This proposal is designed to identify the structural features of complex carbohydrates which are required for the endocytosis of plasma glycoproteins by the Gal/GalNAc lectin of mammalian liver. Four types of ligand probes will be employed: 1) available synthetic, homogenous oligosaccharides, resembling those found on plasma glycoproteins; 2) naturally derived oligosaccharides of known purity and branching pattern; 3) ligands with multiple oligosaccharides synthetically linked together to mimic the arrangement of oligosaccharides on glycoproteins; and 4) serum albumin derivatives with a single oligosaccharide attached to the sulfhydryl function. The naturally derived oligosaccharides will be purified from glycoproteins as tryptic glycopeptides using reversed-phase high performance liquid chromatography. The oligosaccharides, released by N-glycosidase will be further purified using normal phase HPLC. The structure of the purified oligosaccharides will be determined using 400 mHz PMR and fast atom bombardment mass spectrometry. Binding parameters will be measured and data analyzed using an exact mathematical model and statistics (Hardy, M.R., Townsend, R.R., Parkurst, S.M., and Lee, Y.C. (1985) Biochemistry 24, 22-28). The kinetics of ligand internalization, exocytosis, internal and external receptor-ligand uncoupling, and degradation will be measured and compared to computer simulation of complex kinetic processes' (Barshop, B.A., Wrenn, R.F., and Freiden, C. (1983) Anal. Biochem. 130, 134.) These data should allow deduction of carbohydrate structural requirements for this plasma proteins catabolism pathway. The regulation of plasma glycoprotein concentration by the liver has direct relevance to a number of human diseases. The coagulation glycoproteins (fibrinogen, prothrombin, etc.) play key roles in the events associated with vascular thrombosis (myocardial infarction and cerebral thrombosis). The transport glycoproteins, for example, transferrin and ceruloplasmin, have etiological significance for hemochromatosis and Wilson's disease, respectively. The lack of protease inhibitors in plasma has been linked to cirrhosis and emphysema. Further, the effectiveness of replacement therapy using specific plasma components may be improved if additional basic information on the heptic Gal/GalNAc was available. The proposed experiments utilize highly purified naturally derived and synthetic ligands to yield basic information on plasma protein homeostasis which can be utilized to formulate rational diagnostic and therapeutic strategies for the above health problems.
Showing the most recent 10 out of 12 publications
