The primary object of the proposal is to continue the studies of the structure, function and trafficking of human lysosomal membrane glycoproteins. In the past few years, we have greatly advanced our knowledge of two related major lysosomal membrane glycoproteins, lamp-1 and lamp-2. In particular, we now know about their growth structures, the roles of carbohydrates in their stability, and the critical involvement of the cytoplasmic tyrosine in their lysosomal delivery. By studying another lysosomal membrane glycoprotein, Limp II, a novel Leu-IIe motif was identified as a lysosomal delivery signal. We have also demonstrated that highly metastic tumor cells express more lamp molecules than low metastic cells and that the higher the amount of cell surface lamp molecules, the stronger the adhesion to activated endothelial cells. Such adhesion was found to be inhibited by a soluble lamp-1 containing sialyl Le-x structure. Based on these findings, three major areas for further study are the following: 1) Elucidation of critical structural elements for maintaining the stability of lamp-1 and lamp-2. The studies will involve replacing various domains of lamp molecules in order to identify the structural elements important for the stability of lamp molecules. In particular, the roles of O-glycans and the hinge-region will be addressed. 2) Elucidation of roles of carbohydrates attached to lamp molecules in tumor metastasis. The studies will determine if those tumor cells expressing more lamp molecules on the cell surface are more metastatic, and if metastasis can be inhibited by a soluble lamp-1 containing sialyl Le-x structures. 3) Further understanding of trafficking of lysosomal membrane glycoproteins. The studies will further elucidate the critical amino acid residues involved in newly discovered Tyr-containing and Leu-IIe containing motifs for lysosomal delivery. The studies will also be aimed at differentiating the two sorting pathways to lysosomes; direct intracellular sorting versus the endocytosis pathway via the plasma membrane.
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