Lysosome-associated membrane proteins: characterization of LAMP-1 of macrophage P388 and mouse embryo 3T3 cultured cells.
Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, has been identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anit-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromatography with 1D4B monoclonal antibody, yielding a homogeneous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the two purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [35S]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent Mr 92,000, and then converted to heterogeneous mature forms differing in average Mr from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [35S]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent molecular weight of the molecules or the differences between the various cell lines. beta-D-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002460:Cell Line; D005347:Fibroblasts / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D006026:Glycoside Hydrolases / Q000378:metabolism; D006027:Glycosides / Q000494:pharmacology; D007525:Isoelectric Focusing; D007941:Leukemia P388 / Q000378:metabolism*; D007942:Leukemia, Experimental / Q000378:metabolism*; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000032:analysis*; D008264:Macrophages / Q000032:analysis*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011506:Proteins; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D006023:Glycoproteins; D006027:Glycosides; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D011506:Proteins; C018589:lysosomal proteins; C010807:xylosides; D008985:Monensin; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin