The long term goal of this research program is the characterization of the structures, metabolism and function of connective tissue associated protein-carbohydrate complexes as these parameters define the remodeling of connective tissue in development and in disease. Lysosomal enzymes play a key role in remodeling process(es). The recent efforts of this laboratory have emphasized studies concerned with the characterization and function of two membrane-associated glycoproteins involved in the uptake and intracellular transport of lysosomal enzymes, the so-called phosphomannosyl receptors (PMR-1, 215 kDa; and PMR-2, 41-46 kDa). The present research study proposes to continue and to extend these efforts. Primary emphasis will be placed on establishing the properties and function(s) of the novel, low molecular weight phosphomannosyl receptor PMR-2. The structural features of the N-linked oligosaccharides associated with lysosomal enzymes and the phosphomannosyl receptor(s) will be examined in detail to define their roles in the binding of ligand to receptor. Ligand oriented studies will focus on 1) establishing the positional acceptor specificity of the UDP-N-acetylglucosamine: glycoprotein N-acetylglucosamine 1-P phosphotransferase, a key enzyme in the synthesis of the phosphomannosyl recognition marker and 2) on establishing the role of carbohydrates present on lysosomal aggregates in potentiating receptor-ligand interaction and intracellular transport of such aggregates. Receptor oriented studies will exploit the observation that oligosaccharides N-linked to PMR-2 participate in ligand recognition. The effect of oligosaccharide structure(s) and position of N-glycosylation on the polypeptide core of the receptor in potentiating ligand binding will be examined. In separate studies, and as an initial step in determining the structure of the receptor(s) in membranes, efforts will be initiated to chemically define the nature and position of the ligand binding sites on the polypeptide cores of PMR-1 and PMR-2. Finally, experiments are proposed to examine the feasibility of coupling mannose 6-P to antioxidant enzymes. The coupled enzymes will serve as models for the specific targeting of therapeutic agents into connective tissue-associated cells containing phosphomannosyl receptors. """"""""State of the art"""""""" carbohydrate, protein and immunochemical techniques will be utilized in the conduct of the proposed studies.
