U.S. patent application number 10/042523 was filed with the patent office on 2002-10-17 for mammalian hyaluronan synthases, nucleic acids and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Briskin, Michael J..
Application Number | 20020151026 10/042523 |
Document ID | / |
Family ID | 24548240 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151026 |
Kind Code |
A1 |
Briskin, Michael J. |
October 17, 2002 |
Mammalian hyaluronan synthases, nucleic acids and uses thereof
Abstract
The present invention relates to an isolated or recombinant
nucleic acid which encodes a mammalian hyaluronan synthase (e.g.
human). The present invention also relates to a host cell
comprising the nucleic acid encoding mammalian hyaluronan synthase.
The present invention also relates to a method for producing a
mammalian hyaluronan synthase comprising introducing into a host
cell a nucleic acid construct comprising a nucleic acid which
encodes a mammalian hyaluronan synthase, whereby a recombinant host
cell is produced having said coding sequence operably linked to at
least one expression control sequence; and maintaining the host
cells produced in a suitable medium under conditions whereby the
nucleic acid is expressed. The present invention also relates to an
antibody or functional portion thereof which binds mammalian
hyaluronan synthase. The present invention also relates to a method
of detecting mammalian hyaluronan synthase in a sample comprising
contacting a sample with an antibody which binds hyaluronan
synthase under conditions suitable for specific binding of said
antibody to the mammalian hyaluronan synthase; and detecting
antibody-mammalian hyaluronan synthase. The invention further
relates to a method of using hyaluronan synthase to make
hyaluronan.
Inventors: |
Briskin, Michael J.;
(Lexington, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
24548240 |
Appl. No.: |
10/042523 |
Filed: |
October 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10042523 |
Oct 19, 2001 |
|
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|
08635552 |
Apr 22, 1996 |
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Current U.S.
Class: |
435/200 ;
435/320.1; 435/325; 435/69.1; 435/84; 536/23.2; 536/53 |
Current CPC
Class: |
C12N 9/1051
20130101 |
Class at
Publication: |
435/200 ;
435/69.1; 435/320.1; 435/325; 536/23.2; 435/84; 536/53 |
International
Class: |
C12P 019/26; C12N
009/24; C12P 021/02; C12N 005/06; C08B 037/00 |
Claims
We claim:
1. An isolated or recombinant nucleic acid which encodes a
mammalian hyaluronan synthase.
2. The nucleic acid of claim 1 wherein the hyaluronan synthase is
human.
3. The nucleic acid of claim 1 comprising SEQ ID NO: 1.
4. The nucleic acid of claim 1 wherein said nucleic acid hybridizes
under stringent conditions with a second nucleic acid having a
nucleotide sequence of SEQ ID NO: 1.
5. The nucleic acid of claim 1 wherein the nucleic acid encodes the
amino acid sequence of SEQ ID NO: 2.
6. A recombinant nucleic acid construct comprising a nucleic acid
of claim 1.
7. The recombinant nucleic acid construct of claim 6 comprising SEQ
ID NO: 1.
8. The recombinant nucleic acid construct of claim 6 wherein the
nucleic acid encodes the amino acid sequence of SEQ ID NO: 2.
9. The recombinant nucleic acid construct of claim 6 wherein the
nucleic acid is operably linked to an expression control
sequence.
10. A host cell comprising the nucleic acid of claim 1.
11. The host cell of claim 10 wherein the nucleic acid is operably
linked to an expression control sequence, whereby mammalian
hyaluronan synthase is expressed when the host cell is maintained
under conditions suitable for expression.
12. A method for producing a mammalian hyaluronan synthase
comprising: a) introducing into a host cell a nucleic acid
construct comprising a nucleic acid which encodes a mammalian
hyaluronan synthase, whereby a recombinant host cell is produced
having said coding sequence operably linked to at least one
expression control sequence; and b) maintaining the host cells
produced in step a) under conditions whereby the nucleic acid is
expressed.
13. An antibody or functional portion thereof which binds mammalian
hyaluronan synthase.
14. A method of detecting mammalian hyaluronan synthase in a sample
comprising: a) contacting a sample with an antibody which binds
hyaluronan synthase under conditions suitable for specific binding
of said antibody to the mammalian hyaluronan synthase; and b)
detecting an antibody-mammalian hyaluronan synthase complex.
15. A method of producing hyaluronan comprising maintaining a host
cell of claim 10 under conditions whereby hyaluronan is
produced.
16. The method of claim 15, comprising isolating hyaluronan thereby
produced.
Description
BACKGROUND
[0001] Hyaluronan is a constituent of the extracellular matrix of
connective tissue, and is actively synthesized during wound healing
and tissue repair to provide a framework for ingrowth of blood
vessels and fibroblasts. Changes in the serum concentration of
hyaluronan are associated with inflammatory and degenerative
arthropathies such as rheumatoid arthritis. In addition, hyaluronan
has been implicated as an important substrate for migration of
adhesion of leukocytes during inflammation.
[0002] Hyaluronan (hyaluronic acid, HA) is a high molecular mass
polysaccharide that has ubiquitous distribution in the
extracellular matrix, with highest concentrations in soft
connective tissue. It is a linear polysaccharide comprising
alternating glucuronic acid and N-acetylglucosamine residues linked
by .beta.-1-3 and .beta.-1-4 glycosidic bonds (Laurent, T. C. et
al. (1986), "The properties and turnover of hyaluronan." Functions
of proteoglycans (Symposium, C.F., Ed. 124, Chichester, England).
By interacting with other matrix molecules, such as chondroitin
sulfate proteoglycans, hyaluronan provides stability and elasticity
to the extracellular matrix. Hyaluronan has several physiochemical
and biological functions such as space filling, lubrication, and
providing a hydrated matrix through which cells can migrate (Toole,
B. P. et al., Hyaluronate-cell interactions. The role of the
extracellular matrix in development, (Trelstad, R.L., Ed., Alan R.
Liss, New York (1984); Laurent, T. C. et al., Faseb J. 6:2397-2404
(1992)). Interaction of hyaluronan with the leukocyte cell surface
receptor CD44 has been shown to contribute to organ specific
leukocyte homing and migration (Jalkanen, S. T. et al., J. Cell.
Biol., 105:893-990 (1987); Aruffo, A., et al., Cell 61:1303-1313
(1990); Culty, M. et al., J. Cell. Biol., 111:2765-2774 (1990);
Miyake, K. et al., J. Exp. Med. 172:69-75 (1990); Sherman, L. et
al., Current Opinions in Cell Biology, 6:726-733 (1994)).
Hyaluronan synthesis has been suggested to be required for cellular
proliferation (Brecht, M. et al., Biochem. J. 239:445-450 (1986);
Hronowski, L. and Anastassiades, T. P., J. Biol. Chem.
255:9210-9217 (1980); Matuoka, K. et al., J. Cell Biol.
104:1105-1115 (1987); Mian, N., Biochem. J. 237:333-342 (1986);
Tomida, M. et al., J. Cell Physiol. 86:121-130 (1975)), and
over-expression of receptors for hyaluronan, including a receptor
for hyaluronan mediated motility (RHAMM) and CD44, correlates with
increased levels of tumor metastasis (Gunthert, U., Curr. Topics
Microbiol. Immunol. 184:47-63 (1993); Hall, C. L. et al., Cell
82:19-28 (1995); Turley, E. A., Cancer and Metastasis Reviews
11:1233-1241 (1992)). Purified preparations of hyaluronan exhibit
unique viscoelastic properties, and as a consequence of these
characteristics have been used in viscoelastic surgery and
viscosupplementation (Balazs, E. A., and Denninger, J. L., Clinical
uses of hyaluronan, The biology of hyaluronan, Ciba foundation
symposium, Wiley, Chichester, England (1989)). Hyaluronan is
synthesized mainly by mesenchymal cells and the accumulation of HA
is an early event in tissue repair. The serum level of hyaluronan
is elevated in inflammatory settings such as rheumatoid arthritis,
osteoarthritis, liver cirrhosis, Werner's syndrome, renal failure
and psoriasis (Laurent, T. C. et al., Faseb J. 6:2397-2404 (1992);
Laurent, T. C. Annals of Medicine 28:in press (1996)).
