title: A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.
abstract: The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB's preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre; Magdalena.Hagn@petermac.org.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Hagn
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre.
authors.forename: Vivien R
authors.initials: VR
authors.lastname: Sutton
authors.affiliation: Cancer Immunology Program, Peter MacCallum Cancer Centre; joe.trapani@petermac.org.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001224:Aspartic Acid / Q000378:metabolism; D003124:Colorimetry / Q000379:methods*; D005563:Formic Acid Esters / Q000378:metabolism; D053804:Granzymes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007694:Killer Cells, Natural / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000032:analysis / Q000378:metabolism*; D059748:Proteolysis; D051381:Rats; D012697:Serine Endopeptidases / Q000032:analysis / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000201:enzymology / Q000378:metabolism
chemical_list: D005563:Formic Acid Esters; D009842:Oligopeptides; C000345:t-butyloxycarbonyl group; D001224:Aspartic Acid; D053804:Granzymes; D012697:Serine Endopeptidases