administration of
the morphogen stimulates bone formation in osteoporotic bone.
6.2 Histomorphometric Analysis of Morphogen on the Tibia Diaphysis
in Ovariectomized(OVX) Rats
Fifteen female Long-Evans rats weighing about 160 g were
ovariectomized (OVX) to produce an osteoporotic condition and five
rats were sham operated (Charles River Laboratories, Wilmington,
Mass.) as described for Example 8. Food and water were provided ad
libitum. Twenty-two days after ovariectomy, the rats were divided
into four groups: (A) sham-operated (1 ml of PBS by intravenous
injection through tail vein (5 rats); (B) OVX, into which nothing
was injected (5 rats); (C) OVX, receiving about 1 mg of
17.beta.E.sub.2 by intravenous injection through the tail vein (5
rats), and (D) OVX, receiving about 1 .beta.g of morphogen by
intravenous injection through the tail vein (5 rats). In this
example, OP-1 was morphogen tested.
The rats were injected daily as described for seven days, except no
injections were given on the thirteenth day. The rats then were
sacrificed on the nineteenth day. The tibial diaphyseal long bones
then were removed and fixed in ethanol and histomorphometric
analysis was carried out using standard procedures well known in
the art. The results are shown in Table V.
TABLE V
__________________________________________________________________________
(A) (B) (C) (D) MEASUREMENT CONTROL OVX OVX + E.sub.2 OVX + OP-1
__________________________________________________________________________
Longitudinal Growth 20.2 .+-. 0.3 19.4 .+-. 0.2 4.9 .+-. 0.5 17.9
.div. 0.9 Rate (.mu.m/day) Cancellous Bone 20.2 .+-. 1.5 13.0 .+-.
1.6 13.7 .+-. 2.1 16.6 .+-. 1.8 Volume (BV/TV, bone vol/total vol)
Cancellous Bone 16.2 .+-. 1.8 9.6 .+-. 0.9 11.5 .+-. 1.1 12.2 .+-.
0.7 Perimeter (mm) Labeled Cancellous 35.5 .+-. 1.5 51.9 .+-. 5.6
58.0 .+-. 4.2 39.2 .+-. 1.9 Perimeter (%) Mineral Apposition 1.76
.+-. 0.14 2.25 .+-. 0.16 1.87 .+-. 0.08 1.86 .+-. 0.20 Rate
(.mu.m/day)
__________________________________________________________________________
The results presented in Table V confirm the results of Example
6.1, namely that intravenous injection of OP-1 into ovariectomized
rats stimulates bone growth for bone which had been lost due to the
drop in estrogen within the individual rat. Specifically, the
inhibition of cancellous bone volume in OVX rats is repaired by the
systemically provided morphogen. In addition, in morphogen-treated
rats the labelled cancellous perimeter and mineral apposition rate
now return to levels measured in the control, sham-operated rats.
Moreover, morphogen treatment does not inhibit longitudinal bone
growth, unlike estrogen treatment, which appears to inhibit bone
growth significantly. Accordingly, systemic administration of a
morphogen in therapeutically effective concentrations effectively
inhibits loss of bone mass in a mammal without inhibiting natural
bone formation.
Example 7.
Identification of Morphogen-Expressing Tissue
Determining the tissue distribution of morphogens may be used to
identify different morphogens expressed in a given tissue, as well
as to identify new, related morphogens. Tissue distribution also
may be used to identify useful morphogen-producing tissue for use
in screening and identifying candidate morphogen-stimulating
agents. The morphogens (or their mRNA transcripts) readily are
identified in different tissues using standard methodologies and
minor modifications thereof in tissues where expression may be low.
For example, protein distribution may be determined using standard
Western blot analysis or immunofluorescent techniques, and
antibodies specific to the morphogen or morphogens of interest.
Similarly, the distribution of morphogen transcripts may be
determined using standard Northern hybridization protocols and
transcript-specific probes.
Any probe capable of hybridizing specifically to a transcript, and
distinguishing the transcript of interest from other, related
transcripts may be used. Because the morphogens described herein
share such high sequence homology in their active, C-terminal
domains, the tissue distribution of a specific morphogen transcript
may best be determined using a probe specific for the pro region of
the immature protein and/or the N-terminal region of the mature
protein. Another useful sequence is the 3' non-coding region
flanking and immediately following the stop codon. These portions
of the sequence vary substantially among the morphogens of this
invention, and accordingly, are specific for each protein. For
example, a particularly useful Vgr-1-specific probe sequence is the
PvuII-SacI fragment, a 265 bp fragment encoding both a portion of
the untranslated pro region and the N-terminus of the mature
sequence (see Lyons et al. (1989) PNAS 86: 4554-4558 for a
description of the cDNA sequence). Similarly, particularly useful
mOP-1-specific probe sequences are the BstX1l-BglI fragment, a 0.68
Kb sequence that covers approximately two-thirds of the mOP-1 pro
region; a StuI-StuI fragment, a 0.2 Kb sequence immediately
upstream of the 7-cysteine domain; and the Ear1-Pst1 fragment, an
0.3 Kb fragment containing a portion of the 3'untranslated sequence
(See Seq. ID No. 18, where the pro region is defined essentially by
residues 30-291.) Similar approaches may be used, for example, with
hOP-1 (Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and
22.)
Using these morphogen-specific probes, which may be synthetically
engineered or obtained from cloned sequences, morphogen transcripts
can be identified in mammalian tissue, using standard methodologies
well known to those having ordinary skill in the art. Briefly,
total RNA is prepared from various adult murine tissues (e.g.,
liver, kidney, testis, heart, brain, thymus and stomach) by a
standard methodology such as by the method of Chomczyaski et al.
((1987) Anal. Biochem 162: 156-159) and described below. Poly
(A)+RNA is prepared by using oligo (dT)-cellulose chromatography
(e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.). Poly
(A)+RNA (generally 15 .mu.g) from each tissue is fractionated on a
1% agarose/formaldehyde gel and transferred onto a Nytran membrane
(Schleicher & Schuell). Following the transfer, the membrane is
baked at 80.degree. C. and the RNA is cross-linked under UV light
(generally 30 seconds at 1 mW/cm.sup.2). Prior to hybridization,
the appropriate probe is denatured by heating. The hybridization is
carried out in a lucite cylinder rotating in a roller bottle
apparatus at approximately 1 rev/min for approximately 15 hours at
37.degree. C. using a hybridization mix of 40% formamide,
5.times.Denhardts, 5.times.SSPE, and 0.1% SDS. Following
hybridization, the non-specific counts are washed off the filters
in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.
Examples demonstrating the tissue distribution of various
morphogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1,
and OP-2 in developing and adult tissue are disclosed in co-pending
U.S. Ser. No. 08/404,113, and in Ozkaynak, et al., (1991) Biochem.
Biophys. Res. Commn. 179: 116-123, and Ozkaynak, et al. (1992)
(JBC, in press), the disclosures of which are incorporated herein
by reference. Using the general probing methodology described
herein, northern blot hybridizations using probes specific for
these morphogens to probe brain, spleen, lung, heart, liver and
kidney tissue indicate that kidney-related tissue appears to be the
primary expression source for OP-1, with brain, heart and lung
tissues being secondary sources. Lung tissue appears to be the
primary tissue expression source for Vgr-1, BMP5, BMP4 and BMP3.
Lower levels of Vgr-1 also are seen in kidney and heart tissue,
while the liver appears to be a secondary expression source for
BMP5, and the spleen appears to be a secondary expression source
for BMP4. GDF-1 appears to be expressed primarily in brain
tissue.
Of particular relevance to the present application, OP-1 also is
detected in adult rat stomach and gut tissue. Moreover, OP-2
appears to be expressed primarily in early embryonic tissue.
Specifically, northern blots of murine embryos and 6-day post-natal
animals shows abundant OP2 expression in 8-day embryos. Expression
is reduced significantly in 17-day embryos and is not detected in
post-natal animals.
In addition, labelled soluble OP-1 (iodinated with .sup.125 I,
using standard labelling procedures well known in the art) and
injected into the rat tail vein also is localized to the stomach
tissue within 30 minutes of injection.
Example 8.
Detecting Morphogenic Protein in Solution by Immunoassay
Morphogens are readily detected in solution with a standard
immunoassay, using a polyclonal or monoclonal antibody specific for
that protein and standard Western blot, ELISA (enzyme-linked
immunoabsorbant assay) or other immunoassay technique well known in
the art. A currently preferred, exemplary protocol for an ELISA
assay, as well as means for generating morphogen-specific antibody
are presented below. Standard protocols for antibody production,
Western blot and other immunoassays also are described, for
example, in Molecular Cloning A Laboratory Manual, Sambrook et al.,
eds. 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
Standard ELISA technique is described, for example, by Engvall
(1980) Methods Enzymol. 70: 419-439.
8.1 Morphogen-Specific Antiserum
Polyclonal antibody was prepared as follows. Each rabbit was given
a primary immunization of 100 .mu.g/500 .mu.l E. coli-produced OP-1
monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with
500 .mu.l Complete Freund's Adjuvant. The antigen was injected
subcutaneously at multiple sites on the back and flanks of the
animal. The rabbit was boosted after a month in the same manner
using incomplete Freund's Adjuvant. Test bleeds are taken from the
ear vein seven days later. Two additional boosts and test bleeds
were performed at monthly intervals until antibody against OP-1 was
detected in the serum using an ELISA assay. Then, the rabbit was
boosted monthly with 100 .mu.g of antigen and bled (15 ml per
bleed) at days seven and ten after boosting.
8.2 Morphogen-Specific Antibody
Monoclonal antibody specific for a given morphogen was prepared as
follows. A mouse was given two injections of E. coli produced OP-1
monomer. The first injection contains 100 .mu.g of OP-1 in complete
Freund's adjuvant and was given subcutaneously. The second
injection contained 50 .mu.g of OP-1 in incomplete adjuvant and was
given intraperitoneally. The mouse then received a total of 230
.mu.g of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four
intraperitoneal injections at various times over an eight month
period. One week prior to fusion, both mice were boosted
intraperitoneally with 100 .mu.g of OP-1 (307-431) and 30 .mu.g of
the N-terminal peptide (Ser293-Asn309-Cys) conjugated through the
added cysteine to bovine serum albumin with SMCC crosslinking
agent. This boost was repeated five days (IP), four days (IP),
three days (IP) and one day (IV) prior to fusion. The mouse spleen
cells then were fused to commercially available myeloma cells at a
ratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the
cell fusion plated and screened for OP-1-specific antibodies using
OP-1 (307-431) as antigen. The cell fusion and monoclonal screening
then were according to standard procedures well described in
standard texts widely available in the art e.g., Maniatis et al.
Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Press.
8.3 Morphogen ELISA
1 .mu.g/100 .mu.l of affinity-purified polyclonal rabbit IgG
specific for OP-1 was added to each well of a 96-well plate and
incubated at 37.degree. C. for an hour. The wells were washed four
times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH
8.2, containing 0.1% Tween 20. To minimize non-specific binding,
the wells are blocked by filling completely with 1% bovine serum
albumin (BSA) in BSB and incubating for 1 hour at 37.degree. C. The
wells are then washed four times with BSB containing 0.1% Tween 20.
A 100 .mu.l aliquot of an appropriate dilution of each of the test
samples of cell culture supernatant was added to each well in
triplicate and incubated at 37.degree. C. for 30 min. After
incubation, 100 .mu.l biotinylated rabbit anti-OP-1 serum (stock
solution is about 1 mg/ml and diluted 1:400 in BSB containing 1%
BSA before use) are added to each well and incubated at 37.degree.
C. for 30 min. The wells were then washed four times with BSB
containing 0.1% Tween 20. 100 .mu.l strepavidin-alkaline (Southern
Biotechnology Associates, Inc. Birmingham, Alabama, diluted 1:2000
in BSB containing 0.1% Tween 20 before use) was added to each well
and incubated at 37.degree. C. for 30 min. The plates were washed
four times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50 .mu.l
substrate (ELISA Amplification System Kit, Life Technologies, Inc.,
Bethesda, Md.) was added to each well incubated at room temperature
for 15 min. Then, 50 .mu.l amplifier (from the same amplification
system kit) is added and incubated for another 15 min at room
temperature. The reaction was stopped by the addition of 50 .mu.l
0.3 M sulphuric acid. The OD at 490 nm of the solution in each well
was recorded. To quantitate OP-1 in culture media, an OP-1 standard
curve was performed in parallel with the test samples.
The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
present embodiments are therefore to be considered in all respects
as illustrative and not restrictive, the scope of the invention
being indicated by the appended claims rather than by the foregoing
description, and all changes which come within the meaning and
range of equivalency of the claims are therefore intended to be
embraced therein.
__________________________________________________________________________
# SEQUENCE LISTING - (1) GENERAL INFORMATION: - (iii) NUMBER OF
SEQUENCES: 33 - (2) INFORMATION FOR SEQ ID NO:1: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 97 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..97
#/label= GENERIC-SEQ-1FORMATION: #"EACH XAA INDICATES ONE OF THE 20
NATURALLY #L-ISOMER, ALPHA-AMINO ACIDS, OR A DERIVATIVE THEREOF" -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa # 15 - Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xa - #a Xaa Cys Xaa Xaa Xaa # 30 - Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa # 45 -
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Cys Cys Xaa Xaa
# 60 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa
Xaa Xaa #80 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa
Xaa Cys Xaa Cys # 95 - Xaa - (2) INFORMATION FOR SEQ ID NO:2: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 97 amino (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY: Protein (B)
LOCATION: 1..97 #/label= GENERIC-SEQ-2FORMATION: #"EACH XAA
INDICATES ONE OF THE 20 NATURALLY #L-ISOMER, ALPHA-AMINO ACIDS, OR
A DERIVATIVE THEREOF" - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: -
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa
# 15 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xa - #a Xaa Cys Xaa
Xaa Xaa # 30 - Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa
Xaa Xaa Xaa Xaa # 45 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa -
#a Xaa Cys Cys Xaa Xaa # 60 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa #80 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xa - #a Xaa Xaa Cys Xaa Cys # 95 - Xaa - (2)
INFORMATION FOR SEQ ID NO:3: - (i) SEQUENCE CHARACTERISTICS: #acids
(A) LENGTH: 97 amino (B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..97 #/label=
GENERIC-SEQ-3FORMATION: #AWHEREIN EACH XAA IS INDEPENDENTLY
SELECTED FROM #ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE
SPECI - #FICATION " - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - Leu
Tyr Val Xaa Phe Xaa Xaa Xaa Gly Trp Xa - #a Xaa Trp Xaa Xaa Ala #
15 - Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gl - #y Xaa Cys Xaa
Xaa Pro # 30 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Al - #a Xaa
Xaa Xaa Xaa Leu # 45 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa -
#a Xaa Cys Cys Xaa Pro # 60 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu
Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa #80 - Val Xaa Leu Xaa Xaa Xaa Xaa
Xaa Met Xaa Va - #l Xaa Xaa Cys Gly Cys # 95 - Xaa - (2)
INFORMATION FOR SEQ ID NO:4: - (i) SEQUENCE CHARACTERISTICS: #acids
(A) LENGTH: 102 amino (B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..102 #/label=
GENERIC-SEQ-4FORMATION: #AWHEREIN EACH XAA IS INDEPENDENTLY
SELECTED FROM #ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE
SPECI - #FICATION" - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - Cys
Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe Xa - #a Xaa Xaa Gly Trp Xaa #
15 - Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Al - #a Xaa Tyr Cys
Xaa Gly # 30 - Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa
Xaa Asn His Ala # 45 - Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xa -
#a Xaa Xaa Xaa Xaa Xaa # 60 - Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa
Xaa Xa - #a Xaa Xaa Leu Xaa Xaa #80 - Xaa Xaa Xaa Xaa Xaa Val Xaa
Leu Xaa Xaa Xa - #a Xaa Xaa Met Xaa Val # 95 - Xaa Xaa Cys Gly Cys
Xaa 100 - (2) INFORMATION FOR SEQ ID NO:5: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 139 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..139
#/note= "HOP-1 (MATURE FORM)"ON: - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:5: - Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Ar - #g Ser Lys
Thr Pro Lys # 15 - Asn Gln Glu Ala Leu Arg Met Ala Asn Val Al - #a
Glu Asn Ser Ser Ser # 30 - Asp Gln Arg Gln Ala Cys Lys Lys His Glu
Le - #u Tyr Val Ser Phe Arg # 45 - Asp Leu Gly Trp Gln Asp Trp Ile
Ile Ala Pr - #o Glu Gly Tyr Ala Ala # 60 - Tyr Tyr Cys Glu Gly Glu
Cys Ala Phe Pro Le - #u Asn Ser Tyr Met Asn #80 - Ala Thr Asn His
Ala Ile Val Gln Thr Leu Va - #l His Phe Ile Asn Pro # 95 - Glu Thr
Val Pro Lys Pro Cys Cys Ala Pro Th - #r Gln Leu Asn Ala Ile # 110 -
Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Va - #l Ile Leu Lys Lys Tyr
# 125 - Arg Asn Met Val Val Arg Ala Cys Gly Cys Hi - #s # 135 - (2)
INFORMATION FOR SEQ ID NO:6: - (i) SEQUENCE CHARACTERISTICS: #acids
(A) LENGTH: 139 amino (B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix) FEATURE:
(A) NAME/KEY: Protein (B) LOCATION: 1..139 #/note= "MOP-1 (MATURE
FORM)"ON: - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Ar - #g Ser Lys Thr Pro Lys # 15 - Asn
Gln Glu Ala Leu Arg Met Ala Ser Val Al - #a Glu Asn Ser Ser Ser #
30 - Asp Gln Arg Gln Ala Cys Lys Lys His Glu Le - #u Tyr Val Ser
Phe Arg # 45 - Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pr - #o Glu
Gly Tyr Ala Ala # 60 - Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Le -
#u Asn Ser Tyr Met Asn #80 - Ala Thr Asn His Ala Ile Val Gln Thr
Leu Va - #l His Phe Ile Asn Pro # 95 - Asp Thr Val Pro Lys Pro Cys
Cys Ala Pro Th - #r Gln Leu Asn Ala Ile # 110 - Ser Val Leu Tyr Phe
Asp Asp Ser Ser Asn Va - #l Ile Leu Lys Lys Tyr # 125 - Arg Asn Met
Val Val Arg Ala Cys Gly Cys Hi - #s # 135 - (2) INFORMATION FOR SEQ
ID NO:7: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 139
amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear - (ii) MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY:
Protein (B) LOCATION: 1..139 #/note= "HOP-2 (MATURE FORM)"ON: -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - Ala Val Arg Pro Leu Arg
Arg Arg Gln Pro Ly - #s Lys Ser Asn Glu Leu # 15 - Pro Gln Ala Asn
Arg Leu Pro Gly Ile Phe As - #p Asp Val His Gly Ser # 30 - His Gly
Arg Gln Val Cys Arg Arg His Glu Le - #u Tyr Val Ser Phe Gln # 45 -
Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pr - #o Gln Gly Tyr Ser Ala
# 60 - Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Le - #u Asp Ser Cys
Met Asn #80 - Ala Thr Asn His Ala Ile Leu Gln Ser Leu Va - #l His
Leu Met Lys Pro # 95 - Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Th -
#r Lys Leu Ser Ala Thr # 110 - Ser Val Leu Tyr Tyr Asp Ser Ser Asn
Asn Va - #l Ile Leu Arg Lys His # 125 - Arg Asn Met Val Val Lys Ala
Cys Gly Cys Hi - #s # 135 - (2) INFORMATION FOR SEQ ID NO:8: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 139 amino (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY: Protein (B)
LOCATION: 1..139 #/note= "MOP-2 (MATURE FORM)"ON: - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:8: - Ala Ala Arg Pro Leu Lys Arg Arg Gln Pro
Ly - #s Lys Thr Asn Glu Leu # 15 - Pro His Pro Asn Lys Leu Pro Gly
Ile Phe As - #p Asp Gly His Gly Ser # 30 - Arg Gly Arg Glu Val Cys
Arg Arg His Glu Le - #u Tyr Val Ser Phe Arg # 45 - Asp Leu Gly Trp
Leu Asp Trp Val Ile Ala Pr - #o Gln Gly Tyr Ser Ala # 60 - Tyr Tyr
Cys Glu Gly Glu Cys Ala Phe Pro Le - #u Asp Ser Cys Met Asn #80 -
Ala Thr Asn His Ala Ile Leu Gln Ser Leu Va - #l His Leu Met Lys Pro
# 95 - Asp Val Val Pro Lys Ala Cys Cys Ala Pro Th - #r Lys Leu Ser
Ala Thr # 110 - Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Va - #l Ile
Leu Arg Lys His # 125 - Arg Asn Met Val Val Lys Ala Cys Gly Cys Hi
- #s # 135 - (2) INFORMATION FOR SEQ ID NO:9: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 101 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..101
#/note= "CBMP-2A(FX)"NFORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:9: - Cys Lys Arg His Pro Leu Tyr Val Asp Phe Se - #r Asp Val
Gly Trp Asn # 15
- Asp Trp Ile Val Ala Pro Pro Gly Tyr His Al - #a Phe Tyr Cys His
