Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors

Abstract

Granzyme A (GrA) and B (GrB) together with perforin are the main constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytotoxic proteins are released to deliver a lethal hit during contact between the CTL or NK cell and target cell. With the use of an enzyme-linked immunosorbent assay for antigenic levels, we showed in a recent study that plasma of patients with activated CTLs and NK cells contain elevated levels of extracellular GrA. In this study, we determined the form and proteolytic capacity of this extracellular GrA detected in plasma. With the use of various assays, we show that part of the extracellular GrA circulates in the mature conformation and is bound to proteoglycans that protect it against inactivation by protease inhibitors, such as antithrombin III and -2-macroglobulin, whereas another part of GrA circulates as a complex with antithrombin III. Finally, with the use of a novel assay for active GrA, we demonstrate that some plasma samples with high levels of extracellular GrA contain active GrA. These results suggest that various forms of extracellular GrA occur in vivo and that the regulation of GrA activity may be modified by proteoglycans. These data support the notion that granzymes may exert extracellular functions distant from the site of CTL or NK cell interaction with their target cells.

Granzyme A Contributes to Inflammatory Arthritis in Mice Through Stimulation of Osteoclastogenesis

“…The contribution of GzmA to the development of joint pathology seems to be independent of perforin. This finding confirms the physiologic relevance of previous studies in which extracellular GzmA was detected in both serum and synovial fluid from humans with RA (17,18). Paralleling the studies in humans, we found that extracellular GzmA was significantly increased in mouse serum and joints in the present studies.…”

Detection of soluble human granzyme K in vitro and in vivo

“…The same elution pattern was observed for GzmA and B. This difference in elution pattern at high-and low-salt conditions has been shown previously for GzmA and seems to be typical for proteins complexed to proteoglycans [2,24]. Although the constituents of the macromolecular weight complex detected at physiologic conditions were not determined, results are consistent with previous reports showing that granzymes are packaged in cytotoxic granules being non-covalently bound to proteoglycans, and that these complexes are relatively stable under physiologic conditions but dissociate at high salt concentrations [25,26].…”

“…To compare the levels of GzmK with GzmA and B, we additionally analyzed plasma samples from rheumatoid arthritis (RA) patients as well as from patients with viral infections, as these have previously been associated with elevated plasma levels of GzmA and B [2,23]. In RA patients, only GzmA levels were found to be significantly elevated, while plasma levels of GzmB and K were similar to or lower than those of healthy controls (Fig.…”

“…Although granule exocytosis results in the delivery of the granule contents to the target cell, detectable levels of granzyme A (GzmA) and B are also present in plasma samples from healthy controls, and levels are significantly higher in patients suffering from viral, bacterial or parasitic infections [1][2][3][4]. These extracellular granzymes may have originated from the immunological synapse, from which they diffuse into the extracellular space during target cell killing.…”

Sickle cells produce functional immune modulators and cytotoxics

“…27,28 Any leakage of these cytolytic products from circulating SSRBCs did not appear to induce acute or chronic toxicity likely because granzyme in plasma is degraded by blood borne protease inhibitors antithrombin III and a-2-macroglobulin. 34 Thus, despite their cytolytic properties, mice bearing granzyme and perforin transgenes in their SSRBCs showed durable transgene expression with no untoward acute or chronic effects of these proteins.…”