U.S. patent application number 10/352683 was filed with the patent office on 2003-07-31 for carbohydrate ligands specific for mhc molecules.
Invention is credited to Rothenberg, Barry E..
Application Number | 20030143620 10/352683 |
Document ID | / |
Family ID | 23374368 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143620 |
Kind Code |
A1 |
Rothenberg, Barry E. |
July 31, 2003 |
Carbohydrate ligands specific for MHC molecules
Abstract
The present invention provides a substantially purified
carbohydrate ligand that specifically binds to a leczyme. The
invention also provides methods to identify a carbohydrate ligand
that specifically binds to a leczyme or a leczyme that specifically
binds to a carbohydrate ligand. The invention further provides
methods to identify a peptide that binds to the carbohydrate ligand
binding site of a leczyme. The present invention provides methods
to isolate a carbohydrate ligand or a leczyme and to identify a
carbohydrate ligand or a leczyme that modifies the function of a
cell and to obtain such functionally modified cells. The invention
further provides methods to modify a cell to express a carbohydrate
ligand by introducing an expression vector encoding a leczyme into
the cell. The invention also provides methods to modulate the
immune response to an antigen by administering the antigen and a
carbohydrate ligand. In addition, the invention further provides
methods to treat a disease state involving a leczyme by
administering a carbohydrate ligand that binds the leczyme or by
administering a leczyme that has a similar binding specificity to
the leczyme involved in the disease state. The invention further
provides methods to diagnose a genetic basis for hemochromatosis by
detecting a mutation in a class I MHC molecule that reduces it's
ability to associate with .beta..sub.2 microglobulin.
Inventors: |
Rothenberg, Barry E.; (Del
Mar, CA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
23374368 |
Appl. No.: |
10/352683 |
Filed: |
January 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10352683 |
Jan 28, 2003 |
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09732363 |
Dec 6, 2000 |
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6511807 |
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09732363 |
Dec 6, 2000 |
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08349883 |
Dec 6, 1994 |
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5674681 |
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Current U.S.
Class: |
435/6.16 ;
514/44R; 514/54 |
Current CPC
Class: |
C07K 14/70539 20130101;
C12Q 2600/156 20130101; G01N 33/573 20130101; A61K 38/00 20130101;
C12Q 1/6881 20130101; A61P 37/04 20180101; C07K 14/4726 20130101;
C12N 9/00 20130101; A61P 37/00 20180101; C07K 14/47 20130101; A61P
29/00 20180101; A61P 3/00 20180101; A61P 31/00 20180101 |
Class at
Publication: |
435/6 ; 514/44;
514/54 |
International
Class: |
C12Q 001/68; A61K
048/00; A61K 031/715 |
Claims
I claim:
1. A composition, comprising a substantially purified carbohydrate
ligand that specifically binds to a leczyme.
2. The composition of claim 1, further comprising a polypeptide or
lipid moiety bonded to said carbohydrate ligand.
3. A method of identifying a carbohydrate ligand that binds to a
leczyme, comprising the steps of: a. contacting a sample containing
a carbohydrate ligand with a leczyme suspected to bind such a
ligand; and b. detecting whether binding of the ligand and the
leczyme has occurred.
4. The method of claim 3, wherein said leczyme is an MHC-derived
gene product.
5. The method of claim 4, wherein said MHC-derived gene product is
a class I molecule.
6. The method of claim 4, wherein said MHC-derived gene product is
a class II molecule.
7. The method of claim 4, wherein said MHC-derived gene product is
an nonclassical class I molecule.
8. The method of claim 3, wherein said leczyme is a non-MHC derived
gene product.
9. The method of claim 3, wherein said carbohydrate ligand is
bonded to a polypeptide or lipid moiety.
10. The method of claim 3, wherein said carbohydrate ligand or said
leczyme contains a detectable label.
11. The method of claim 3, wherein said carbohydrate ligand or said
leczyme is associated with a cell.
12. A method of identifying a leczyme that binds to a carbohydrate
ligand, comprising the steps of: a. contacting a sample containing
a leczyme with a carbohydrate ligand suspected to bind such a
leczyme; and b. detecting whether binding of the leczyme and the
ligand has occurred.
13. The method of claim 12, wherein said leczyme is an MHC-derived
gene product.
14. The method of claim 12, wherein said leczyme is a non-MHC
derived gene product.
15. The method of claim 12, wherein said carbohydrate ligand is
bonded to a polypeptide or lipid moiety.
16. The method of claim 12, wherein said carbohydrate ligand or
said leczyme contains a detectable label.
17. The method of claim 12, wherein said carbohydrate ligand or
said leczyme is associated with a cell.
18. A method of purifying a carbohydrate ligand that specifically
binds to a reagent, comprising the steps of: a. contacting a sample
containing a carbohydrate ligand with a reagent capable of binding
such a ligand to form a ligand-reagent complex; b. separating the
complex from the rest of the sample; and c. dissociating the ligand
from the complex to obtain the purified ligand.
19. The method of claim 18, wherein said reagent is a leczyme.
20. The method of claim 18, wherein said reagent is an
antibody.
21. The method of claim 18, wherein said leczyme is an MHC-derived
gene product.
22. The method of claim 18, wherein said leczyme is a non-MHC
derived gene product.
23. The method of claim 18, wherein said carbohydrate ligand is
bonded to a polypeptide or lipid moiety.
24. A method of purifying a leczyme that specifically binds to a
carbohydrate ligand, comprising the steps of: a. contacting a
sample containing a leczyme with a carbohydrate ligand capable of
binding such a leczyme to form a ligand-leczyme complex; b.
separating the complex from the rest of the sample; and c.
dissociating the leczyme from the complex to obtain the purified
leczyme.
25. The method of claim 24, wherein said leczyme is an MHC-derived
gene product.
26. The method of claim 24, wherein said leczyme is a non-MHC
derived gene product.
27. The method of claim 24, wherein said carbohydrate ligand is
bonded to a polypeptide or lipid moiety.
28. A method of identifying a carbohydrate ligand that modifies the
function of a leczyme-expressing cell, comprising the steps of: a.
contacting a sample containing such a carbohydrate ligand with a
leczyme-expressing cell; and b. subsequently assaying the cell to
determine it's function.
29. The method of claim 28, wherein said leczyme is an MHC-derived
gene product.
30. The method of claim 28, wherein said leczyme is a non-MHC
derived gene product.
31. The method of claim 28, wherein said carbohydrate ligand is
bonded to a polypeptide or lipid moiety.
32. A method of identifying a leczyme that modifies the function of
a carbohydrate ligand-expressing cell, comprising the steps of: a.
contacting a sample containing such a leczyme with a carbohydrate
ligand-expressing cell; and b. subsequently assaying the cell to
determine it's function.
33. The method of claim 32, wherein said leczyme is an MHC-derived
gene product.
34. The method of claim 32, wherein said leczyme is a non-MHC
derived gene product.
35. A method of modifying the function of a leczyme-expressing
cell, comprising contacting the cell with a carbohydrate ligand
that binds the leczyme.
36. The method of claim 35, wherein said leczyme is an MHC-derived
gene product.
37. The method of claim 35, wherein said leczyme is a non-MHC
derived gene product.
38. The method of claim 35, wherein said carbohydrate ligand is
bonded to a polypeptide or lipid moiety.
39. A method of modifying the function of a carbohydrate
ligand-expressing cell, comprising contacting the cell with a
leczyme that binds the ligand.
40. The method of claim 39, wherein said leczyme is an MHC-derived
gene product.
41. The method of claim 39, wherein said leczyme is a non-MHC
derived gene product.
42. A method of identifying a peptide that binds to a carbohydrate
ligand binding site of a leczyme, comprising the steps of: a.
contacting a leczyme and a carbohydrate ligand known to bind the
leczyme with a test sample containing a peptide to be identified;
b. determining the amount of carbohydrate ligand bound to the
leczyme after said reacting; and c. comparing the amount of
carbohydrate ligand bound in the test sample with the amount of
carbohydrate ligand bound in a control sample, wherein a decreased
amount of carbohydrate ligand bound in the test sample relative to
the amount of carbohydrate ligand bound in the control sample
indicates binding of the peptide to the leczyme.
43. The method of claim 42, wherein said peptide and said leczyme
are contacted prior to contacting said carbohydrate ligand.
44. The method of claim 42, wherein said carbohydrate ligand or
said leczyme contains a detectable label.
45. The method of claim 42, wherein said leczyme or said
carbohydrate ligand is associated with a cell.
46. The method of claim 42, wherein said test sample contains a
peptide library.
47. A method of modifying a cell to produce a carbohydrate ligand,
comprising introducing an expression vector encoding a leczyme into
the cell to obtain expression of the leczyme, wherein said
expression results in production of the carbohydrate ligand by the
cell.
48. The method of claim 47, wherein said leczyme is an MHC-derived
gene product.
49. The method of claim 47, wherein said leczyme is a non-MHC
derived gene product.
50. A method for modulating an immune response in an individual to
an antigen, comprising administering the carbohydrate ligand of
claim 1 and the antigen.
51. The method of claim 50, wherein said administering results in
an increase in the immune response to the antigen.
52. The method of claim 50, wherein said administering results in a
decrease in the immune response to the antigen.
53. The Method of claim 50, wherein said administering further
comprises an immune suppressing agent.
54. The method of claim 50, wherein said antigen and said
carbohydrate ligand are covalently bonded.
55. The method of claim 50, wherein an adjuvant is administered
along with said antigen and said carbohydrate ligand.
56. A method for treating a disease state involving a leczyme,
comprising administering an effective amount of the carbohydrate
ligand of claim 1.
57. The method of claim 56, wherein said disease state is an
MHC-linked disease.
58. The method of claim 56, wherein said MHC-linked disease is an
autoimmune disease.
59. The method of claim 56, wherein said MHC-linked disease is
hemochromatosis.
60. The method of claim 56, wherein said disease state is an
infection.
61. The method of claim 56, wherein said disease state is
transplantation rejection.
62. A method for treating a disease state involving a leczyme,
comprising administering an effective amount of a leczyme having a
similar binding specificity for a carbohydrate ligand as the
leczyme involved in the disease state.
63. The method of claim 62, wherein said leczyme is an MHC-derived
gene product.
64. The method of claim 62, wherein said leczyme is a non-MHC
derived gene product.
65. The method of claim 62, wherein said disease. state is an
MHC-linked disease.
66. The method of claim 65, wherein said MHC-linked disease is an
autoimmune disease.
67. The method of claim 65, wherein said MHC-linked disease is
hemochromatosis.
68. The method of claim 62, wherein said disease state is an
infection.
69. The method of claim 62, wherein said disease state is
transplantation rejection.
70. A method for detecting a genetic predisposition for
hemochromatosis, comprising detecting a mutation in the heavy chain
of a class I MHC molecule that reduces the ability of said heavy
chain to associate with .beta..sub.2 microglobulin.
71. The method of claim 70, wherein said mutation eliminates a
signal for the addition of a phosphate group.
72. The method of claim 70, wherein said mutation eliminates the
ability of a phosphate group in said heavy chain to be
de-phosphorylated in a cell.
73. A method for diagnosing hemochromatosis resulting from a
reduction in the ability of a heavy chain of an MHC class I
molecule to bind .beta..sub.2 microglobulin, comprising the steps
of: a. isolating a class I MHC heavy chain from an individual to be
tested; b. contacting the heavy chain with .beta..sub.2
microglobulin under conditions suitable for associating a class I
MHC heavy chain with .beta..sub.2 microglobulin; and c. detecting
the association of said heavy chain with said .beta..sub.2
microglobulin, wherein a reduced association of said heavy chain
with .beta..sub.2 microglobulin compared to the association of a
control heavy chain with .beta..sub.2 microglobulin is diagnostic
for hemochromatosis.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to the field of
immunological disorders and, more specifically, to major
histocompatibility complex transplantation molecules.
[0003] 2. Background Information
[0004] The major histocompatibility complex (MHC) codes for a
variety of gene products, many of which play a central role in the
body's defense against pathogenic organisms. Such molecules include
the classical transplantation antigens and structurally related
molecules, proteins for transport of foreign peptides within cells,
serum complement proteins, the lymphokines tumor necrosis .alpha.
and tumor necrosis .beta., cytochromes and heat shock proteins.
[0005] The classical transplantation antigens, encoded for by genes
in the MHC, are a highly polymorphic group of molecules that were
originally discovered for their role in determining rejection of
foreign transplanted cells and tissue. An extensive body of
experimental work has since supported a role for the classical
transplantation antigens in self-recognition. In the current
paradigm, transplantation antigens serve to present peptides
derived from both self and foreign proteins, for recognition by
cells of the immune system.
[0006] Two distinct groups of antigens, class I and class II
antigens, are encoded by genes within the MHC. Class I antigens are
expressed on virtually all nucleated cells in the body and play a
role in the mediation of immune responses based on cytotoxic
thymus-derived (T) lymphocyte mediated cell killing. Cytotoxic T
lymphocytes play a role in killing of virus infected cells and
tumor cells. The class I MHC molecule is composed of a 45
kiloDalton (kDa) heavy chain associated non-covalently with a 12
kDa protein known as .beta..sub.2 microglobulin (.beta..sub.2M).
The present paradigm characterizes class I antigens as presenting
peptide fragments derived from both self and foreign proteins
synthesized endogenously within the cell.
