CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells
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...
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Veröffentlicht in: | The Journal of biological chemistry 1991-02, Vol.266 (5), p.3239-3245 |
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Zusammenfassung: | 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)49980-2 |