Gly # 30 - Glu Cys Pro Phe Pro Leu Ala Asp His Leu As - #n Ser Thr
Asn His Ala # 45 - Ile Val Gln Thr Leu Val Asn Ser Val Asn Se - #r
Lys Ile Pro Lys Ala # 60 - Cys Cys Val Pro Thr Glu Leu Ser Ala Ile
Se - #r Met Leu Tyr Leu Asp #80 - Glu Asn Glu Lys Val Val Leu Lys
Asn Tyr Gl - #n Asp Met Val Val Glu # 95 - Gly Cys Gly Cys Arg 100
- (2) INFORMATION FOR SEQ ID NO:10: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 101 amino (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix)
FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..101 #/note=
"CBMP-2B(FX)"NFORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- Cys Arg Arg His Ser Leu Tyr Val Asp Phe Se - #r Asp Val Gly Trp
Asn # 15 - Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Al - #a Phe Tyr
Cys His Gly # 30 - Asp Cys Pro Phe Pro Leu Ala Asp His Leu As - #n
Ser Thr Asn His Ala # 45 - Ile Val Gln Thr Leu Val Asn Ser Val Asn
Se - #r Ser Ile Pro Lys Ala # 60 - Cys Cys Val Pro Thr Glu Leu Ser
Ala Ile Se - #r Met Leu Tyr Leu Asp #80 - Glu Tyr Asp Lys Val Val
Leu Lys Asn Tyr Gl - #n Glu Met Val Val Glu # 95 - Gly Cys Gly Cys
Arg 100 - (2) INFORMATION FOR SEQ ID NO:11: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 102 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..102
#/note= "DPP(FX)"ER INFORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:11: - Cys Arg Arg His Ser Leu Tyr Val Asp Phe Se - #r Asp Val
Gly Trp Asp # 15 - Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Al - #a
Tyr Tyr Cys His Gly # 30 - Lys Cys Pro Phe Pro Leu Ala Asp His Phe
As - #n Ser Thr Asn His Ala # 45 - Val Val Gln Thr Leu Val Asn Asn
Asn Asn Pr - #o Gly Lys Val Pro Lys # 60 - Ala Cys Cys Val Pro Thr
Gln Leu Asp Ser Va - #l Ala Met Leu Tyr Leu #80 - Asn Asp Gln Ser
Thr Val Val Leu Lys Asn Ty - #r Gln Glu Met Thr Val # 95 - Val Gly
Cys Gly Cys Arg 100 - (2) INFORMATION FOR SEQ ID NO:12: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 102 amino (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY: Protein (B)
LOCATION: 1..102 #/note= "VGL(FX)"ER INFORMATION: - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:12: - Cys Lys Lys Arg His Leu Tyr Val Glu
Phe Ly - #s Asp Val Gly Trp Gln # 15 - Asn Trp Val Ile Ala Pro Gln
Gly Tyr Met Al - #a Asn Tyr Cys Tyr Gly # 30 - Glu Cys Pro Tyr Pro
Leu Thr Glu Ile Leu As - #n Gly Ser Asn His Ala # 45 - Ile Leu Gln
Thr Leu Val His Ser Ile Glu Pr - #o Glu Asp Ile Pro Leu # 60 - Pro
Cys Cys Val Pro Thr Lys Met Ser Pro Il - #e Ser Met Leu Phe Tyr #80
- Asp Asn Asn Asp Asn Val Val Leu Arg His Ty - #r Glu Asn Met Ala
Val # 95 - Asp Glu Cys Gly Cys Arg 100 - (2) INFORMATION FOR SEQ ID
NO:13: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 102 amino
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY: Protein
(B) LOCATION: 1..102 #/note= "VGR-1(FX)" INFORMATION: - (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:13: - Cys Lys Lys His Glu Leu Tyr
Val Ser Phe Gl - #n Asp Val Gly Trp Gln # 15 - Asp Trp Ile Ile Ala
Pro Lys Gly Tyr Ala Al - #a Asn Tyr Cys Asp Gly # 30 - Glu Cys Ser
Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala # 45 - Ile
Val Gln Thr Leu Val His Val Met Asn Pr - #o Glu Tyr Val Pro Lys #
60 - Pro Cys Cys Ala Pro Thr Lys Val Asn Ala Il - #e Ser Val Leu
Tyr Phe #80 - Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Ty - #r Arg
Asn Met Val Val # 95 - Arg Ala Cys Gly Cys His 100 - (2)
INFORMATION FOR SEQ ID NO:14: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 106 amino (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix)
FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..106 #/note= "GDF-1
(FX)"INFORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: - Cys
Arg Ala Arg Arg Leu Tyr Val Ser Phe Ar - #g Glu Val Gly Trp His #
15 - Arg Trp Val Ile Ala Pro Arg Gly Phe Leu Al - #a Asn Tyr Cys
Gln Gly # 30 - Gln Cys Ala Leu Pro Val Ala Leu Ser Gly Se - #r Gly
Gly Pro Pro Ala # 45 - Leu Asn His Ala Val Leu Arg Ala Leu Met Hi -
#s Ala Ala Ala Pro Gly # 60 - Ala Ala Asp Leu Pro Cys Cys Val Pro
Ala Ar - #g Leu Ser Pro Ile Ser #80 - Val Leu Phe Phe Asp Asn Ser
Asp Asn Val Va - #l Leu Arg Gln Tyr Glu # 95 - Asp Met Val Val Asp
Glu Cys Gly Cys Arg # 105 - (2) INFORMATION FOR SEQ ID NO:15: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 5 amino (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: peptide - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: -
Cys Xaa Xaa Xaa Xaa 1 5 - (2) INFORMATION FOR SEQ ID NO:16: - (i)
SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 1822 base (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: cDNA - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
49..1341 #/product= "HOP-1"R INFORMATION: - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:16: #CAC GTG 57AGCCCGG AGCCCGGGTA GCGCGTAGAG
CCGGCGCG ATG # Met - # His Val # 1 - CGC TCA CTG CGA GCT GCG GCG
CCG CAC AGC TT - #C GTG GCG CTC TGG GCA 105 Arg Ser Leu Arg Ala Ala
Ala Pro His Ser Ph - #e Val Ala Leu Trp Ala # 15 - CCC CTG TTC CTG
CTG CGC TCC GCC CTG GCC GA - #C TTC AGC CTG GAC AAC 153 Pro Leu Phe
Leu Leu Arg Ser Ala Leu Ala As - #p Phe Ser Leu Asp Asn # 35 - GAG
GTG CAC TCG AGC TTC ATC CAC CGG CGC CT - #C CGC AGC CAG GAG CGG 201
Glu Val His Ser Ser Phe Ile His Arg Arg Le - #u Arg Ser Gln Glu Arg
# 50 - CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TT - #G GGC TTG CCC
CAC CGC 249 Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Le - #u Gly Leu
Pro His Arg # 65 - CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TC - #G
GCA CCC ATG TTC ATG 297 Pro Arg Pro His Leu Gln Gly Lys His Asn Se
- #r Ala Pro Met Phe Met # 80 - CTG GAC CTG TAC AAC GCC ATG GCG GTG
GAG GA - #G GGC GGC GGG CCC GGC 345 Leu Asp Leu Tyr Asn Ala Met Ala
Val Glu Gl - #u Gly Gly Gly Pro Gly # 95 - GGC CAG GGC TTC TCC TAC
CCC TAC AAG GCC GT - #C TTC AGT ACC CAG GGC 393 Gly Gln Gly Phe Ser
Tyr Pro Tyr Lys Ala Va - #l Phe Ser Thr Gln Gly 100 1 - #05 1 - #10
1 - #15 - CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TT - #C CTC ACC
GAC GCC GAC 441 Pro Pro Leu Ala Ser Leu Gln Asp Ser His Ph - #e Leu
Thr Asp Ala Asp # 130 - ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CA
- #T GAC AAG GAA TTC TTC 489 Met Val Met Ser Phe Val Asn Leu Val
Glu Hi - #s Asp Lys Glu Phe Phe # 145 - CAC CCA CGC TAC CAC CAT CGA
GAG TTC CGG TT - #T GAT CTT TCC AAG ATC 537 His Pro Arg Tyr His His
Arg Glu Phe Arg Ph - #e Asp Leu Ser Lys Ile # 160 - CCA GAA GGG GAA
GCT GTC ACG GCA GCC GAA TT - #C CGG ATC TAC AAG GAC 585 Pro Glu Gly
Glu Ala Val Thr Ala Ala Glu Ph - #e Arg Ile Tyr Lys Asp # 175 - TAC
ATC CGG GAA CGC TTC GAC AAT GAG ACG TT - #C CGG ATC AGC GTT TAT 633
Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Ph - #e Arg Ile Ser Val Tyr
180 1 - #85 1 - #90 1 - #95 - CAG GTG CTC CAG GAG CAC TTG GGC AGG
GAA TC - #G GAT CTC TTC CTG CTC 681 Gln Val Leu Gln Glu His Leu Gly
Arg Glu Se - #r Asp Leu Phe Leu Leu # 210 - GAC AGC CGT ACC CTC TGG
GCC TCG GAG GAG GG - #C TGG CTG GTG TTT GAC 729 Asp Ser Arg Thr Leu
Trp Ala Ser Glu Glu Gl - #y Trp Leu Val Phe Asp # 225 - ATC ACA GCC
ACC AGC AAC CAC TGG GTG GTC AA - #T CCG CGG CAC AAC CTG 777 Ile Thr
Ala Thr Ser Asn His Trp Val Val As - #n Pro Arg His Asn Leu # 240 -
GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GG - #G CAG AGC ATC AAC CCC
825 Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gl - #y Gln Ser Ile Asn
Pro # 255 - AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CC - #C CAG AAC
AAG CAG CCC 873 Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pr - #o Gln
Asn Lys Gln Pro 260 2 - #65 2 - #70 2 - #75 - TTC ATG GTG GCT TTC
TTC AAG GCC ACG GAG GT - #C CAC TTC CGC AGC ATC 921 Phe Met Val Ala
Phe Phe Lys Ala Thr Glu Va - #l His Phe Arg Ser Ile # 290
- CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AA - #C CGC TCC AAG ACG
CCC 969 Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln As - #n Arg Ser Lys
Thr Pro # 305 - AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GT - #G GCA
GAG AAC AGC AGC 1017 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Va -
#l Ala Glu Asn Ser Ser # 320 - AGC GAC CAG AGG CAG GCC TGT AAG AAG
CAC GA - #G CTG TAT GTC AGC TTC 1065 Ser Asp Gln Arg Gln Ala Cys
Lys Lys His Gl - #u Leu Tyr Val Ser Phe # 335 - CGA GAC CTG GGC TGG
CAG GAC TGG ATC ATC GC - #G CCT GAA GGC TAC GCC 1113 Arg Asp Leu
Gly Trp Gln Asp Trp Ile Ile Al - #a Pro Glu Gly Tyr Ala 340 3 - #45
3 - #50 3 - #55 - GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CC - #T
CTG AAC TCC TAC ATG 1161 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pr
- #o Leu Asn Ser Tyr Met # 370 - AAC GCC ACC AAC CAC GCC ATC GTG
CAG ACG CT - #G GTC CAC TTC ATC AAC 1209 Asn Ala Thr Asn His Ala
Ile Val Gln Thr Le - #u Val His Phe Ile Asn # 385 - CCG GAA ACG GTG
CCC AAG CCC TGC TGT GCG CC - #C ACG CAG CTC AAT GCC 1257 Pro Glu
Thr Val Pro Lys Pro Cys Cys Ala Pr - #o Thr Gln Leu Asn Ala # 400 -
ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AA - #C GTC ATC CTG AAG AAA
1305 Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser As - #n Val Ile Leu