[0007] The class I molecules were discovered for their role in
transplantation and were termed the "classical" class I molecules,
to distinguish them from a later discovered group of class I
molecules termed the "nonclassical" class I molecules. Genes
encoding the nonclassical class I MHC molecules consist of the
majority of genes so far identified in the MHC locus. Nonclassical
class I MHC molecules are overall structurally related to the
classical class I MHC transplantation antigens in having extensive
sequence homology and a heavy chain noncovalently associated with
.beta..sub.2M. Nonclassical class I MHC molecules are, in general,
less polymorphic than the classical class I MHC molecules and are
more circumscribed in their tissue distribution. Several types of
nonclassical class I molecules are expressed principally in the
gastrointestinal (GI) tract, raising questions regarding their
function, if any in the immune system.
[0008] MHC class II antigens are expressed principally by
specialized antigen presenting cells in the body. Such cells are
limited to the antibody producing B lymphocyte as well as
macrophages and dendritic cells distributed in various tissues of
the body. The class II molecule on the cell-surface is composed of
an .alpha. chain of 33 kDa and a .beta. chain of 28 kDa associated
noncovalently. Class II molecules as presently understood function
principally to present peptides derived from self or foreign
proteins to a specialized class of T lymphocyte that supports the
development of cytotoxic T lymphocytes, provides immunity to fungal
infections and assists B lymphocytes in the generation of
protective antibody responses to encapsulated bacterial infections.
MHC class II antigens present peptide fragments derived from
proteins taken up by cells from the surrounding environment, in
contrast to classical class I molecules, which present peptides
derived from endogenously synthesized proteins.
[0009] A variety of human autoimmune diseases have been shown to be
associated more frequently in the population with individuals who
inherit certain genes of the MHC. For many of these diseases, the
association is localized to the region of the MHC encoding class II
histocompatibility antigens. These diseases are not inherited by
simple mendelian segregation of MHC genes, since only one sibling
of a set of identical twins may have the disease. This feature
suggests that other genetic factors or environmental factors have
roles in the development of autoimmunity, with genes in the MHC
playing a significant part of the process.
[0010] The current paradigm for MHC gene function provides several
theories to explain a role for MHC genes in autoimmune disease.
They include the inappropriate expression of class II MHC molecules
in cells eliciting the autoimmune response or aberrant recognition
of self-peptides by particular MHC gene products. Such theories,
however, remain to be proven. In addition, the current paradigm
fails to provide a useful hypothesis to explain the basis for an
MHC-associated iron storage disease known as hemochromatosis. This
disease is known from animal studies and from the genomic structure
of several class I genes to involve an MHC encoded class I molecule
since deletion of the .beta..sub.2M gene in these animals results
in the disease.
[0011] Thus, there exists a need to develop new approaches to the
treatment of MHC associated diseases. The present invention is
based on a new paradigm for the role of class I and class II
antigens and other broadly related molecules in self-recognition
and in regulation of the immune system. This paradigm provides that
self-recognition molecules have a central function to recognize and
modify carbohydrate structures. Thus, the present invention
provides new methods for identifying carbohydrate ligands for
self-recognition molecules and utilizing such ligands to treat
diseases involving aberrant self-recognition such as autoimmune
diseases, inflammatory diseases or susceptibility to infections and
provides related advantages as well.
SUMMARY OF THE INVENTION
[0012] The present invention provides a substantially purified
carbohydrate ligand that specifically binds to a leczyme. In
addition, the invention provides methods to identify a carbohydrate
ligand that specifically binds to a leczyme or a leczyme that
specifically binds to a carbohydrate ligand. The invention further
provides methods to identify a peptide that binds to the
carbohydrate ligand binding-site of a leczyme.
[0013] The present invention also provides methods for isolating a
carbohydrate ligand that binds to a leczyme or for isolating a
leczyme that binds to a carbohydrate ligand. The invention further
provides methods to identify a carbohydrate ligand or a leczyme
that can modify the function of a cell and to obtain such
functionally modified cells.
[0014] The invention also provides methods for modifying a cell to
produce a carbohydrate ligand by introducing an expression vector
encoding a leczyme into the cell, wherein the expression of the
leczyme produces the carbohydrate ligand.
[0015] The invention also provides methods for modulating an immune
response to an antigen by administering the antigen and a
carbohydrate ligand.
[0016] The invention also provides methods for treating a disease
state involving a leczyme by administering an effective amount of a
carbohydrate ligand that binds to the leczyme involved in the
disease state or by administering an effective amount of a leczyme
that has a similar binding specificity to the leczyme involved in
the disease state.
[0017] The invention further provides methods to diagnose a genetic
basis for hemochromatosis by detecting a mutation in a class I MHC
molecule that reduces it's ability to associate with .beta..sub.2
microglobulin.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention results from a profound new paradigm
for the function of self-recognition molecules in organisms
including mammals. The new paradigm holds that many types of
self-recognition molecules heretofore known as peptide recognition
and presentation structures have a more central function in the
recognition and modification of carbohydrate-based molecules.
Although the current paradigm does not exclude recognition of
peptide that is bound to a carbohydrate, such as a peptide derived
from a glycoprotein, the current paradigm provides that it is the
peptide rather than the carbohydrate that is bound by the
self-recognition receptor molecule. Thus, self-recognition
molecules of the new paradigm have the ability to specifically bind
a substrate carbohydrate structure and chemically modify it either
by catalyzing further addition of carbohydrate or by catalyzing
chemical modification of the existing carbohydrate. Additionally,
after enzymatic modification, the self-recognition molecule can
specifically bind with equivalent or greater affinity to the
modified carbohydrate structure than to the substrate originally
recognized.
[0019] A molecule whose function includes the enzymatic
modification of carbohydrate and the recognition of the enzymatic
product has been termed a "leczyme" based on the combination of
having both lectin-binding and enzymatic activity in the same base
molecule. The present invention provides that leczyme function is
characteristic of many peptide recognition molecules that are well
known in the art. Such molecules include the class I and class II
MHC encoded molecules and other members of the immunoglobulin gene
superfamily (IgGSF) of molecules. In addition, leczyme function
also can be associated with nonclassical class I molecules.
[0020] As used herein, "leczyme" defines a cellular protein, which
can catalyze the chemical modification of a substrate resulting in
a product with additional carbohydrate or chemically modified
carbohydrate. Leczymes also can catalyze chemical modifications of
a carbohydrate molecule such as phosphorylation, acetylation,
carboxylation or sulfation. A leczyme can enzymatically modify
other leczymes or can modify non-leczyme molecules. In addition, a
leczyme can be expressed in the cytoplasm, on the cell-surface or
can be secreted from a cell and recognize its' enzymatically
modified product either expressed on a cell-surface or secreted
from a cell.
[0021] A leczyme can exhibit enzymatic activity and carbohydrate
binding activity in the same isoform of the molecule or these
activities can reside separately in different isoforms of the
molecule. For example, differential RNA splicing of a leczyme can
result in an enzymatically active isoform of the leczyme which
contains a signal(s) directing the leczyme to sites an the cell
normally associated with glycosylation, such as the endoplasmic
reticulum or the golgi complex. Differential RNA splicing can also
result in an isoform of the leczyme that exhibits carbohydrate
recognition capability and contains a signal(s) directing the
receptor to the cell-surface or to export from the cell.
Alternatively, a leczyme expressed either in the cell or on the
cell-surface can contain both enzymatic activity as well as
carbohydrate recognition capability in the same molecule.
[0022] Leczyme function can be resident in the groove formed at the
top of MHC encoded classical class I or class II molecule, which is
characterized in the current paradigm as a peptide-binding groove.
The new paradigm provides that the groove functions principally to
recognize a carbohydrate structure. In addition, a leczyme such as
a classical class I or class II molecule is also endowed with the
ability to catalyze the chemical modification of the carbohydrate
structure it recognizes and to recognize the modified product.
[0023] The present invention provides compositions of substantially
purified carbohydrate ligands that can bind to a leczyme. As used
herein, the term "carbohydrate ligand" or "ligand" means a
sugar-based molecule where the sugar is a part of the ligand that
is recognized by the leczyme. A carbohydrate ligand can comprise
one or more sugar residues. Multiple sugar residues of a
carbohydrate ligand can be linked in either a straight chain or
branched chain configuration.
[0024] Carbohydrate ligands composed of multiple sugar residues can
vary in the type and location of the linkage between each residue.
Sugar residues useful for producing a carbohydrate ligand include,
for example, glucose, galactose, fucose, mannose and sialic acid.
Sugar residues of a ligand also can be acetylated, phosphorylated
or sulfated by chemically processes well known in the art. A
carbohydrate ligand also can be chemically bonded to other
molecules such as a lipid, glycolipid, protein, glycoprotein,
proteoglycan, glucosaminoglycan or an organic molecule. Such
additional molecules can provide the carbohydrate ligand with
features such as increased binding to the leczyme or increased
stability in vivo.
[0025] A carbohydrate ligand can be multivalent in nature by having
more than one carbohydrate ligand attached to a backbone structure.
The backbone structure can be a natural protein such as a serum
albumin or can be a synthetic molecule such as a synthetic peptide.
Approaches to link multiple carbohydrate ligands to a backbone
structure are known in the art and include, for example,
biotin-avidin linkage (Rothenberg et al., Proc. Natl. Acad. Sci.
(USA) 90:11939-11943 (1993), which is incorporated herein by
reference).
[0026] The knowledge that a self-recognition molecule is a leczyme
and that it has been selected through evolution to recognize and
modify a carbohydrate structure such as a carbohydrate ligand
provides new methods to treat disease states resulting from such
self-recognition leczymes. Such disease states include, for
example, autoimmunity, hemochromatosis, inflammation,
transplantation rejection, and infections. In many of the above
disease states, disease results from aberrant recognition of
self-carbohydrate structures by lymphocytes. Thus, the
administration of a carbohydrate ligand that can bind to the
aberrant self-recognition molecule of an individual provides a
means to disrupt the aberrant self-recognition cycle mediating the
disease.
[0027] A variety of leczymes exist that differ in their ability to
modify particular types of molecules. This difference results from
differences in the specificity of the lectin binding site that
leczymes have for their substrate. Thus, a part of the leczyme
structure is a recognition site for the substrate. The catalytic
site of a leczyme can be the same site as the substrate recognition
site or can be a site different from the substrate recognition
site. After modification of the substrate, the leczyme can exhibit
similar or greater binding affinity for the modified substrate over
the original substrate due to coordinate binding by both the
substrate recognition site and the catalytic site of the leczyme or
by multivalency of the ligand.
[0028] Leczymes utilized in the present invention include a broad
group of structurally related molecules, many of which are
contained within the IGSF. The IgGSF series of genes share an
evolutionary homology (ie. common ancestor) but are not necessarily
functionally related, genetically linked or coordinately regulated.
The products of the IgGSF have been defined by the presence of one
or more regions homologous to the basic structural unit of
immunoglobulin (Ig), known as the Ig homology unit. These units are
characterized by a primary amino acid sequence of about 70-100
residues in length and include an essentially invariant disulfide
bridge spanning 50-70 residues in length and several other
relatively conserved residues that maintain a tertiary structure
known as the Ig fold (for review see Hunkapiller and Hood, Adv.
Immunol., 44:1-63, (1989)).
[0029] The genes of the IgGSF encode many molecules with known
immunological function, such as the immunoglobulins, T lymphocyte
receptors, classical and nonclassical MHC molecules, various T
lymphocyte and B lymphocyte cell-surface molecules or
.beta..sub.2M. In addition, the IgGSF encodes several cell-surface
molecules known to function as receptors for cell-cell adhesion.
Such adhesion molecules include, for example, the neural cell
adhesion molecule carcinoembryonic antigen. Those IgGSF molecules
devoted exclusively to mediating cell adhesion or immunological
recognition such as immunoglobulins or the T cell receptor are not
a leczyme.
[0030] Leczymes of he IgGSF are encoded by genes located within the
MHC region. In humans, the MHC is in a continuous stretch of DNA
located on the short arm of chromosome 6. The entire MHC in humans
is called the HLA complex. In mice, the MHC is located on
chromosome 17 and contains the H-2, Q, T and M complexes. As used
herein, the term "MHC-derived gene product" means any molecule that
contains at least one polypeptide encoded for by a gene located
within the MHC. Leczymes that are MHC-derived gene products include
class I and class II molecules. Class I and class II molecules that
are leczymes in humans are encoded by genes within the HLA-D region
such as HLA-DP, HLA-DN, HLA-DM, HLA-DO, HLA-DQ or HLA-DR, or the
various alleles of HLA-A, HLA-B and HLA-C loci, or the HLA-X,
HLA-E, HLA-J, HLA-H, HLA-G and HLA-F genes.
[0031] Leczymes that are class I MHC molecules contain a 45 kDa
polymorphic heavy chain or .alpha. chain associated noncovalently
with a small nonpolymorphic protein called .beta..sub.2M. The heavy
chain is an MHC-encoded gene product located in or near the A, B or
C regions of the human HLA complex and within or near the K or D/L
regions of the Mouse H-2 complex. Although .beta..sub.2M is encoded
by a gene located outside the MHC and on a different chromosome,
the heavy chain of the class I molecule is encoded by a gene
located within the MHC, thereby including a class I molecule within
the definition of an MHC-encoded gene product.
[0032] Leczymes that are class II MHC molecules are MHC-derived
gene products composed of a 34 kDa .alpha. chain associated
noncovalently with a 28 kDa .beta. chain. An additional chain
called the invariant chain is transiently associated with the class
II heterodimer during transport to the plasma membrane of the
cell.
