Experiments will investigate the structure and formation of the collagen fibrils of hyaline cartilage from mammalian and avian species. The fibrils are assembled from type II, type IX and type XI collagen and are associated with several non-collagenous proteins including decorin, fibromodulin and cartilage matrix protein (CMP). Extensive use will be made to high affinity monoclonal antibodies (mAbs) specific for each collagen type for which the location of different epitopes along the collagen molecule is known. Monoclonal antibodies will be prepared using different approaches including i) use of synthetic peptides coupled to keyhole limpet hemocyanin (KLH), ii) adjuvants that heighten the immunogenicity of synthetic peptides, iii) expression of non-collagenous domains from cDNA constructs in E. Coli and use of the expressed protein as an immunogen. for type II collagen, monoclonal antibodies are already available that recognize epitopes at three different locations along the length of the molecule. One of these antibodies is of special interest since its epitope is located in a short amino acid sequence of the amino- teleopeptide. Both the amino- and carboxyl-telopeptides of type II collagen will be synthesized and coupled to KLH for the preparation of additional monoclonal antibodies. These antibodies will be used i) to analyze cross-links between type II and type IX collagen by rotary shadowing to determine if type IX collagen is parallel or antiparallel to type II collagen. ii) to look for non-collagenous proteins that are cross-linked to the collagen fibril and are released from the surface of the fibril by bacterial collagenase digestion. iii) to develop an antibody to the cross-linking peptides such that the epitope is lost when cross-linking occurs. iv) to develop in vitro model systems of collagen fibril formation by chondrocytes and to investigate which monoclonal antibodies inhibit fibril assembly. For type XI collagen the amino- terminal non-collagenous domains will be cloned from bovine cartilage cDNA, expressed in E. coli, and used as an immunogen to prepare monoclonal antibodies. These monoclonal antibodies will be used to investigate the role of type XI collagen in fibril structure and during fibrillogenesis. Using synthetic peptides, monoclonal antibodies will be developed to the chains of mammalian type IX collagen. These antibodies will be used to block incorporation of type IX collagen onto the surface of the fibril and to investigate if wider collagen fibrils result from the absence of type IX collagen. Synthetic peptides for the carboxyl-termini of the three chains will be prepared in order to determine if sufficient information is present in these short sequences to promote the assembly of the triple helix. The proposed experiments will provide new information on the structure and organization of cartilage matrix and will provide new reagents to analyze the degenerative diseases of cartilage and genetic diseases involving abnormal cartilage development in man.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR030481-21
Application #
6511817
Study Section
Special Emphasis Panel (NSS)
Program Officer
Tyree, Bernadette
Project Start
1985-09-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2004-03-31
Support Year
21
Fiscal Year
2002
Total Cost
$245,170
Indirect Cost
Name
University of Alabama Birmingham
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Le Goff, Magali M; Lu, Hongbin; Ugarte, Marta et al. (2012) The vitreous glycoprotein opticin inhibits preretinal neovascularization. Invest Ophthalmol Vis Sci 53:228-34
Bishop, P N; Takanosu, M; Le Goff, M et al. (2002) The role of the posterior ciliary body in the biosynthesis of vitreous humour. Eye (Lond) 16:454-60
Takanosu, M; Boyd, T C; Le Goff, M et al. (2001) Structure, chromosomal location, and tissue-specific expression of the mouse opticin gene. Invest Ophthalmol Vis Sci 42:2202-10
Henry, S P; Takanosu, M; Boyd, T C et al. (2001) Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family. J Biol Chem 276:12212-21
Li, S W; Takanosu, M; Arita, M et al. (2001) Targeted disruption of Col11a2 produces a mild cartilage phenotype in transgenic mice: comparison with the human disorder otospondylomegaepiphyseal dysplasia (OSMED). Dev Dyn 222:141-52
Xu, Y; Gurusiddappa, S; Rich, R L et al. (2000) Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1. J Biol Chem 275:38981-9
Paassilta, P; Pihlajamaa, T; Annunen, S et al. (1999) Complete sequence of the 23-kilobase human COL9A3 gene. Detection of Gly-X-Y triplet deletions that represent neutral variants. J Biol Chem 274:22469-75
Tiller, G E; Warman, M L; Gong, Y et al. (1998) Physical and linkage mapping of the gene for the alpha3 chain of type IX collagen, COL9A3, to human chromosome 20q13.3. Cytogenet Cell Genet 81:205-7
Birk, D E; Mayne, R (1997) Localization of collagen types I, III and V during tendon development. Changes in collagen types I and III are correlated with changes in fibril diameter. Eur J Cell Biol 72:352-61
Mechling, D E; Gambee, J E; Morris, N P et al. (1996) Type IX collagen NC1 domain peptides can trimerize in vitro without forming a triple helix. J Biol Chem 271:13781-5

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