Lys Lys # 415 - TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TG - #C CAC
TAGCTCCTCC 1351 Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cy - #s His
420 4 - #25 4 - #30 - GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT
CCATTGCTCG CC - #TTGGCCAG 1411 - GAACCAGCAG ACCAACTGCC TTTTGTGAGA
CCTTCCCCTC CCTATCCCCA AC - #TTTAAAGG 1471 - TGTGAGAGTA TTAGGAAACA
TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CA - #GTGGCAGC 1531 - ATCCAATGAA
CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AA - #AAAACAAC 1591 -
GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TG -
#CACGGACT 1651 - CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA
CCCAGCCGTG GG - #AGGAAGGG 1711 - GGCGTGGCAA GGGGTGGGCA CATTGGTGTC
TGTGCGAAAG GAAAATTGAC CC - #GGAAGTTC 1771 # 1822CACAATA AAACGAATGA
ATGAAAAAAA AAAAAAAAAA A - (2) INFORMATION FOR SEQ ID NO:17: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 431 amino (B) TYPE:
amino acid (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: - Met His Val Arg Ser Leu
Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala # 15 - Leu Trp Ala Pro
Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser # 30 - Leu Asp
Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser # 45 -
Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu
# 60 - Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser
Ala Pro # 80 - Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val
Glu Glu Gly Gly # 95 - Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Ty -
#r Lys Ala Val Phe Ser # 110 - Thr Gln Gly Pro Pro Leu Ala Ser Leu
Gln As - #p Ser His Phe Leu Thr # 125 - Asp Ala Asp Met Val Met Ser
Phe Val Asn Le - #u Val Glu His Asp Lys # 140 - Glu Phe Phe His Pro
Arg Tyr His His Arg Gl - #u Phe Arg Phe Asp Leu 145 1 - #50 1 - #55
1 - #60 - Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Al - #a Ala Glu
Phe Arg Ile # 175 - Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp As - #n
Glu Thr Phe Arg Ile # 190 - Ser Val Tyr Gln Val Leu Gln Glu His Leu
Gl - #y Arg Glu Ser Asp Leu # 205 - Phe Leu Leu Asp Ser Arg Thr Leu
Trp Ala Se - #r Glu Glu Gly Trp Leu # 220 - Val Phe Asp Ile Thr Ala
Thr Ser Asn His Tr - #p Val Val Asn Pro Arg 225 2 - #30 2 - #35 2 -
#40 - His Asn Leu Gly Leu Gln Leu Ser Val Glu Th - #r Leu Asp Gly
Gln Ser # 255 - Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Ar - #g His
Gly Pro Gln Asn # 270 - Lys Gln Pro Phe Met Val Ala Phe Phe Lys Al
- #a Thr Glu Val His Phe # 285 - Arg Ser Ile Arg Ser Thr Gly Ser
Lys Gln Ar - #g Ser Gln Asn Arg Ser # 300 - Lys Thr Pro Lys Asn Gln
Glu Ala Leu Arg Me - #t Ala Asn Val Ala Glu 305 3 - #10 3 - #15 3 -
#20 - Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Ly - #s Lys His Glu
Leu Tyr # 335 - Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Tr - #p Ile
Ile Ala Pro Glu # 350 - Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cy
- #s Ala Phe Pro Leu Asn # 365 - Ser Tyr Met Asn Ala Thr Asn His
Ala Ile Va - #l Gln Thr Leu Val His # 380 - Phe Ile Asn Pro Glu Thr
Val Pro Lys Pro Cy - #s Cys Ala Pro Thr Gln 385 3 - #90 3 - #95 4 -
#00 - Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp As - #p Ser Ser Asn
Val Ile # 415 - Leu Lys Lys Tyr Arg Asn Met Val Val Arg Al - #a Cys
Gly Cys His # 430 - (2) INFORMATION FOR SEQ ID NO:18: - (i)
SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 1873 base (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: cDNA - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
104..1393 #/product= "MOP1 (CDNA)"RMATION: - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:18: - CTGCAGCAAG TGACCTCGGG TCGTGGACCG
CTGCCCTGCC CCCTCCGCTG CC - #ACCTGGGG 60 - CGGCGCGGGC CCGGTGCCCC
GGATCGCGCG TAGAGCCGGC GCG ATG CA - #C GTG CGC 115 # Met His Val Ar
- #g # 1 - TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GT - #G GCG CTC
TGG GCG CCT 163 Ser Leu Arg Ala Ala Ala Pro His Ser Phe Va - #l Ala
Leu Trp Ala Pro # 20 - CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TT -
#C AGC CTG GAC AAC GAG 211 Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp
Ph - #e Ser Leu Asp Asn Glu # 35 - GTG CAC TCC AGC TTC ATC CAC CGG
CGC CTC CG - #C AGC CAG GAG CGG CGG 259 Val His Ser Ser Phe Ile His
Arg Arg Leu Ar - #g Ser Gln Glu Arg Arg # 50 - GAG ATG CAG CGG GAG
ATC CTG TCC ATC TTA GG - #G TTG CCC CAT CGC CCG 307 Glu Met Gln Arg
Glu Ile Leu Ser Ile Leu Gl - #y Leu Pro His Arg Pro # 65 - CGC CCG
CAC CTC CAG GGA AAG CAT AAT TCG GC - #G CCC ATG TTC ATG TTG 355 Arg
Pro His Leu Gln Gly Lys His Asn Ser Al - #a Pro Met Phe Met Leu #
80 - GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AG - #C GGG CCG GAC
GGA CAG 403 Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Se - #r Gly Pro
Asp Gly Gln #100 - GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AG - #T
ACC CAG GGC CCC CCT 451 Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Se
- #r Thr Gln Gly Pro Pro # 115 - TTA GCC AGC CTG CAG GAC AGC CAT
TTC CTC AC - #T GAC GCC GAC ATG GTC 499 Leu Ala Ser Leu Gln Asp Ser
His Phe Leu Th - #r Asp Ala Asp Met Val # 130 - ATG AGC TTC GTC AAC
CTA GTG GAA CAT GAC AA - #A GAA TTC TTC CAC CCT 547 Met Ser Phe Val
Asn Leu Val Glu His Asp Ly - #s Glu Phe Phe His Pro # 145 - CGA TAC
CAC CAT CGG GAG TTC CGG TTT GAT CT - #T TCC AAG ATC CCC GAG 595 Arg
Tyr His His Arg Glu Phe Arg Phe Asp Le - #u Ser Lys Ile Pro Glu #
160 - GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG AT - #C TAT AAG GAC
TAC ATC 643 Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Il - #e Tyr Lys
Asp Tyr Ile 165 1 - #70 1 - #75 1 - #80 - CGG GAG CGA TTT GAC AAC
GAG ACC TTC CAG AT - #C ACA GTC TAT CAG GTG 691 Arg Glu Arg Phe Asp
Asn Glu Thr Phe Gln Il - #e Thr Val Tyr Gln Val # 195 - CTC CAG GAG
CAC TCA GGC AGG GAG TCG GAC CT - #C TTC TTG CTG GAC AGC 739 Leu Gln
Glu His Ser Gly Arg Glu Ser Asp Le - #u Phe Leu Leu Asp Ser # 210 -
CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TT - #G GTG TTT GAT ATC ACA
787 Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Le - #u Val Phe Asp Ile
Thr # 225 - GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CG - #G CAC AAC
CTG GGC TTA 835 Ala Thr Ser Asn His Trp Val Val Asn Pro Ar - #g His
Asn Leu Gly Leu # 240 - CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AG
- #C ATC AAC CCC AAG TTG 883 Gln Leu Ser Val Glu Thr Leu Asp Gly
Gln Se - #r Ile Asn Pro Lys Leu 245 2 - #50 2 - #55 2 - #60 - GCA
GGC CTG ATT GGA CGG CAT GGA CCC CAG AA - #C AAG CAA CCC TTC ATG 931
Ala Gly Leu Ile Gly Arg His Gly Pro Gln As - #n Lys Gln Pro Phe Met
# 275 - GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CT - #C CGT AGT ATC
CGG TCC 979 Val Ala Phe Phe Lys Ala Thr Glu Val His Le - #u Arg Ser
Ile Arg Ser # 290 - ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TC - #C
AAG ACG CCA AAG AAC 1027 Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Se
- #r Lys Thr Pro Lys Asn # 305 - CAA GAG GCC CTG AGG ATG GCC AGT
GTG GCA GA - #A AAC AGC AGC AGT GAC 1075 Gln Glu Ala Leu Arg Met
Ala Ser Val Ala Gl - #u Asn Ser Ser Ser Asp # 320 - CAG AGG CAG GCC
TGC AAG AAA CAT GAG CTG TA - #C GTC AGC TTC CGA GAC 1123 Gln Arg
Gln Ala Cys Lys Lys His Glu Leu Ty - #r Val Ser Phe Arg Asp 325 3 -
#30 3 - #35 3 - #40 - CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GA -
#A GGC TAT GCT GCC TAC 1171 Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro
Gl - #u Gly Tyr Ala Ala Tyr # 355 - TAC TGT GAG GGA GAG TGC GCC TTC
CCT CTG AA - #C TCC TAC ATG AAC GCC 1219 Tyr Cys Glu Gly Glu Cys
Ala Phe Pro Leu As - #n Ser Tyr Met Asn Ala # 370 - ACC AAC CAC GCC
ATC GTC CAG ACA CTG GTT CA - #C TTC ATC AAC CCA GAC 1267 Thr Asn
His Ala Ile Val Gln Thr Leu Val Hi - #s Phe Ile Asn Pro Asp # 385 -
ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CA - #G CTC AAC GCC ATC TCT
1315 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gl - #n Leu Asn Ala
Ile Ser # 400 - GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC AT - #C CTG
AAG AAG TAC AGA 1363 Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Il -
#e Leu Lys Lys Tyr Arg 405 4 - #10 4 - #15 4 - #20 - AAC ATG GTG
GTC CGG GCC TGT GGC TGC CAC TA - #GCTCTTCC TGAGACCCTG 1413 Asn Met
Val Val Arg Ala Cys Gly Cys His # 430 - ACCTTTGCGG GGCCACACCT
TTCCAAATCT TCGATGTCTC ACCATCTAAG TC - #TCTCACTG 1473 - CCCACCTTGG
CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TC - #CCAACCGG 1533
- AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CT -
#TTCCTTCT 1593 - GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA
CGCCTAAGAG CA - #GGAAAAAT 1653 - GTCTGCCAGG AAAGTGTCCA GTGTCCACAT
GGCCCCTGGC GCTCTGAGTC TT - #TGAGGAGT 1713 - AATCGCAAGC CTCGTTCAGC
TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GG - #CGCTGGCG 1773 - TCTGTGTTGA
AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TA - #AAACCCAT 1833 #
1873 AAAA AAAAAAAAAA AAAAGAATTC - (2) INFORMATION FOR SEQ ID NO:19:
- (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 430 amino (B)
TYPE: amino acid (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: - Met His Val Arg Ser
Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala # 15 - Leu Trp Ala
Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser # 30 - Leu
Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser #
45 - Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu
Gly Leu # 60 - Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His
Asn Ser Ala Pro # 80 - Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al -
#a Val Glu Glu Ser Gly # 95 - Pro Asp Gly Gln Gly Phe Ser Tyr Pro
Tyr Ly - #s Ala Val Phe Ser Thr # 110 - Gln Gly Pro Pro Leu Ala Ser
Leu Gln Asp Se - #r His Phe Leu Thr Asp # 125 - Ala Asp Met Val Met
Ser Phe Val Asn Leu Va - #l Glu His Asp Lys Glu # 140 - Phe Phe His
Pro Arg Tyr His His Arg Glu Ph - #e Arg Phe Asp Leu Ser 145 1 - #50
1 - #55 1 - #60 - Lys Ile Pro Glu Gly Glu Arg Val Thr Ala Al - #a
Glu Phe Arg Ile Tyr # 175 - Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn
Gl - #u Thr Phe Gln Ile Thr # 190 - Val Tyr Gln Val Leu Gln Glu His
Ser Gly Ar - #g Glu Ser Asp Leu Phe # 205 - Leu Leu Asp Ser Arg Thr
Ile Trp Ala Ser Gl - #u Glu Gly Trp Leu Val # 220 - Phe Asp Ile Thr
Ala Thr Ser Asn His Trp Va - #l Val Asn Pro Arg His 225 2 - #30 2 -
#35 2 - #40 - Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Le - #u Asp
Gly Gln Ser Ile # 255 - Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg Hi
- #s Gly Pro Gln Asn Lys # 270 - Gln Pro Phe Met Val Ala Phe Phe
Lys Ala Th - #r Glu Val His Leu Arg # 285 - Ser Ile Arg Ser Thr Gly
Gly Lys Gln Arg Se - #r Gln Asn Arg Ser Lys # 300 - Thr Pro Lys Asn
Gln Glu Ala Leu Arg Met Al - #a Ser Val Ala Glu Asn 305 3 - #10 3 -
#15 3 - #20 - Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Ly - #s His
Glu Leu Tyr Val # 335 - Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Il
- #e Ile Ala Pro Glu Gly # 350 - Tyr Ala Ala Tyr Tyr Cys Glu Gly
Glu Cys Al - #a Phe Pro Leu Asn Ser # 365 - Tyr Met Asn Ala Thr Asn
His Ala Ile Val Gl - #n Thr Leu Val His Phe # 380 - Ile Asn Pro Asp
Thr Val Pro Lys Pro Cys Cy - #s Ala Pro Thr Gln Leu 385 3 - #90 3 -
#95 4 - #00 - Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Se - #r Ser
Asn Val Ile Leu # 415 - Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cy
- #s Gly Cys His # 430 - (2) INFORMATION FOR SEQ ID NO:20: - (i)
SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 1723 base (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: cDNA - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
490..1695 - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: - GGCGCCGGCA
GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TG - #GCACGGCA 60 -
GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GC -
#AACAGCTC 120 - CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC
GCCCCTGCGC TG - #CTCGGACC 180 - GCGGCCACAG CCGGACTGGC GGGTACGGCG
GCGACAGAGG CATTGGCCGA GA - #GTCCCAGT 240 - CCGCAGAGTA GCCCCGGCCT
CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CA - #GGAGCCAG 300 - GACAGGTGTC
GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CC - #GCCCGTCC 360 -
CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GG -
#GCGTCCCC 420 - AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA
GCGCCCCAGC TG - #AGCGCCCC 480 - CGGCCTGCC ATG ACC GCG CTC CCC GGC
CCG CTC TGG - # CTC CTG GGC CTG 528 #Gly Pro Leu Trp Leu Leu Gly
Leu # 10 - GCG CTA TGC GCG CTG GGC GGG GGC GGC CCC GG - #C CTG CGA
CCC CCG CCC 576 Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gl - #y Leu
Arg Pro Pro Pro # 25 - GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GA -
#G CGC CGG GAC GTG CAG 624 Gly Cys Pro Gln Arg Arg Leu Gly Ala Arg
Gl - #u Arg Arg Asp Val Gln # 45 - CGC GAG ATC CTG GCG GTG CTC GGG
CTG CCT GG - #G CGG CCC CGG CCC CGC 672 Arg Glu Ile Leu Ala Val Leu
Gly Leu Pro Gl - #y Arg Pro Arg Pro Arg # 60 - GCG CCA CCC GCC GCC
TCC CGG CTG CCC GCG TC - #C GCG CCG CTC TTC ATG 720 Ala Pro Pro Ala
Ala Ser Arg Leu Pro Ala Se - #r Ala Pro Leu Phe Met # 75 - CTG GAC
CTG TAC CAC GCC ATG GCC GGC GAC GA - #C GAC GAG GAC GGC GCG 768 Leu
Asp Leu Tyr His Ala Met Ala Gly Asp As - #p Asp Glu Asp Gly Ala #
90 - CCC GCG GAG CGG CGC CTG GGC CGC GCC GAC CT - #G GTC ATG AGC
TTC GTT 816 Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Le - #u Val Met
Ser Phe Val # 105 - AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CA - #C
CAG GAG CCC CAT TGG 864 Asn Met Val Glu Arg Asp Arg Ala Leu Gly Hi
- #s Gln Glu Pro His Trp 110 1 - #15 1 - #20 1 - #25 - AAG GAG TTC
CGC TTT GAC CTG ACC CAG ATC CC - #G GCT GGG GAG GCG GTC 912 Lys Glu
Phe Arg Phe Asp Leu Thr Gln Ile Pr - #o Ala Gly Glu Ala Val # 140 -
ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CC - #C AGC ATC CAC CTG CTC
960 Thr Ala Ala Glu Phe Arg Ile Tyr Lys Val Pr - #o Ser Ile His Leu
Leu # 155 - AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GT - #G GTC CAG
GAG CAG TCC 1008 Asn Arg Thr Leu His Val Ser Met Phe Gln Va - #l
Val Gln Glu Gln Ser # 170 - AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT
CT - #T CAG ACG CTC CGA GCT 1056 Asn Arg Glu Ser Asp Leu Phe Phe
Leu Asp Le - #u Gln Thr Leu Arg Ala # 185 - GGA GAC GAG GGC TGG CTG
GTG CTG GAT GTC AC - #A GCA GCC AGT GAC TGC 1104 Gly Asp Glu Gly
Trp Leu Val Leu Asp Val Th - #r Ala Ala Ser Asp Cys 190 1 - #95 2 -
#00 2 - #05 - TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CT - #C CGC
CTC TAT GTG GAG 1152 Trp Leu Leu Lys Arg His Lys Asp Leu Gly Le -
#u Arg Leu Tyr Val Glu # 220 - ACT GAG GAC GGG CAC AGC GTG GAT CCT
GGC CT - #G GCC GGC CTG CTG GGT 1200 Thr Glu Asp Gly His Ser Val
Asp Pro Gly Le - #u Ala Gly Leu Leu Gly # 235 - CAA CGG GCC CCA CGC
TCC CAA CAG CCT TTC GT - #G GTC ACT TTC TTC AGG 1248 Gln Arg Ala
Pro Arg Ser Gln Gln Pro Phe Va - #l Val Thr Phe Phe Arg # 250 - GCC
AGT CCG AGT CCC ATC CGC ACC CCT CGG GC - #A GTG AGG CCA CTG AGG
1296 Ala Ser Pro Ser Pro Ile Arg Thr Pro Arg Al - #a Val Arg Pro
Leu Arg # 265 - AGG AGG CAG CCG AAG AAA AGC AAC GAG CTG CC - #G CAG
GCC AAC CGA CTC 1344 Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu Pr -
#o Gln Ala Asn Arg Leu 270 2 - #75 2 - #80 2 - #85 - CCA GGG ATC
TTT GAT GAC GTC CAC GGC TCC CA - #C GGC CGG CAG GTC TGC 1392 Pro
Gly Ile Phe Asp Asp Val His Gly Ser Hi - #s Gly Arg Gln Val Cys #
300 - CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GA - #C CTC GGC TGG
CTG GAC 1440 Arg Arg His Glu Leu Tyr Val Ser Phe Gln As - #p Leu
Gly Trp Leu Asp # 315 - TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TA
- #T TAC TGT GAG GGG GAG 1488 Trp Val Ile Ala Pro Gln Gly Tyr Ser
Ala Ty - #r Tyr Cys Glu Gly Glu # 330 - TGC TCC TTC CCA CTG GAC TCC
TGC ATG AAT GC - #C ACC AAC CAC GCC ATC 1536 Cys Ser Phe Pro Leu
Asp Ser Cys Met Asn Al - #a Thr Asn His Ala Ile # 345 - CTG CAG TCC
CTG GTG CAC CTG ATG AAG CCA AA - #C GCA GTC CCC AAG GCG 1584 Leu
Gln Ser Leu Val His Leu Met Lys Pro As - #n Ala Val Pro Lys Ala 350
3 - #55 3 - #60 3 - #65 - TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC
TC - #T GTG CTC TAC TAT GAC 1632 Cys Cys Ala Pro Thr Lys Leu Ser
Ala Thr Se - #r Val Leu Tyr Tyr Asp # 380 - AGC AGC AAC AAC GTC ATC
CTG CGC AAA CAC CG - #C AAC ATG GTG GTC AAG 1680 Ser Ser Asn Asn
Val Ile Leu Arg Lys His Ar - #g Asn Met Val Val Lys # 395 # 172 -
#3TCAGCC CGCCCAGCCC TACTGCAG Ala Cys Gly Cys His 400 - (2)
INFORMATION FOR SEQ ID NO:21: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 402 amino (B) TYPE: amino acid (D) TOPOLOGY:
linear - (ii) MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:21: - Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Le - #u Gly
Leu Ala Leu Cys # 15 - Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pr -
#o Pro Pro Gly Cys Pro # 30 - Gln Arg Arg Leu Gly Ala Arg Glu Arg
Arg As - #p Val Gln Arg Glu Ile # 45 - Leu Ala Val Leu Gly Leu Pro
Gly Arg Pro Ar - #g Pro Arg Ala Pro Pro # 60 - Ala Ala Ser Arg Leu
Pro Ala Ser Ala Pro Le - #u Phe Met Leu Asp Leu # 80 - Tyr His Ala
Met Ala Gly Asp Asp Asp Glu As - #p Gly Ala Pro Ala Glu # 95 - Arg
Arg Leu Gly Arg Ala Asp Leu Val Met Se - #r Phe Val Asn Met Val #
110 - Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pr - #o His Trp Lys
Glu Phe # 125 - Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Gl - #u Ala
Val Thr Ala Ala # 140 - Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile Hi
- #s Leu Leu Asn Arg Thr 145 1 - #50 1 - #55 1 - #60 - Leu His Val
Ser Met Phe Gln Val Val Gln Gl - #u Gln Ser Asn Arg Glu # 175 - Ser
Asp Leu Phe Phe Leu Asp Leu Gln Thr Le - #u Arg Ala Gly Asp Glu #
190 - Gly Trp Leu Val Leu Asp Val Thr Ala Ala Se - #r Asp Cys Trp
Leu Leu
# 205 - Lys Arg His Lys Asp Leu Gly Leu Arg Leu Ty - #r Val Glu Thr
Glu Asp # 220 - Gly His Ser Val Asp Pro Gly Leu Ala Gly Le - #u Leu
Gly Gln Arg Ala 225 2 - #30 2 - #35 2 - #40 - Pro Arg Ser Gln Gln
Pro Phe Val Val Thr Ph - #e Phe Arg Ala Ser Pro # 255 - Ser Pro Ile
Arg Thr Pro Arg Ala Val Arg Pr - #o Leu Arg Arg Arg Gln # 270 - Pro
Lys Lys Ser Asn Glu Leu Pro Gln Ala As - #n Arg Leu Pro Gly Ile #
285 - Phe Asp Asp Val His Gly Ser His Gly Arg Gl - #n Val Cys Arg
Arg His # 300 - Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Tr - #p Leu
Asp Trp Val Ile 305 3 - #10 3 - #15 3 - #20 - Ala Pro Gln Gly Tyr
Ser Ala Tyr Tyr Cys Gl - #u Gly Glu Cys Ser Phe # 335 - Pro Leu Asp
