title: Characterization of a trypsin-like serine protease of activated B cells mediating the cleavage of surface proteins.
abstract: Activated B cells may cleave their surface receptors due to the proteolytic activity on the cell membrane or in its vicinity. We attempted to isolate and characterize the protease(s) responsible for this cleavage. Zymograms prepared from the supernatant and the plasma membrane fraction of activated human B cells and BL41/95 cell line exhibited a 85-90 kDa doublet band with protease activity, while that of resting B cells did not. Soybean trypsin inhibitor (STI), Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and EDTA treatment abolished the activity of this protease. The excess of Zn(2+) ions in EDTA did not restore the enzymatic activity, while it was completely recovered in the presence of Ca(2+). We affinity-purified a 85-90 kDa protease from the supernatant of BL41/95 cells using STI coupled to Sepharose 4B beads, and measured its kinetic parameters. For the arginyl substrate K(M) was 358+/-59 microM and for the lysyl substrate 582+/-103 microM. TLCK and benzamidine inhibited the protease at micromolar, while STI at nanomolar concentrations. Both the inhibition profile and the substrate specificity suggest that it is a trypsin-like serine protease. We assume that the 85-90 kDa serine protease expressed on and secreted by activated B cells and BL41/95 cell line is responsible for the cleavage of various membrane proteins, including Fcgamma receptors; thus it may play a crucial role in regulating B cell's function.
authors.affiliation: Research Group of Immunology of Hungarian Academy of Science at L. Eötvös University, Budapest, Hungary.
authors.forename: Anna
authors.initials: A
authors.lastname: Biró
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Hérincs
authors.forename: Erzsébet
authors.initials: E
authors.lastname: Fellinger
authors.forename: László
authors.initials: L
authors.lastname: Szilágyi
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Barad
authors.forename: János
authors.initials: J
authors.lastname: Gergely
authors.forename: László
authors.initials: L
authors.lastname: Gráf
authors.forename: Gabriella
authors.initials: G
authors.lastname: Sármay
mesh_terms: D001402:B-Lymphocytes / Q000201:enzymology*; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008213:Lymphocyte Activation; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
chemical_list: D014107:Tosyllysine Chloromethyl Ketone; D004492:Edetic Acid; D012697:Serine Endopeptidases