[0003] Hyaluronan is synthesized by a membrane bound synthase;
monosaccharide and disaccharide residues are added to the reducing
end of the polysaccharide as it protrudes through the plasma
membrane (Prehm, P., Biochem. J. 211:181-189 (1983); Prehm, P.,
Biochem. J. 220:597-600 (1984)). Regulation of hyaluronan
biosynthesis has been studied in several tissue culture systems.
Factors involved in tissue growth and repair such as different
isoforms of platelet derived growth factor (PDGF-AA, PDGF-BB),
epidermal growth factor (EGF), basic fibroblast growth factor
(bFGF), and transforming growth factor .beta.(TGF-.beta.), all
exhibit stimulatory activity on hyaluronan biosynthesis (Heldin, P.
et al., Biochem. J. 258, 919-922 (1992)).
[0004] A cDNA encoding a bacterial hyaluronan synthase has been
cloned from Streptococcus pyogenes (hasA) (DeAngelis, J. P. et al.,
J. Biol. Chem. 268, 19181-19184 (1993)). Other related genes with
N-acetylglucosaminyl transferase activity have been isolated from
the nitrogen fixing bacteria Rhizobium (nodC) and chitin synthases
(Chs) from Saccharomyces (DeAngelis, P. L. et al., Biochem.
Biophys. Res. Comm. 199:1-10 (1994)). A putative vertebrate
homolog, (DG42), was cloned from Xenopus laevis and has also been
speculated to be a glycosaminoglycan synthetase (Rosa, F. et al.,
Develop. Biol. 129:114-123 (1988)). To date, however, a mammalian
hyaluronan synthase gene has not been identified.
SUMMARY OF THE INVENTION
[0005] The present invention relates to isolated and/or recombinant
nucleic acids which encode a mammalian hyaluronan synthase (e.g.,
human). In one embodiment, the nucleic acid of the present
invention comprises SEQ ID NO: 1. In another embodiment, the
invention relates to a nucleic acid wherein said nucleic acid
hybridizes under stringent conditions with a second nucleic acid
having a nucleotide sequence of SEQ ID NO: 1.
[0006] The present invention also relates to a host cell comprising
a nucleic acid encoding mammalian hyaluronan synthase. In a
particular embodiment, the host cell comprises nucleic acid
encoding mammalian hyaluronan synthase which is operably linked to
an expression control sequence, whereby mammalian hyaluronan
synthase is expressed when the host cell is maintained under
conditions suitable for expression.
[0007] The present invention also relates to a method for producing
a mammalian hyaluronan synthase comprising introducing into a host
cell a nucleic acid construct comprising a nucleic acid which
encodes a mammalian hyaluronan synthase, whereby a recombinant host
cell is produced having said coding sequence operably linked to an
(i.e., at least one) expression control sequence; and maintaining
the host cells produced in a suitable medium under conditions
whereby the nucleic acid is expressed.
[0008] The present invention also relates to an antibody or
functional portion thereof (e.g., an antigen binding portion such
as an Fv, Fab, Fab', or F(ab').sub.2 fragment) which binds
mammalian hyaluronan synthase.
[0009] The present invention also relates to a method of detecting
mammalian hyaluronan synthase in a sample comprising contacting a
sample with an antibody which binds hyaluronan synthase under
conditions suitable for specific binding of said antibody to the
mammalian hyaluronan synthase; and detecting antibody-mammalian
hyaluronan synthase.
[0010] The invention further relates to a method of using
hyaluronan synthase to make hyaluronan.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1A is a graph illustrating that CHO cells tranfected
with human hyaluronan synthase cDNA synthesize hyaluronic acid;
media and cell lysates were combined and then incubated overnight
in the absence (.smallcircle. - - - .smallcircle.) or presence
(.circle-solid. - - - .circle-solid.) of 10U Streptomyces
hyaluronidase/ml and subjected to chromatography on Sephadex G-50
columns; Streptomyces hyaluronidase-sensitive radioactivity
represents synthesized hyaluronan.
[0012] FIG. 1B is a graph illustrating that CHO cells not
transfected with human hyaluronan synthase cDNA produce very little
high molecular weight streptomyces hyaluronidase-sensitive
material.
[0013] FIG. 2 is an illustration of the nucleotide sequence (SEQ ID
NO: 1) and deduced protein sequence (SEQ ID NO: 2) determined from
human hyaluronan synthase cDNA clone 30C; cysteine residues are
circled and a conserved motif, B(X.sub.7)B, believed to be
important for binding hyaluronan is lightly outlined; consensus
phosphorylation sequences for protein kinase C (RHLT, KYT and RWLS)
and cAMP dependent protein kinases (RWS) are outlined in bold; also
shown with a bold underline at position 2066 is a consensus
polyadenylation signal, AATAAA. (Standard single letter amino acid
codes are used.)
[0014] FIG. 3A is an amino acid alignment of the human hyaluronan
synthase protein sequence (SEQ ID NO: 2) with the DG42 sequence
from Xenopus laevis (SEQ ID NO: 3) and hasA sequence of
Streptococcus pyogenes (SEQ ID NO: 4) prepared using the DNAStar
program and the Clustal method with default parameters for gap
penalties.
[0015] FIG. 3B is a comparison of Kyte-Doolittle hydrophilicity
profiles of human hyaluronan synthase, DG42 and hasA.
[0016] FIG. 3C is a proposed structure of human hyaluronan
synthase, indicating approximate boundaries of transmembrane
regions and intra- and extracellular loops; a hyaluronan binding
motif (HBM), B(X.sub.7)B, is indicated at the amino portion of a
large predicted intracellular loop; approximate locations of
protein kinase C consensus sites are indicated by open circles,
while a single cAMP dependent kinase site is shown as a filled
circle.
[0017] FIG. 4A is a Northern blot probed with the full length
insert of the human hyaluronan synthase cDNA clone 30C; the blot
was subsequently stripped and reprobed with a M-actin cDNA as a
control.
[0018] FIG. 4B is a Southern blot initially hybridized with
full-length human hyaluronan synthase cDNA, washed at 50.degree.
C., and exposed overnight; a considerable amount of background was
seen although specific bands could be detected; subsequently the
blot was stripped and probed with a 450 bp Sac II fragment
encompassing the 3' end of the cDNA; this probe gave a similar
pattern with less background (likely due to a lower GC
content).
DETAILED DESCRIPTION OF THE INVENTION
[0019] Proteins and Peptides
[0020] The present invention relates to isolated and/or recombinant
(including, e.g., essentially pure) proteins or polypeptides
designated mammalian hyaluronan synthase and variants of mammalian
hyaluronan synthase. In a preferred embodiment, the isolated and/or
recombinant proteins of the present invention have at least one
property, activity or function characteristic of a mammalian
hyaluronan synthase (as defined herein), such as activity in the
synthesis of hyaluronan and/or ability to confer of cell adhesion
by the lymphocyte receptor CD44 (i.e., human CD44 or a mammalian
homolog thereof).
[0021] Proteins or polypeptides referred to herein as "isolated"
are proteins or polypeptides purified to a state beyond that in
which they exist in mammalian cells. "Isolated" proteins or
polypeptides include proteins or polypeptides obtained by methods
described herein, similar methods or other suitable methods,
including essentially pure proteins or polypeptides, proteins or
polypeptides produced by chemical synthesis (e.g., synthetic
peptides), or by combinations of biological and chemical methods,
and recombinant proteins or polypeptides which are isolated. The
proteins can be obtained in an isolated state of at least about 50%
by weight, preferably at least about 75% by weight, and more
preferably, in essentially pure form. Proteins or polypeptides
referred to herein as "recombinant" are proteins or polypeptides
produced by the expression of recombinant nucleic acids.
[0022] As used herein "mammalian hyaluronan synthase" refers to
naturally occurring or endogenous mammalian hyaluronan synthase
proteins, to proteins having an amino acid sequence which is the
same as that of a naturally occurring or endogenous corresponding
mammalian hyaluronan synthase (e.g., recombinant proteins), and to
functional variants of each of the foregoing (e.g., functional
fragments and/or mutants produced via mutagenesis and/or
recombinant techniques). Accordingly, as defined herein, the term
includes mature mammalian hyaluronan synthase, glycosylated or
unglycosylated mammalian hyaluronan synthase proteins, polymorphic
or allelic variants, and other isoforms of mammalian hyaluronan
synthase (e.g., produced by alternative splicing or other cellular
processes), and functional fragments.
[0023] Naturally occurring or endogenous mammalian hyaluronan
synthase proteins include wild type proteins such as mature
mammalian hyaluronan synthase, polymorphic or allelic variants and
other isoforms which occur naturally in mammals (e.g., primate,
preferably human, murine, bovine). Such proteins can be recovered
from a source which naturally produces mammalian hyaluronan
synthase, for example. These mammalian proteins having the same
amino acid sequence as naturally occurring or endogenous
corresponding mammalian hyaluronan synthase, are referred to by the
name of the corresponding mammal. For example, as described herein,
where the corresponding mammal is human, the protein is designated
as a human hyaluronan synthase (HAS), such as recombinant human
hyaluronan synthase produced in a suitable host cell.
[0024] "Functional variants" of mammalian hyaluronan synthase
include functional fragments, functional mutant proteins, and/or
functional fusion proteins. Generally, fragments or portions of
mammalian hyaluronan synthase encompassed by the present invention
include those having a deletion (i.e., one or more deletions) of an
amino acid (i.e., one or more amino acids) relative to the mature
mammalian hyaluronan synthase (such as N-terminal, C-terminal or
internal deletions). Fragments or portions in which only contiguous
amino acids have been deleted or in which non-contiguous amino
acids have been deleted relative to mature mammalian hyaluronan
synthase are also envisioned.
[0025] Generally, mutants or derivatives of mammalian hyaluronan
synthase, encompassed by the present invention include natural or
artificial variants differing by the addition, deletion and/or
substitution of one or more contiguous or non-contiguous amino acid
residues, or modified polypeptides in which one or more residues is
modified, and mutants comprising one or more modified residues.
Preferred mutants are natural or artificial variants of mammalian
hyaluronan synthase differing by the addition, deletion and/or
substitution of one or more contiguous or non-contiguous amino acid
residues.
[0026] A "functional fragment or portion", "functional mutant"
and/or "functional fusion protein" of a mammalian hyaluronan
synthase refers to an isolated and/or recombinant protein or
oligopeptide which has at least one property, activity and/or
function characteristic of a mammalian hyaluronan synthase, such as
activity or function characteristic of a mammalian hyaluronan
synthase (as defined herein), such as activity in the synthesis of
hyaluronan and/or ability to confer cell adhesion by the lymphocyte
receptor CD44.
[0027] Suitable fragments or mutants can be identified by
screening. For example, the N-terminal, C-terminal, or internal
regions of the protein can be deleted in a step-wise fashion and
the resulting protein or polypeptide can be screened using a
suitable binding or adhesion assay. Where the resulting protein
displays activity in the assay, the resulting protein ("fragment")
is functional. Information regarding the structure and function of
other hyaluronan synthases (e.g., hasA, DG42), and of HAS as shown
herein, provides a basis for dividing HAS into functional
domains.
[0028] The term variant also encompasses fusion proteins,
comprising a mammalian hyaluronan synthase (e.g., mature mammalian
hyaluronan synthase) as a first moiety, linked to a second moiety
not occurring in the mammalian hyaluronan synthaseas found in
nature. Thus, the second moiety can be an amino acid, oligopeptide
or polypeptide. The first moiety can be in an N-terminal location,
C-terminal location or internal to the fusion protein. In one
embodiment, the fusion protein comprises a mammalian hyaluronan
synthase or portion thereof as the first moiety, and a second
moiety comprising a linker sequence and affinity ligand (e.g., an
enzyme, an antigen, epitope tag).
[0029] Examples of "mammalian hyaluronan synthase" proteins include
proteins having an amino acid sequence as set forth or
substantially as set forth in FIG. 2 (SEQ ID NO: 2) and functional
portions thereof. In a preferred embodiment, a mammalian hyaluronan
synthase or variant has an amino acid sequence which has at least
about 50% identity, more preferably at least about 75% identity,
and still more preferably at least about 90% identity, to the
protein shown in FIG. 2 (SEQ ID NO: 2).
[0030] Method of Producing Recombinant Proteins
[0031] Another aspect of the invention relates to a method of
producing a mammalian hyaluronan synthase or variant (e.g.,
portion) thereof. Recombinant protein can be obtained, for example,
by the expression of a recombinant DNA molecule encoding a
mammalian hyaluronan synthase or variant thereof in a suitable host
cell, for example.
[0032] Constructs suitable for the expression of a mammalian
hyaluronan synthase or variant thereof are also provided. The
constructs can be introduced into a suitable host cell, and cells
which express a recombinant mammalian hyaluronan synthase or
variant thereof, can be produced and maintained in culture. Such
cells are useful for a variety of purposes, and can be used in the
production of protein for characterization, isolation and/or
purification, (e.g., affinity purification), and as immunogens, for
instance. Suitable host cells can be procaryotic, including
bacterial cells such as E. coli, B. subtilis and or other suitable
bacteria (e.g., Streptococci) or eucaryotic, such as fungal or
yeast cells (e.g., Pichia pastoris, Aspergillus species,
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora
crassa) or other lower eucaryotic cells, and cells of higher
eucaryotes such as those from insects (e.g., Sf9 insect cells) or
mammals (e.g., Chinese hamster ovary cells (CHO), COS cells, HuT 78
cells, 293 cells). (See, e.g., Ausubel, F. M. et al., eds. Current
Protocols in Molecular Biology, Greene Publishing Associates and
John Wiley & Sons Inc., (1993)).
[0033] Host cells which produce a recombinant mammalian hyaluronan
synthase or variants thereof can be produced as follows. For
example, a nucleic acid encoding all or part of the coding sequence
for the desired protein can be inserted into a nucleic acid vector,
e.g., a DNA vector, such as a plasmid, virus or other suitable
replicon for expression. A variety of vectors are available,
including vectors which are maintained in single copy or multiple
copy, or which become integrated into the host cell chromosome.
[0034] The transcriptional and/or translational signals of a
mammalian hyaluronan synthase gene can be used to direct
expression. Alternatively, suitable expression vectors for the
expression of a nucleic acid encoding all or part of the coding
sequence of the desired protein are available. Suitable expression
vectors can contain a number of components, including, but not
limited to one or more of the following: an origin of replication;
a selectable marker gene; one or more expression control elements,
such as a transcriptional control element (e.g., a promoter, an
enhancer, terminator), and/or one or more translation signals; a
signal sequence or leader sequence for membrane targeting or
secretion (of mammalian origin or from a heterologous mammal or
non-mammalian species). In a construct, a signal sequence can be
provided by the vector, the mammalian hyaluronan synthase coding
sequence, or other source.
[0035] A promoter can be provided for expression in a suitable host
cell. Promoters can be constitutive or inducible. The promoter is
operably linked to a nucleic acid encoding the mammalian hyaluronan
synthase or variant thereof, and is capable of directing expression
of the encoded polypeptide in the host cell. A variety of suitable
promoters for procaryotic (e.g., lac, tac, T3, T7 promoters for E.
coli) and eucaryotic (e.g., yeast alcohol dehydrogenase (ADH1),
SV40, CMV) hosts are available.
