The overall objective of this proposal is to develop methods for synthesis of biologically active protein-protein conjugates containing an enzyme and a cell surface receptor-binding protein for use as probes to study the mechanism of cell surface receptor-mediated protein transports. Initially optimal conditions for preparing conjugate between an enzyme and asialoorosomucoid either through a disulfide or thioether linkage will be studied using methyl 5-bromovalerimidate/thiosulfate or m-maleimidobenzoyl N-hydroxysuccinimide ester in conjuction with methyl 5-bromovalerimidate/ thiosulfate or m-maleimidobenzoyl N-hydroxysuccinimide ester in conjunction with methy 4-mercaptoburyrimidate as cross-linking reagents. Large quantity of each conjugate will then be synthesized, purified by gel filtration followed by affinity chromatography on columns with immobilized asialoglycoprotein receptor, and characterized for biological activities and molecular size. Conjugates radioactively labelled solely on enzymatic moiety will then by synthesized accordingly and used to study the uptake by rat liver organ cultured explants of the labelled enzyme moiety in comparison with that of unconjugated labelled enzyme under the effect of unlabelled asialoglycoproteins. Observation of uptake of the conjugated enzyme that is both competitively inhibited by excess asialoglycoprotein and at a rate substantially higher than that of unconjugated enzyme will be indicative of uptake through an asialoglycoprotein receptor-mediated transport process, and therefore validates the usefulness of the conjugates in studying receptor-mediated protein transport. Hybrid conjugates between the enzymatically active and the receptor-binding subunits of diphtheria toxin and ricin will be prepared subsequently and used for studying the mechanism of transport of these toxins. Finally conjugate of lysosomal enzymes to human plasma low density lipoprotein will be prepared and used for studying the mechansims by which lysosomal enzymes can be transported and function in fibroblast lysosomes while low density lipoprotein is degraded. The information obtained from these experiments will be useful for determining whether or not it is possible to utilize various receptor-mediated protein transport systems for introducing lysosomal enzymes into deficient cells for enzyme replacement therapy and toxin active fragment for cancer chemotherapy.
NIH ExPorter
NIH ExPorter