Ser Cys Met Asn Ala Thr Asn Hi - #s Ala Ile Leu Gln Ser # 350 - Leu
Val His Leu Met Lys Pro Asn Ala Val Pr - #o Lys Ala Cys Cys Ala #
365 - Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Ty - #r Tyr Asp Ser
Ser Asn # 380 - Asn Val Ile Leu Arg Lys His Arg Asn Met Va - #l Val
Lys Ala Cys Gly 385 3 - #90 3 - #95 4 - #00 - Cys His - (2)
INFORMATION FOR SEQ ID NO:22: - (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1926 base (B) TYPE: nucleic acid (C)
STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
cDNA - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 93..1289
#/product= "MOP2 CDNA"FORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:22: - GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC
CC - #GACCAGCT 60 - ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG
CGT - # CCC GGG CCA 113 #Met Ala Met Arg Pro Gly Pro # 1 5 - CTC
TGG CTA TTG GGC CTT GCT CTG TGC GCG CT - #G GGA GGC GGC CAC GGT 161
Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Le - #u Gly Gly Gly His Gly
# 20 - CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CG - #C CTG GGA GCG
CGC GAG 209 Pro Arg Pro Pro His Thr Cys Pro Gln Arg Ar - #g Leu Gly
Ala Arg Glu # 35 - CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GT - #G
CTC GGG CTA CCG GGA 257 Arg Arg Asp Met Gln Arg Glu Ile Leu Ala Va
- #l Leu Gly Leu Pro Gly # 55 - CGG CCC CGA CCC CGT GCA CAA CCC GCC
GCT GC - #C CGG CAG CCA GCG TCC 305 Arg Pro Arg Pro Arg Ala Gln Pro
Ala Ala Al - #a Arg Gln Pro Ala Ser # 70 - GCG CCC CTC TTC ATG TTG
GAC CTA TAC CAC GC - #C ATG ACC GAT GAC GAC 353 Ala Pro Leu Phe Met
Leu Asp Leu Tyr His Al - #a Met Thr Asp Asp Asp # 85 - GAC GGC GGG
CCA CCA CAG GCT CAC TTA GGC CG - #T GCC GAC CTG GTC ATG 401 Asp Gly
Gly Pro Pro Gln Ala His Leu Gly Ar - #g Ala Asp Leu Val Met # 100 -
AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT AC - #C CTG GGC TAC CAG GAG
449 Ser Phe Val Asn Met Val Glu Arg Asp Arg Th - #r Leu Gly Tyr Gln
Glu # 115 - CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA AC - #C CAG ATC
CCT GCT GGG 497 Pro His Trp Lys Glu Phe His Phe Asp Leu Th - #r Gln
Ile Pro Ala Gly 120 1 - #25 1 - #30 1 - #35 - GAG GCT GTC ACA GCT
GCT GAG TTC CGG ATC TA - #C AAA GAA CCC AGC ACC 545 Glu Ala Val Thr
Ala Ala Glu Phe Arg Ile Ty - #r Lys Glu Pro Ser Thr # 150 - CAC CCG
CTC AAC ACA ACC CTC CAC ATC AGC AT - #G TTC GAA GTG GTC CAA 593 His
Pro Leu Asn Thr Thr Leu His Ile Ser Me - #t Phe Glu Val Val Gln #
165 - GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TT - #T TTG GAT CTT
CAG ACG 641 Glu His Ser Asn Arg Glu Ser Asp Leu Phe Ph - #e Leu Asp
Leu Gln Thr # 180 - CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CT - #G
GAC ATC ACA GCA GCC 689 Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Le
- #u Asp Ile Thr Ala Ala # 195 - AGT GAC CGA TGG CTG CTG AAC CAT
CAC AAG GA - #C CTG GGA CTC CGC CTC 737 Ser Asp Arg Trp Leu Leu Asn
His His Lys As - #p Leu Gly Leu Arg Leu 200 2 - #05 2 - #10 2 - #15
- TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GA - #T CCT GGC CTG GCT
GGT 785 Tyr Val Glu Thr Ala Asp Gly His Ser Met As - #p Pro Gly Leu
Ala Gly # 230 - CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CA - #G CCT
TTC ATG GTA ACC 833 Leu Leu Gly Arg Gln Ala Pro Arg Ser Arg Gl - #n
Pro Phe Met Val Thr # 245 - TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG
GC - #C CCT CGG GCA GCG AGA 881 Phe Phe Arg Ala Ser Gln Ser Pro Val
Arg Al - #a Pro Arg Ala Ala Arg # 260 - CCA CTG AAG AGG AGG CAG CCA
AAG AAA ACG AA - #C GAG CTT CCG CAC CCC 929 Pro Leu Lys Arg Arg Gln
Pro Lys Lys Thr As - #n Glu Leu Pro His Pro # 275 - AAC AAA CTC CCA
GGG ATC TTT GAT GAT GGC CA - #C GGT TCC CGC GGC AGA 977 Asn Lys Leu
Pro Gly Ile Phe Asp Asp Gly Hi - #s Gly Ser Arg Gly Arg 280 2 - #85
2 - #90 2 - #95 - GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AG - #C
TTC CGT GAC CTT GGC 1025 Glu Val Cys Arg Arg His Glu Leu Tyr Val Se
- #r Phe Arg Asp Leu Gly # 310 - TGG CTG GAC TGG GTC ATC GCC CCC
CAG GGC TA - #C TCT GCC TAT TAC TGT 1073 Trp Leu Asp Trp Val Ile
Ala Pro Gln Gly Ty - #r Ser Ala Tyr Tyr Cys # 325 - GAG GGG GAG TGT
GCT TTC CCA CTG GAC TCC TG - #T ATG AAC GCC ACC AAC 1121 Glu Gly
Glu Cys Ala Phe Pro Leu Asp Ser Cy - #s Met Asn Ala Thr Asn # 340 -
CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG AT - #G AAG CCA GAT GTT GTC
1169 His Ala Ile Leu Gln Ser Leu Val His Leu Me - #t Lys Pro Asp
Val Val # 355 - CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AG - #T GCC
ACC TCT GTG CTG 1217 Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Se -
#r Ala Thr Ser Val Leu 360 3 - #65 3 - #70 3 - #75 - TAC TAT GAC
AGC AGC AAC AAT GTC ATC CTG CG - #T AAA CAC CGT AAC ATG 1265 Tyr
Tyr Asp Ser Ser Asn Asn Val Ile Leu Ar - #g Lys His Arg Asn Met #
390 - GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CC - #CAGCATCC
TGCTTCTACT 1319 Val Val Lys Ala Cys Gly Cys His 395 - ACCTTACCAT
CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT TA - #TCATAGCT 1379 -
CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA AA -
#ATTCTGGT 1439 - CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA
TCACCCCGCC CT - #CTCCATCC 1499 - TCCTACCCCA AGCATAGACT GAATGCACAC
AGCATCCCAG AGCTATGCTA AC - #TGAGAGGT 1559 - CTGGGGTCAG CACTGAAGGC
CCACATGAGG AAGACTGATC CTTGGCCATC CT - #CAGCCCAC 1619 - AATGGCAAAT
TCTGGATGGT CTAAGAAGGC CGTGGAATTC TAAACTAGAT GA - #TCTGGGCT 1679 -
CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA CA -
#TACACTTA 1739 - GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT
TGTTAGAAAA AG - #AATCAGAG 1799 - CCAGGTATAG CGGTGCATGT CATTAATCCC
AGCGCTAAAG AGACAGAGAC AG - #GAGAATCT 1859 - CTGTGAGTTC AAGGCCACAT
AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA AA - #AAAAAAAC 1919 # 1926 - (2)
INFORMATION FOR SEQ ID NO:23: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 399 amino (B) TYPE: amino acid (D) TOPOLOGY:
linear - (ii) MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:23: - Met Ala Met Arg Pro Gly Pro Leu Trp Leu Le - #u Gly
Leu Ala Leu Cys # 15 - Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pr -
#o His Thr Cys Pro Gln # 30 - Arg Arg Leu Gly Ala Arg Glu Arg Arg
Asp Me - #t Gln Arg Glu Ile Leu # 45 - Ala Val Leu Gly Leu Pro Gly
Arg Pro Arg Pr - #o Arg Ala Gln Pro Ala # 60 - Ala Ala Arg Gln Pro
Ala Ser Ala Pro Leu Ph - #e Met Leu Asp Leu Tyr # 80 - His Ala Met
Thr Asp Asp Asp Asp Gly Gly Pr - #o Pro Gln Ala His Leu # 95 - Gly
Arg Ala Asp Leu Val Met Ser Phe Val As - #n Met Val Glu Arg Asp #
110 - Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Ly - #s Glu Phe His
Phe Asp # 125 - Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Th - #r Ala
Ala Glu Phe Arg # 140 - Ile Tyr Lys Glu Pro Ser Thr His Pro Leu As
- #n Thr Thr Leu His Ile 145 1 - #50 1 - #55 1 - #60 - Ser Met Phe
Glu Val Val Gln Glu His Ser As - #n Arg Glu Ser Asp Leu # 175 - Phe
Phe Leu Asp Leu Gln Thr Leu Arg Ser Gl - #y Asp Glu Gly Trp Leu #
190 - Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Tr - #p Leu Leu Asn
His His # 205 - Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Th - #r Ala
Asp Gly His Ser # 220 - Met Asp Pro Gly Leu Ala Gly Leu Leu Gly Ar
- #g Gln Ala Pro Arg Ser 225 2 - #30 2 - #35 2 - #40 - Arg Gln Pro
Phe Met Val Thr Phe Phe Arg Al - #a Ser Gln Ser Pro Val # 255 - Arg
Ala Pro Arg Ala Ala Arg Pro Leu Lys Ar - #g Arg Gln Pro Lys Lys #
270 - Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pr - #o Gly Ile Phe
Asp Asp # 285 - Gly His Gly Ser Arg Gly Arg Glu Val Cys Ar - #g Arg
His Glu Leu Tyr # 300 - Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Tr
- #p Val Ile Ala Pro Gln 305 3 - #10 3 - #15 3 - #20 - Gly Tyr Ser
Ala Tyr Tyr Cys Glu Gly Glu Cy - #s Ala Phe Pro Leu Asp # 335 - Ser
Cys Met Asn Ala Thr Asn His Ala Ile Le - #u Gln Ser Leu Val His #
350 - Leu Met Lys Pro Asp Val Val Pro Lys Ala Cy - #s Cys Ala Pro
Thr Lys # 365 - Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Se - #r Ser
Asn Asn Val Ile # 380 - Leu Arg Lys His Arg Asn Met Val Val Lys Al
- #a Cys Gly Cys His 385 3 - #90 3 - #95 - (2) INFORMATION FOR SEQ
ID NO:24: - (i) SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 1368
base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear - (ii) MOLECULE TYPE: cDNA - (ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..1365 - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: -
ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GT - #T GCA GTG CTC GCC TCC
48 Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Va - #l Ala Val Leu Ala
Ser # 15 - CTG GGA CTC GGA ATG GTT CTG CTC ATG TTC GT - #G GCG ACC
ACG CCG CCG 96 Leu Gly Leu Gly Met Val Leu Leu Met Phe Va - #l Ala
Thr Thr Pro Pro # 30
- GCC GTT GAG GCC ACC CAG TCG GGG ATT TAC AT - #A GAC AAC GGC AAG
GAC 144 Ala Val Glu Ala Thr Gln Ser Gly Ile Tyr Il - #e Asp Asn Gly
Lys Asp # 45 - CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GA - #C GAC
AAG CTG GAC GTC 192 Gln Thr Ile Met His Arg Val Leu Ser Glu As - #p
Asp Lys Leu Asp Val # 60 - TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC
GC - #C GAA CGG CCG ACG CAC 240 Ser Tyr Glu Ile Leu Glu Phe Leu Gly
