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Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells.

ABSTRACT: Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic "exchange-adsorption" model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm.

SUBMITTER: Bird CH

PROVIDER: S-EPMC1190293 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells.

Bird Catherina H CH Sun Jiuru J Ung Kheng K Karambalis Diana D Whisstock James C JC Trapani Joseph A JA Bird Phillip I PI

Molecular and cellular biology 20050901 17

Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 re ...[more]

PMID: 16107729

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Project description:Granzymes (Gzm) are serine proteases, contained into the secretory granules of cytotoxic cells, responsible for the cell-mediated cytotoxicity (CMC) against tumor cells and intracellular pathogens such as virus and bacteria. In fish, they have received little attention to their existence, classification or functional characterization. Therefore, we aimed to identify and evaluate their functional and transcriptomic relevance in the innate CMC activity of two relevant teleost fish species, gilthead seabream and European sea bass. Afterwards, we wanted to focus on their regulation upon nodavirus (NNV) infection, a virus that causes great mortalities to sea bass specimens while seabream is resistant. In this study, we have identified genes encoding GzmA and GzmB in both seabream and sea bass, as well as GzmM in seabream, which showed good phylogenetic relation to their mammalian orthologs. In addition, we found enzymatic activity related to tryptase (GzmA and/or GzmK), aspartase (GzmB), metase (GzmM), or chymase (GzmH) in resting head-kidney leucocytes (HKLs), with the following order of activity: GzmA/K ~ GzmM >> GzmH >>> GzmB. In addition, during innate CMC assays consisting on HKLs exposed to either mock- or NNV-infected target cells, though all the granzyme transcripts were increased only the tryptase activity did. Thus, our data suggest a high functional activity of GzmA/K in the innate CMC and a marginal one for GzmB. Moreover, GzmB activity was detected into target cells during the CMC assays. However, the percentage of target cells with GzmB activity after the CMC assays was about 10-fold lower than the death target cells, demonstrating that GzmB is not the main inductor of cell death. Moreover, in in vivo infection with NNV, gzm transcription is differently regulated depending on the fish species, genes and tissues. However, the immunohistochemistry study revealed an increased number of GzmB stained cells and areas in the brain of seabream after NNV infection, which was mainly associated with the lesions detected. Further studies are needed to ascertain the molecular nature, biological function and implication of fish granzymes in the CMC activity, and in the antiviral defense in particular.

Project description:PurposeConventional chemotherapy and enucleation usually fail to cure advanced retinoblastoma. We investigated the retinoblastoma immune microenvironment and the efficacy of the combination of dinutuximab and CD16-expressing NK-92MI (NK-92MIhCD16-GFP) cells on retinoblastoma cells in this study.Patients and methodsImmunohistochemistry and flow cytometry (FC) were performed to assess the expression level of GD2 in retinoblastoma tissues and cells. Gene set enrichment analysis (GSEA), immunohistochemisrztry and immunocytochemistry were conducted to assess the retinoblastoma immune microenvironment and the integrity of the blood-retinal barrier (BRB). After overexpressing CD16 in NK-92MI cells, fluorescence-activated cell sorting (FACS) was applied to select the positive subpopulation. LDH assays and FC were used to detect LDH release and apoptosis in retinoblastoma cells subjected to a combination of dinutuximab and NK-92MIhCD16-GFP cells. Finally, the release of perforin-granzyme B and the expression of CD107a in NK-92MIhCD16-GFP stimulated by retinoblastoma cells were assessed via enzyme-linked immunosorbent assays (ELISAs) and FC in the presence of dinutuximab or an isotype control.ResultsGD2 was heterogeneously expressed in retinoblastoma tissues and cell lines and positively correlated with proliferation and staging. GSEA revealed the immunosuppressive status of retinoblastoma microenvironment. The immune cell profile of retinoblastoma tissues and vitreous bodies suggested BRB destruction. LDH release and apoptosis in retinoblastoma cells caused by NK-92MIhCD16-GFP cells were significantly enhanced by dinutuximab. Finally, the release of perforin-granzyme B and the expression of CD107a in NK-92MIhCD16-GFP cells stimulated by retinoblastoma cells were obviously increased by dinutuximab.ConclusionThis study indicates that retinoblastoma impairs the integrity of the BRB and contributes to dysregulated immune cell infiltrates. GD2 is a specific target for natural killer (NK) cell-based immunotherapy and that the combination of dinutuximab and NK-92MIhCD16-GFP cells exerts potent antitumor effects through antibody-dependent cell-mediated cytotoxicity.

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Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells.

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Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells.

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