The long term objectives of this project are to define the nature of the interactions between cartilage aggregating proteoglycans, link protein and hyaluronic acid and transfer this information to other systems, such as those of related lymphocyte hyaluronate-receptors. The complex of these three components forms the bulk of cartilage by virtue of its ability to bind large amounts of water. The complex is constrained within a network of collagen fibrils and together, the proteoglycan aggregate and collagen enable cartilage to absorb shock. The proteoglycan aggregate probably plays a role in limiting the diffusion of degradative enzymes, and may play a role in facilitating the diffusion of slats and low molecular weight molecules. Link protein is a 40-50 kDa glycoprotein which stabilizes the cartilage proteoglycan aggregate. The link protein and the region of proteoglycan (G1) which binds to hyaluronic acid (HA) are similar in structure. They both have a region which is in the immunoglobulin fold family, which has been shown to be the general site for interaction between link protein and proteoglycan, and they both have a unique structure which has been shown to bind to hyaluronate. This research project aims to analyze the required amino acids in link protein and G1 in order to define individual binding sites. To do this, an evolutionary remote proteoglycan aggregate (from shark) will be analyzed. The amino acid sequence of shark link protein (LP) and shark proteoglycan G1 will be completed and compared to the same structures in mammalian aggregates with a view to modeling both the surface topology and the binding sites in these molecules. Bovine link protein and aggrecan G1 will be chemically cross-linked and the cross-linked peptides isolated and identified, defining the relative orientation of LP and G1 and thus the exact nature of the binding site between them. This study will also determine whether LP and G1 are folded similarly or differently from each other. Analysis of the structure of the G2 domain in shark aggregating proteoglycan will also be completed. This structure is similar to the region of G1 which binds to HA, but, at least in mammals, does not bind to HA. Comparison of G2 structures will enable us to define the """"""""framework"""""""" residues in this domain. By comparing the G1 domains with the G2 domains, it will be possible to estimate which residues are involved in the function of G1 and which are simply required for folding. This will eventually require site-directed mutagenesis, expression of individual domains and physical chemistry. The studies proposed here will enable meaningful site directed mutagenesis experiments to be designed. These analyses will have broader relevance to both closely related proteoglycans from brain (neurocan), fibroblasts (versican), and also more remotely related, lymphocyte and fibroblast cell surface, hyaluronate-binding, proteoglycans (CD44 and TSG-6). All of these recently described molecules contain a related hyaluronate binding domain.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR035322-08
Application #
2079047
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1989-04-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of South Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Tampa
State
FL
Country
United States
Zip Code
33612
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Neame, P J; Butler, W T (1996) Posttranslational modification in rat bone osteopontin. Connect Tissue Res 35:145-50
Cheng, H; Caterson, B; Neame, P J et al. (1996) Differential distribution of lumican and fibromodulin in tooth cementum. Connect Tissue Res 34:87-96
Bengtsson, E; Neame, P J; Heinegard, D et al. (1995) The primary structure of a basic leucine-rich repeat protein, PRELP, found in connective tissues. J Biol Chem 270:25639-44
Barry, F P; Rosenberg, L C; Gaw, J U et al. (1995) N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature and immature bovine cartilage. J Biol Chem 270:20516-24
Neame, P J; Barry, F P (1994) The link proteins. EXS 70:53-72
Neame, P J; Sommarin, Y; Boynton, R E et al. (1994) The structure of a 38-kDa leucine-rich protein (chondroadherin) isolated from bovine cartilage. J Biol Chem 269:21547-54
Barry, F P; Neame, P J; Sasse, J et al. (1994) Length variation in the keratan sulfate domain of mammalian aggrecan. Matrix Biol 14:323-8
Neame, P J; Barry, F P (1993) The link proteins. Experientia 49:393-402
Neame, P J; Young, C N; Brock, C W et al. (1993) Pleiotrophin is an abundant protein in dissociative extracts of bovine fetal epiphyseal cartilage and nasal cartilage from newborns. J Orthop Res 11:479-91

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