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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 mouse mast cell protease-4, the 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 (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 with 4 M HCl, desalted on properties and ultrastructure, and that these effects are associated PD10 columns, and concentrated. A portion of GAG chains were treated with defective storage of heparin within granules. Collectively, with HNO2 (pH 1.5) to degrade heparin/HS chains (15). HNO2-treated or our findings suggest that MC granule composition is a result of untreated GAGs (10,000 cpm in 100 ml) were mixed with 150 mg blue a dynamic electrostatic relationship between oppositely charged dextran (marker of void volume; Sigma-Aldrich) and applied to a Super- dex 200 column (GE Healthcare). The column was eluted with 1 M NaCl/ compounds. 0.05 M Tris-HCl (pH 7.4) at 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed by scintillation counting. Materials and Methods For anion-exchange chromatography, GAG samples were mixed with 2.25 mg standard pig mucosal heparin and 0.5 mg standard chondroitin Reagents sulfate A. Samples (10,000 cpm in 1 ml) were applied to a HiTrap DEAE FF The chromogenic substrates S-2288 (for detection of -like activity/ column (GE Healthcare) that was washed with 50 mM NaAc/50 mM LiCl tryptase) and S-2586 (for detection of -like activity/chymase) (pH 4) and then eluted at 0.5 ml/min with a linear gradient of LiCl (from 50 were from Chromogenix (Milano, Italy), and M-2245 (for detection of CPA mM to 3 M) in 50 mM NaAc (pH 4). Twenty-five microliters of each Downloaded from activity) was from Bachem (Bubendorf, Switzerland). fraction was assayed with the carbazole method to detect the unlabeled standard heparin and CS; the rest (475 ml) was analyzed by scintillation Mice counting. Mice triple-deficient in mMCP-4, mMCP-6, and CPA3 were generated Disaccharide analysis by intercrossing mMCP-42/2 (11), mMCP-62/2 (12), and CPA32/2 mice (13), all on a C57BL/6 genetic background. The mMCP-62/2 strain used is Samples were prepared as previously described followed by reverse-phase mMCP-7–sufficient (12), whereas the WT C57BL/6 mice lack expression ion-pair (RPIP)-HPLC analysis (18). of mMCP-7 (14). Mice were genotyped by PCR using the following primer http://www.jimmunol.org/ combinations: mMCP-4 (Mcpt4) (forward primer, 59-CAA GGT Statistical analysis 9 9 CCA ACT AAC TCC CTT TGT GCT CC-3 , wild-type [WT] reverse, 5 - The raw data were exported to the Microsoft Office Excel 2007 software 9 GGT GAT CTC CAG ATG GGC CAT GTA AGG GCG-3 , knockout [KO] program, where an unpaired t test was performed. Results shown are from 9 9 reverse, 5 -GGG CCA GCT CAT TCC TCC CAC TCA TGA TCT-3 ), individual experiments, representative of at least two individual experi- 9 mMCP-6 gene (Tpsb2) (forward primer, 5 -TTT AGC TGG ACT CAG ments. A p value #0.05 was considered statistically significant. GCT GTG CTC CTC ACT-39, WT reverse, 59-CTC CTG AAT TGG AGC TAA CCC TGG GAT TCT-39, KO reverse, 59-GAC CAT GTG ATC GCG CTT CT-39), and CPA3 gene (Cpa3) (forward primer, 59-GGA CTG TTC Results ATC CCC AGG AAC C-39, reverse 1, 59-CTG GCG TGC TTT TCA TTC Generation of mice with deficiency in multiple MC proteases