[0033] Leczymes can be expressed on the cell-surface by virtue of
having a transmembrane region and cytoplasmic tail, as in the case
of the classical transplantation antigens. Leczymes also can be
linked to the cell-surface in a manner similar to some nonclassical
class I molecules. For example, many of the nonclassical class I Qa
and Tla molecules are linked to the cell-surface by a
phosphatidylinositol (PI) linkages, and the product of the Q10 gene
appears to be secreted (Devlin et al., EMBO J. 4:369-374 (1985)).
The majority of Qa and Tla antigens lack the classical class I
cytoplasmic exons including the phosphorylation site in exon seven
(Thor et al., J. Immunol., 151:211-224 (1993)), although the
transmembrane domain and the seventh exon is present in Q1 and Q2
gene products.
[0034] The MHC class I heavy chain is organized into three external
domains (.alpha.1, .alpha.2 and .alpha.3), each containing about 90
amino acids each, a transmembrane domain of about 40 amino acids
and a cytoplasmic anchor segment of about 30 amino acids.
.beta..sub.2M is similar in size and in organization to the
external .alpha.3 domain of the heavy chain. X-ray crystallographic
analysis of the extracellular portion of the MHC class I molecule
shows that the .alpha.1 and .alpha.2 domains interact and are most
external to the cell membrane while the .alpha.3 and .beta..sub.2M
domains interact and are more proximal to the cell membrane. The
interacting .alpha.1 and .alpha.2 domains form a platform
containing a deep groove or cleft located on the top surface of the
molecule.
[0035] The current paradigm for the function of the classical class
I MHC molecule interprets the groove at the top of the molecule as
a peptide binding site. The site is sufficiently large enough to
bind a peptide of about 8-20 residues in length and present both
self and foreign-derived peptides for recognition by certain T
lymphoid cells. Extensive research has shown that the MHC classical
class I molecule can bind peptide of about the length of the
groove. In addition, the x-ray crystallographic analysis of a
classical class I molecule purified from a cell indicated that a
peptide was resident in the groove. However, as described above,
the new paradigm in the present invention provides that the peptide
binding groove of the classical class I molecule MHC is suited for
binding a carbohydrate ligand.
[0036] Leczymes that are a class II MHC molecule share significant
structural features with a class I molecule. The class II molecule
is a membrane bound glycoprotein that contains external domains, a
transmembrane segment, and a cytoplasmic anchor segment. The
.alpha. chain contains two external domains labelled .alpha.1 and
.alpha.2 and the .beta. chain contains two external domains .beta.1
and .beta.2 domain. X-ray crystallography shows that the .alpha.2
and .beta.2 domains interact as a membrane proximal structure
analogous to the .alpha.3 domain and .beta..sub.2M domain
interaction of the class I molecule. The .alpha.2 and .beta.2
domains of a class II molecule that together form a cleft at the
top of the molecule that is very similar to the cleft formed by the
.alpha.2 and .alpha.3 domains of a class I heavy chain. Extensive
evidence indicates that the groove in the class II molecule can
bind and present both self and foreign peptides for recognition by
T lymphoid cells. Peptides have been isolated from the class II
molecule that are from 13-18 amino acids in length, slightly longer
that the octomeric or nonomeric peptides commonly isolated from MHC
classical class I molecules. As discussed above, the new paradigm
of the present invention provides that the peptide binding groove
in the class II MHC molecule, like the groove in the classical
class I MHC molecule is suited for binding a carbohydrate
ligand.
[0037] Leczymes also are encoded by nonclassical class I genes. In
the mouse, genes encoding leczymes are located in the MHC regions
Q, T and M downstream of the classical histocompatibility antigens.
There are similar regions in humans coding for known nonclassical
class I molecules such as HLA-F and HLA-G. The nonclassical class I
genes are overall less polymorphic than the classical class I genes
and show different patterns of expression. The Q, T and M complex
genes of mice consist of approximately 45 genes, coding for
non-polymorphic differentiation antigens with limited tissue
distribution.
[0038] Leczymes which are nonclassical class I MHC molecules
exhibit limited tissue distribution in comparison with leczymes
that are classical class I MHC molecules. For example, the Qa and
Tla antigens, the products of the Q and T genes, are expressed on
subpopulations of lymphocytes (for review, see Flaherty et al.
Critical Reviews in Immunology, 10:131-175 (1990)). Previously, no
convincing function had been assigned to the products of the
nonclassical class I genes, although they have been suggested as
possible restriction elements for .gamma..delta. T cells (Hershberg
et al. Proc. Nat. Acad. Sci (USA), 87:9727-31 (1993)). The Qa and
TLa antigens have also been reported to be expressed on intestinal
epithelium (Wu et al, J. Exp. Med., 174:213-218 (1991); Hershberg
et al., Proc. Natl. Acad. Sci. (USA) 87:9727-97231 (1990); Wang et
al., Immunogenet., 38:370-372 (1993)) where their function was
unknown. The new paradigm of the present invention provides that
these nonclassical class I molecules are leczymes.
[0039] The nonclassical class I molecule Q2, produced by a gene
within the mouse MHC, is an example of a leczyme that is involved
in iron transport (see Example I). The gene for Q2 is located in a
head to head relationship with another gene most likely encoding a
mucin. Both genes share a single promoter region, located between
the genes, the promoter being analogous in structure to the
.beta.-globin promoter involved in iron metabolism. The coordinated
regulation of these two genes can be readily understood in view of
the receptor/ligand and receptor/substrate interactions defined as
leczyme function in the new paradigm. Interestingly, the Q2 gene is
distinguished from other nonclassical class I genes in being highly
polymorphic with Q2 molecules of different strains of mice
differing significantly in amino acid sequence. Despite these
differences, the Q2 molecules from separate strains of mice all
function as a receptor for their co-regulated gene product since,
as a leczyme, Q2 can enzymatically modify it's ligand/substrate in
accordance with the lectin recognition and enzymatic function of
each Q2 gene product and can recognize the resulting product. Thus,
the combined enzymatic/recognition capability of a leczyme as
defined in the new paradigm maintains receptor/ligand relationships
in the face of extensive genetic polymorphism.
[0040] Leczymes exist with a variety of enzymatic activities. For
example, a leczyme can have as a glycosyl transferase enzymatic
activity that results in the catalytic transfer of a glycosyl group
(mono or oligosaccharide) from a glycosylnucleotide to an acceptor
molecule such as a protein, carbohydrate or lipid. However, not all
glycosyl transferases are leczymes. In fact, very few such enzymes
would be leczymes since the overwhelming majority of
glycosyltransferases are restricted to expression in the
endoplasmic reticulum and golgi complex of the cell.
[0041] There is currently only one glycosyl transferase
(.beta.1,4-galactosyltransferase) that is previously known to be
expressed in both the cytoplasm and on the cell, for a review see.
Shur, Curr. Opin. in Cell Biol., 5:854-863 (1993)). This enzyme has
both carbohydrate recognition capability and carbohydrate catalytic
activity and has been implicated in a variety of cell-cell and
cell-matrix interactions. One hallmark of the cell-surface
expressed form of .beta.1,4-galactosyltransferase is that it no
longer retains binding activity for the product it generates after
enzymatic modification (Miller et al., Nature, 357:590-593 (1992)).
Thus, this particular transferase is not a leczyme because it fails
to exhibit recognition for it's enzymatic product.
[0042] A Leczyme of the IgGSF can be encoded by a gene located
outside the MHC. For example, CD-1 is a product of the IgGSF gene
that is related in structure to the class I MHC molecule but the
CD-1 heavy chain is encoded by a gene outside the MHC. The T-6 CD-1
molecule is expressed by a specialized antigen presenting cell in
the skin (Langerhan's cell) and can be internalized along with MHC
class II antigen, indicating an immunological function for T-6.
[0043] The present invention provides a composition, comprising a
substantially purified carbohydrate ligand that is specifically
bound by a leczyme. As used herein, the term "substantially
purified" means a carbohydrate ligand that is relatively free from
other contaminating molecules such as lipids, proteins, nucleic
acids, carbohydrates or other molecules normally associated with a
carbohydrate ligand in a cell or tissue. A substantially purified
carbohydrate ligand can be obtained, for example, using well known
biochemical methods of purification of a carbohydrate source or by
chemical or enzymatic synthesis.
[0044] A carbohydrate ligand of the present invention can include
known forms of carbohydrate containing molecules such as
glycoproteins, proteoglycans, glycolipids or mucopolysaccharides
that have N-linked or O-linked forms of glycosylation. The
proteoglycans include, for example, mucins and those proteoglycans
glycosylated with hyaluronate, chondroitin sulfate, heparin,
heparan sulfate or dermatin sulfate. Glycolipids that contain
carbohydrate ligands include, for example, acylglycerol, a
sphingoid or a ceramide.
[0045] A sample containing a carbohydrate ligand can be obtained
from a variety of sources such as from fluids, tissues or cells.
These sources can be from any plant species or any animal such as a
mammal or any organism. A source of carbohydrate ligand can also
include a cell that has been modified by introducing into the cell
an expression vector that encodes a leczyme or a protein that when
expressed contains a carbohydrate ligand.
[0046] A sample containing a carbohydrate ligand can be obtained
from a chemically produced library of carbohydrates. Such libraries
can be made by mixing carbohydrates from natural sources and from
enzymatically-produced sources. In addition, individual
carbohydrates from the library can be tagged with a detectable
label such as a fluorescent label to assist in structural
determination of the carbohydrate ligand.
[0047] A sample containing a carbohydrate ligand can be processed
to further purify the ligand by methods well known in the art. Such
methods include, for example, purification of glycoconjugates,
labelling Of glycoconjugates by chemical or metabolic means,
release of oligosaccharides from glycoconjugates and
characterization of the structure of the released carbohydrate
(see, for example, Ausabal et al, In Current Protocols in Molecular
Biology Vol. 2, chapter 17, (Green Publishing Associates and Wiley
Interscience, New York, 1994); Fukuda and Kobata, Glycobiology: A
practical Approach, (IRL Press, New York, 1993), both of which are
incorporated herein by reference). In addition, these methods are
useful for structural characterization, including sequencing of the
carbohydrate ligand. Elucidation of the structure of a carbohydrate
ligand purified from a tissue or a cell can enable future
production or the ligand by direct chemical synthesis or enzymatic
synthesis or purification from a natural source.
[0048] The present invention provides methods to identify a
carbohydrate ligand that can bind to a leczyme. In this method, a
sample containing a carbohydrate ligand is contacted with a leczyme
suspected of binding to the ligand under suitable conditions to
allow specific binding of the ligand to the leczyme. Suitable
conditions include, for example, an appropriate buffer
concentration and pH and time and temperature that permits binding
of the particular leczyme and the carbohydrate ligand. After a
suitable reaction period, the amount of carbohydrate ligand bound
to the leczyme can be determined, for example, by attaching a
detectable moiety such as a radionuclide or a fluorescent label to
the carbohydrate ligand and measuring the amount of label that is
associated with the leczyme after any unbound carbohydrate ligand
has been removed from the ligand-leczyme complex.
[0049] As used herein, "detectable label" means a molecule whose
presence can be detected due to a physical, chemical or biological
characteristic of the molecule. Detectable labels include, for
example, radioisotopes, fluorescent molecules, enzyme/substrate
systems, or visually detectable molecules. Methods for detectably
labelling a carbohydrate molecule are well known in the art, and
include, for example, reduction with NaB(.sup.3H).sub.4 or
synthesis with radiolabelled sugars (see, for example, Varki,
surpa, 1994 and Rothenberg et al., Proc. Natl. Acad. Sci. (USA),
90:11939-11943 (1993), both of which are incorporated herein by
reference, and Fukuda and Kobata, supra 1993). In addition, kits
for the preparation of a labelled carbohydrate molecule are readily
available from commercial sources such as Oxford GlycoSystems
(Rosedale, N.Y.).
[0050] Methods to remove unbound labelled ligand from the
ligand-leczyme complex depend, for example, on attaching the
leczyme to a solid support. Solid supports useful in the present
invention and methods to attach proteins to such supports are well
known in the art (see for example Harlow and Lane, Antibodies: A
laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1988), which is incorporated herein by reference).
Such solid supports include, for example, Sepharose, agarose or
polystyrene.
[0051] After a suitable reaction period and after any unbound label
has been removed from the support by, for example, washing, the
amount of label attached to the solid support provides a direct
measurement of the amount of carbohydrate ligand bound to the
leczyme on the support. Alternatively, the amount of labelled
carbohydrate ligand bound to the support can be indirectly
determined after the reaction period by measuring the amount of
unbound label and subtracting this from the amount of label added
at the start of the reaction.
[0052] To accurately determine the amount of labelled ligand that
binds specifically to the leczyme, a control reaction can be
performed where all conditions are the same as in the binding
reaction between the labelled ligand and leczyme except that the
leczyme is not included in the control reaction or the leczyme is
replaced by an irrelevant protein such as a serum albumin. The
control reaction determines the amount of binding of the labelled
carbohydrate ligand that occurs nonspecifically such as the amount
of labelled ligand that binds to the solid support rather than to
the leczyme on the solid support. Thus, it is necessary to subtract
the nonspecific binding value obtained from the control reaction
from the binding value obtained from the reaction that included
both the labelled carbohydrate ligand and the leczyme to determine
the amount of ligand that specifically bound the leczyme under the
conditions tested.
