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Purification and characterization of Granzyme K from human IL-2 activated cell line

Human cytotoxic cells such as natural killer (NK) cells and cytotoxic T lymphocytes (CTL) mediate their function by exocytosis of granules. These cytotoxic granules have been shown to contain a poreforming protein, perforin and 5 serine proteases called granzynes (A, B, K, H and M) among other proteins. The structure and function of granzyme A and B have been extensively characterized but little work has been done on the remaining granzymes. The ability to purify native and catalytically active forms of granzyme K (GrK) will facilitate further study of its physiological role, since it has been recently shown in our laboratory that GrK may play important role in inflammatory responses such as sepsis. In order to identify and characterize GrK, we have successfully developed polyclonal antibodies against peptide sequence unique for GrK. The specificity of the antibody was demonstrated by its reactivity against human recombinant GrK in Western blot. In this study, GrK was partially purified from the cytotoxic granules obtained from the human IL-2 activated NK cell line (92 MI). These granules were isolated following disrupting the cells by differential gradient centrifugation and subsequent extraction with hypotonic detergent containing buffer followed by buffer containing high salt (salt extract). The protein of expected size was enriched in the salt extract and was further analyzed by Western blot and for its biological activity by cleaving synthetic substrate BLT (Z-Arg-Sbzl). Western blot analysis of the salt extract using affinity purified antibody and comercialy available monoclonal antibody against GrK, revealed a specific band corresponding in size to rGrK (28 kDa). In the BLT activity assay, the most activity of GrK was found in the salt extract. Recently, the 28 kDa protein together with several proteins of higher molecular size were detected by these antibodies in the culture media suggesting that different forms of GrK may be secreted by growing cells in vitro. Current efforts are focused on further development of efficient purification strategy for GrK from biological sources.

Cytotoxic cells; NK cells; Granzym K; purification; characterization

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