Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases.
APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca2(+)-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40 kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a molecular mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn2+, but not Ca2+, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn2(+)-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.
Mariani
S M
SM
Division of Immunogenetics, German Cancer Research Center, Heidelberg.
Matiba
B
B
Bäumler
C
C
Krammer
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000502:physiology*; D008969:Molecular Sequence Data; D011817:Rabbits; D013601:T-Lymphocytes / Q000378:metabolism*; D015854:Up-Regulation; D019014:fas Receptor
D000954:Antigens, Surface; D007106:Immune Sera; D019014:fas Receptor; D008666:Metalloendopeptidases