The long term goal of this research program is to examine the structure, function, and metabolism of connective tissue-associated, protein- carbohydrate complexes as these parameters define the remodeling of connective tissue during development and in disease. Lysosomal enzymes play a key role in remodeling process(es). During the past 15 years, the efforts of this laboratory have emphasized studies on the characterization and properties of three receptors that recognize ligands (largely lysosomal acid hydrolases) that carry covalently-bound mannose -phosphate (Man-6-P) residues; 1) a membrane-associated cation dependent glycoprotein receptor (CI-MPR/IGF-II, equals approximately 215 kDa that contains two sites that bind Man-6-P ligands and a separate site that binds IGF-II); 2) a soluble, developmentally regulated CI-MPR/IGF-II (equals approximately 205 kDa); and 3) a membrane associated, cation """"""""enhanced"""""""" Man-~P receptor (CD-MPR) that contains a single Man-6-P-binding site. The new studies proposed will emphasize attempts to solve the crystal structure of the soluble CI-MPR/IGF-II receptor by X-ray diffraction and computer assisted analysis. The soluble receptor has the same ligand- binding properties and specificity as the membrane-associated receptor. Studies will be conducted on the crystallized receptor and on truncated, soluble, functional fragments constructed by molecular biology procedures. The truncated fragments will be used to probe the fine structure of each ligand binding site independent of on another and independent of other polypeptide portions of the receptor. The effects of glycosylation on the stability, folding and function of the receptor will be probed by expressing appropriately-constructed cDNAs in COS cells (yielding glycosylated fragments) and in bacteria (yielding nonglycosylated fragments). In conjunction with these studies, a novel series of photo affinity probes will be used to establish the position and nature of each Man-6-P binding site on the soluble receptor. A second goal of this proposal is concerned with cellular events that regulate ligand recognition by the Man-6-P receptors and the intracellular trafficking of lysosomal hydrolases. These studies include: 1) attempts to isolate a putative developmentally-regulated proteinase that catalyzes the formation of soluble CI-MPR/IGF-II; 2) efforts to characterize the mechanism(s) involved in the formation of lysosomal enzyme complexes and their role in the intracellular trafficking of acid hydrolases; and 3) defining the positional specificity of UDP-N -acetylglucosamine: glycoprotein N-acetylglucosamine- 1 -phosphotransferase, a key enzyme in ligand/receptor recognition. Knowledge gained from the these efforts will enhance our understanding of the mechanism(s) by which the Man-6-P receptors participate in the intracellular trafficking of lysosomal hydrolases and hence regulation of the remodeling of connective tissue in health and disease, and may provide novel and specific means for targeting therapeutic agents.