[0053] An advantage of using a solid support is that the labelled
ligand can be added in excess relative to the leczyme, making it
possible to identify lower levels of binding affinity between the
carbohydrate ligand and the leczyme. Methods such as Scatchard
analysis are well known in the art for determining the binding
affinity between two molecules, both of which can be in solution or
one of which can be attached to a solid support. Equilibrium
dialysis is an example of a method where the binding of a ligand to
leczyme can be determined when both molecules are in solution.
[0054] Methods to measure the binding of a labelled carbohydrate
ligand to a leczyme also can be performed when the leczyme is
associated with a cell. In a manner analogous to the use of solid
supports, cells that express the leczyme on the cell-surface can
bind the labelled carbohydrate ligand and, after a suitable
reaction period, the cells can be separated from the unbound ligand
by methods well known in the art such as by centrifugation or
filtration. Cells that express a leczyme in the cytoplasm can also
be used to detect binding of a carbohydrate ligand to the leczyme
provided the cell membrane has been sufficiently permeabilized to
allow access of the carbohydrate ligand to the leczyme in the cell.
Methods that use cells in binding assays such as antigen-antibody
binding assays are well known in the art (see, for example, Harlow
and Lane supra, 1988) and are generally applicable to binding
assays between a carbohydrate ligand and a leczyme.
[0055] A leczyme-expressing cell can be a cell that naturally
expresses the leczyme such as a lymphocyte that expresses a class I
or class II MHC encoded leczyme or can be a cell that expresses the
leczyme as a result of introducing an expression vector encoding
the leczyme into the cell. Leczyme-expressing cells can be obtained
from in vivo sources by methods well known in the art such as
mechanical disruption of tissue or digestion of tissue by enzymes
to release cells from their surrounding matrix (see for example,
Freshney Culture of Animal Cells (Alan R. Liss, New York, 1993),
which is incorporated herein by reference). A leczyme-expressing
cell can be a cell line that is available from public cell
repositories such as from the American Type Culture Collection.
[0056] It is well known in the art that the binding between two
molecules can be performed when either of the two molecules
contains a detectable label. Thus, the identification of a
detectably labelled carbohydrate ligand that binds to a leczyme
attached to a solid support or a cell also can be performed if the
leczyme contains the detectable label and the carbohydrate ligand
is attached to a solid support or expressed by a cell. A leczyme
can be detectably labelled using methods for labelling a protein,
which are well know in the art and include, for example,
biotinylation or incorporation of radioisotopic labelled
precursors. A carbohydrate ligand-expressing cell can be a cell
obtained from tissues or organs or can be a cell line such as a
cell line available from a public repository.
[0057] Methods for attaching a carbohydrate ligand to a solid
support depend on the chemical nature of the ligand. Thus,
attachment can be accomplished through the carbohydrate moiety or
other molecule bonded to the carbohydrate ligand attachment via
chemistry suitable for attaching carbohydrate, peptide or lipid
structures to a solid support. Methods to attach carbohydrates,
proteins or lipids to various types of solid supports are well
known in the art.
[0058] The binding of a carbohydrate ligand to a leczyme can be
determined without the need for a detectable label by measuring a
physical characteristic of the either the ligand or the leczyme
such as absorption of ultraviolet radiation. Such methods for
quantitating a protein or carbohydrate by physical characteristics
are well known in the art. The ability to follow a physical
characteristic of the ligand or leczyme can be applied to binding
assays that use a solid support or an expressing cell or when both
molecules are in solution. The binding of a carbohydrate ligand to
a leczyme also can be evaluated if the ligand is a substrate for
the enzymatic activity of the leczyme. In this case, binding can be
measured by following substrate conversion kinetics measured, for
example, by the Michealis-Menten equation (Devlin, Textbook of
Biochemistry (Wiley-Liss Inc. New York, 1992), which is
incorporated herein by reference).
[0059] Methods for identifying a carbohydrate ligand that binds a
leczyme can be performed using a single purified carbohydrate
ligand or a limited number of carbohydrate ligands, which can be
purified by conventional procedures as described above or can be
purified by binding to a reagent. A purified carbohydrate ligand
can also be detectably labelled by methods disclosed herein. A
carbohydrate ligand that is not purified, such as one that is in a
sample containing other molecules, can be used in a binding assay
provided it is attached to a solid support or is expressed by a
cell and binding is determined by detecting binding of a leczyme.
In this case, if the non-purified carbohydrate ligand can bind the
leczyme, the sample containing the ligand can be subjected to
purification and subsequent binding assays to obtain the
carbohydrate ligand in a purified state.
[0060] Purified leczymes can be obtained from cells by classical
methods for protein or glycoprotein purification such as methods
known in the art for purifying class I or class II molecules.
Leczymes also can be obtained from cells that have been modified by
molecular biological techniques to enable expression of a leczyme.
A gene encoding a leczyme can be cloned into an expression vector
and then introduced into a host cell. Vectors are well known in the
art and include, for example, cloning vectors and expression
vectors, as well as plasmids or viral vectors (see, for example,
Goedell, Methods in Enzymology, vol. 185 (Academic Press, New York,
1990), which is incorporated herein by reference). A baculovirus
vector is an example of a vector that can be used to express a
leczyme in insect cells and result in expression of new
carbohydrate ligands on the cell.
[0061] A vector comprising a nucleic acid molecule encoding a
leczyme also can contain a promoter or enhancer element, which can
be constitutive or inducible and, if desired, can be tissue
specific. Host cells also are known in the art and an appropriate
host cell can be selected for the particular vector to be used. For
example, a baculovirus transfer vector can be used with baculovirus
DNA to infect insect cell lines such as SF21 cells. Cloning of such
transformed cells to produce a stable cell line can provide a
source of the expressed leczyme or can provide a source of
carbohydrate ligand modified by the expressed leczyme.
[0062] The gene encoding a leczyme can be expressed as a fusion
protein to assist in purification or in further downstream
Processing of the leczyme. For example, the leczyme can be produced
as a chimeric protein fused to the CH2 or CH3 domain that
constitutes the Fc binding region of an immunoglobulin molecule, as
was performed previously for expressing the CD22.beta. lectin
(Stamenkovic et al. Cell, 66:1133-1144 (1991)). The use of Protein
A from Staphylococcus aureus bound to a solid support, which is
readily available from commercial sources, can be used to purify
the Fc containing chimeric leczyme. In addition, the solid support
containing the chimeric leczyme can be used directly to evaluate
binding of a carbohydrate ligand.
[0063] The present invention provides methods to identify a leczyme
that binds a carbohydrate ligand. In this method, a sample
containing a leczyme is contacted with a carbohydrate ligand
suspected of binding to the leczyme under suitable conditions to
allow specific binding of the ligand to the leczyme. The methods
that have been described above for identifying a carbohydrate
ligand that binds to a leczyme can also be used to identify a
leczyme that binds to a carbohydrate ligand. Leczymes to be
identified for binding include, for example, a purified leczyme or
a leczyme contained within a complex mixture such as a mixture of
proteins expressed from a cDNA expression library. Methods to
produce a cDNA expression library are well known in the art (see,
for example, Sambrook et al, Molecular Cloning: A laboratory manual
(Cold Spring Harbor Laboratory Press 1989), which is incorporated
herein by reference).
[0064] The present invention provides methods of purifying a
carbohydrate ligand that specifically binds to a reagent. In these
methods, a sample containing the carbohydrate ligand is contacted
with the reagent under suitable conditions to allow formation of a
ligand-reagent complex. Suitable conditions includes, for example,
an appropriate buffer concentration and pH and time and temperature
that permits binding of the carbohydrate ligand to the reagent. The
ligand-reagent complex is then separated from the rest of the
sample by a separation method such as by washing, and the ligand is
dissociated from the complex.
[0065] As used herein, "reagent" means a chemical or biological
molecule that can specifically bind to a carbohydrate ligand. For
example, a leczyme that binds to a carbohydrate ligand is a reagent
that can be used to purify that ligand. Also, an antibody can be a
reagent if it can react specifically with the carbohydrate, protein
or lipid portion of a carbohydrate ligand.
[0066] Purification of the carbohydrate ligand can be accomplished
if the reagent is attached to a solid support such as agarose,
Sepharose or plastic. Methods for coupling a protein or a
carbohydrate to a solid support, disclosed above for detecting the
binding of a carbohydrate ligand to a leczyme, also are useful for
attaching a reagent to a solid support.
[0067] Methods to dissociate a carbohydrate ligand from a
ligand-reagent complex can depend on the nature of the reagent. For
example, if the reagent is a leczyme, then a method for
dissociating the complex can involve competitive inhibition of the
complex with a sugar structure that has binding affinity for the
same site in the leczyme that binds the carbohydrate ligand. Other
well known treatments that are useful for dissociating a
carbohydrate ligand from a reagent include, for example, extremes
in pH, high salt concentration or chaotrophic agents (see, for
example, Harlow and Lane, supra, 1988), which is incorporated
herein by reference and Varki, supra, 1994). Carbohydrate ligands
purified by the above disclosed methods are suitable for structural
analysis as described above, in order to enable future production
of the ligand by chemical or enzymatic synthesis.
[0068] An antibody that specifically binds to a carbohydrate ligand
can be produced to the carbohydrate or a protein moiety or a lipid
moiety, if such moieties are bonded to the ligand. An antibody
specific for the peptide backbone of carbohydrate ligand such as
the peptide backbone of a mucin can be useful for purifying a
source of mucin from different cells or from different individuals,
since the peptide backbone can be more conserved between peptide
containing carbohydrates than the carbohydrate portions of these
molecules. Methods for producing antibodies such as polyclonal
antibodies, monoclonal antibodies, antibody fragments or the like,
that are specific for protein, carbohydrate or lipid are well known
in the art (see, for example, Harlow and Lane supra, 1988).
[0069] The present invention provides methods for purifying a
leczyme that specifically binds to a carbohydrate ligand. In these
methods, a sample containing the leczyme is contacted with a
carbohydrate ligand under suitable conditions to allow formation of
a ligand-leczyme complex. Suitable conditions includes, for
example, an appropriate buffer concentration and pH and time and
temperature that permits binding of the leczyme the carbohydrate.
The ligand-reagent complex is then separated from the rest of the
sample by a method such as by washing, and the leczyme is
dissociated from the complex.
[0070] Purification of the leczyme can be accomplished if the
carbohydrate ligand is attached to a solid support such as agarose,
Sepharose or plastic. Methods for coupling a carbohydrate ligand to
a solid support, such as those disclosed above for detecting the
binding of a carbohydrate ligand to a leczyme, are useful for
attaching a carbohydrate ligand to a solid support. Methods for
dissociating the leczyme from the ligand-leczyme complex can
utilize the methods disclosed herein for dissociating a
carbohydrate ligand from a ligand-leczyme complex.
[0071] The present invention provides methods to identify a
carbohydrate ligand that modifies the function of a
leczyme-expressing cell by contacting a sample containing a
carbohydrate ligand with the cell under suitable conditions, which
allow specific binding of the ligand to the leczyme on the cell.
After a suitable period of time to allow for binding of the ligand
to the leczyme, the cells are evaluated to determine their
function. A carbohydrate ligand that modifies the function of a
leczyme-expressing cell is one that when contacted with the cell
results in a function that differs from the function of the same
type of cell that had not contacted the ligand.
[0072] As used herein, "function" in reference to a cell includes
any activity that can be detected for a cell. The function of a
cell can vary with the nature of the cell in question. For example,
the function of a T lymphocyte can include activities such as the
production of certain cytokines, acquisition of cell mediated
lympholysis, ability to mediate antibody dependent cell mediated
cytotoxicity or the ability to help B lymphocytes to produce
antibody. Thus, a particular carbohydrate ligand that can bind to a
leczyme on a T lymphocyte and subsequently effect the function of
the cell can do so by increasing or decreasing any of the above T
lymphocyte functions.
[0073] Contacting a carbohydrate ligand with a leczyme-expressing
cell can be performed in vitro in a cell culture medium. Methods
for measuring the function of lymphoid cells or other cells are
well known in the art (see for example, Colligan et al., Curr.
Protocols in Immunol. (Greene Publishing Associates and Wiley
Interscience, New York, 1992); Mishell and Shiigi, Selected Meth.
in Cell. Immunol. (W. H. Freeman and Co., New York, 1980), each of
which are incorporated herein by reference)
[0074] The present invention also provides methods to identify a
leczyme that modifies the function of a carbohydrate
ligand-expressing cell. Methods described above for identifying a
carbohydrate ligand that modifies the function of a
leczyme-expressing cell are also useful for identifying a leczyme
that modifies the function of a carbohydrate ligand-expressing
cell.
[0075] The present invention provides methods to modify is the
function of a leczyme-expressing cell by contacting the cell with a
carbohydrate ligand that binds the leczyme. In addition, the
invention provides methods to modify the function of a carbohydrate
ligand-expressing cell by contacting the cell with a leczyme that
binds the ligand. The identification of either a carbohydrate
ligand or a leczyme that can modify the function of a cell has both
in vitro and in vivo uses. For example, ligands or leczymes capable
of decreasing or increasing the functional activity of cell that is
involved in a disease state can be administered to an individual to
treat the disease.
[0076] The present invention provides methods to identify a peptide
that can bind to the carbohydrate ligand binding-site of a leczyme.
These methods involve contacting a sample containing a peptide or
peptides to be tested with a leczyme under suitable conditions to
enable binding of peptide to the leczyme. Subsequently, the leczyme
is reacted with a carbohydrate ligand known to bind to the leczyme.
The reaction is performed under conditions suitable for the
carbohydrate ligand to bind to the leczyme. Alternatively, the
peptide, leczyme and carbohydrate ligand can be added together at
the start of the reaction.
