Leukosialin (CD43) is proteolytically cleaved from stimulated HMC-1 cells.
Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA). The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA. Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA. In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6). PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation. The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane. Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43. In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors. Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant. Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced. In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells. Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells.
Weber
S
S
Department of Dermatology, Virchow Clinics, Humboldt-Universität zu Berlin, Germany.
Babina
M
M
Hermann
B
B
Henz
B M
BM
D015703:Antigens, CD; D002460:Cell Line; D004306:Dose-Response Relationship, Immunologic; D015536:Down-Regulation / Q000187:drug effects / Q000276:immunology; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051917:Leukosialin; D008407:Mast Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015703:Antigens, CD; D051917:Leukosialin; D011480:Protease Inhibitors; C465014:SPN protein, human; D012795:Sialoglycoproteins; D010450:Endopeptidases; D013755:Tetradecanoylphorbol Acetate