title: CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells.
abstract: To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A. (1987) Proc. Natl. Acad. Sci. U.S. A. 84, 3365-3369) to allow cloning of intracellular antigens. A megakaryocyte expression cDNA library was transiently transfected into MOP-8 mouse fibroblasts cultured on polyvinylidene difluoride membranes. Individual cells expressing intracellular CD63 were identified by autoradiography. cDNA was extracted from positive spots and reintroduced into Escherichia coli. After two screening rounds, a CD63 cDNA clone was isolated as assessed by immunofluorescence and Western blot analysis. The single long open reading frame of 238 amino acids contained four putative transmembrane regions and three N-glycosylation sites. The CD63 gene was expressed in a wide variety of cells. Surprisingly, CD63 was identical to ME491, an antigen reported as a melanoma-associated antigen (Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer Res. 48, 2955-2962). By immunoelectron microscopy, co-localization with the lysosomal glycoproteins lamp-1 and -2 identified CD63 as a novel lysosomal membrane glycoprotein. CD63 was not related to the lysosomal glycoprotein family but contained the putative lysosomal targeting signal Gly-Tyr in its short cytoplasmic tail.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Metzelaar
authors.forename: P L
authors.initials: PL
authors.lastname: Wijngaard
authors.forename: P J
authors.initials: PJ
authors.lastname: Peters
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: H K
authors.initials: HK
authors.lastname: Nieuwenhuis
authors.forename: H C
authors.initials: HC
authors.lastname: Clevers
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D014162:Transfection
chemical_list: D015703:Antigens, CD; C554587:Cd63 protein, mouse; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30