[0077] The carbohydrate ligand can be added directly to the mixture
containing the peptide and leczyme or can be added after any
unbound peptide has been removed from the leczyme. After the end of
the reaction, the amount of carbohydrate that bound to the leczyme
is determined and compared to the amount of carbohydrate ligand
that bound to leczyme in a control sample that did not contain
peptide. If the amount of carbohydrate ligand that bound to the
leczyme in the sample containing peptide is less than the amount of
carbohydrate ligand that bound to the leczyme in the control
sample, then it can be concluded that the peptide had bound to the
carbohydrate ligand binding site of the leczyme and is therefore a
peptide mimetope of the carbohydrate ligand.
[0078] A peptide mimetope can be identified in an assay format that
utilizes a carbohydrate ligand containing a detectable label and a
leczyme that is bound to a solid support or is expressed by a cell.
Methods disclosed herein for identifying a carbohydrate ligand that
bind to a leczyme are useful to generate the assay format for
identifying a peptide mimetope of a carbohydrate ligand.
[0079] A defined peptide sequence can be chemically synthesized or
produced by biological methods, such as by recombinant DNA
techniques (see, for example, Sambrook et al., supra, 1989). A
complex mixture of peptides also can be used to identify a peptide
mimetope. Such complex mixtures can include, for example, a mixture
of defined sequences, or can be a semi-random or random library of
sequences. Methods to generate peptide libraries by such methods as
chemical synthesis on a bead or a microtiter plate or biological
production such as on the surface of a bacteriophage are well known
an the arc (see, for example, Huse et al., Science 246:1275-1281
(1989), which is incorporated herein by reference).
[0080] A peptide that can bind to the carbohydrate ligand binding
site of a leczyme can also have some of the functional
characteristics of a carbohydrate ligand and thus be considered a
functional mimetope of the carbohydrate ligand. Such peptide
mimetopes can be used to modify the function of a cell and also can
be used to treat a disease state that involves a leczyme that can
bind to the mimetope.
[0081] The present invention provides methods to modify a cell to
produce a carbohydrate ligand, comprising introducing an expression
vector encoding a leczyme into a cell to obtain expression of the
leczyme, which results in production of the carbohydrate ligand by
the cell. Cells producing a particular carbohydrate ligand are
useful to provide unique types of ligands, which can be purified
from the cells. In addition, such cells are useful in binding
assays to identify a leczyme that binds the ligand.
[0082] The present invention provides methods for modulating an
immune response in an individual, such as a human or other animal,
using an antigen for which the immune response is desired and a
carbohydrate ligand that binds to a leczyme. As leczymes include,
for example, the major histocompatibility complex molecules, that
are involved in presentation of foreign molecules for recognition
by cells of the immune system, injection of a carbohydrate ligand
and an antigen can modulate an immune response. A used herein,
"modulate" means increase or decrease. An increase in the immune
response can be obtained by administering a carbohydrate ligand
bound to antigen such chat the antigen is targeted via the leczyme
to an antigen presenting cell.
[0083] An antigen can be associated with a carbohydrate ligand by
covalently bonding the antigen to carbohydrate or to any protein or
lipid of the ligand using methods well known in the art. The actual
method to covalently couple the antigen to the carbohydrate ligand
will depend on the nature of each molecule to be coupled and
whether the coupling procedure is detrimental to the any critical
antigenic determinants of the antigen or the capability of the
carbohydrate ligand to bind its target leczyme. Such detrimental
effects can be readily evaluated in binding assays as described
above.
[0084] More than a single antigen molecule or more than a single
carbohydrate ligand can be coupled together to produce an
immunogen. Such molecules can be made multivalent for either or
both of the antigen or the carbohydrate ligand and can be used for
eliciting a greater immune response than an immunogen containing a
single molecule of antigen and a single molecule of a carbohydrate
ligand.
[0085] Methods to increase an immune response in an individual are
well known to those in the art and require optimization of
parameters such as dose, route of administration, use of an
adjuvant, or schedule of administration (see, for example, Harlow
and Lane, chapter 5, supra, 1988). An increased immune response
obtained after administering an antigen and a carbohydrate ligand
is achieved when the immune response parameter has increased by a
statistically significant level over the level of the parameter
manifested prior to administration of the antigen and carbohydrate
ligand.
[0086] The immune response parameters that can increase after
administering an antigen associated with a carbohydrate ligand
include an antibody-mediated response or a cellular-mediated
response. Methods to measure antibody immune responses are well
known to those in the art and include, for example, detection of
immunoglobulins by both in vitro and in vivo methods (see for
example Harlow and Lane, supra, 1988). Methods to measure cellular
immune responses are also well known in the art and include in vivo
methods such as skin testing for delayed hypersensitivity and in
vitro methods such as direct cell cytotoxicity or cell activation
assays (see, for example, Coligand et al. supra, 1992; Mishell and
Shiigi, supra, 1980).
[0087] An antigen associated with a carbohydrate ligand can be used
to decrease an immune response to the antigen and can be
particularly useful for treating a deleterious immune response such
as an autoimmune disease state. Methods for decreasing an immune
response can, under some conditions result in a prolonged state of
specific immunological unresponsiveness to the antigen, commonly
referred to as a state of tolerance to the antigen.
[0088] Decreasing an immune response to an antigen by administering
the antigen bonded to a carbohydrate ligand can be accomplished
using methods well known in the art to suppress or tolerize an
individual to an antigen. Such methods include, for example,
administration of low doses, monomeric and nonaggregated forms of
the antigen and carbohydrate ligand or administration orally. In
addition, a decreased immune response can be obtained by
administering the antigen and carbohydrate ligand concurrently with
an immunosuppressive agent such as cyclosporin A, FK506 or
antibodies to a particular T lymphocyte cell-surface receptor.
Methods for using such agents to decrease the immune response to an
antigen in humans or animals are well known in the art.
[0089] The present invention provides methods for treating a
disease state involving a leczyme, by administering an effective
amount of a carbohydrate ligand that binds to the leczyme. As used
herein, the term "disease state" includes any diseases, whether
genetic or acquired, provided a leczyme plays a role in the disease
process. Such disease states include inflammation, transplantation
rejection, and also includes diseases having both a genetic and an
environmental basis such as iron storage diseases, autoimmunity or
cancer. In addition, a disease state includes diseases resulting
from an infectious agent such as a virus, bacteria, yeast or
parasite. The ability of an infectious agent to enter and infect
cells of the host can occur by binding to leczyme or carbohydrate
ligand expressed on the cells of the host. A peptide mimetope for a
carbohydrate can also be used to treat a disease state that
involves a leczyme for which the mimetope can bind.
[0090] The present invention provides methods for treating a
disease state involving a leczyme by administering a leczyme having
a similar binding specificity for a carbohydrate ligand as the
leczyme involved in the disease state. The disease states useful
for treatment by a leczyme include those described above for
treatment by a carbohydrate ligand. Thus, aberrant
self-recognition, mediated by a leczyme in a diseased individual,
can be treated by administration of a leczyme. Such a leczyme can
bind to the natural carbohydrate ligand detected on a target cell
by the aberrant self-reactive leczyme-expressing cell, and,
therefore, block the ability of the self-reactive
leczyme-expressing cell to recognize and react aberrantly towards
the target cell.
[0091] The present invention provides methods for treating an iron
metabolic disorder known as hemochromatosis. Defects in iron
metabolism can have a basis in leczyme function. In elevated
concentrations, iron is a toxic inorganic molecule that has been
implicated in the pathophysiology of a number of common diseases.
These include but are not limited to cancer (Stevens et al, N.
Engl. J. Med., 319:1047 (1988); Stevens, et al., Med. Oncol. Tumor
Pharmacother, 7:177-181 (1990)), heart disease (Kannel, et al,
1976; Sullivan, Lancet, 1:1293-1294 (1981); Salonen, et al,
Circulation, 36:803-811 (1992)), reperfusion injury (Zweier, J.
Biol. Chem., 263:1353-1357 (1988)) and rheumatoid arthritis (Blake
et al., Arthritis Rheum., 27:495-501 (1984)). There is no argument
that severe iron overload results in a constellation of
pathologies, collectively called hemochromatosis, the most common
genetic disease affecting man.
[0092] Hemochromatosis results from enhanced absorption of iron
from the GI tract by active transport but the underlying metabolic
defect is currently unknown. Identification of the genes
responsible for the absorption of iron, and developing an animal
model in which iron overload is due to active enhanced absorption
of iron from the GI tract, would greatly facilitate understanding
hemochromatosis and increase knowledge about the general mechanisms
of iron metabolism. The present invention provides the results from
a new animal model and data from humans that indicate a role for an
MHC-encoded leczyme in the pathogenesis of hemochromatosis.
[0093] Hemochromatosis is not usually brought to clinical attention
until symptoms develop, and several studies have indicated that
removal of the iron after the development of tissue damage does not
necessarily improve the organ function (Cundy, et al., Clin.
Endocrinol., 38:617-620 (1993); Westera et al., Am. J. Clin. Path.,
99:39-44 (1993)). Hemochromatosis is an underdiagnosed and
undertreated disease that would benefit greatly from early
diagnosis and an effective treatment (for reviews see Edwards et
al., Hosp. Pract. Suppl., 3:30-36 (1991); Edwards and Kushner, N.
Engl. J. Med., 328:1616-1620 (1993)).
[0094] Untreated hemochromatosis is characterized by iron overload
of parenchymal cells, which is toxic and the probable cause of
various complications including hepatopathy (including cirrhosis,
and liver cancer), arthropathy, hypogonadotropic hypogonadism,
marrow aplasia, skin disorders, diabetes mellitus, and
cardiomyopathy (for review see Halliday and Powell, Iron and Human
Disease, Lauffer, R B, (ed). 131-160 (1992)). There are reportedly
1.5 to 2 million active cases of hemochromatosis within the U.S.,
with approximately 25% of late diagnosed or untreated patients
developing hepatomas.
[0095] In untreated hemochromatosis, iron is universally deposited
in the hepatocytes of the liver, and elevated saturation of
transferrin with elevated serum ferritin levels combined with liver
biopsy provides the best diagnostic test currently available
(Fairbanks, Hosp. Pract., 26:17-24 (1991)). The iron is found
primarily in the cytoplasm of hepatocytes, and by electron
microscopy in lysosomal vacuoles, and in more severe cases, iron is
deposited in mitochondria (for review see Iancu, Ped. Pathol.,
10:281-296 (1990)). Other liver toxins such as alcohol and
hepatitis exacerbate the damage caused by the iron deposition
(Piperno et al., J. Hepat., 16:364-368 (1992)). Patients with
hemochromatosis are advised not to drink alcohol, because of
increased liver damage, or to smoke tobacco products, as iron
deposition can also occur in the lungs.
[0096] Hemochromatosis is an autosomal recessive disease in which
the responsible gene(s) is ligand to the A locus of the human MHC
(HLA complex), located on human chromosome 6 (Simon and Brissot,
Hepatol., 6:116-124 (1988)). Linkage to human HLA-A3 has been
documented in approximately 73% of cases. However, other genetic
loci also have been implicated, especially in African (Gorduke et
al., N. Engl. J. Med., 326:95-100 (1992)) and African-American
populations (Barton et al., Blood, 35:95a (1993))
[0097] Hemochromatosis is the most common genetic malady in humans
far exceeding cystic fibrosis, phenylketonuria and muscular
dystrophy combined (Leggett et al., Clin. Chem., 36:1350-1355
(1990)). One explanation for the high incidence of this genetic
disease may be that results from different mutations in multiple
linked genes that produces a similar phenotype. Hemochromatosis
occurs most frequently in populations of European origin with a
frequency in homozygotes and heterozygotes of approximately 0.3 and
13%, respectively.
[0098] Several markers, including the recently described D6S105,
have been identified in the human MHC locus and have narrowed the
genomic location of the hemochromatosis gene to within 1
centimorgan of the A locus (Jazwinska et al., Am. J. Hum. Genet.,
53:347-352 (1993)), and possibly centromeric to HLA-F (Gasparini,
et al., Hum. Mol. Genet., 5:571-576 (1993)). Others have reported
candidate (HC) genes located 20-200 kb telomeric to HLA-A (el
Kahloun et al., Hum. Mol. Genet., 2:55-60 (1993)). While several of
these candidate genes were thought to be single copy, three of the
genes, termed HCG II, IV and VII, were found to be multicopy genes.
Thus, despite the advances made in determining the location of the
HC gene, it has not yet been isolated.
[0099] Animal models for iron overload exist, however, these models
are not entirely suitable for the study of hemochromatosis since
they do not reflect enhanced iron absorption from the gut by active
transport. Mice homozygous for deletion of the gene encoding
.beta..sub.2M (.beta..sub.2-/-mice (Koller et al., Science,
248:1227-1230 (1990); Zijlstra et al., Nature, 344:742-746 (1990))
provide an excellent animal model for the study or hemochromatosis.
These animals lack detectable class I proteins on the cell-surface,
although biochemical labeling shows that class I gene products are
being synthesized. Activated lymphocytes from .beta..sub.2-/-
animals can be lysed by activated natural killer (NK) cells, again
suggesting a deficiency in class I expression (Liao et al.,
Science, 253:199-202 (1991)). These mice were originally developed
to study the role of .beta..sub.2M in development. While the mice
developed and bred normally, they failed to generate significant
numbers of CD8+ T cells. Consequently, these mice have been
intensely studied from an immunologic perspective.
[0100] .beta..sub.2-/-mice combat viral infections relatively well,
although the course of the infection is longer than in normal
animals (Eichelberger et al., J. Exp Med., 174:875-878 (1991);
Muller et al., Nature, 255:1576-1579 (1992)). They reject
allografts (Zijlistra et al., J. Exp. Med., 175:885-889 (1992)) and
show higher levels of Ig production and faster class switching of
antibody types than normal mice. Although CD8+ T cells are low to
undetectable at birth, studies have shown that the animals can
generate CD8+ T cells, and a cytotoxic CD8+ T cell response can be
mounted under appropriate circumstances (Apasov and Stikovsky, J.
Immunol., 152:2087-2097 (1994). Another significant abnormality
reported in these animals is that they develop hyperglycemia
(glucose>300 mg/dl) in old age (greater than 2 years), It has
been suggested that the onset of diabetes in the .beta..sub.2-/-
mice is related to autoimmunity (Faustman et al., Science,
254:1756-1761 (1991)), however this explanation has been disputed
(Serreze et al., Diabetes, 43:505-509 (1994); Wicker et al.,
Diabetes, 43:500-504 (1994)).
[0101] .beta..sub.2-/-mice can develop iron overload that is
similar to human hemochromatosis. .beta..sub.2-/-mice can
spontaneously develop hepatomas. This observation combined with the
molecular biology data of the .beta.-GAP genes (see Example I),
suggested that the mice would develop iron overload. Histochemical
examination of tissues from these mice, confirmed this hypothesis.
Iron was found deposited in the liver of all animals, and in the
kidneys, spleen and lungs of some of the animals. In addition, 16%
of the animals developed liver disease, having either hepatomas or
liver necrosis. Thus, the clinical findings for the
.beta..sub.2-/-deficient mice are sufficiently similar to the
pathology of hemochromatosis to make the .beta..sub.2-/-mouse an
attractive model for the study of a mechanism underlying human
hemochromatosis. More importantly, the .beta..sub.2-/-mice
demonstrate that .beta..sub.2M plays a role in this disease.
[0102] The ability of .beta.-GAP promoters to co-regulate both the
.beta.-GAP gene and a nonclassical class I gene that encodes
leczyme, both of which are expressed in the intestine, supports a
role for a class I leczyme in hemochromatosis. The nonclassical
class I gene regulated by the .beta.-GAP promoter is a leczyme that
can recognize and modify a carbohydrate structure associated with
the .beta.-GAP gene product, the latter of which directly or
indirectly binds iron (ie. .beta.-GAP can be an iron carrier).
Disruption of .beta..sub.2M expression results in a loss of
regulation of the leczyme function provided by the nonclassical
class I molecule, leading to iron overload and hemochromatosis.
[0103] A carbohydrate ligand or a leczyme of the present invention
can be used to prepare a medicament for the treatment of a disease
state such as hemochromatosis, autoimmune disease, transplantation
rejection, inflammation or infection. Autoimmune diseases that can
be treated by the present invention include systemic autoimmune
diseases such as ankylosing spondylitis, multiple sclerosis,
rheumatoid arthritis, slceroderma, Sjogren's syndrome or systemic
lupus erythematosus, and organ-specific autoimmune diseases such as
Addison's disease, Goodpasture's syndrome, Grave's disease,
Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura,
myasthenia gravis or pernicious anemia. As hemochromatosis in
humans is likely mediated by a .beta.-GAP promoter-driven leczyme,
then treatment with a carbohydrate ligand, leczyme or competing
molecule with the same or similar binding specificity as the
leczyme involved in the disease can be used to modulate the disease
process. A carbohydrate ligand that binds the nonclassical class I
leczyme involved in hemochromatosis can be administered to inhibit
binding to the .beta.-GAP iron carrier.
[0104] A process to follow for using a carbohydrate ligand to treat
a disease such as autoimmunity can first require identification of
the lecyzme that is involved in the disease process. Subsequently,
a candidate carbohydrate ligand that can bind to the leczyme is
identified by methods disclosed herein. Thus, such candidate
carbohydrate ligands can then be tested in vitro to identify those
efficient at blocking the autoimmune reaction exhibited when the
leczyme on autoreactive immune cells from the diseased individual
recognizes a carbohydrate molecule expressed on the cells of the
individual that is the target of the autoreactive cell. The
autoimmune reaction can be measured by an increase in a cell
function such as cell proliferation or release of cytokines (see
for example, Coligan et al. supra, 1992; Mishell and Shiigi, supra,
1980). The best candidate carbohydrate ligands can then be used as
a medicament to treat the disease.
[0105] The methods disclosed herein for the treatment of
hemochromatosis are also suitable for the treatment of many other
medical diseases or complication resulting from iron overload.
Since multiple leczyme genes are involved in mediating control of
iron metabolism, the type of mutation, its location in the gene and
the number and type of leczyme genes mutated in an individual are
factors that can effect the extent of iron overload in an
individual. As the extent of iron overload exhibited by an
individual is dependent on the above factors, then the methods
disclosed herein to treat hemochromatosis are also applicable for
treating other diseases resulting from iron overload. Such diseases
include, for example, hepatopathy (including cirrhosis, and liver
cancer), arthropathy, hypogonadotropic hypogonadism, marrow
aplasia, skin disorders, diabetes mellitus, and cardiomyopathy (for
review see Halliday and Powell, Iron and Human Disease, Lauffer, R
B, (ed). 131-160 (1992)).
[0106] In order to modulate hemochromatosis or other iron storage
disease, the carbohydrate ligand or mimetope is administered in an
effective amount. The total effective amount can be administered to
a subject as a single dose, either as a bolus or by infusion over a
relatively short period of time, or can be administered using a
fractionated treatment protocol, in which the multiple doses are
administered over a more prolonged period of time. One skilled in
the art would know that the concentration of carbohydrate ligand
required to obtain an effective dose in a subject depends on many
factors including the age and general health of the subject as well
as the route of administration and the number of treatments to be
administered and the chemical form of the carbohydrate ligand. In
view of these factors, the skilled artisan would adjust the
particular amount so as to obtain an effective amount for the
subject being treated.
[0107] A carbohydrate ligand or a leczyme also is useful in vivo
for the treatment of autoimmune diseases involving a leczyme. In
autoimmune disease, a leczyme expressed on a lymphoid cell can
recognize a self-carbohydrate ligand as foreign carbohydrate
ligand, resulting in immune-directed destruction of cells
expressing the self-carbohydrate ligand. Thus, administration of a
carbohydrate ligand, mimetope or other competing molecule that can
bind to the leczyme involved in aberrant self-recognition can block
lymphoid cell recognition or activation leading to a reduction in
symptoms or cessation of autoimmune disease. Alternatively,
administration of a leczyme that has the same or similar binding
specificity for the self-carbohydrate ligand recognized by a
leczyme of the autoreactive lymphoid cell can also be used to treat
the autoimmune disease.
[0108] A carbohydrate ligand or a leczyme can be used to treat a
disease state resulting from an infectious agent such as a virus,
bacterium, yeast or parasite. Infectious agents have evolved to
express their own external receptors that can recognize
carbohydrate structures or leczymes on the cell-surface, enabling
entry of the agent into the cell to be infected. Thus,
administration of an appropriate carbohydrate ligand or a leczyme
to an individual exposed to an infectious agent can block the
binding of the agent to target cells, subsequently inhibiting the
extent of infection and thereby reducing the spread of the
disease.
[0109] A carbohydrate ligand or a leczyme of the present invention
can be used to treat transplantation rejection. Since rejection is
based on the recognition of foreign molecules by lymphocytes of the
transplant recipient, then treatment with a carbohydrate ligand
that can bind to the leczyme of the transplant recipient's
lymphocyte that is involved in foreign antigen recognition can
inhibit recognition leading to transplantation rejection. Also,
administration of a leczyme that has the same or similar binding
site specificity as the leczyme of a transplant recipient's
lymphocyte involved in foreign antigen recognition can inhibit
recognition leading to transplantation rejection.
[0110] A carbohydrate ligand or leczyme of the present invention is
particularly useful when administered as a pharmaceutical
composition containing a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known in the art and
include, for example, aqueous solutions such as a physiologically
buffered saline or other solvents or vehicles such as glycols,
glycerol, oils such as olive oil or injectable organic esters.
[0111] A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize or to increase the absorption of a carbohydrate ligand or
leczyme. Such physiologically acceptable compounds include, for
example, carbohydrates, such as glucose, sucrose, dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins or other stabilizers or
excipients. One skilled in the art would know that the choice of a
pharmaceutically acceptable carrier, including a physiologically
acceptable compound, depends, for example, on the route of
administration of the composition.
[0112] One skilled in the art would know that a pharmaceutical
composition containing a carbohydrate ligand or leczyme can be
administered to a subject by various routes including, for example,
by direct instillation, orally or parenterally, such as
intravenously, intramuscularly, subcutaneously or
intraperitoneally. The composition can be administered by injection
or by intubation. The pharmaceutical composition also can be
incorporated, if desired, into liposomes or microspheres or can be
microencapsulated in other polymer matrices (Gregoriadis, Liposome
Technology, Vol. 1 (CRC Press, Boca Raton, Fla., 1984), which is
incorporated herein by reference). Liposomes, for example, which
consist of phospholipids or other lipids, are nontoxic,
physiologically acceptable and metabolizable carriers that are
relatively easy to make and administer.
[0113] An expression vector encoding a leczyme can be administered
in vivo to treat a disease state resulting from a leczyme. For
example, a disease state resulting from a mutated leczyme, such as
anemia, can be treated by administering an expression vector
encoding a functional leczyme involved in iron transport and
obtaining expression of the vector in cells of the digestive
tract.
[0114] The level of expression of a particular leczyme in a cell
can have a impact on the nature of a carbohydrate ligand expressed
by the cell. If expression of a particular carbohydrate ligand is
involved in a disease process, the ligand can be eliminated from a
cell by reducing the expression of the leczyme responsible for
producing the ligand. Thus, an expression vector can contain an
exogenous nucleic acid molecule encoding an antisense nucleotide
sequence that is complementary to a nucleotide sequence encoding a
portion of a leczyme such that when introduced into a cell under
the appropriate conditions, the expression vector can produce an
antisense nucleic acid molecule, which can selectively hybridize to
the leczyme gene or message in a cell and, thereby, affect the
expression of the leczyme in the cell. For example, the antisense
nucleic acid molecule can hybridize to a leczyme gene in the cell
and can reduce or inhibit transcription of the leczyme gene. Also,
the antisense molecule can hybridize to the message encoding the
leczyme in the cell and can reduce or inhibit translation,
processing and cell stability or half-life of the RNA.
[0115] Expression vectors also can be used to effect the expression
of a leczyme or of a carbohydrate ligand involved in a disease
state by introducing into a cell an exogenous nucleic acid molecule
encoding a ribozyme that can specifically cleave RNA encoding the
leczyme or peptide backbone of a carbohydrate ligand. Thus, by
introducing the ribozyme into cells involved in a disease process,
one can reduce expression of the leczyme or carbohydrate ligand
involved in the disease and therefore reduce or inhibit the disease
process. An antisense nucleic acid molecule or a ribozyme can be
chemically synthesized and incorporated into an expression vector
using recombinant DNA techniques. The antisense nucleic acid
molecule or ribozyme also can be added directly to a cell without
having been incorporated into an expression vector.
[0116] Methods for introducing an expression vector into cell are
well known in the art. Such methods are described in Sambrook et
al, supra, 1989; Kriegler M. Gene Transfer and Expression: A
Laboratory Manual (W. H. Freeman and Co. New York, N.Y. (1990),
both of which are incorporated herein by reference) and, for
example, include transfection methods such as calcium phosphate,
electroporation, lipofection, or viral infection.
[0117] Recombinant viral rectors are available for introducing an
exogenous nucleic acid molecule into a mammalian cell and include,
for example, adenovirus, herpesvirus and retrovirus-derived
vectors. For example, a viral vector encoding a leczyme can be
packaged into a virus to enable delivery of the genetic information
and expression of these leczyme in gastrointestinal epithelial
cells following infection by the virus. Also, a recombinant virus
which contains an antisense sequence or a ribozyme specific for a
nucleotide sequence encoding a leczyme can introduced into a cell
in an individual to inhibit a disease state mediated by the leczyme
or a leczyme with a similar carbohydrate binding specificity.
[0118] Recombinant viral infection can be more selective than
direct DNA delivery due to the natural ability of a virus to infect
only certain types of cells. This natural ability for selective
viral infection can be exploited to limit infection to only certain
cell types within a mixed cell population. For example,
adenoviruses can be used to restrict viral infection principally to
cells of epithelial origin. In addition, a retrovirus can be
modified by recombinant DNA techniques to enable expression of a
unique receptor or ligand that provides further specificity to
viral gene delivery. Retroviral delivery systems that provide high
infection rates, stable genetic integration and high levels of
exogenous gene expression are well known in the art.
[0119] As described above, recombinant viral delivery systems exist
that provide the means to deliver genetic information into a
selected type of cell. The choice of viral system will depend on
the desired cell type to be targeted, while the choice of vector
will depend on the intended application. Recombinant viral vectors
are readily available to those in the art and can be easily
modified by one skilled in the art using standard recombinant DNA
methods (see, for example, Krieger, Gene Transfer and Expression: A
Laboratory Manual, (W. H. Freeman. and Company, 1990); Goeddel,
Methods in Enzymology, vol. 185, (Academic Press, 1990); and
Stoker, In Molec. Virol., A Practical Approach (eds. Davison and
Elliott, IRL Press, 1993), all three of which are incorporated
herein by reference).
