The overall objective of this research program is to elucidate the mechanisms which regulate the expression of proteoglycans by focussing on the structure and synthesis of the prototype proteoglycan of cartilage. These extracellular macromolecules, along with hyaluronic acid and collagen, exhibit changing patterns in expression during development of cartilage tissue. As well, alterations n 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. Specific questions to be addressed and hypothesis to be tested include: 1) elucidating the role of specific amino acid sequences (recognition or consensus) in marking sites of carbohydrate substitutions in core proteins, 2) exploring the structural organization of core protein domains and the relatedness of core protein families expressed by different cells and tissues, 3) defining the mechanisms by which the intracellular processing machinery is organized and controlled 4) establishing the significance of gene structure to the diversity and ontogeny of the characteristic domain organization of proteoglycans, 5) defining the mechanism of action of specific enzymes responsible for initiating and processing proteoglycans. These will be accomplished by a combinational approach using direct amino acid sequencing of carbohydrate-substituted peptides, nucleotide sequencing of cDNA clones coding for these regions of core proteins and synthesizing artificial peptides to be tested as model acceptors. Biosynthetic studies will take advantage of a newly developed method for preparing permeabilized cells that can be labeled directly with nucleotide sugar precursors. The mechanism of action of relevant enzymes will be examined using appropriate peptide fragments and newly synthesized substrate analogs.
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