Ile Al - #a Glu Arg Pro Thr His # 80 - CTG AGC AGC CAC CAG TTG TCG
CTG AGG AAG TC - #G GCT CCC AAG TTC CTG 288 Leu Ser Ser His Gln Leu
Ser Leu Arg Lys Se - #r Ala Pro Lys Phe Leu # 95 - CTG GAC GTC TAC
CAC CGC ATC ACG GCG GAG GA - #G GGT CTC AGC GAT CAG 336 Leu Asp Val
Tyr His Arg Ile Thr Ala Glu Gl - #u Gly Leu Ser Asp Gln # 110 - GAT
GAG GAC GAC GAC TAC GAA CGC GGC CAT CG - #G TCC AGG AGG AGC GCC 384
Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Ar - #g Ser Arg Arg Ser Ala
# 125 - GAC CTC GAG GAG GAT GAG GGC GAG CAG CAG AA - #G AAC TTC ATC
ACC GAC 432 Asp Leu Glu Glu Asp Glu Gly Glu Gln Gln Ly - #s Asn Phe
Ile Thr Asp # 140 - CTG GAC AAG CGG GCC ATC GAC GAG AGC GAC AT - #C
ATC ATG ACC TTC CTG 480 Leu Asp Lys Arg Ala Ile Asp Glu Ser Asp Il
- #e Ile Met Thr Phe Leu 145 1 - #50 1 - #55 1 - #60 - AAC AAG CGC
CAC CAC AAT GTG GAC GAA CTG CG - #T CAC GAG CAC GGC CGT 528 Asn Lys
Arg His His Asn Val Asp Glu Leu Ar - #g His Glu His Gly Arg # 175 -
CGC CTG TGG TTC GAC GTC TCC AAC GTG CCC AA - #C GAC AAC TAC CTG GTG
576 Arg Leu Trp Phe Asp Val Ser Asn Val Pro As - #n Asp Asn Tyr Leu
Val # 190 - ATG GCC GAG CTG CGC ATC TAT CAG AAC GCC AA - #C GAG GGC
AAG TGG CTG 624 Met Ala Glu Leu Arg Ile Tyr Gln Asn Ala As - #n Glu
Gly Lys Trp Leu # 205 - ACC GCC AAC AGG GAG TTC ACC ATC ACG GTA TA
- #C GCC ATT GGC ACC GGC 672 Thr Ala Asn Arg Glu Phe Thr Ile Thr
Val Ty - #r Ala Ile Gly Thr Gly # 220 - ACG CTG GGC CAG CAC ACC ATG
GAG CCG CTG TC - #C TCG GTG AAC ACC ACC 720 Thr Leu Gly Gln His Thr
Met Glu Pro Leu Se - #r Ser Val Asn Thr Thr 225 2 - #30 2 - #35 2 -
#40 - GGG GAC TAC GTG GGC TGG TTG GAG CTC AAC GT - #G ACC GAG GGC
CTG CAC 768 Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Va - #l Thr Glu
Gly Leu His # 255 - GAG TGG CTG GTC AAG TCG AAG GAC AAT CAT GG - #C
ATC TAC ATT GGA GCA 816 Glu Trp Leu Val Lys Ser Lys Asp Asn His Gl
- #y Ile Tyr Ile Gly Ala # 270 - CAC GCT GTC AAC CGA CCC GAC CGC
GAG GTG AA - #G CTG GAC GAC ATT GGA 864 His Ala Val Asn Arg Pro Asp
Arg Glu Val Ly - #s Leu Asp Asp Ile Gly # 285 - CTG ATC CAC CGC AAG
GTG GAC GAC GAG TTC CA - #G CCC TTC ATG ATC GGC 912 Leu Ile His Arg
Lys Val Asp Asp Glu Phe Gl - #n Pro Phe Met Ile Gly # 300 - TTC TTC
CGC GGA CCG GAG CTG ATC AAG GCG AC - #G GCC CAC AGC AGC CAC 960 Phe
Phe Arg Gly Pro Glu Leu Ile Lys Ala Th - #r Ala His Ser Ser His 305
3 - #10 3 - #15 3 - #20 - CAC AGG AGC AAG CGA AGC GCC AGC CAT CCA
CG - #C AAG CGC AAG AAG TCG 1008 His Arg Ser Lys Arg Ser Ala Ser
His Pro Ar - #g Lys Arg Lys Lys Ser # 335 - GTG TCG CCC AAC AAC GTG
CCG CTG CTG GAA CC - #G ATG GAG AGC ACG CGC 1056 Val Ser Pro Asn
Asn Val Pro Leu Leu Glu Pr - #o Met Glu Ser Thr Arg # 350 - AGC TGC
CAG ATG CAG ACC CTG TAC ATA GAC TT - #C AAG GAT CTG GGC TGG 1104
Ser Cys Gln Met Gln Thr Leu Tyr Ile Asp Ph - #e Lys Asp Leu Gly Trp
# 365 - CAT GAC TGG ATC ATC GCA CCA GAG GGC TAT GG - #C GCC TTC TAC
TGC AGC 1152 His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gl - #y Ala
Phe Tyr Cys Ser # 380 - GGC GAG TGC AAT TTC CCG CTC AAT GCG CAC AT
- #G AAC GCC ACG AAC CAT 1200 Gly Glu Cys Asn Phe Pro Leu Asn Ala
His Me - #t Asn Ala Thr Asn His 385 3 - #90 3 - #95 4 - #00 - GCG
ATC GTC CAG ACC CTG GTC CAC CTG CTG GA - #G CCC AAG AAG GTG CCC
1248 Ala Ile Val Gln Thr Leu Val His Leu Leu Gl - #u Pro Lys Lys
Val Pro # 415 - AAG CCC TGC TGC GCT CCG ACC AGG CTG GGA GC - #A CTA
CCC GTT CTG TAC 1296 Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Al -
#a Leu Pro Val Leu Tyr # 430 - CAC CTG AAC GAC GAG AAT GTG AAC CTG
AAA AA - #G TAT AGA AAC ATG ATT 1344 His Leu Asn Asp Glu Asn Val
Asn Leu Lys Ly - #s Tyr Arg Asn Met Ile # 445 # 1368GG TGC CAT TGA
Val Lys Ser Cys Gly Cys His # 455 - (2) INFORMATION FOR SEQ ID
NO:25: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 455 amino
(B) TYPE: amino acid (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: - Met Ser Gly
Leu Arg Asn Thr Ser Glu Ala Va - #l Ala Val Leu Ala Ser # 15 - Leu
Gly Leu Gly Met Val Leu Leu Met Phe Va - #l Ala Thr Thr Pro Pro #
30 - Ala Val Glu Ala Thr Gln Ser Gly Ile Tyr Il - #e Asp Asn Gly
Lys Asp # 45 - Gln Thr Ile Met His Arg Val Leu Ser Glu As - #p Asp
Lys Leu Asp Val # 60 - Ser Tyr Glu Ile Leu Glu Phe Leu Gly Ile Al -
#a Glu Arg Pro Thr His # 80 - Leu Ser Ser His Gln Leu Ser Leu Arg
Lys Se - #r Ala Pro Lys Phe Leu # 95 - Leu Asp Val Tyr His Arg Ile
Thr Ala Glu Gl - #u Gly Leu Ser Asp Gln # 110 - Asp Glu Asp Asp Asp
Tyr Glu Arg Gly His Ar - #g Ser Arg Arg Ser Ala # 125 - Asp Leu Glu
Glu Asp Glu Gly Glu Gln Gln Ly - #s Asn Phe Ile Thr Asp # 140 - Leu
Asp Lys Arg Ala Ile Asp Glu Ser Asp Il - #e Ile Met Thr Phe Leu 145
1 - #50 1 - #55 1 - #60 - Asn Lys Arg His His Asn Val Asp Glu Leu
Ar - #g His Glu His Gly Arg # 175 - Arg Leu Trp Phe Asp Val Ser Asn
Val Pro As - #n Asp Asn Tyr Leu Val # 190 - Met Ala Glu Leu Arg Ile
Tyr Gln Asn Ala As - #n Glu Gly Lys Trp Leu # 205 - Thr Ala Asn Arg
Glu Phe Thr Ile Thr Val Ty - #r Ala Ile Gly Thr Gly # 220 - Thr Leu
Gly Gln His Thr Met Glu Pro Leu Se - #r Ser Val Asn Thr Thr 225 2 -
#30 2 - #35 2 - #40 - Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Va -
#l Thr Glu Gly Leu His # 255 - Glu Trp Leu Val Lys Ser Lys Asp Asn
His Gl - #y Ile Tyr Ile Gly Ala # 270 - His Ala Val Asn Arg Pro Asp
Arg Glu Val Ly - #s Leu Asp Asp Ile Gly # 285 - Leu Ile His Arg Lys
Val Asp Asp Glu Phe Gl - #n Pro Phe Met Ile Gly # 300 - Phe Phe Arg
Gly Pro Glu Leu Ile Lys Ala Th - #r Ala His Ser Ser His 305 3 - #10
3 - #15 3 - #20 - His Arg Ser Lys Arg Ser Ala Ser His Pro Ar - #g
Lys Arg Lys Lys Ser # 335 - Val Ser Pro Asn Asn Val Pro Leu Leu Glu
Pr - #o Met Glu Ser Thr Arg # 350 - Ser Cys Gln Met Gln Thr Leu Tyr
Ile Asp Ph - #e Lys Asp Leu Gly Trp # 365 - His Asp Trp Ile Ile Ala
Pro Glu Gly Tyr Gl - #y Ala Phe Tyr Cys Ser # 380 - Gly Glu Cys Asn
Phe Pro Leu Asn Ala His Me - #t Asn Ala Thr Asn His 385 3 - #90 3 -
#95 4 - #00 - Ala Ile Val Gln Thr Leu Val His Leu Leu Gl - #u Pro
Lys Lys Val Pro # 415 - Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Al
- #a Leu Pro Val Leu Tyr # 430 - His Leu Asn Asp Glu Asn Val Asn
Leu Lys Ly - #s Tyr Arg Asn Met Ile # 445 - Val Lys Ser Cys Gly Cys
His # 455 - (2) INFORMATION FOR SEQ ID NO:26: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 104 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (ix) FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..104
#/label= BMP3 OTHER INFORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:26: - Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Al - #a Asp Ile
Gly Trp Ser # 15 - Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Al - #a
Tyr Tyr Cys Ser Gly # 30 - Ala Cys Gln Phe Pro Met Pro Lys Ser Leu
Ly - #s Pro Ser Asn His Ala # 45 - Thr Ile Gln Ser Ile Val Ala Arg
Ala Val Gl - #y Val Val Pro Gly Ile # 60 - Pro Glu Pro Cys Cys Val
Pro Glu Lys Met Se - #r Ser Leu Ser Ile Leu #80 - Phe Phe Asp Glu
Asn Lys Asn Val Val Leu Ly - #s Val Tyr Pro Asn Met # 95 - Thr Val
Glu Ser Cys Ala Cys Arg 100 - (2) INFORMATION FOR SEQ ID NO:27: -
(i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 102 amino (B)
TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -
(ii) MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY: Protein
(B) LOCATION: 1..102 #/label= BMP5 OTHER INFORMATION: - (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:27: - Cys Lys Lys His Glu Leu Tyr
Val Ser Phe Ar - #g Asp Leu Gly Trp Gln # 15 - Asp Trp Ile Ile Ala
Pro Glu Gly Tyr Ala Al - #a Phe Tyr Cys Asp Gly # 30 - Glu Cys Ser
Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala # 45 - Ile
Val Gln Thr Leu Val His Leu Met Phe Pr - #o Asp His Val Pro Lys #
60 - Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Il - #e Ser Val Leu
Tyr Phe #80 - Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Ty - #r Arg
Asn Met Val Val # 95 - Arg Ser Cys Gly Cys His 100 - (2)
INFORMATION FOR SEQ ID NO:28: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 102 amino (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix)
FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..102 #/label= BMP6
OTHER INFORMATION:
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: - Cys Arg Lys His Glu
Leu Tyr Val Ser Phe Gl - #n Asp Leu Gly Trp Gln # 15 - Asp Trp Ile
Ile Ala Pro Lys Gly Tyr Ala Al - #a Asn Tyr Cys Asp Gly # 30 - Glu
Cys Ser Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala #
45 - Ile Val Gln Thr Leu Val His Leu Met Asn Pr - #o Glu Tyr Val
Pro Lys # 60 - Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Il - #e Ser
Val Leu Tyr Phe #80 - Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Ty -
#r Arg Trp Met Val Val # 95 - Arg Ala Cys Gly Cys His 100 - (2)
INFORMATION FOR SEQ ID NO:29: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 102 amino (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix)
FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..102 #/label= OPX)
OTHER INFORMATION: #"WHEREIN XAA AT EACH POS'N IS INDEPENDENTLY
#FROM THE RESIDUES OCCURING AT THE CORRESPONDING #IN THE C-TERMINAL
SEQUENCE OF MOUSE OR HUMAN OP - #1 OR OP2 (SEQ. - # ID NOS.
