Cellular distribution of a GPI-anchored complement regulatory protein CD59: homodimerization on the surface of HeLa and CD59-transfected CHO cells.
Human glycosyl phosphatidylinositol-anchored protein CD59 was solubilized in detergent-insoluble complexes (DICs) and in post-nuclear pellets by a two-step solubilization procedure using Triton X-100 and octylglucoside. CD59 molecules are recovered in both fractions, the amount being greater in the latter fraction in all cell types tested. Specific labeling of surface CD59 molecules revealed that the CD59 detected in DICs originated from intracellular compartments, whereas that in post-nuclear pellets was in part derived from the cell surface. Cross-linking of surface proteins with chemical cross-linker followed by Western blotting with anti-CD59 antibody revealed cross-linked products with molecular masses of 28-36 kDa on HeLa and human CD59 cDNA-transfected CHO cells; the CD59-associating molecules were estimated to be 13-18 kDa in size. The cross-linked products were extracted in the post nuclear pellets, and CD59 existed mainly as a cross-linked form on the cell surface. Two-dimensional electrophoresis of the cross-linked products revealed no trace of molecules other than CD59. The cross-linked products showed the same N-terminal sequences as CD59 and a strikingly similar amino acid composition to that of CD59. Thus, most likely, the cross-linked products are CD59 dimers. The finding that CD59 localized on outer membranes is all in the form of dimers suggests the importance of dimerization for CD59 functioning.
Hatanaka
M
M
Department of Clinical Pathology, Osaka Medical College, Daigaku-cho, Takatsuki, Osaka 569-8686.
Seya
T
T
Miyagawa
S
S
Matsumoto
M
M
Hara
T
T
Tanaka
K
K
Shimizu
A
A
D000818:Animals; D018957:CD59 Antigens / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017830:Octoxynol; D012995:Solubility; D013501:Surface-Active Agents; D014162:Transfection
D018957:CD59 Antigens; D017261:Glycosylphosphatidylinositols; D013501:Surface-Active Agents; D017830:Octoxynol