Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency Mirjana Grujic, Gabriela Calounova, Inger Eriksson, Thorsten Feyerabend, Hans-Reimer Rodewald, Elena This information is current as Tchougounova, Lena Kjellén and Gunnar Pejler of October 1, 2021. J Immunol 2013; 191:3931-3938; Prepublished online 23 August 2013; doi: 10.4049/jimmunol.1301441 http://www.jimmunol.org/content/191/7/3931 Downloaded from References This article cites 29 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/191/7/3931.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency Mirjana Grujic,* Gabriela Calounova,*,1 Inger Eriksson,†,1 Thorsten Feyerabend,‡ Hans-Reimer Rodewald,‡ Elena Tchougounova,x Lena Kjelle´n,† and Gunnar Pejler* Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule com- pounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through Downloaded from a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron mi- croscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic http://www.jimmunol.org/ proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge. The Journal of Immunology, 2013, 191: 3931–3938. ast cells (MCs) are versatile effector cells of the im- is widely recognized that the preformed granule compounds that mune system, contributing both to adaptive and innate are released upon MC degranulation are key actors in MC-me- M immune responses (1). A hallmark feature of MCs is diated events (1–3). Hence, the MC secretory granules are of cru- their high content of cytoplasmic secretory granules, which con- cial importance for the biological actions of MCs, but despite this stitute the dominating morphological criterion for identifying MCs notion there is only limited knowledge of the mechanisms that regulate their composition. as such. When MCs are activated they can undergo degranulation, by guest on October 1, 2021 a process in which the contents of the secretory granules are re- Serglycin is composed of a small (∼17 kDa) core protein to which leased to the extracellular space (2). The granules contain a panel negatively charged glycosaminoglycans (GAGs) are attached, with of preformed compounds, including bioactive amines (e.g., his- the GAGs attached being of either heparin/heparan sulfate (HS) tamine), preformed cytokines (e.g., TNF), a number of MC- or chondroitin sulfate (CS) type (5). Heparin/HS is built up by re- specific proteases (3, 4), and proteoglycans of serglycin type (5, peating hexuronic acid (HexA)-glucosamine (GlcN) disaccharide 6). Although in many cases the exact mechanisms by which MCs units, where the HexA residues can be either glucuronic acid or contribute in pathological settings are incompletely understood, it iduronic acid and where the GlcN residues can be either N-sulfated or N-acetylated. Additionally, the HexA residues may be 2-O-sul- fated and the GlcN units can carry 6-O- and, rarely, 3-O-sulfate *Department of Anatomy, Physiology, and Biochemistry, Swedish University of groups (7). CS chains are built up by repeating HexA-N-acetylga- Agricultural Sciences, 75123 Uppsala, Sweden; †Department of Medical Biochem- lactosamine (GalNAc) disaccharide units, in which the HexA resi- istry and Microbiology, Uppsala University, 75237 Uppsala, Sweden; ‡Division for Cellular Immunology, German Cancer Research Center, D-69210 Heidelberg, Ger- dues can be 2-O-sulfated and the GalNAc residues may carry 4-O- many; and xDepartment of Immunology, Genetics, and Pathology, Rudbeck Labora- and/or 6-O-sulfate groups (8). Importantly, both heparin/HS and CS tory, Uppsala University, 75185 Uppsala, Sweden exhibit a large extent of heterogeneity, both with regard to chain 1G.C. and I.E. contributed equally to this work. length and to the extent and pattern of sulfation (7, 8). Received for publication May 31, 2013. Accepted for publication July 1, 2013. In previous studies we have shown that serglycin proteoglycan This work was supported by grants from the Swedish Research Council, the Swedish promotes the storage of a number of positively charged secretory Cancer Foundation, and by Formas and King Gustaf V’s 80-Year Anniversary Fund. granule–localized MC proteases, including chymases (mouse MC H.-R.R. received support from European Research Council Advanced Grant 233074 and Sonderforschungsbereich Grant 938-project L. protease [mMCP]-4 and mMCP-5), tryptase (mMCP-6), and car- Address correspondence and reprint requests to Prof. Gunnar Pejler, Swedish Uni- boxypeptidase A3 (CPA3) (3, 4, 9, 10). A likely explanation for versity of Agricultural Sciences, Department of Anatomy, Physiology and Biochem- these observations is that negatively charged GAG chains attached istry, BMC, Box 575, 75123 Uppsala, Sweden. E-mail address: [email protected] to the serglycin core protein interact electrostatically with positive Abbreviations used in this article: BMMC, bone marrow–derived mast cell; CPA, charge displayed by its binding partners, that is, the proteases (5, carboxypeptidase; CS, chondroitin sulfate; GAG, glycosaminoglycan; GalNAc, N- acetylgalactosamine; GlcN, glucosamine; HexA, hexuronic acid; HS, heparan sul- 6). Although these findings have established a role for negatively fate; KO, knockout; MC, mast cell; mMCP, mouse mast cell protease; PCMC, peri- charged proteoglycans of serglycin type in regulating the storage toneal cell–derived mast cell; RPIP, reverse-phase ion-pair; TEM, transmission of granule compounds in MCs, little is known of the dynamics in electron microscopy; WT, wild-type. regulating MC secretory granule composition. For example, one Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 important question is whether there is an interdependence of the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301441 3932 PROTEASE-DEFICIENT MAST CELLS various granule compounds such that a reduction of positive DNA was removed by centrifugation (2000 3 g, 10 min, 4˚C) and smaller charge will affect the granule composition in an analogous fashion DNA fragments were further digested by 300 U DNAse I from bovine as does the reduction in negative charge density. To address this pancreas (Sigma-Aldrich). The lysed cells were subsequently applied to DEAE Sephacel columns. Bound proteoglycans were eluted with PBS/3 M issue, in this study we generated mice deficient in multiple posi- NaCl/0.5% Triton X-100, and 10 ml of each fraction (300 ml/fraction) was tively charged proteases that are stored within MC granules, that mixed with 2 ml OptiPhase HiSafe 3 scintillation mixture and analyzed by is, mMCP-4, mMCP-5, mMCP-6, and CPA3. Indeed, we show scintillation counting. Fractions containing [35S]-labeled proteoglycans that the absence of multiple MC proteases results in severe effects were pooled, desalted through PD10 columns (GE Healthcare), and treated with 0.5 M NaOH overnight at 4˚C to release free GAG chains from the on granule integrity, as reflected by distorted granule staining proteoglycans. The samples were neutralized