[0120] The present invention provides methods for diagnosing a
genetic predisposition for hemochromatosis or other iron storage
diseases based on a leczyme by detecting a mutation in the heavy
chain of a class I MHC molecule encoded for by a gene in the MHC
locus. These methods can be used to diagnose an individual having
the symptoms of an iron storage disease. A positive diagnosis of
mutation in an individual's heavy chain is useful to verify the
underlying cause of the disease and by identifying the particular
leczyme that is mutated. The identification of the mutated leczyme
can be used with the methods disclosed herein to identify a
carbohydrate suitable for treating the disease.
[0121] An individual who does not have an iron storage disease, but
is suspected of inheriting a mutation that can predispose the
individual to develop an iron storage disease later in life can
also benefit from having their class I molecules tested for
mutation by the methods disclosed herein.
[0122] A mutation that is diagnostic for the disease is one that
results in a significantly reduced affinity of the heavy chain for
human .beta..sub.2M. For example, a mutation in a nonclassical
class I heavy chain that results in deletion of a signal for
phosphorylation is a mutation that is diagnostic for
hemochromatosis since a properly phosphorylated heavy chain is
necessary for the chain to interact with .beta..sub.2M. Consensus
amino acid sequences that signal a cell to phosphorylate a serine
or a threonine residue in a polypeptide are well known in the art.
A mutation that is diagnostic for hemochromatosis also can occur in
a region of the heavy chain that is near to a phosphorylation site.
Such a mutation can reduce the ability of the heavy chain to
associate with .beta..sub.2M if the phosphate group added to this
site cannot be removed in a cell.
[0123] Methods to detect a phosphorylation site mutation in a
nonclassical class I heavy chain can be based either on analysis of
the protein or the nucleic acid encoding the protein. For protein
determination, the nonclassical class I molecule can be purified
from a source of cells or body fluids of an individual and the
heavy chain can be isolated from .beta..sub.2M. Methods to purify a
class I MHC molecule and isolate the heavy chain from .beta..sub.2M
are well known in the art. The isolated heavy chain can then be
subjected to amino acid sequencing, peptide mapping or other such
protein analyses to determine if the sequence a phosphorylation
site has been mutated. Such methods for protein determination are
well known to those in the art.
[0124] A mutation in a nucleic acid sequence can be detected by
various methods to analyze nucleic acids such as by nucleic acid
sequencing, polymerase chain reaction or hybridization. Such
methods are well known to those in the art (see, for example,
Sambrook et al, supra, 1989; Hames and Higgins Nucleic Acid
Hybridisation: a practical approach (IRL Press, New York, 1985),
both of which are incorporated herein by reference).
[0125] Methods to detect decreased binding of a mutated heavy chain
with .beta..sub.2M can be used for diagnosing an iron storage
disease such as hemochromatosis. In these methods, the heavy chain
of an class I MHC molecule is isolated from an individual and
contacted with .beta..sub.2M under conditions suitable for a
non-mutated such heavy chain to associate with .beta..sub.2M. A
control reaction, which contains a non-mutant form of the same or
similar class I heavy chain to the one being tested for a mutation
is performed in parallel. After contacting the heavy chain with
.beta..sub.2M, the reaction is incubated under suitable conditions,
including, for example, an appropriate buffer concentration and pH
and time and temperature, which is sufficient for the control heavy
chain to associate with .beta..sub.2M. The heavy chain being tested
from the individual is considered to have a mutation diagnostic for
an iron storage disease when the fraction of this heavy chain that
associates with .beta..sub.2M is significantly less than the
fraction of control heavy chain that associates with
.beta..sub.2M.
[0126] The association of a class I heavy chain with .beta..sub.2M
can be detected, for example, by attaching one of the molecules to
a solid support and attaching a detectable label such as a
radionuclide or a fluorescent label to the other molecule and
measuring the amount of detectable label that is associated with
the solid support, wherein the amount of label detected indicates
the amount of association of the heavy chain with
.beta..sub.2M.
[0127] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Cloning and Expression of the .beta.-GAP Genes
[0128] This example provides an approach to identify and clone
leczyme genes from various species of animal to elucidate their
role in iron metabolic diseases.
[0129] Cloning of the Mouse .beta.-Gap Genes
[0130] Genomic .lambda. libraries were constructed by partial Hae
III digestion of DNA from A/J and Balb/c mouse liver and cloning
the fragments into the vector Charon 4A. The libraries were
screened with the S15 probe, isolated from the H-2L.sup.d gene
(Margulies et al., Nature, 295:168-170 (1982), which is
incorporated herein by reference)). S15 is a 3' class I MHC mouse
probe and consists of 522 base pairs including 36 base pairs of
exon 4 encoding the alpha-3 domain and 486 base pairs of intron
(Evans et al., Proc. Natl. Acad. Sci. (USA), 79:1994-1998 (1982)).
Probes were prepared by excising the insert from M13 RF or pUC18,
purifying the fragment from disulfide cross-linked acrylamide gels
(Hansen, Anal. Biochem. 116:146-151 (1981)), and labeling with
.sup.32P to a specific activity of >10.sup.8 cpm/.mu.g by nick
translation (Rigby et al., J. Mol. Biol. 113:237-251 (1977)).
Libraries were screened using standard colony hybridization
techniques (for details see Sambrook et al., supra, 1988).
[0131] Seventeen unique .lambda. clones were isolated from the
libraries and were subjected to restriction enzyme digestion
mapping. BamHI digestion and gel electrophoresis of these clones
revealed five from the A/J strain and one from the Balb/c stain
that contained a unique 500 base pair (bp) BamHI restriction
fragment (BB500). The six clones containing the unique fragment
were subjected to BAMHl digestion, the BB500 fragment was gel
purified and subcloned into M13 vector Mp18 and mp19
(Yanisch-Perron et al., Gene 33:103-119 (1985)). DNA sequences were
determined by the chain termination method (Sanger, et al., Proc.
Natl. Acad. Sci. (USA) 74:5463-5467 (1977)) using .sup.35S-ATP.
Reactions were analyzed on 6% urea-polyacrylamide gradient gels
(Biggins et al., Proc. Natl. Acad. Sci. (USA) 80:3963-3965 (1983)).
DNA sequences were assembled and analyzed using the University of
Wisconsin Computer Group Programs (Devereux, et al., Nucleic Acids
Res. 12:387-395 (1984)) run on a VAX-11/785 computer.
[0132] DNA sequence comparisons demonstrated that the BB500
fragments share greater than 93% sequence homology. A region within
the BB500 fragment shows 100% sequence homology between the
.lambda. clones and has been termed .beta.-GAP (globin analogous
promoter) since it is a regulatory motif that shares sequence
homology with mouse, rabbit or human .beta.-globin promoters (for a
detailed comparison see ahead). There is close similarity between
all six fragments (called .beta.-GAP1-6) with the minor exception
of .beta.-GAP4 where an 8 base pair sequence AAGAGGAG, immediately
downstream of a CCAAT element, has been deleted. There are other
minor differences between these sequences, and the .lambda. clones
they have been isolated from demonstrate different restriction
patterns confirming that the various .lambda. clones contain unique
sequences and are not a cloning artifact. Thus, the A/J strain
mouse contains at least five highly homologous .beta.-GAP sequences
within its genome.
[0133] Mapping the .beta.-GAP Sequences Map to Chromosome 17 in the
Mouse
[0134] Southern blotting was used to determine if the BB500
sequence could identify genes located on chromosome 17 of the
mouse. DNA from several Chinese hamster ovary (CHO) mouse somatic
hybrid cell lines were evaluated by Southern blotting using the
.beta.-GAP6 BB500 probe. Genomic DNA was isolated from cultured
cells, digested with EcoR1, electrophoresed on 0.8% agarose gels
and transferred to a nitrocellulose membrane. Hybridization with
the BB500 probe was carried out in the presence of dextran sulfate
under the conditions described by Meinkof and Wahl (Anal. Biochem.
138:267-284 (1985)) with a final wash in 0.2.times. sodium chloride
sodium citrate buffer, pH 7.0 (SSC) at 60.degree. C.
[0135] The BB500 probe hybridized with the HM27 cell line
containing the DNA from mouse chromosomes 15 and 17 and revealed
the same banding pattern as with total genomic BALB/c DNA. The cell
line HM65 that lacks BALB/c chromosome 17 was devoid of
hybridizable bands, indicating that the probe did not bind
nonspecifically to CHO DNA. DNA from other CHO cell lines
containing mouse chromosomes other than chromosome 17 were examined
by Southern blotting with the BB500 probe and were found to be
negative (not shown). These results indicate that the .beta.-GAP
sequences all map to chromosome 17 in the mouse.
[0136] Mapping the .beta.-GAP Sequences to the Murine Q/TL
Complex
[0137] The fact that the .beta.-GAP sequences were isolated from
the mouse genome provided several powerful tools to precisely map
the location of the sequences. First, the murine MHC is highly
characterized, particularly with respect to the nonclassical class
I region and, secondly, congenic strains of mice exist where the
position of genes in the MHC can be pinpointed. Congenic strains
were originally developed by breeding strains of inbred mice
together. Subsequent generations of chromosomal crossing over has
produced a number of strains which contain a portion of the MHC
from one strain and the remainder of the MHC from another strain.
Consequently, it is possible to compare restriction fragment length
polymorphism (RFLP) between the strains, and determine if the
banding patterns are linked to a given MHC locus (for review see
Klein, Natural History of the Major Histocompatibility Complex,
50-73 (1986)). RFLP analysis was performed by obtaining purified
genomic DNA from the various mouse strains, digesting the DNA with
EcoRI and performing Southern hybridization with the .beta.-GAP6
BB500 probe as described above. The Southern blot showed that the
probe identified up to ten different bands from the DNA of the
mouse strains tested (Table 1). Four of these bands, 30 kb, 20 kb,
16 kb and 10.5 kb, were mapped within the MHC locus. The RFLP
analysis indicated that there were at least four to six copies of
the .beta.-GAP sequences/genome depending on the strain of mouse
tested. In addition genetic analysis of the RFLP patterns indicated
that the 30 kb and 10.5 kb .beta.-GAP bands mapped to Q region
between Q1 and Q4 while the 20 kb and 16 kb .beta.-GAP bands mapped
to the T region. In addition, two of the .beta.-GAP sequences that
did not demonstrate RFLP polymorphism were mapped telomeric to the
classical class I genes.
[0138] Locating the .beta.-GAP Sequences Directly Adjacent to
Nonclassical Class I Genes
[0139] The two of the .beta.-GAP gene sequences that were mapped to
the Q region between Q1 and Q4, were directly linked to Q1 and Q2
by DNA sequence analysis of Q1 and Q2 genes isolated from a C57BL/6
(H-2.degree.) .lambda. library. Sequencing showed that both the Q1
and Q2 genes are associated in a head to head configuration with an
unknown gene (currently defined as the .beta.-GAP gene) with both
genes transcriptionally regulated by a single promoter/enhancer
region having two promoters defined by a pair of CAAT and TATA
boxes located about 25 bp apart on opposite strands of the DNA.
Thus, having intact promoters and a common regulatory region, the
class I and .beta.-GAP genes would be transcribed from opposite
strands, with the class I genes Q1 or Q2 transcribed from 5' to 3'
on the top strand and the .beta.-GAP gene transcribed from 5' to 3'
on the bottom strand.
[0140] The sequence analysis of Q1 and Q2 genes from C57BL/6, as
well as a TL gene from A/J (H-2.sup.a, Watts et al. EMBO J.
8:1749-1759 (1980)) indicated that .beta.-GAP promoter and
regulatory regions had replaced the typical classical class I-type
5' regulatory sequences known to be involved in the regulation of
classical class I genes. The .beta.-GAP promoter is an active
promoter since it is known that the Q2 gene expresses a gene
product that can be detected in the intestine (Wang et al.,
Immunogenet., 38:370-372 (1993)). These results indicate that the
.beta.-GAP promoter regulates the expression of some nonclassical
class I genes.
1TABLE 1 COMPARISON OF SOUTHERN BLOT ANALYSIS OF EcoRI DIGESTS OF
MURINE DNA USING THE BB500 LOW COPY NUMBER PROBE WITH GENETIC MAPS
OF VARIOUS ALLOGENEIC AND CONGENIC STRAINS. MHC REGION EcoRI BAND
SIZE (kb) STRAIN K D Q T 30 20 16 14.5 13 10.5 9.2 8.0 7.8 7.5 B6,
B10 b b b b + + + + + B6, K1 b b k k + + + + + B6, K2 b b bk k + +
+ + + AKR k k k k + + + + + B6, K3 k k b a + + + + B6, K4 k k k a +
+ + + + B6-H-2.sup.k k k k k + + + + + B6-T1a.sup.a b b ba a + + +
+ + A/J k d a a + + + + + + Balb/cJ d d d d + + + + + + B10, A k d
a a + + + + + + A-T1a.sup.b k d a b + + + + +
[0141] Head to head gene structure with co-regulation of the genes
has been previously described in organisms ranging from bacteria to
humans, indicating that co-regulation is a widely adopted strategy.
