Proteoglycans in cell-mediated cytotoxicity. Identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis.

Abstract

A clone of natural killer (NK) cells (JTB18) was found to be ultrastructurally similar to peripheral blood large granular lymphocytes (LGL). These cells incorporated [35S]sulfate into cell-associated proteoglycan molecules, which were then isolated by CsCl density gradient centrifugation. As assessed by gel filtration chromatography, the native 35S-labeled proteoglycan and its beta-eliminated 35S-labeled glycosaminoglycans were of Mr approximately 200,000 and 50,000, respectively. The 35S-labeled proteoglycans were resistant to proteolysis, since their Mr were apparently not altered by incubation with either pronase or S. aureus V8 protease. The purified NK cell 35S-labeled proteoglycans were degraded by approximately 90% to 35S-labeled disaccharides with either chondroitinase ABC or AC. High performance liquid chromatographic analysis of the digests revealed these disaccharides to be composed entirely of chondroitin sulfate A (glucuronic acid----N-acetylgalactosamine-4SO4). Whole 35S-labeled cells incubated with chondroitinase ABC failed to release 35S-labeled disaccharides into the supernatant, and x-ray energy-dispersive analysis revealed that sulfur-containing molecules were present in the intracellular granules, thereby localizing the NK cell-associated proteoglycan primarily in the granules of the cell, rather than on the plasma membrane. The 35S-labeled cloned NK cells incubated for 30 min to 4 h with K562 tumor cell targets at a 0.5:1 ratio exocytosed a mean of 49% of the granular 35S-labeled proteoglycans during the first 60 min of the culture. Proteoglycan release was maximal with an effector/target cell ratio of 0.5:1 for JTB18:K562. Significant proteoglycan release from JTB18 NK cells was also obtained with other sensitive target cells such as REX, Molt4, and CEM, but not with cells such as KG1 and Laz156, which have been shown previously to be resistant to killing by this NK cell. Thus, protease-resistant intracellular proteoglycans with chondroitin sulfate A side chains are specifically exocytosed from the granules of human NK effector cells upon contact with sensitive targets, suggesting that these proteoglycans may be involved in the mechanism of cytotoxicity.

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

“…Inhibition of granule release results in decreased or complete abolition of target cell lysis (Kopcow et al, 2005). Incubation with K562 target cells induces NK cells to expel the lytic granule proteins (MacDermott et al 1985;Nagatake et al 1995). The magnitude of the affect was different when observed by the different assays.…”

Delayed Contraction of the CD8+ T Cell Response toward Lymphocytic Choriomeningitis Virus Infection in Mice Lacking Serglycin

“…Among various granzymes, granzyme B (GrB) 3 and granzyme A (GrA) are the most abundant (5,7,8). Additional components of the cytotoxic granules include chemokines (9), various lysosomal proteases and lysosomal membrane proteins (10,11), and finally, proteoglycans (12,13), i.e., high molecular weight molecules composed of a protein core in which one or more sulfated and thereby negatively charged glycosaminoglycan chains are attached (reviewed in Refs. 14,15).…”

Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

“…Recently, proteoglycans of the chondroitin sulfate A type have been identified in the granules of NK cells and shown to be released from cells during cytolysis (25,26). As shown here, proteoglycans may also be used as a granule marker for CTLL .…”

“…Proteoglycans of CTLL-R8 cell granules can also be visualized on polyacrylamide gels stained with Alcian Blue, which reveal several bands of different molecular masses (profile not shown here). Because proteoglycans have previously been speculated to exert a protective function during cell-mediated killing, tentatively through their binding to PFP/perforin released from cells (25,26), we tested the effects of heparin and chondroitin sulfate on the hemolytic activity mediated by PFP. Only heparin inhibited hemolysis at high concentrations (0.5 mg/ml), whereas chondroitin sulfate (and the three subtypes A, B, and C) produced no effect in the wide concentration range tested in our experiments.…”