5,6,7&8 OR 16,18, 20&22" - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:29: - Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xa - #a Asp Leu
Gly Trp Xaa # 15 - Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Al - #a
Tyr Tyr Cys Glu Gly # 30 - Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met
As - #n Ala Thr Asn His Ala # 45 - Ile Xaa Gln Xaa Leu Val His Xaa
Xaa Xaa Pr - #o Xaa Xaa Val Pro Lys # 60 - Xaa Cys Cys Ala Pro Thr
Xaa Leu Xaa Ala Xa - #a Ser Val Leu Tyr Xaa #80 - Asp Xaa Ser Xaa
Asn Val Xaa Leu Xaa Lys Xa - #a Arg Asn Met Val Val # 95 - Xaa Ala
Cys Gly Cys His 100 - (2) INFORMATION FOR SEQ ID NO:30: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 97 amino (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: protein - (ix) FEATURE: (A) NAME/KEY: Protein (B)
LOCATION: 1..97 #/label= GENERIC-SEQ-5FORMATION: #AWHEREIN EACH XAA
IS INDEPENDENTLY SELECTED FROM #ONE OR MORE SPECIFIED AMINO ACIDS
AS DEFINED IN THE SPECI - #FICATION" - (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:30: - Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xa - #a Xaa
Trp Xaa Xaa Xaa # 15 - Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gl -
#y Xaa Cys Xaa Xaa Pro # 30 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn
His Al - #a Xaa Xaa Xaa Xaa Xaa # 45 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xa - #a Xaa Cys Cys Xaa Pro # 60 - Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Leu Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa #80 - Val Xaa Leu
Xaa Xaa Xaa Xaa Xaa Met Xaa Va - #l Xaa Xaa Cys Xaa Cys # 95 - Xaa
- (2) INFORMATION FOR SEQ ID NO:31: - (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 102 amino (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (ix)
FEATURE: (A) NAME/KEY: Protein (B) LOCATION: 1..102 #/label=
GENERIC-SEQ-6FORMATION: #AWHEREIN EACH XAA IS INDEPENDENTLY
SELECTED FROM #ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE
SPECI - #FICATION" - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: - Cys
Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xa - #a Xaa Xaa Gly Trp Xaa #
15 - Xaa Trp Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Al - #a Xaa Tyr Cys
Xaa Gly # 30 - Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa
Xaa Asn His Ala # 45 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa -
#a Xaa Xaa Xaa Xaa Xaa # 60 - Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa
Xaa Xa - #a Xaa Xaa Leu Xaa Xaa #80 - Xaa Xaa Xaa Xaa Xaa Val Xaa
Leu Xaa Xaa Xa - #a Xaa Xaa Met Xaa Val # 95 - Xaa Xaa Cys Xaa Cys
Xaa 100 - (2) INFORMATION FOR SEQ ID NO:32: - (i) SEQUENCE
CHARACTERISTICS: #pairs (A) LENGTH: 1247 base (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE
TYPE: cDNA - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 84..1199
#/product= "GDF-1"R INFORMATION: - (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:32: - GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC
CT - #GCGCACTC 60 - TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG
CA - #A GGT CCC TGC 110 #Gly Pro Cys Pro Pro Pro Gln Gln # 5 1 -
GGC CAC CAC CTC CTC CTC CTC CTG GCC CTG CT - #G CTG CCC TCG CTG CCC
158 Gly His His Leu Leu Leu Leu Leu Ala Leu Le - #u Leu Pro Ser Leu
Pro # 25 - CTG ACC CGC GCC CCC GTG CCC CCA GGC CCA GC - #C GCC GCC
CTG CTC CAG 206 Leu Thr Arg Ala Pro Val Pro Pro Gly Pro Al - #a Ala
Ala Leu Leu Gln # 40 - GCT CTA GGA CTG CGC GAT GAG CCC CAG GGT GC -
#C CCC AGG CTC CGG CCG 254 Ala Leu Gly Leu Arg Asp Glu Pro Gln Gly
Al - #a Pro Arg Leu Arg Pro # 55 - GTT CCC CCG GTC ATG TGG CGC CTG
TTT CGA CG - #C CGG GAC CCC CAG GAG 302 Val Pro Pro Val Met Trp Arg
Leu Phe Arg Ar - #g Arg Asp Pro Gln Glu # 70 - ACC AGG TCT GGC TCG
CGG CGG ACG TCC CCA GG - #G GTC ACC CTG CAA CCG 350 Thr Arg Ser Gly
Ser Arg Arg Thr Ser Pro Gl - #y Val Thr Leu Gln Pro # 85 - TGC CAC
GTG GAG GAG CTG GGG GTC GCC GGA AA - #C ATC GTG CGC CAC ATC 398 Cys
His Val Glu Glu Leu Gly Val Ala Gly As - #n Ile Val Arg His Ile
#105 - CCG GAC CGC GGT GCG CCC ACC CGG GCC TCG GA - #G CCT GTC TCG
GCC GCG 446 Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser Gl - #u Pro Val
Ser Ala Ala # 120 - GGG CAT TGC CCT GAG TGG ACA GTC GTC TTC GA - #C
CTG TCG GCT GTG GAA 494 Gly His Cys Pro Glu Trp Thr Val Val Phe As
- #p Leu Ser Ala Val Glu # 135 - CCC GCT GAG CGC CCG AGC CGG GCC
CGC CTG GA - #G CTG CGT TTC GCG GCG 542 Pro Ala Glu Arg Pro Ser Arg
Ala Arg Leu Gl - #u Leu Arg Phe Ala Ala # 150 - GCG GCG GCG GCA GCC
CCG GAG GGC GGC TGG GA - #G CTG AGC GTG GCG CAA 590 Ala Ala Ala Ala
Ala Pro Glu Gly Gly Trp Gl - #u Leu Ser Val Ala Gln # 165 - GCG GGC
CAG GGC GCG GGC GCG GAC CCC GGG CC - #G GTG CTG CTC CGC CAG 638 Ala
Gly Gln Gly Ala Gly Ala Asp Pro Gly Pr - #o Val Leu Leu Arg Gln 170
1 - #75 1 - #80 1 - #85 - TTG GTG CCC GCC CTG GGG CCG CCA GTG CGC
GC - #G GAG CTG CTG GGC GCC 686 Leu Val Pro Ala Leu Gly Pro Pro Val
Arg Al - #a Glu Leu Leu Gly Ala # 200 - GCT TGG GCT CGC AAC GCC TCA
TGG CCG CGC AG - #C CTC CGC CTG GCG CTG 734 Ala Trp Ala Arg Asn Ala
Ser Trp Pro Arg Se - #r Leu Arg Leu Ala Leu # 215 - GCG CTA CGC CCC
CGG GCC CCT GCC GCC TGC GC - #G CGC CTG GCC GAG GCC 782 Ala Leu Arg
Pro Arg Ala Pro Ala Ala Cys Al - #a Arg Leu Ala Glu Ala # 230 - TCG
CTG CTG CTG GTG ACC CTC GAC CCG CGC CT - #G TGC CAC CCC CTG GCC 830
Ser Leu Leu Leu Val Thr Leu Asp Pro Arg Le - #u Cys His Pro Leu Ala
# 245 - CGG CCG CGG CGC GAC GCC GAA CCC GTG TTG GG - #C GGC GGC CCC
GGG GGC 878 Arg Pro Arg Arg Asp Ala Glu Pro Val Leu Gl - #y Gly Gly
Pro Gly Gly 250 2 - #55 2 - #60 2 - #65 - GCT TGT CGC GCG CGG CGG
CTG TAC GTG AGC TT - #C CGC GAG GTG GGC TGG 926 Ala Cys Arg Ala Arg
Arg Leu Tyr Val Ser Ph - #e Arg Glu Val Gly Trp # 280 - CAC CGC TGG
GTC ATC GCG CCG CGC GGC TTC CT - #G GCC AAC TAC TGC CAG 974 His Arg
Trp Val Ile Ala Pro Arg Gly Phe Le - #u Ala Asn Tyr Cys Gln # 295 -
GGT CAG TGC GCG CTG CCC GTC GCG CTG TCG GG - #G TCC GGG GGG CCG CCG
1022 Gly Gln Cys Ala Leu Pro Val Ala Leu Ser Gl - #y Ser Gly Gly
Pro Pro # 310 - GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC AT - #G CAC
GCG GCC GCC CCG 1070 Ala Leu Asn His Ala Val Leu Arg Ala Leu Me -
#t His Ala Ala Ala Pro # 325 - GGA GCC GCC GAC CTG CCC TGC TGC GTG
CCC GC - #G CGC CTG TCG CCC ATC 1118 Gly Ala Ala Asp Leu Pro Cys
Cys Val Pro Al - #a Arg Leu Ser Pro Ile 330 3 - #35 3 - #40 3 - #45
- TCC GTG CTC TTC TTT GAC AAC AGC GAC AAC GT - #G GTG CTG CGG CAG
TAT 1166 Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Va - #l Val Leu
Arg Gln Tyr # 360 - GAG GAC ATG GTG GTG GAC GAG TGC GGC TGC CG - #C
TAACCCGGGG CGGGCAGGGA 1219 Glu Asp Met Val Val Asp Glu Cys Gly Cys
Ar - #g # 370 # 1247 AATG CCGCGTGG - (2) INFORMATION FOR SEQ ID
NO:33: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 372 amino
(B) TYPE: amino acid (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: - Met Pro Pro
Pro Gln Gln Gly Pro Cys Gly Hi - #s His Leu Leu Leu Leu # 15 - Leu
Ala Leu Leu Leu Pro Ser Leu Pro Leu Th - #r Arg Ala Pro Val Pro #
30 - Pro Gly Pro Ala Ala Ala Leu Leu Gln Ala Le - #u Gly Leu Arg
Asp Glu # 45 - Pro Gln Gly Ala Pro Arg Leu Arg Pro Val Pr - #o Pro
Val Met Trp Arg # 60 - Leu Phe Arg Arg Arg Asp Pro Gln Glu Thr Ar -
#g Ser Gly Ser Arg Arg # 80 - Thr Ser Pro Gly Val Thr Leu Gln Pro
Cys Hi - #s Val Glu Glu Leu Gly # 95 - Val Ala Gly Asn Ile Val Arg
His Ile Pro As - #p Arg Gly Ala Pro Thr # 110 - Arg Ala Ser Glu Pro
Val Ser Ala Ala Gly Hi - #s Cys Pro Glu Trp Thr # 125 - Val Val Phe
Asp Leu Ser Ala Val Glu Pro Al - #a Glu Arg Pro Ser Arg # 140 - Ala
Arg Leu Glu Leu Arg Phe Ala Ala Ala Al - #a Ala Ala Ala Pro Glu 145
1 - #50 1 - #55 1 - #60 - Gly Gly Trp Glu Leu Ser Val Ala Gln Ala
Gl - #y Gln Gly Ala Gly Ala # 175 - Asp Pro Gly Pro Val Leu Leu Arg
Gln Leu Va - #l Pro Ala Leu Gly Pro # 190 - Pro Val Arg Ala Glu Leu
Leu Gly Ala Ala Tr - #p Ala Arg Asn Ala Ser # 205 - Trp Pro Arg Ser
Leu Arg Leu Ala Leu Ala Le - #u Arg Pro Arg Ala Pro
# 220 - Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Le - #u Leu Leu Val
Thr Leu 225 2 - #30 2 - #35 2 - #40 - Asp Pro Arg Leu Cys His Pro
Leu Ala Arg Pr - #o Arg Arg Asp Ala Glu # 255 - Pro Val Leu Gly Gly
Gly Pro Gly Gly Ala Cy - #s Arg Ala Arg Arg Leu # 270 - Tyr Val Ser
Phe Arg Glu Val Gly Trp His Ar - #g Trp Val Ile Ala Pro # 285 - Arg
Gly Phe Leu Ala Asn Tyr Cys Gln Gly Gl - #n Cys Ala Leu Pro Val #
300 - Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Le - #u Asn His Ala
Val Leu 305 3 - #10 3 - #15 3 - #20 - Arg Ala Leu Met His Ala Ala
Ala Pro Gly Al - #a Ala Asp Leu Pro Cys # 335 - Cys Val Pro Ala Arg
Leu Ser Pro Ile Ser Va - #l Leu Phe Phe Asp Asn # 350 - Ser Asp Asn
Val Val Leu Arg Gln Tyr Glu As - #p Met Val Val Asp Glu # 365 - Cys
Gly Cys Arg 370
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