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.