[0036] In addition, the expression vectors typically comprise a
selectable marker for selection of host cells carrying the vector,
and in the case of a replicable expression vector, an origin of
replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and may be used in
procaryotic (e.g., .beta.-lactamase gene (ampicillin resistance),
Tet gene for tetracycline resistance) and eucaryotic cells (e.g.,
neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin,
or hygromycin resistance genes). Dihydrofolate reductase marker
genes permit selection with methotrexate in a variety of hosts.
Genes encoding the gene product of auxotrophic markers of the host
(e.g., LEU2, URA3, HIS3) are often used as selectable markers in
yeast. Use of viral (e.g., baculovirus) or phage vectors, and
vectors which are capable of integrating into the genome of the
host cell, such as retroviral vectors, are also contemplated. The
present invention also relates to cells carrying these expression
vectors.
[0037] For example, a nucleic acid encoding a mammalian hyaluronan
synthase or variant thereof can be incorporated into a vector,
operably linked to one or more expression control elements, and the
construct can be introduced into host cells which are maintained
under conditions suitable for expression, whereby the encoded
polypeptide is produced. The construct can be introduced into cells
by a method appropriate to the host cell selected (e.g.,
transformation, transfection, electroporation, infection). For
production of a protein, host cells comprising the construct are
maintained under conditions appropriate for expression, (e.g., in
the presence of inducer, suitable media supplemented with
appropriate salts, growth factors, antibiotic, nutritional
supplements, etc.). The encoded protein (e.g., human hyaluronan
synthase) can be isolated from the host cells or medium.
[0038] Fusion proteins can also be produced in this manner. For
example, some embodiments can be produced by the insertion of a
mammalian hyaluronan synthase cDNA or portion thereof into a
suitable expression vector, such as Bluescript.RTM.II
SK+/-(Stratagene), pGEX-4T-2 (Pharmacia), pcDNA-3 (Invitrogen) and
pET-15b (Novagen). The resulting construct can then be introduced
into a suitable host cell for expression. Upon expression, fusion
protein can be isolated or purified from a cell lysate by means of
a suitable affinity matrix (see e.g., Current Protocols in
Molecular Biology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26,
pp. 16.4.1-16.7.8 (1991)). In addition, affinity labels provide a
means of detecting a fusion protein. For example, the cell surface
expression or presence in a particular cell fraction of a fusion
protein comprising an antigen or epitope affinity label can be
detected by means of an appropriate antibody.
[0039] Nucleic Acids, Constructs and Vectors
[0040] The present invention relates to isolated and/or recombinant
(including, e.g., essentially pure) nucleic acids (e.g.,
polynucleotides) having sequences which encode a mammalian
hyaluronan synthase or variant thereof as described herein.
[0041] Nucleic acids referred to herein as "isolated" are nucleic
acids separated away from the nucleic acids of the genomic DNA or
cellular RNA of their source of origin (e.g., as it exists in cells
or in a mixture of nucleic acids such as a library), and may have
undergone further processing. "Isolated" nucleic acids include
nucleic acids obtained by methods described herein, similar methods
or other suitable methods, including essentially pure nucleic
acids, nucleic acids produced by chemical synthesis, by
combinations of biological and chemical methods, and recombinant
nucleic acids which are isolated (see e.g., Daugherty, B. L. et
al., Nucleic Acids Res., 19(9):2471-2476 (1991); Lewis, A. P. and
J. S. Crowe, Gene, 101: 297-302 (1991)). Nucleic acids referred to
herein as "recombinant" are nucleic acids which have been produced
by recombinant DNA methodology, including those nucleic acids that
are generated by procedures which rely upon a method of artificial
recombination, such as the polymerase chain reaction (PCR) and/or
cloning into a vector using restriction enzymes. "Recombinant"
nucleic acids are also those that result from recombination events
that occur through the natural mechanisms of cells, but are
selected for after the introduction to the cells of nucleic acids
designed to allow and make probable a desired recombination
event.
[0042] In one embodiment, the nucleic acid or portion thereof
encodes a protein or polypeptide having at least one property,
activity or function characteristic of a mammalian hyaluronan
synthase (as defined herein), such as activity or function
characteristic of a mammalian hyaluronan synthase (as defined
herein), such as activity in the synthesis of hyaluronan and/or
ability to mediate cell adhesion by the lymphocyte receptor
CD44.
[0043] The present invention also relates more specifically to
isolated and/or recombinant nucleic acids or a portion thereof
having sequences which encode mammalian hyaluronan synthase or
variants thereof.
[0044] The invention relates to isolated and/or recombinant nucleic
acids that are characterized by:
[0045] (1) their ability to hybridize to (a) a nucleic acid
encoding a mammalian hyaluronan synthase, such as a nucleic acid
having a nucleotide sequence as set forth or substantially as set
forth in FIG. 2 (SEQ ID NO: 1); (b) the complement of (a); or (c)
portions of either of the foregoing (e.g., a portion comprising the
open reading frame); or
[0046] (2) by their ability to encode a polypeptide having the
amino acid sequence of a mammalian hyaluronan synthase (e.g., SEQ
ID NO: 2); or
[0047] (3) by both characteristics.
[0048] In one embodiment, the nucleic acid shares at least about
50% nucleotide sequence similarity to the nucleotide sequences
shown in FIG. 2 (SEQ ID NO: 1). More preferably, the nucleic acid
shares at least about 75% nucleotide sequence similarity, and still
more preferably, at least about 90% nucleotide sequence similarity,
to the sequence shown in FIG. 2 (SEQ ID NO: 1).
[0049] Isolated and/or recombinant nucleic acids meeting these
criteria comprise nucleic acids having sequences identical to
sequences of naturally occurring mammalian hyaluronan synthase or
variants of the naturally occurring sequences. Such variants
include mutants differing by the addition, deletion or substitution
of one or more residues, modified nucleic acids in which one or
more residues are modified (e.g., DNA or RNA analogs), and mutants
comprising one or more modified residues.
[0050] A nucleic acid of the present invention may be in the form
of RNA or in the form of DNA (e.g., cDNA, genomic DNA, and
synthetic DNA). The DNA may be double-stranded or single-stranded,
and if single stranded may be the coding strand or non-coding
(anti-sense) strand. The coding sequence which encodes the mature
polypeptide may be identical to the coding sequence shown in FIG. 2
(SEQ ID NO: 1) or that of the cDNA in clone 30C or may be a
different coding sequence which coding sequence, as a result of the
redundancy or degeneracy of the genetic code, encodes the same,
mature polypeptides as the DNA of FIG. 2 (SEQ ID NO: 2) or the cDNA
in clone 30C.
[0051] The polynucleotide which encodes a mature polypeptide
encoded by the cDNA of clone 30C may include: only the coding
sequence of a mature polypeptide; the coding sequence for a mature
polypeptide and additional coding sequence such as a leader or
secretory sequence; the coding sequence for a mature polypeptide
(and optionally additional coding sequence) and non-coding
sequence, such as introns or non-coding sequence 5' and/or 3' of
the coding sequence.
[0052] Nucleic acids of the present invention, including those
which hybridize to a selected nucleic acid as described above, can
be detected or isolated under high stringency conditions or
moderate stringency conditions, for example. "High stringency
conditions" and "moderate stringency conditions" for nucleic acid
hybridizations are explained at pages 2.10.1-2.10.16 (see
particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in
Molecular Biology (Ausubel, F. M. et al., eds., Vol. 1, Suppl. 26,
1991), the teachings of which are hereby incorporated by reference.
Factors such as probe length, base composition, percent mismatch
between the hybridizing sequences, temperature and ionic strength
influence the stability of nucleic acid hybrids. Thus, high or
moderate stringency conditions can be determined empirically, and
depend in part upon the characteristics of the known nucleic acid
(e.g., DNA) and the other nucleic acids to be assessed for
hybridization thereto.
