Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-02-26
2004-07-06
Low, Christopher S. F. (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C514S04400A
Reexamination Certificate
active
06759528
ABSTRACT:
BACKGROUND OF THE INVENTION
Fibrils of the abundant collagen types I and II incorporate monomers of the low abundance fibrillar collagen types V and XI, respectively, which play a role in regulating type I collagen fibrillogenesis in vivo (1,2). Type V collagen helps regulate the size and shape of type I/V heterotypic fibrils (3-5). In some cases of classical Ehlers-Danlos Syndrome (EDS), a heritable connective tissue disorder, mutations in type V collagen genes (6-10) give rise to type I collagen fibrils of abnormal shape and diameter and cause connective tissue fragility, particularly in skin and joints. In chondrodysplasia, defects in a type XI collagen gene give rise to abnormal type II collagen fibrils (11).
Fibrillar collagens are synthesized as procollagen precursors with N- and C-propeptides that are proteolytically processed to yield mature monomers. Type V collagen is widely distributed in vertebrate tissues as an &agr;1(V)
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&agr;2(V) heterotrimer (12,13) that helps regulate the diameters of fibrils of the abundant collagen type I. Previously, mutations in the human COL5A1 and COL5A2 genes, which encode the pro-&agr;1(V) and pro-&agr;2(V) chains, respectively, have been identified as the underlying defects in cases of the heritable connective tissue disorder classical EDS (Ehlers-Danlos Syndrome) (formerly EDS types I and II, see Reference (Ref.) 76). However, both COL5A1 and COL5A2 have been excluded in some cases of classical type I (EDS I), while a locus has yet to be identified for the hypermobility type of EDS (formerly EDS type III), a condition marked by gross joint laxity, recurrent joint dislocation, and chronic diffuse musculoskeletal pain not attributable to joint involvement.
Another type V collagen is an &agr;1(V)&agr;2(V)&agr;3(V) heterotrimer, isolated primarily from placenta (17,18), but also reported in uterus, skin, and synovial membranes (12,19-21). The &agr;1(V)&agr;2(V)&agr;3(V) heterotrimer has remained poorly characterized but has a lower melting temperature than the &agr;1(V)
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&agr;2(V) heterotrimer and may be incorporated into heterotypic fibrils. Type XI collagen, in the form of an &agr;1(XI)&agr;2(XI)&agr;3(XI) heterotrimer (22), was first characterized as a minor collagen of cartilage. However, findings of type XI chains in noncartilaginous tissues (23), of type V chains in cartilage (24), and of cross-type heterotrimers composed of &agr;2(V) and &agr;1(XI) chains (25,26) now suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner.
Complete primary structures of the type V/XI procollagen chains pro-&agr;1(V), pro-&agr;2(V), pro-&agr;1(XI), and pro-&agr;2(XI) are known (27-35). The pro-&agr;3(XI) chain is thought to be an alternatively spliced product of the gene that encodes the pro-&agr;1 chain of type II collagen (13, 24). Full-length cDNA sequences have provided not only the inferred primary structure of each chain, but have also provided probes that have allowed fine mapping of the expression domains of cognate mRNAs (27,36-41). Such studies are important, as the low levels of collagen type V/XI chains have limited biochemical and histochemical analyses of expression in developing and adult tissues. Nucleic acid probes have also enabled those studies which established the causal links between defects in type V/XI chains and genetic diseases (6-11).
Of the fibrillar procollagen chains, only the pro-&agr;3(V) remains largely uncharacterized at the nucleotide and amino acid level. The &agr;3(V) chain exhibits only limited distribution in mammals and is believed to be the least abundant fibrillar (type V/XI) collagen chain. The limited distribution may reflect a more specialized role than those of the other type V/XI chains. It is the only fibrillar (type V/XI) collagen or procollagen chain for which neither complete primary structure nor nucleic acid probes are available. About a third of the amino acid sequence of the major collagenous domain of the &agr;3(V) chain was determined by N-terminal sequencing of proteolytic fragments (42). Nevertheless, a true understanding of the nature of mammalian type V/XI collagen and its roles in development, physiology, disease and treatment requires characterization the pro-&agr;3(V) and &agr;3(V) chains.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that mammalian &agr;3(V) polypeptides and variants thereof are disclosed, as are recombinant materials, including genetic constructs, and methods for their production. The invention is further summarized in that polynucleotides that encode the polypeptides and the variants are also disclosed. The invention is still further summarized in that investigative, diagnostic and therapeutic compositions and methods employing the polypeptides, polynucleotides and related materials, such as antibodies, sense- or antisense oligonucleotides and polynucleotides, and the like, are also disclosed. The chromosomal map positions in humans and mice of the polynucleotides that encode the mammalian &agr;3(V) polypeptides are also disclosed.
It is an object of the present invention to enable production of large quantities of mammalian &agr;3(V) polypeptide chains for research, diagnostic and therapeutic use.
It is an advantage of the present invention that collagen comprising mammalian pro-&agr;3(V) or &agr;3(V) chains can be synthesized for any such use.
Other objects, features and advantages of the present invention will become apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, a “mammalian &agr;3(V) polypeptide” refers to a modified or unmodified polypeptide having an amino acid sequence characteristic of those shown in SEQ ID NO:2 and SEQ ID NO:4, or a novel fragment thereof, especially a fragment that is antigenic or has a biological activity. Preferably, a mammalian &agr;3(V) polypeptide exhibits at least one biological activity of mammalian &agr;3(V) procollagen or collagen. A mammalian &agr;3(V) polypeptide can be a mature protein or a larger protein that can include native or non-native amino acid sequences at the N- or C-terminus or both, a propeptide sequence, or other sequence attached to the mature polypeptide sequence. These sequences can include amino acid sequences that assist in purification, detection, or stabilization of the mammalian &agr;3(V) polypeptide.
Within the scope of the invention are polypeptides that have at least 80% amino acid identity to that of either SEQ ID NO:2 or SEQ ID NO:4 over its entire length, and more particularly polypeptides having at least 90% identity, or more preferably at least 95% identity, to that of SEQ ID NO:2 or SEQ ID NO:4, when the sequences are aligned to obtain the highest order match using published techniques. Most preferred are polypeptides having between 97 and 99% amino acid identity to that of SEQ ID NO:2 or SEQ ID NO:4. The term “identity” is given its art recognized meaning. Sequence identity can be determined, for example, using the methods disclosed by Devereux et al. (83), incorporated herein by reference in its entirety.
An polypeptide is, e.g., 80% “identical” if it contains up to 20 amino acid sequence differences, changes or alterations (including substitutions, deletions, or insertions) per each 100 amino acids in reference sequences SEQ ID NO:2 or SEQ ID NO:4. The differences, changes or alterations can be at any position in the amino acid sequence of either polypeptide and can be interspersed as individual changes or contiguous differences.
A “mammalian &agr;3(V) polynucleotide” refers to a polynucleotide that encodes any mammalian &agr;3(V) polypeptide, or a polynucleotide fragment thereof, or a complement of any of the foregoing. A polynucleotide can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A modified pol
Greenspan Daniel S.
Imamura Yasutada
Liu Samuel W.
Low Christopher S. F.
Wisconsin Alumni Research Foundation
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