Mammalian tolloid-like gene and protein

Details Mammalian tolloid-like gene and protein Mammalian tolloid-like gene and protein

1999-02-01

2001-10-02

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06297011

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to the field of bone morphogenetic proteins and more particularly to a gene in the BMP-1/Tld family of genes.
Bone formation in mammals such as mice and humans is governed by a set of bone morphogenetic proteins (BMP). Of the seven BMPs known to participate in osteogenesis, six (designated BMP-2 through BMP-7) belong to the TGF-&bgr; super family. The seventh BMP (designated BMP-1) is not TGF-&bgr;-like, but instead appears to derive from a different gene family. The BMP-1 gene family members typically contain the following domains: an astacin-like metalloprotease domain, one or more EGF-like motifs which in other proteins are thought to bind Ca
++
, and a number of CUB domains. A CUB domain is a motif that mediates protein-protein interactions in complement components C1s/C1s which has also been identified in various proteins involved in developmental processes. BMP-1 was described, at the nucleotide sequence level, by Wozney, J. M., et al.,
Science
242:1528-1534 (1988).
The mammalian BMP-1 domain structure is shared by proteins found in other non-mammalian species. These proteins include Drosophila tolloid (Tld) (Shimell, M. J.,
Cell
67:469-481 (1991)), a tolloid-like Drosophila gene product (Tlr-1 or tolkin) (Nguyen, T.,
Dev. Biol
. 166:569-586 (1994) and Finelli, A. L., et al.,
Genetics
141:271-281 (1995)), a sea urchin BMP-1 homolog (suBMP-1) (Hwang, S.-P., et al.,
Development
120:559-568 (1994)), two related sea urchin developmental gene products, SpAN and BP10 (Reynolds, S. D., et al.,
Development
114:769-786 (1992) and Lepage, T., et al.,
Development
114:147-164 (1992)), a Xenopus BMP-1 (xBMP-1) (Maeno, M. et al.,
Gene
134:257-261 (1993) and a mammalian tolloid (mTld) (Takahara, K. et al.,
J. Biol. Chem
. 269:32572-32578 (1994)). A tolloid-like gene (xolloid) obtained from Xenopus has been briefly mentioned in passing in a article reviewing the astacin family of metalloproteases. Bond, J. S. and R. J. Benynon,
Protein Science
4:1247-1261 at 1249 (1995), but data relating to the gene itself has not been published. Some of the nucleic acid sequences of the genes that encode these proteins are known. The mammalian BMP1 gene encodes both the BMP-1 protein and the mTld protein, albeit on two distinct, alternately spliced mRNA molecules. The papers mentioned in this paragraph are incorporated herein by reference.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that a novel mammalian-tolloid-like gene product (mTll) and its cognate gene, which is distinct from mTld and from all other known BMP-1-related proteins and genes, are described. The murine and human versions of the gene are reported.
It is an object of the present invention to provide a gene and gene product involved in the deposition of extracellular matrix in vertebrates (e.g., in osteogenesi).
It is another object of the present invention to target molecule for rational development of a drug for inhibiting activity of the tolloid-like genes to treat fibrosis, scarring, keloids, surgical adhesions, and the like.
It is yet another object of the present invention to provide a recombinant DNA construct, and a protein encoded by the construct, for use in accelerated wound and fracture healing.
It is still another object of the present invention to provide a marker gene that maps to the central portion of mouse chromosome 8.
It is yet another object of the present invention to provide a marker gene that maps to the 4q32-4q33 region of human chromosome 4.
It is still another object of the present invention to provide a nucleotide sequence that functions as a probe for a non-BMP-1 bone morphogenetic protein gene in mammalian cells.
It is a feature of the present invention that the murine gene described contains a novel simple sequence repeat in the 3′-untranslated region of the gene.
Other objects, features, and advantages of the present invention will become apparent upon consideration of the following detailed description considered in conjunction with the accompanying drawings. dr
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1
presents a map of the murine mTll cDNA including 5′- and 3′-untranslated portions thereof. Also aligned beneath the mTll cDNA for comparison are schematic representations of cDNA clones of related genes, drawn to the same scale as mTll. Portions of the cDNA corresponding to domains of the gene product are highlighted. Stippled, darkly shaded, striped, lightly shaded, and black boxes represent signal peptide, proregion, metalloprotease, CUB, and EGF domains, respectively. White boxes represent domains unique to the various proteins. Wavy lines represent 5′- and 3′-untranslated regions. Abbreviations: mTld, mammalian tolloid; mBMP-1, mammalian BMP-1; xBMP-1, xenopus BMP-1; suBMP-1, sea urchin BMP-1; Tld, Drosophila tolloid; Tlr-1, Drosophila tolloid-related gene; SpAN and BP10, related sea urchin developmental genes. Restriction enzymes include: Bg, BglII; C, ClaI; E, EcoRI; H, HincII; N, NcoI; S, SmaI; St, StuI.
FIG. 2
aligns the amino acid sequence of the disclosed mTll gene to that of the mTld gene. The domain structure common to both proteins is shown schematically. Domains are represented as in FIG.
1
. Alignment was performed using the GAP program (Genetics Computer Group, Madison, Wis.), with a GAP weight of 3.0 and GAP length weight of 0.1, with some additional manual alignment of putative signal peptide sequences. Cysteines are boxed, potential Asn-linked glycosylation sites are underlined and the metalloendopeptidase active site motif HEXXH is enclosed by a dashed box.
FIG. 3
shows a schematic map of the central portion of mouse chromosome 8.The Map Manager program (Manly K., “A Macintosh Program for Storage and Analysis of Experimental Genetic Mapping Data,”
Mammalian Genome
4:301-313 (1993) compared segregation data for Tll and for other loci from the TJL BSS backcross panel, performed the linkage analysis and generated the map. The TJL BSS backcross panel data are available on the Jackson Laboratories Public Data Base (http://www.jax.org/Resources/documents/cmdata).


REFERENCES:
Bond, Judith S. et al., “The Astacin Family of Metalloendopeptidases,”Protein Science, 4:1247-1261 (1995).
Crystal, Ronald,Science, 270:404-409 (1995).
Greenspan, D.S., “Mus Musculus Mammalian Tolloid-like Protein mRNA”, Genbank Accession No. U34042 (1996).
Johnson, George, “The Chicken with the Duck's Feet: It's All in the Biochemical Signal,”The New York Times Science(May 21,1996).
Kessler, Efrat, et al., “Bone Morphogenetic Protein-1: Type 1 Procollagen C-Proteinase”,Science, 271: 360-362 (1996).
Mastrangelo et al.,Seminars in Oncology, 23(1): 4-21 (1996).
Takahara, Kazuhiko, et al., Bone Morphgenetic Protein-1 and a Mammalian Tolloid Homologue (mTld) Are Encoded by Alternatively Spliced Transripts Which Are Differentially Expressed in Some Tissues,The Journal of Biological Chemistry, 269(51): 32572-32578 (1994).
Takahara, Kazuhiko et al., “Structural Organization and Genetic Localization of the Human Bone Morphogenetic Protein 1/Mammalian Tolloid Gene”,Genomics, 29(1): 9-15 (1995).
Takahara, Kazuhiko et al., “Characterization of a Novel Gene Product (Mammalian Tolloid-Like) with High Sequence Similarity to the Mammalian Tolloid/Bone Morphogenetic Protein-1”,Genomics, 34(2): 157-165 (1996).

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