[0053] Isolated and/or recombinant nucleic acids that are
characterized by their ability to hybridize (e.g., under high or
moderate stringency conditions) to (a) a nucleic acid encoding a
mammalian hyaluronan synthase (for example, the nucleic acid
depicted in FIG. 2 (SEQ ID NO: 1); (b) the complement of the
nucleic acids of (a), (c) or a portion thereof, can also encode a
protein or polypeptide having at least one property, activity or
function characteristic of a mammalian hyaluronan synthase (as
defined herein), such as activity in the synthesis of hyaluronan
and/or ability to mediate cell adhesion by the lymphocyte receptor
CD44, and in a preferred embodiment encode polypeptides which
retain substantially the same biological function or activity as
the mature polypeptide encoded by the cDNA of FIG. 2 (SEQ ID NO: 1)
or the cDNA of clone 30C.
[0054] Nucleic acids of the present invention can be used in the
production of proteins or polypeptides. For example, a nucleic acid
(e.g., DNA) encoding a mammalian hyaluronan synthase can be
incorporated into various constructs and vectors created for
further manipulation of sequences or for production of the encoded
polypeptide in suitable host cells as described above.
[0055] A further embodiment of the invention is antisense nucleic
acid, which is complementary, in whole or in part, to a target
molecule comprising a sense strand, and can hybridize with the
target molecule. The target can be DNA, or its RNA counterpart
(i.e., wherein T residues of the DNA are U residues in the RNA
counterpart). When introduced into a cell, antisense nucleic acid
can inhibit the expression of the gene encoded by the sense strand.
Antisense nucleic acids can be produced by standard techniques.
[0056] In a particular embodiment, the antisense nucleic acid is
wholly or partially complementary to and can hybridize with a
target nucleic acid, wherein the target nucleic acid can hybridize
to a nucleic acid having the sequence of the complement of the
strand shown in FIG. 2 (SEQ ID NO: 1). For example, antisense
nucleic acid can be complementary to a target nucleic acid having
the sequence shown as the open reading frame in FIG. 2 (SEQ ID NO:
1) or to a portion thereof sufficient to allow hybridization. In
another embodiment, the antisense nucleic acid is wholly or
partially complementary to and can hybridize with a target nucleic
acid which encodes a mammalian hyaluronan synthase.
[0057] The nucleic acids can also be used as probes (e.g., in in
situ hybridization) to assess associations between inflammatory
settings (e.g., rheumatoid arthritis, osteoarthritis, liver
cirrhosis, Werner's syndrome, renal failure and psoriasis) and
increased expression of mammalian hyaluronan synthase in affected
tissues or serum. The nucleic acids can also be used as probes to
detect and/or isolate (e.g., by hybridization with RNA or DNA)
polymorphic or allelic variants, for example, in a sample (e.g.,
inflamed tissue) obtained from a host (e.g. mammalian). Moreover,
the presence or frequency of a particular variant in a sample(s)
obtained from one or more affected hosts, as compared with a
sample(s) from normal host(s), can be indicative of an association
between an inflammatory setting and a particular variant, which in
turn can be used in the diagnosis of the condition.
[0058] As described in the exemplification, functional expression
cloning was used to identify a cDNA encoding human hyaluronan
synthase, and it was demonstrated that this gene can confer
activity both in the synthesis of hyaluronan and as a mediator of
cell adhesion by the lymphocyte receptor CD44. A human hyaluronan
synthase (HAS) cDNA was isolated by a functional expression cloning
approach. Transfection of CHO cells conferred hyaluronidase
sensitive adhesiveness of a mucosal T cell line via the lymphocyte
hyaluronan receptor, CD44, as well as increased hyaluronan levels
in the cultures of transfected cells. The HAS amino acid sequence
shows homology to the hasA gene product of Streptococcus pyogenes
and a putative glycosaminoglycan synthetase from xenopus laevis.
Expression of HAS message parallels tissues where high levels of
hyaluronan synthesis occur, indicating that transcription of
synthase mRNA is a critical component of hyaluronate synthesis.
[0059] Utilities
[0060] Mammalian hyaluronan synthases of the present invention can
be used to produce hyaluronan. Hyaluronan has a variety of uses,
including use in cosmetics and pharmaceuticals (see e.g.,
EPO,443,043 B1 and U.S. Pat. No. 5,015,577 the teachings of which
are each incorporated herein by reference). Hyaluronan or
pharmaceutical compositions comprising hyaluronan are useful for
treating wounds or surgical incisions and can reduce or prevent
hypertrophic scars and keloid formation, and in eye surgery as a
replacement for vitreous fluid, for example.
[0061] For example, a mammalian hyaluronan synthase can be
expressed in a suitable host cell under conditions appropriate for
production of hyaluronan to occur (e.g., in suitable medium
comprising any required precursors). Isolated or purified
hyaluronan synthase can also be used to prepare hyaluronan from
precursors (e.g., UDP-glucuronic acid and
UDP-N-aceytl-glucosamine).
[0062] The present invention also provides antibodies which (1) can
bind a "mammalian hyaluronan synthase" in vitro and/or in vivo;
and/or (2) can inhibit an activity or function characteristic of a
"mammalian hyaluronan synthase", such as hyaluronan synthesis.
Preferably the antibodies are capable of selective binding of
mammalian hyaluronan synthase in vitro and/or in vivo (e.g., bind
selectively to mammalian hyaluronan synthase expressed in ovary
and/or spleen, thymus, prostate, etc. (e.g., as assessed
immunohistologically)).
[0063] Preferably, the antibodies can bind a mammalian (e.g. human)
hyaluronan synthase with high affinity (for example, a Ka in the
range of about 1-10 nM, or a Kd in the range of about 133 10.sup.-8
to 1.times.10.sup.-10 mol.sup.-1).
[0064] The antibodies of the present invention are useful in a
variety of applications, including processes, research, diagnostic
and therapeutic applications. For instance, they can be used to
isolate and/or purify mammalian hyaluronan synthase or variants
thereof (e.g., by affinity purification or other suitable methods),
and to study mammalian hyaluronan synthase structure (e.g.,
conformation) and function.
[0065] The antibodies of the present invention can also be used to
modulate mammalian hyaluronan synthase function in diagnostic
(e.g., in vitro) or therapeutic applications. For instance,
antibodies can act as inhibitors of (reduce or prevent) hyaluronan
synthesis, thereby inhibiting process mediated by hyaluronan such
as cell adhesion and metastasis.
[0066] In addition, antibodies of the present invention can be used
to detect and/or measure the level of a mammalian hyaluronan
synthase in a sample (e.g., tissues or body fluids, such as an
inflammatory exudate, blood, serum, bowel fluid, or on cells
transfected with a nucleic acid of the present invention). For
example, a sample (e.g., tissue and/or fluid) can be obtained from
a host (e.g., mammalian) and a suitable immunological method can be
used to detect and/or measure mammalian hyaluronan synthase levels,
including methods such as enzyme-linked immunosorbent assays
(ELISA), including chemiluminescence assays, radioimmunoassay, and
immunohistology. In one embodiment, a method of detecting a
selected mammalian hyaluronan synthase in a sample is provided,
comprising contacting a sample with an antibody which binds an
isolated mammalian hyaluronan synthase under conditions suitable
for specific binding of said antibody to the selected mammalian
hyaluronan synthase, and detecting antibody-mammalian hyaluronan
synthase complexes which are formed.
[0067] In an application of the method, antibodies reactive with a
mammalian hyaluronan synthase can be used to analyze normal versus
inflamed tissues in mammals for mammalian hyaluronan synthase
reactivity and/or expression (e.g., immunohistologically). Thus,
the antibodies of the present invention permit immunological
methods of assessment of expression of primate (e.g., human
mammalian hyaluronan synthase) in normal versus inflamed tissues,
through which the presence of disease, disease progress and/or the
efficacy of anti-mammalian hyaluronan synthase therapy in
inflammatory disease can be assessed.