(Brickman et al., J. Molec. Biol., 212:669-682 (1990); Xu and
Doolittle, Proc. Natl. Acad. Sci. (USA), 87: 2097-2101 (1990);
Lennard and Fried, Molec. Cell. Biol., 11:1281-1294 (1991);
Heikkila et al., J. Biol. Chem., 268:24677-24682 (1993); Fererjian
and Kafatos, Dev. Biol., 161:37-47 (1994); Sun and Kitchingman,
Nucleic Acids Res., 22:861-868 (1994)). In both prokaryotic and
eukaryotic systems, interaction between, or linkage in a metabolic
pathway of two gene products has been suggested (Galvalas, et al.,
Mol. Cell. Biol., 13:4784-4792 (1993); Lightfoot et al., Br. J
Cancer, 69:264-2673 (1994)). It should be noted that in the
.beta.-GAP clones so far studied, the Q1 and Q2 genes still
possesses their own CAAT and TATA elements, and it is only the
typical classical class I regulatory enhancer regions which are
absent.
[0142] Conservation of the .beta.-GAP Sequences Across Species
[0143] To demonstrate that the .beta.-GAP sequences are conserved,
and that various species, including human, contain multiple copies
of these genes a "Zoo blot" of various species of genomic DNAs was
digested with EcoRI and analyzed by Southern blotting using the
murine .beta.-GAP6 BB500 probe. Under low stringency the blot
showed detection of a multiplicity of bands in DNA from human, rat,
mouse, dog, rabbit and monkey. This indicates that multiple copies
of the .beta.-GAP sequences were found in many species including
human. In addition, the conservation of the .beta.-GAP multigene
family predates speciation of murine and human and therefore is not
the product of a recent gene duplication or rearrangement. The
demonstration of interspecies sequence homology is significant
because, in general, exons and regulatory regions tend to be
conserved. Thus, the pattern of specific regions of retained
homology suggests that the .beta.-GAP sequences are retained by
selective pressure.
[0144] Homology Between the .beta.-GAP Sequences and the Promoters
for .beta.-Globin
[0145] Sequences within all six of the 500 bp .beta.-GAP clones
show striking sequence and positional homology to mouse, rabbit and
human .beta.-globin promoter regulatory elements. Important
regulatory elements within a 106 bp region of the .beta.-globin
promoter have been characterized (Myers et al., Science 232:613-618
(1986); Stuve and Myers, Mol. Cell Biol. 6:3350-3358 (1990)). Using
saturation mutagenesis and 5' deletion promoters, Myers and his
colleagues constructed a series of mutants that were used to
identify four regulatory sequences. The four regulatory motifs were
located between positions -95 and -26 which contain a CACCC element
(positions -95 to -87), CCAAT and TATA box motifs at positions -79
to -72 and -30 to -26, respectively, and a 11 bp repeat element
located between the CCAAT and TATA boxes (positions -53 to -32)
that contains 2 imperfect duplicated repetitive elements
(.beta.DRE). The fact that these .beta.DRE are essential for the
expression of globin genes has been shown by deletional
studies.
[0146] Comparison of the six 500 bp .beta.-GAP sequences with the
.beta.-globin promoter sequences from various species showed
several striking sequence homologies to .beta.-globin regulatory
elements. Analysis of the .beta.-GAP sequence in this region
revealed 5 regulatory motifs found in the .beta.-globin promoters.
These include the 5' CACCC erythroid element between positions -127
to -123, CCAAT and TATA box motifs between positions -109 to -105
and -30 to -26, respectively, the cap consensus sequence positions
-13 to -10, and a fifth and more complex regulatory element
involved a .beta.-globin .beta.DRE of a 10 and 11 bp sequence (base
pair numbering was determined from sequence alignments with gaps
inserted and does not reflect the true base pair position from the
transcriptional start site).
[0147] In all the .beta.-GAP clones, two of the four .beta.DRE
regulatory motifs were flanked by the CCAAT and TATA elements
between positions -54 and -32, while two other .beta.DREs were
found immediately upstream of the TATA box (positions -11 to +1 and
+3 to +12). All of these .beta.DRE were conserved in sequence, and
moreover, two of them were conserved in position (-54 and -32). It
is significant that the .beta.DREs conserved in .beta.-GAP were
conserved in globins from multiple species (mouse, rabbit, chicken
and human) covering more than 100 million years of evolution. This
observation of evolutionary conservation indicates the .beta.-GAP
genes are old genes.
[0148] A final putative regulatory motif from the .beta.-GAP clones
was AGATAA (nucleotides -82 to -77), which is identical to the DNA
consensus sequence for the transcriptional binding factors NF-E1.
This family of DNA binding proteins (NF-E1a, b, and c) are involved
in the erythroid and/or T-cell specific expression of many genes,
including mouse and chicken adult .beta.-globin, the heme pathway
enzyme porphobilinogen (PGB) deaminase, the T-cell receptor and the
leukemia virus HTLV III.
[0149] A closer inspection of the regions of homology between the
.beta.-GAP and mouse .beta.-globin promoters reveals several
features: 1) 18 of 26 base pairs match at positions -35 to -10
encompassing the consensus TATA motif (Bucher, J. Mol. Biol.
212:563-578 (1990)); 2) a region encompassing the .beta.-GAP CCAAT
box, positions -113 to -109 contains the .beta.-globin regulatory
element CACCC which has been shown to be essential for the
appropriate expression of .beta.-globin in erythroid cells; 3) a
perfect match of the CCAAT element exists at positions -109 to
-105; 4) the fourth matching region encompasses a .beta.DRE
element, located between the CCAAT and TATA boxes at positions -64
to -45 (this region contains 16 of 19 bp matches with no gaps); and
5) a consensus cap site sequence as defined by Bucher (Bucher,
supra, 1990) and a putative transcriptional start site is
identified at nucleotides -13 to +1.
[0150] Several other putative regulatory sequences are apparent in
the .beta.-GAP promoter. Between positions -68 and -37 and
beginning 5 nucleotides distal of the TATA element are 4
palindromes. The 5 base pair repeat TCAGA appears twice within 24
base pairs. These repeats flank and are found within a globin-like
imperfect direct repeat element (positions -57 to -47). Two longer
palindromes with imperfect dyad symmetry of 12 bp, and 15 bp,
positions -67 to -56 and -51 to -37, respectively, contain smaller
internal palindromes of 7 bp, CCTCAGG (-66 to -60) and 5 bp repeat,
TCAGA (-46 to -42), respectively. This .beta.-GAP 33 bp
.beta.DRE-like region combining the two large 12 and 15 bp
imperfect palindromes, the .beta.-globin imperfect direct repeat
element and the two TCAGA palindromic repeats shows about 50%
(16/33) nucleotide sequence homology to the mouse .beta.-globin
promoter.
[0151] Expression of Genes Immediately Downstream from the
.beta.-GAP Sequences in the Gastrointestinal Tract
[0152] The pattern of specific regions of retained homology between
the .beta.-globin regulatory motifs and .beta.-GAP promoters
suggests: 1) the sequences have diverged from a common ancestral
gene; and 2) the preserved regions in the .beta.-GAP sequences play
a critical role in the regulation of expression of their respective
genes. Furthermore, the homology to promoters for genes intimately
involved in iron metabolism, the occurrence of erythroid specific
regulatory sequences, and the close proximity of these genes to the
human locus responsible for hemochromatosis, indicates a role for
the .beta.-GAP genes in iron metabolism.
[0153] To demonstrate that the .beta.-GAP promoters regulate
downstream messages, it is imperative to show that the associated
genes encode transcribable messages. Moreover, such messages should
be expressed in tissues involved in iron absorption, i.e. the
gastrointestinal tract, if they are to be involved in the
pathogenesis of hemochromatosis.
[0154] Northern blotting was performed with poly A+ RNA from
various organs including the gastrointestinal tract. The blot was
developed using two probes derived from a .beta.-GAP (Q2.sup.b)
cosmid clone. Total cellular RNA was prepared by the TRIzol.TM.
Reagent method according to the manufacturer's instruction
(Gibco/BRL, Gaitherburg, Md.). poly A+ or mRNA was purified by
oligo dT cellulose chromatography (Strategene, San Diego, Calif.).
RNA was analyzed on formaldehyde-agarose gels and transferred to
Zeta Bind membranes as previously described (Evans, et al., Proc.
Natl. Acad. Sci. (USA) 81:5532-5536 (1984)). The cosmid clone
containing a .beta.-GAP sequence that was used or the probe was
obtained from a .lambda. library. The clone was digested with ApaLI
and KpnI to yield a 10 kb fragment. The fragment was partially
digested with BamHI to yield a 2 kb probe encompassing the
.beta.-GAP sequences and a 8 kb probe piece further downstream
containing the coding sequences for a .beta.-GAP gene.
[0155] Northern blotting with the 2 kb probe showed the presence of
polydisperse messages produced in tissues from stomach, duodenum,
jejunum, spleen and liver, principally of 5 kb and 8 kb in size.
The kidney showed less polydispersity with only the 8 kb band
predominating. These results indicate that .beta.-GAP promoter and
upstream .beta.-GAP coding sequences are expressed in the
gastrointestinal tract and are associated with members of a
multigene family of which the 5 Kb message of the jejunum is most
prominent. The fact that this probe also recognized a band in the
liver, spleen, kidney and stomach, suggested that related members
of a .beta.-GAP family can be functioning in other tissues. The
downstream 8 Kb probe identified a band about 5 kb in jejunum which
was absent in from the kidney polyA+ RNA. This result indicates
that downstream .beta.-GAP coding sequences are less conserved and
can be restricted in expression.
[0156] The size and complexity of the .beta.-GAP mRNA products
detected by northern blotting is consistent with .beta.-GAP genes
coding for a family of large proteins. These characteristics are
more like those of a mucin protein family rather than an ion
transport family of molecules. The homology to .beta.-globin
promoters, the occurrence of erythroid specific regulatory
sequences and close proximity of nonclassical class I and
.beta.-GAP genes to the locus responsible for hemochromatosis in
humans, an inheritable disease of iron metabolism indicates a role
for the .beta.-GAP genes and the nonclassical class I genes in iron
metabolism. With this information in hand and the facts disclosed
herein that .beta..sub.2M-knockout mice have an unusually high
incidence of hepatomas led to the understanding that these mice
have a metabolic and pathological condition similar to
hemochromatosis.
[0157] Isolation of Murine .beta.-GAP cDNAs From a Mouse Jejunal
Library.
[0158] The 2 kb .beta.-GAP cosmid probe was used to screen a mouse
jejunal cDNA library (Strategene .lambda. ZAP Express kit).
Northern blots suggested that the messages recognized by the
.beta.-GAP probe were abundant (bands were visible after only three
hours of exposure) and this observation was confirmed upon
screening the library. Approximately 0.5% of the clones gave a
positive signal on the initial screening. 30 positive clones were
picked and rescreened, and 26 positive clones were picked from the
secondary screen. The murine clones ranged in size from
approximately 2 kb to >8 kb, and the size of the inserts
corresponded to the bands seen by northern blotting with the 2 kb
probe.
[0159] DNA purified from the selected murine cDNA clones were
digested with EcoRI and subjected to southern blot analysis. The
blot was probed with the 2 kb .beta.-GAP cosmid probe, and 5 were
found to be positive indicating they contained .beta.-GAP genes.
These .beta.-GAP clones are nearly full length cDNA since they were
quite large and since they were isolated with a 5' .beta.-GAP
probe.
[0160] Cloning of the Human .beta.-Gap Genes
[0161] A human genomic DNA library produced in sCOS cosmid vector
was prepared as described previously for producing a mouse genomic
library in sCOS (Strategene, San Diego Calif.). The isolation of
the human .beta.-GAP genes from the human sCOS cosmid library was
performed by screening clones with a class I MHC probe. The probe
was generated from exons 4 and 5 of the HLA-A2 gene, which encodes
the highly conserved .beta..sub.2M binding domain and the
transmembrane region. Twenty five putative clones containing class
I sequences were detected, and the Cosmids from these clones were
purified, cut with the restriction enzyme EcoRI, run on a 0.7%
agarose gel and blotted onto a charged nylon membrane. The blot was
hybridized with the class I probe, striped and rehybridized with
the 2 Kb .beta.-GAP probe. Three unique clones were found that
reacted with both the murine .beta.-GAP probe and the human class I
probe. This result indicates that the human .beta.-GAP genes can be
isolated and have a genomic structure with a closely linked class I
gene as was observed in mice and rats.
EXAMPLE II
.beta..sub.2M Knockout Mice Develop Iron Overload Similar to
Hemochromatosis
[0162] This example provides a method to analyze iron deficiency in
an animal model where an MHC-encoded leczyme function has been
genetically deleted. In addition, these mice are useful for
evaluating the in vivo utility of carbohydrate ligands on the
treatment of hemochromatosis and various iron related diseases such
as atherosclerosis, arthritis or cancer.
[0163] The data concerning iron overload in the .beta..sub.2M
knockout mice is contained in Rothenberg and Voland, 1994.
Histologic examination of tissues from 12-18 month old knockout
mice, contained on a standard diet, revealed evidence of hepatic
necrosis. Iron stains of the tissues revealed iron deposition in
the liver of all animals, and in the kidney, or the lung of
approximately 10% of the animals. The standard diet contains 350
mg/kg Ferric carbonate. When animals were placed on a "breeder
diet", which contains in addition to ferric carbonate, 10 mg/kg
ferrous sulfate, iron stores rose dramatically. Iron deposition in
the animals was also age related with the highest levels of iron
seen in the oldest animals. Together these data indicate that the
.beta..sub.2M-knockout mice develop iron overload that is diet and
age related. In addition we have shown that the animals develop
hepatomas and others have reported that older animals develop
diabetes (Faustman et al., Science 254:1756-1761 (1991)). This
constellation of pathologies mirrors human hemochromatosis.
[0164] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *