The overall objective of this research program is to elucidate the mechanisms which regulate the synthesis of proteoglycans. These extracellular macromolecules, along with hyaluronic acid and collagen, exhibit changing patterns in expression during development of cartilage tissue. As well, alterations in these components may underlie certain disorders of cartilage, resulting in abnormal growth, development or function. Thus, understanding the mechanisms which control their expression is a fundamental problem in connective tissue biochemistry.
Our specific aims i nclude: 1) The determination of amino acid sequences for selected regions of proteoglycan core proteins in order to identify primary sequences around glycosylation sites, compare structures of precursor forms and proteoglycans with different glycosaminoglycan substituents and from different sources, and to synthesize specific oligonucleotides in order to clone the core protein gene. Core proteins and subfragments are followed through isolation, deglycosylation, cleavage and sequencing procedures immunologically with antibodies to core protein and with a bioassay using xylosyltransferase acceptor activity; 2) Elucidation of the temporal, topological and chemical events involved in synthesis and processing of the core protein from the initially synthesized precursors to the final secreted proteoglycan. Localization and characterization of the biosynthetic intermediate will involve metabolic labeling, subcellular fractionation, in vitro glycosylation, and chemical and immunological analysis, and 3) Certain aspects of the enzymes responsible for initiation and processing of proteoglycans will also be considered, as a complement to the structure and biosynthetic studies. The mechanism of action of xylosyltransferase will be examined with isolated peptide fragments and synthetic peptides, and an active site-directed covalent inhibitor of nucleotide sugars. The PAPS synthesizing system will be purified and structural relationship of components elucidated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR019622-11
Application #
3155058
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1977-08-01
Project End
1990-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
11
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Domowicz, Miriam S; Mueller, Melissa M; Novak, Todd E et al. (2003) Developmental expression of the HNK-1 carbohydrate epitope on aggrecan during chondrogenesis. Dev Dyn 226:42-50
Schwartz, Nancy B; Domowicz, Miriam (2002) Chondrodysplasias due to proteoglycan defects. Glycobiology 12:57R-68R
Pirok 3rd, E W; Henry, J; Schwartz, N B (2001) cis elements that control the expression of chick aggrecan. J Biol Chem 276:16894-903
Domowicz, M S; Pirok 3rd, E W; Novak, T E et al. (2000) Role of the C-terminal G3 domain in sorting and secretion of aggrecan core protein and ubiquitin-mediated degradation of accumulated mutant precursors. J Biol Chem 275:35098-105
Schwartz, N (2000) Biosynthesis and regulation of expression of proteoglycans. Front Biosci 5:D649-55
Krueger Jr, R C; Kurima, K; Schwartz, N B (1999) Completion of the mouse aggrecan gene structure and identification of the defect in the cmd-Bc mouse as a near complete deletion of the murine aggrecan gene. Mamm Genome 10:1119-25
Kurima, K; Singh, B; Schwartz, N B (1999) Genomic organization of the mouse and human genes encoding the ATP sulfurylase/adenosine 5'-phosphosulfate kinase isoform SK2. J Biol Chem 274:33306-12
Schwartz, N B; Lyle, S; Ozeran, J D et al. (1998) Sulfate activation and transport in mammals: system components and mechanisms. Chem Biol Interact 109:143-51
Immergluck, L C; Domowicz, M S; Schwartz, N B et al. (1998) Viral and cellular requirements for entry of herpes simplex virus type 1 into primary neuronal cells. J Gen Virol 79 ( Pt 3):549-59
Pirok 3rd, E W; Li, H; Mensch Jr, J R et al. (1997) Structural and functional analysis of the chick chondroitin sulfate proteoglycan (aggrecan) promoter and enhancer region. J Biol Chem 272:11566-74

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