Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody

Biochem Biophys Res Commun. 1993 Sep 15;195(2):910-20. doi: 10.1006/bbrc.1993.2131.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal
  • Antigen-Antibody Complex / isolation & purification
  • Blotting, Western
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Granzymes
  • Humans
  • Killer Cells, Natural / enzymology*
  • Leukemia
  • Leukemia, Experimental
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Peptides / chemical synthesis
  • Peptides / immunology
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Serine Endopeptidases / biosynthesis
  • Serine Endopeptidases / immunology
  • Serine Endopeptidases / isolation & purification*
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Antibodies, Monoclonal
  • Antigen-Antibody Complex
  • Peptides
  • Recombinant Proteins
  • GZMB protein, human
  • Granzymes
  • Gzmb protein, mouse
  • Gzmb protein, rat
  • Serine Endopeptidases