The primary object of this proposal is to study the structure, biosynthesis and trafficking of human lysosome-associated membrane proteins, lamp-1 and lamp-2, in non-tumorigenic and tumorigenic cells. We have isolated two major lysosomal membrane glycoproteins with apparent Mr. -120,000, lamp-1 and lamp-2. We found that the glycoproteins are heavily glycosylated by N-linked saccharides, some of which are polylactosaminoglycans. We have also succeeded in isolating cDNAs encoding for both lamp-1 and lamp-2 and our results have shown that they have unique structures. Four major aspects for further studies deal with the following: 1) Elucidation of the structure of lamp-1 and lamp-2. The studies, as comprehensive as possible, will be to understand the chemical structure of lamp-1 and lamp-2. Particular attention will be paid to defining the attachment sites for polylactosaminoglycan and to determine the structures of disulfide bonds. These studies will be complemented by elucidation of the genomic structure of lamp-1 and lamp-2 genes, in order to further understand the structural domains. 2) Elucidation of the biosynthesis of lamp-1 and lamp-2. The studies will be to understand the biosynthesis of polylactosaminoglycan during the processing of the glycoproteins and evaluate the significance of polylactosaminoglycan in cell surface expression of lamp-1 and lamp-2. 3) Identification of molecular signals for targeting of lamp-1 and lamp- 2. Our preliminary results suggest that the cytoplasmic segments of the lysosomal membrane glycoproteins are probably responsible for targeting the molecules to lysosomes. Further studies will be to define the amino acid residues critically involved in this targeting and to demonstrate if the cytoplasmic segment is sufficient for targeting of heterologous reporter molecules. 4) Comparison of the biosynthesis and intracellular movement of lamp-1 and lamp-2 between metastatic and non-metastatic tumor cells. The studies will determine if metastic tumor cells synthesize and express more lamps on cell surface than non-metastatic tumor cells.
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