[0068] An antibody can be administered in an effective amount which
inhibits mammalian hyaluronan synthase activity. For therapy, an
effective amount will be sufficient to achieve the desired
therapeutic and/or prophylactic effect (such as an amount
sufficient to reduce or prevent mammalian hyaluronan
synthase-mediated hyaluronan synthesis). The antibody can be
administered in a single dose or multiple doses. The dosage can be
determined by methods known in the art and is dependent, for
example, upon the individual's age, sensitivity, tolerance and
overall well-being. Suitable dosages for antibodies can be from
0.1-1.0 mg/kg body weight per treatment.
[0069] According to the method, an antibody can be administered to
an individual (e.g., a human) alone or in conjunction with another
agent (administered before, along with or subsequent to
administration of the additional agent).
[0070] A variety of routes of administration are possible
including, but not necessarily limited to parenteral (e.g.,
intravenous, intraarterial, intramuscular, subcutaneous injection),
oral (e.g., dietary), topical, inhalation (e.g., intrabronchial,
intranasal or oral inhalation, intranasal drops), or rectal,
depending on the disease or condition to be treated. Parenteral
administration is a preferred mode of administration.
[0071] Formulation will vary according to the route of
administration selected (e.g., solution, emulsion, capsule). An
appropriate composition comprising the antibody to be administered
can be prepared in a physiologically acceptable vehicle or carrier.
For solutions or emulsions, suitable carriers include, for example,
aqueous or alcoholic/aqueous solutions, emulsions or suspensions,
including saline and buffered media. Parenteral vehicles can
include sodium chloride solution, Ringer's dextrose, dextrose and
sodium chloride, lactated Ringer's or fixed oils. Intravenous
vehicles can include various additives, preservatives, or fluid,
nutrient or electrolyte replenishers (See, generally, Remington's
Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). For
inhalation, the compound can be solubilized and loaded into a
suitable dispenser for administration (e.g., an atomizer, nebulizer
or pressurized aerosol dispenser).
Exemplification
[0072] Plasmids, Monoclonal Antibodies and Cell Lines
[0073] The following plasmids were used as controls in expression
cloning and for functional adhesion assays: pSV-SPORT-1 (GIBCO,
Gaithersburg, Md.) or pcDNA3 (Invitrogen, San Diego, Calif.)
controls and murine MAdCAM-1 in pCDM8 (pCDMAD-7 (Briskin, M. J.,
Nature 363:461-464 (1993)). Monoclonal antibodies used were
anti-murine CD-44 TJB1.7 (a gift from T. Yoshino and E. Butcher,
Stanford, Calif.); anti-murine MAdCAM-1 MECA-367 (Streeter, P. R.
et al., Nature 331:41-46 (1988)); anti-human VCAM-1 2G7 (Graber, N.
J. Immunol. (145):819 (1990)); anti-murine .beta.7 FIB 504 (Andrew,
D. P. et al., J. Immunol. 153:3847-3861 (1994)); and anti-murine
.alpha.4 PS/2 (Miyake, K. J. Exp. Med. 173:599-607 (1991)). Cell
lines used for expression cloning and functional adhesion assays
were: CHO/P (Heffernan, M. and Dennis, J. D. Nucl. Acids Res. 19:85
(1991)) and the murine T cell lymphoma TK1 (Butcher, E. C. et al.,
Eur. J. Immunol. 10:556-561 (1980)).
[0074] cDNA Synthesis and Library Construction
[0075] mRNA was isolated from human lymph nodes using standard
procedures previously described (Briskin, M. J., Nature 363:461-464
(1993)). cDNA was synthesized using the Superscript.TM. lambda
system in conjunction with the pSV-SPORT-1 vector (Gibco,
Gaithersburg, Md.) essentially using the manufacturer's protocol.
The highest molecular weight fractions (>1.5 kb) of cDNA were
ligated into the pSV-SPORT-1 vector and plated in pools at a
density of 5,000 clones/plate on 100 LB agar plates with ampicillin
(50 .mu.g/ml). After incubation overnight, plasmid DNAs were
purified from each plate individually by use of QIAprep spin
columns (QIAGEN, Chatsworth, Calif.) according to manufacturer's
instructions.
[0076] Expression Cloning
[0077] CHO/P cells were seeded into 24 well plates approximately 24
hours prior to transfection at a density of 40,000 cells/well. DNAs
were transiently transfected using the LipofectAMINE.TM. reagent
(GIBCO, Gaithersburg, Md.) as recently described (Shyjan, A. M. et
al., J. Immunol., 156:2851-2857 (1996)).
[0078] For the adhesion assays in the expression cloning screen,
TK1 cells are resuspended at a density of 2.times.10.sup.6/ml in a
cell binding assay buffer previously described (Shyjan, A. M. et
al., J. Immunol. in press (1996)). After incubation at 4.degree. C.
for 15 minutes, 0.25 ml of the TK1 cell suspension
(5.times.10.sup.5 TK1 cells) was added to each well and incubation
on a rocking platform was continued for an additional 30 minutes at
4.degree. C. Plates were washed by gently inverting in a large
beaker of phosphate buffered saline (PBS) followed by inversion in
a beaker of PBS with 1.5% glutaraldehyde for fixation for a minimum
of 1 hour. Wells were then examined microscopically (10.times.
objective) for rosetting of TK1 cells mediated by the pools of cDNA
clones. Pools yielding one or more TK1 rosettes were further
subfractionated three times until individual colonies could be
assayed and the clones conferring adhesion of the TK1 cells were
identified.
[0079] Functional Adhesion Assays
[0080] Assays with purified clones were similar to those performed
in expression cloning with the following exception: as several
wells were to be transfected for antibody inhibition studies, a
master liposome mix with multiples of the wells to be transfected
was first made for each plasmid. On the day of the assay monoclonal
antibodies were incubated with cells at 20 .mu.g/ml or supernatants
(undiluted) at 4.degree. C. for 15 minutes prior to the start of
the assay.
[0081] For adhesion assays with hyaluronan, human umbilical cord
hyaluronan (Calbiochem, San Diego, Calif.) was diluted to 5 mg/ml
in PBS. Streptomyces hyaluronidase (Calbiochem, San Diego, Calif.)
was diluted to 20 TRU/ml in HBSS. TK1 cells were resuspended in
HBSS containing 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 2% serum and 20
mM HEPES at 10.sup.6 cells/ml. Wells of 24-well plates were coated
with 200 .mu.l of hyaluronan and stored at 4.degree. C. overnight.
Wells were rinsed with 0.5 ml PBS three times, and were treated
with 0.25 ml Streptomyces hyaluronidase at final concentrations of
0, 5, 10 and 20 TRU/ml for 1 hour at 37.degree. C. Wells were
rinsed three times with 0.5 ml PBS, blocked with 0.5 ml serum for 1
hour on ice and then rinsed three times with 0.5 ml PBS. TK1 cells
(0.5 ml) were added to each well and plates were incubated with
shaking at 4.degree. C. for 20 minutes.
[0082] For assessment of hyaluronate mediated binding to CHO/P
cells, the transfectants were rinsed with 0.5 ml PBS three times.
Individual wells were treated with 250 .mu.l Streptomyces
hyaluronidase at 0, 5, 10 and 20 TRU/ml (final concentrations) for
1 hour at 37.degree. C. Transfectants were rinsed three times with
0.5 ml PBS. TK1 cells (0.5 ml in the same buffer as described
above) were added to each well and plates were incubated with
shaking at 4.degree. C. for 30 minutes. Wells were rinsed with 0.5
ml PBS three times and viewed under the light microscope. Assays
were fixed as described above and analyzed by examination of
multiple fields and counting both lymphocytes and CHO cells at
10.times. magnification.
[0083] Measurement of Hyaluronic Acid Biosynthesis in CHO Cell
Transfectants
[0084] 0.5.times.10.sup.6 CHO cells seeded in 100 mm plates were
transfected with Lipofectamine reagent according to manufacaturer's
instructions. Tranfections utilized 20 .mu.g of HAS cDNA in pcDNA3
(Invitrogen, San Diego, Calif.) and 160 .mu.l of lipofectamine
reagent. Clone 30C was digested with EcoRI and NotI and the insert
released thereby was cloned into the ECORI and NotI sites of
pcDNA3. Transformants of E. coli XL-1 Blue (Stratagene) or DH10B
(Gibco) containing the resulting construct were obtained.
