Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody.
Enzymatically active granule-associated serine protease ("granzyme") B has been purified from human NK cell lysates, using novel granzyme B-specific monoclonal antibodies. Two antibodies, designated 2C5 and 1D10, were produced following immunization of BALB/c mice with a nineteen amino acid peptide synthesized according to the sequence deduced from a granzyme B cDNA clone. Of several peptide-reactive culture supernatants that resulted from cell fusion of splenocytes with NS-1 myeloma cells, clones 2C5 (IgG2a) and 1D10 (IgG1) produced antibodies which detected a approximately 32kDa molecule in human NK cell lysates by Western blotting. This reactive species was detectable in lysates of IL-2-stimulated peripheral blood mononuclear cells, the human NK leukemia cell line YT, the rat NK leukemia cell line RNK-16, but not in the mouse cytotoxic T cell line CTLL-R8 or a variety of non-cytolytic hemopoietic tumor cell lines. The specificity of reactivity with granzyme B was demonstrated by the reaction of the monoclonal antibody with active granzyme in the lysate of COS-7 cells transfected with human granzyme B cDNA, but not with granzyme H expressed in an identical fashion. Western blotting on Percoll-fractionated IL-2 activated human peripheral blood lymphocyte lysates and YT demonstrated reactivity of the monoclonal antibody with a approximately 32kDa species only in those fractions with granzyme A (BLT esterase) and B (Asp-ase) activities. Moreover, 2C5/1D10 antibodies coupled to Protein A-sepharose beads immunoprecipitated enzymatically active granzyme B from YT cell lysates. Scale up of this procedure should yield a means of purifying the large quantities of natural or recombinant granzyme B required to study the function of this granzyme in cellular cytotoxicity.
Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Australia.
J A
JA
Trapani
K A
KA
Browne
M
M
Dawson
M J
MJ
Smyth
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D053804:Granzymes; D006801:Humans; D007694:Killer Cells, Natural / Q000201:enzymology*; D007938:Leukemia; D007942:Leukemia, Experimental; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010455:Peptides; D011994:Recombinant Proteins; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; C509215:Gzmb protein, rat; D012697:Serine Endopeptidases