Approximately 72 hours after transfection, 440 .mu.g/ml of G418 was
added in fresh media. After the transfected and control (non
transfected) cells had reached subconfluency, the media was
replaced with fresh complete media containing 5 mCi/ml
D-[6-.sup.3H] glucosamine hydrochloride (New England Nuclear,
Boston, Mass., specific activity 33.3 ci/ml, concentration 1
mCi/ml), a precursor of sulfated glucosaminoglycans such as
hyaluronan. The amounts of synthesized hyaluronan in transfected
and control CHO cells were determined after 48 hours of incubation
at 37.degree. C. as follows. Media was collected and the cell
layers were combined with the corresponding media. Aliquots from
each sample were incubated overnight at 37.degree. C. in the
presence or absence of Streptomyces hyaluronidase. Then the samples
were applied on sephadex G-50 superfine columns (100.times.100 mm)
which were equilibrated with 0.05 M sodium acetate, pH 6.0
containing 0.2M NaCl. Newly synthesized [.sup.3H] hyaluronan was
determined as the Streptomyces sensitive radioactivity.
[0085] DNA Sequencing
[0086] Plasmids were sequenced on both strands using
oligonucleotide primers and the sequenase.TM. 7-deaza-dGTP DNA
sequencing kit with sequenase version 2.0 T7 DNA polymerase (United
States Biochemical, Cleveland, Ohio) and .sup.35SdCTP (Amersham
Life Science, Arlington Heights, Ill. and New England Nuclear,
Boston, Mass.) using manufacturer's instructions.
[0087] Northern and Southern Blot Analysis
[0088] Northern blots used were human multiple tissue northerns I
and II (Clontech, Palo Alto, Calif.). Hybridization was performed
with ExpressHyb (Clontech) solution, using manufacturer's
instructions except that a final wash at high stringency
(0.1.times.SSC, 0.1% SDS, 65.degree. C.) for 30 min was added. A
commercially prepared southern blot (Human GENO-BLOT) (Clontech,
Palo Alto, Calif.) was hybridized as described for the Northern
blot with the exception that an initial wash at 50.degree. C. was
exposed and then the blot was subsequently washed at 65.degree. C.
and exposed again. cDNA's were labelled with .alpha..sup.32P-dCTP
by priming with random hexamers. After washing, filters were
exposed to Kodak XAR film with an intensifying screen.
[0089] Results and Discussion
[0090] An expression cloning system was developed to isolate cDNA
clones that encode proteins that confer adhesion of the murine T
cell lymphoma TK1 (Butcher, E. C. et al., Eur. J. Immunol.
10:556-561 (1980)). A human mesenteric lymph node expression
library was constructed that, upon transfection into CHO/P cells,
yielded a cDNA clone, called 30C, that mediated rosetting of TK1
cells to some of the CHO/P transfectants. Transformants of E. coli
XL-1 Blue (Stratagene) containing Clone 30C were obtained. In order
to understand the nature of the observed interaction, the adhesion
assay after pre-incubation of the TK1 line with several antibodies
to adhesion receptors known to be expressed on TK1 cells was
repeated. Binding could be completely inhibited by pre-incubation
of TK1 cells with an antibody to CD44 (Table 1), while other
antibodies (anti-.alpha.4 and anti-.beta.7 integrins (Andrew, D. P.
et al., J. Immunol. 153:3847-3861 (1994); Miyake, K. J. Exp. Med.
173:599-607 (1991)) had no effect.
1TABLE 1 Adhesion of TK1 cells to clone 30C transfectants. Tk1
Binding TK1 after Binding TK1 Binding anti-CD44 after after MAb
anti-a4 Cells/Matrix Tk1 Cell hyaluronidase TJB1.7 MAb PS/2 HAS +++
-- -- +++ Transfectants Mock -- -- -- -- Transfectants Hyaluronate
+++ -- -- +++ TK1 cells bind to CHO/P cells transiently transfected
with clone 30C. Binding is blocked by pretreatment of the
transfectants with hyaluronidase or pretreatment of TK1 cells with
anti-CD44 MAb TJB1.7. Similar results are seen with binding to
immobilized hyaluronate, while TK1 cells do not bind mock
transfectants. A score of "--" indicates that no TK1 cells (above
controls) were observed in those wells while "+++" indicates TK1
rosetting on transfectants # (>5 TK1 cells/CHO/P transfectant)
or a monolayer of cells binding to immobilized hyaluronate. Assays
were all repeated three times with similar results.
[0091] As CD44 is known to be a hyaluronan receptor (Aruffo, A., et
al., Cell 61:1303-1313 (1990); Culty, M. et al., J. Cell. Biol.,
111:2765-2774 (1990); Miyake, K. et al., J. Exp. Med. 172:69-75
(1990)), it was investigated whether the isolated cDNA encoded a
novel CD44 ligand or, alternatively, was involved in de novo
synthesis of hyaluronan. Hyalurondase pretreatment completely
abrogated TK1 binding to the transfectants as well as to hyaluronan
controls (Table 1), indicating that the cloned cDNA mediated
synthesis of HA. Finally, CHO cells were stably transfected with
the 30C cDNA and assessed for their ability to mediate hyaluronan
biosynthesis (FIG. 1A,B). Whereas, untranfected cells produced very
little high molecular weight Streptomyces hyaluronaidase-sensitive
material (FIG. 1B), cell cultures transfected with 30C cDNA
produced a substantial amount of hyaluronan (FIG. 1A).
[0092] The cDNA encoding clone 30C is 2116 nucleotides in length
(FIG. 2) with a short 5' untranslated region of 35 bp and a longer
3' untranslated region of 347 bp. From the first ATG, a predicted
open reading frame of 1734 bp yielding a protein of 578 amino acid
residues is present. Genbank searches of the nucleotide and protein
sequences revealed significant homology with the hasA gene of
Streptococcus pyogenes (DeAngelis, J. P. a. P. H. W., J. Biol.
Chem. 268:19181-19184 (1993)), which was reported to be a
hyaluronan synthase (FIG. 3A,B) and a sequence from Xenopus laevis
called DG42 (FIG. 3A,B) which has also been speculated to be a
glycosaminoglycan synthetase (Rosa, F. et al., Develop. Biol.
129:114-123 (1988)). Amino acid sequence identities between the
predicted protein and these sequences were 22% and 54%,
respectively. Significant similarity was also observed with other
membrane associated proteins with N-acetylyglucosylamino
transferase activity including NodC from Rhizobium and three chitin
synthases from Saccharomyces (Chs) (DeAngelis, P. L. et al.,
Biochem. and Biophys. Res. Comm. 199:1-10 (1994)). The similarities
observed, coupled with the functional adhesion indicate that clone
30C encodes a human homolog of hyaluronan synthase (HS). Using
nomenclature based on the streptococcus gene locus, this human gene
encoding hyaluronan synthase is designated HAS.
[0093] The predicted molecular mass of the HAS protein is 64,793
daltons. Hydrophilicity (Kyte-Doolitle) analysis predicts a
membrane protein with several hydrophobic regions that would be
predicted to span the cell membrane at least four times (FIG.
3A-C). This prediction is in agreement with labeling studies which
suggested that hyaluronan synthase is associated with the plasma
membrane (Prehm, P., Biochem. J. 220:597-600 (1984); Phillipson, L.
H. and Schwartz, N. B. J. Biol. Chem. 259:5017-5023 (1984); Klewes,
L. et al., Biochem J. 290:791-795 (1993); O'Regan, M. et al., Int.
J. Biol. Macromol. 16:283-286 (1994)). Conservation of secondary
structure between hasA, DG42 and HAS, is indicated by similar
hydrophilicity plots. The approximate locations of these regions,
with respect to HAS, are shown in the alignment in FIG. 3A and
their representative hydrophilicity plots are shown in FIG. 3B.
[0094] The estimated number of transmembrane segments would suggest
a structure with a small N-terminal extracellular domain followed
by a long intracellular loop and then three more transmembrane
regions to yield one more small extracellular loop, a small
intracellular loop followed by a C-terminal extracellular extension
(FIG. 3C). Such a model, with the predominant portion of the
protein located intracellularly would be consistent with studies
indicating that hyaluronan biosynthesis occurs at the inner surface
of the plasma membrane (Prehm, P. Biochem. J. 220:597-600 (1984);
Phillipson, L. H., and Schwartz, N. B. J. Biol. Chem. 259:5017-5023
(1984)). This predicted large intercellular loop, is more highly
conserved than the overall protein at 70% (vs 54%) when compared
with DG42, which would imply conservation of a functional domain.
Within the amino terminal portion of this domain lies a motif,
designated B(X.sub.7)B (FIG. 2, 3C), where B is a basic amino acid
(e.g., R, K) and X is any non-acidic residue. This motif has been
found in both RHAMM, link protein and CD44, and mutagenesis studies
has shown that this sequence is required for binding hyaluronan
(Yang, B., et al., EMBO 13:286-296 (1994)). The presence of this
putative hyaluronan binding motif (HBM) in HAS raises the
possibility of a requirement of binding hyaluronan during its
synthesis and prior to transport out of the cell.
[0095] Northern blots probed with the entire human cDNA, revealed a
major transcript of 2.4 kb that was most highly expressed in ovary
and also expressed at significant levels in spleen, thymus,
prostate, testes and large intestine (FIG. 4A). In addition, a less
abundant transcript of approximately 7 kb was also observed in
these tissues and in addition to a faint 9 kb species only
expressed in ovary. Extremely weak expression was observed in small
intestine while peripheral blood leukocytes (PBL) were negative
under the conditions used. Moderate expression was also observed in
heart. The larger transcript observed might be a related gene in
these tissues although a southern blot probed first with both full
length and then a 3' region of HAS cDNA and washed at several
temperatures shows a simple banding pattern suggestive of a single
copy gene (FIG. 4B). It is therefore likely that these larger
species represent unprocessed nuclear precursors, as opposed to
related genes. The expression pattern observed is consistent with
high levels of hyaluronan that are observed in lymphoid tissues,
preovulatory follicles and in perivascular connective tissue and
vessel walls of both atrium and ventricle (Edelstrom, G. A. B. et
al., Histochem. Cytochem., 39:1131-1135 (1991); Laurent, C. et al.,
Cell Tissue Res., 263: 201-205 (1991)) and would indicate that
synthesis of hyaluronan is at least partially regulated by
transcriptional mechanisms. Interestingly, however, expression of
HAS RNA was barely detectable in skeletal muscle under the
conditions used, although histochemical analysis has shown
ubiquitous distribution of hyaluronan in connective tissue and the
septum dividing muscle fibers (Edelstrom, G. A. B. et al.,
Histochem. Cytochem 39:1131-1135 (1991); Laurent, C. et al., Cell
Tissue Res. 263: 201-205 (1991)). This may indicate that turnover
rates of hyaluronan may display great variation in different
tissues.
[0096] Induction of synthase activity by growth factors has been
shown to require protein synthesis and is mediated by a signaling
pathway involving tyrosine phosphorylation and/or activation of
protein kinase C (Heldin, P. et al., Biochem. J. 258, 919-922
(1992); Suzuki, M. et al., Biochem. J. 307:817-821 (1995)) as both
PMA and inhibitors of phosphotyrosine phosphatases can induce
hyaluronan synthesis. Serum alone can also induce synthase activity
and this induction was blocked by protein kinase C inhibitors and
cycloheximide. cAMP has also been implicated in activation and
phosphorlyation of the synthase itself may play a key role in
regulation of its activity (Klewes, L. and Prehm, P., J. of Cell.
Physiol. 160:539-544 (1994)). Examination of hydrophilic regions of
HAS reveals several conserved motifs which are potential substrates
for protein kinase C and cAMP dependent kinases (FIGS. 2, 3C.) and
are likely targets for future mutagenesis studies (Pearson, R. B.
Studies of protein kinase/phosphatase specificity using synthetic
peptides. Protein phosphorylation: A practical approach (Hardie, D.
G., Ed.), Oxford University Press, Oxford (1993)). As observed,
increased expression of the HAS gene in tissues that are known to
produce large quantities of hyaluronan, it is likely that the
regulation of hyaluronan synthesis is mediated by regulation of HAS
gene transcription, in addition to complex regulatory circuits
which involve both alterations in phosphorylation of the synthase
or proteins associated with HAS.
[0097] Previously, a 52 kDa protein was isolated from a
mouse/hamster hybridoma (B6 cells) that was initially reported to
be a mammalian hyaluronan synthase (Klewes, L. et al., Biochem J.
290:791-795 (1993)). This protein was incapable of binding
UDP-Glucuronic acid (UDP-[14C] GlcA) and UDP-N-acetyl glucosamine
(UDP-[3H] GlcNAc) unless complexed to a 60 kDa protein, which may
be the hyaluronan receptor (RHAMM) recently implicated in
fibroblast migration and tumor metastasis (Turley, E. A. et al., J.
Cell Biol., 112:1041-1047 (1991)). This protein cross-reacted with
antibodies against a putative synthase from Streptococcus
equisimilis. The gene encoding this protein was cloned from a
streptoccal library and shown to be related to proteins involved in
oligopeptide processing and transport and showed no homology to the
hasA gene sequence (O'Regan, M. et al., Int. J. Biol. Macromol.
16:283-286 (1994); Lansing, M. et al., Biochem. J. 289:179-184
(1993)). It is likely that the 52 kd protein isolated from the B6
line is a homolog to the streptococcal transport protein and not
the synthase itself. The human hyaluronan synthase cDNA is
therefore the first example of a mammalian gene responsible for
synthesis of hyaluronan.
[0098] Studies in streptococci show that the machinery responsible
for synthesis of hyaluronan is encoded in the has operon which
consists of three genes hasA, B and C (Dougherty, B. P., and van de
Rijn, I. J. Biol. Chem. 269:169-175 (1994); Dougherty, B. P., and
van de Rijn, I. J. Biol. Chem. 268:7118-7124 (1993); Crater, D. L.,
and van de Rijn, I. J. Biol. Chem. 270:18452-18458 (1995)). It has
been demonstrated that HAS is homologous to hasA which encodes
hyaluronan synthase, along with a recently cloned cDNA encoding the
murine synthase (Has) as well. The hasB and C loci encode UDP:Glc
dehydrogenase and UDP-GLc pyrophosphorylase respectively
(Dougherty, B. P., and van de Rijn, I. J. Biol. Chem. 269:169-175
(1994); Dougherty, B. P., and van de Rijn, I. J. Biol. Chem.
268:7118-7124 (1993); Crater, D. L., and van de Rijn, I. J. Biol.
Chem. 270:18452-18458 (1995)). Also demonstrated herein is that
transfection of the HAS cDNA into CHO cells is sufficient to
mediate de novo synthesis of hyaluronan, which indicates that all
of the other factors necessary for hyaluronan biosynthesis such as
those encoded by hasB and C are possibly expressed in CHO cells.
Recent data suggests that hyaluronan can also be synthesized upon
transfection of the synthase into COS cells and a murine preB
lymphoma which suggests that these backgrounds have endogenous
UDP-GLc dehydrogenase and UDP-GLc phosphorylase and expression of
HAS is then the most significant factor in regulating hyaluronan
synthesis in mammalian cells. The identification of this cDNA will
therefore assist further characterization of the molecular events
resulting in synthesis of hyaluronan and its relationship to
cellular migration in wound healing, tumor metastasis and leukocyte
migration.
[0099] Equivalents
[0100] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
Sequence CWU 1
1
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