TGG-39, reverse 2, 59-GTC CGG ACA CGC TGA ACT TG-39). WT and by guest on October 1, 2021 mMCP-4/-6/CPA3–deficient mice were on a C57BL/6J genetic background. To study the role of positively charged proteases in granule Eight- to 10-wk-old mice were used in all experiments. All experiments dynamics, we chose to generate mice with a triple deficiency in were approved by the Local Ethics Committee. chymase, tryptase, and CPA3 by intercrossing mMCP-42/2 (11), 2/2 2/2 Cell culture mMCP-6 (12), and CPA3 (13) mice. The triple deficiency in mMCP-4, mMCP-6, and CPA3 was confirmed by genotyping Bone marrow–derived MCs (BMMCs) (15) and peritoneal cell–derived MCs (PCMCs) (16, 17) were isolated and cultured as previously described. (data not shown). To ascertain that the corresponding protein products were absent, Western blot analysis was carried out. The Western blot analysis analysis was performed on extracts from ear skin tissue and Western blot analysis was performed as described (15). peritoneal cells, with both of these tissues being rich in MCs. As expected, mMCP-4, mMCP-6, and CPA3 were readily detected in b Analysis of -hexosaminidase and protease activities WT peritoneal cell extracts, and mMCP-6 and CPA3 were de- Sample preparation and measurements of enzymatic activities were per- tectable in ear tissue, but they were all absent in corresponding formed as described (15). tissue from the triple KO mice (Fig. 1A). Previous studies have Cell morphology indicated that CPA3 and mMCP-5 show a strong interdependence at the protein level (13, 19), and we therefore decided to assess For toluidine blue staining, peritoneal cells or PCMCs were collected onto whether the levels of mMCP-5 protein were affected in the triple cytospin slides. Samples were fixed with 100% methanol and subsequently stained with toluidine blue solution (0.1% toluidine blue in 0.17 mM NaCl KO mice. As seen in Fig. 1B, mMCP-5 was detected in ear tissue [pH 2]). Samples were rinsed with H2O, dried, and covered with Vector- and in peritoneal cell extracts from WT mice, but was strongly Mount permanent mounting medium (Vector Laboratories, Burlingame, diminished in tissue from triple KO animals, in agreement with CA). Ear tissue was fixed in 4% paraformaldehyde/PBS, embedded in paraf- mMCP-5 being dependent on CPA3 for storage. Hence, at the fin, sectioned, and stained with toluidine blue. Transmission electron micros- copy (TEM) was performed as previously described (9). protein level, the generated mouse strain has a quadruple defi- ciency in mMCP-4, mMCP-5, mMCP-6, and CPA3, that is, all of Proteoglycan analysis the proteases that are known to be stored in complex with ser- Proteoglycan analysis was performed as described with some modifications glycin in MCs (9, 10). (15). Briefly, 107 PCMCs (0.55 3 106 cells/ml) in sulfate-free DMEM The absence of the corresponding proteases was also verified in m 35 medium were labeled biosynthetically with 12.5 Ci/ml of Na2([ S]O4) MCs cultured from bone marrow, that is, BMMCs (data not shown). (PerkinElmer, Waltham, MA). After 24 h the samples were harvested, To study the effects of the protease deletion in MCs with a phe- centrifuged, and the supernatants (corresponding to the medium fraction) were applied to 0.3 ml DEAE Sephacel columns (GE Healthcare, Uppsala, notype that closely resembles that of in vivo–derived MCs, we also Sweden). Pelleted cells were lysed with 500 ml 0.5% Triton X-100 in PBS/ derived MCs by expanding the peritoneal MC population, that is, 2 M NaCl and subsequently diluted to 10 ml with 0.5% Triton X-100. PCMCs (16). As shown in Fig. 1, PCMCs contained high levels of The Journal of Immunology 3933 Downloaded from

FIGURE 1. Levels of proteases in WT and multiprotease-deficient MCs and tissues. Protein extracts were prepared from ear tissue (EAR; corre- sponding to 15 mg tissue), the peritoneal cell compartment (PC; corre- http://www.jimmunol.org/ sponding to 0.36 3 106 cells), and from cultured PCMCs [corresponding to 0.36 3 106 cells (A) or 0.05 3 106 (B)] of WT (+/+) and multiprotease- deficient (2/2) mice as indicated. Samples from protein extracts were subjected to immunoblot analysis for (A) mMCP-6, CPA3, mMCP-4, and (B) mMCP-5 as indicated. n.d., not detected mMCP-4, mMCP-5, mMCP-6, and CPA3, and all of these pro- teases were either absent (mMCP-4, mMCP-6, CPA3; Fig. 1A) or

strongly reduced (mMCP-5; Fig. 1B) in PCMCs developed from by guest on October 1, 2021 multiprotease-deficient mice. MC proteases account for the major part of the total proteolytic activities in skin, peritoneum, and in cultured MCs To evaluate the functional consequences of the multiple protease deficiency, we first assessed the effect of multiple MC protease deficiency on total proteolytic activities in tissues and cultured MCs. As shown in Fig. 2A, trypsin-like activity (measured with S-2288) was readily detected in extracts of total peritoneal cells and ear tissue (Fig. 2B), as well as in extracts from cultured FIGURE 2. Effect of deficiency in multiple MC proteases on total BMMCs and PCMCs from WT mice (Fig. 2C, 2D). In corre- proteolytic activities. Protein extracts were prepared from the peritoneal cell compartment (A), ear skin (B), BMMCs (C), and PCMCs (D). Samples sponding samples from skin, peritoneal cells, and BMMCs from were assayed for total trypsin-like activity using S-2288, total chymo- multiprotease-deficient mice, a profound reduction of trypsin- trypsin-like activity using S-2586, and total CPA activity using M-2245 like activity was seen. However, robust residual trypsin-like activ- (CPA activity toward M2245 results in decreased absorbance). Total ac- ity was seen in multiprotease-deficient PCMCs. Possibly, the high tivities are given as activity per 106 cells (A, C, D) or per milligram wet residual trypsin-like activity in multiprotease-deficient MCs may tissue (B). *p # 0.05, **p # 0.01, ***p # 0.001. be explained by the intact expression of mMCP-7, a serglycin- 2/2 independent tryptase, in the mMCP-6 mouse strain (12). In- proteolytic activities in ear skin tissue, the peritoneal cavity, and in deed, immunoblot analysis verified the presence of mMCP-7 cultured MCs. protein in PCMCs from the multiprotease-deficient mice (data not shown). Chymotrypsin-like activity (measured with S-2586) was The absence of multiple MC proteases does not the affect the clearly detected in ear tissue, peritoneal cell, and PCMC extracts ability of MCs to degranulate from WT mice but was virtually undetectable in corresponding To evaluate whether the multiple protease deficiency affected the tissues/cells from multiprotease-deficient animals (Fig. 2). Fur- ability of MCs to degranulate, we stimulated PCMCs with cal- thermore, whereas CPA-like activity (measured with M-2245) cium ionophore and measured the release of b-hexosaminidase. was clearly detected in WT tissues/cells, CPA-like activity was As seen in Fig. 3, b-hexosaminidase release was similar in WT undetectable in corresponding samples from multiprotease- and multiprotease-deficient PCMCs, suggesting that the simul- deficient mice (Fig. 2). Taken together, these results indicate taneous absence of mMCP-4, mMCP-5, mMCP-6, and CPA3 did that the serglycin-dependent MC proteases account for a major not affect MC functionality in terms of their ability to degra- part of the total trypsin-like, chymotrypsin-like, and CPA-like nulate. The baseline levels of b-hexosaminidase as well as residual 3934 PROTEASE-DEFICIENT MAST CELLS

FIGURE 3. Multiprotease deficiency does not affect the ability of MCs to degranulate. PCMCs from WT or multiprotease-deficient (KO) mice were either left untreated or treated with calcium ionophore A23187 for 150 min. Cell pellets and conditioned media were collected and analyzed for b-hexosaminidase activity (n = 4). *p # 0.05, ***p # 0.001. n.s., Not significant. Downloaded from b-hexosaminidase levels following degranulation were slightly higher in multiprotease-deficient versus WT MCs (Fig. 3). The absence of multiple MC proteases results in distorted granule integrity A widely established method to judge granule integrity is to stain MCs with cationic dyes such as toluidine blue. In mature MCs, such http://www.jimmunol.org/ dyes produce strong metachromatic staining, and we have shown that the metachromatic staining is critically dependent on the presence of serglycin with sulfated heparin side chains within granules (10, 20, 21). As expected, WT MCs in both peritoneum (Fig. 4A) and in ear skin (Fig. 4F) stained strongly with toluidine blue. Furthermore, peritoneal MCs deficient in individual pro- teases showed normal staining properties (Fig. 4B–D). However,

when simultaneously lacking mMCP-4, mMCP-5, mMCP-6, and by guest on October 1, 2021 CPA3, peritoneal MCs exhibited a markedly reduced toluidine blue staining, indicating that the combined protease deficiency FIGURE 4. Multiprotease deficiency causes defective granule integrity caused distorted granule composition. The numbers of peritoneal in MCs. Cytospin slides were prepared from peritoneal cell exudates of A 2/2 B 2/2 C 2/2 D MCs were similar in WT and multiprotease-deficient mice. Strongly WT ( ), mMCP-4 ( ), mMCP-6 ( ), CPA3 ( ), and multi- 2/2 2/2 2/2 E F defective granular staining was also seen in multiprotease-deficient protease-deficient (mMCP-4 ; mMCP-6 ;CPA3 )( ) mice. ( and G) Sections were prepared from ear skin tissue of WT (F) and multi- skin MCs of the ear tissue (Fig. 4G). In fact, the effect of the protease-deficient (G) mice. Cytospin slides and sections were stained with multiprotease deficiency on MC morphology was even more pro- toluidine blue. In (F) and (G), MCs are indicated by arrows. Insets display nounced in ear tissue in comparison with the peritoneal cavity. enlarged images of individual MCs. Note the defective staining of MCs To extend these findings we also performed ultrastructural lacking multiple proteases. Original magnification 3400. analysis using TEM. As depicted in Fig. 5, granules in WT peri- toneal MCs were filled with highly electron dense material that was homogeneously distributed within the granules. In contrast, glycan secretion (not shown), but neither of these observations the granules of multiprotease-deficient MCs had a patchy, pro- reached statistical significance. After liberation of free GAG foundly less homogeneous appearance, and demonstrated an chains from corresponding proteoglycans by alkali treatment, 35 overall reduction in electron density. Hence, the simultaneous ab- a further analysis showed that essentially all of the [ S]-labeled sence of mMCP-4, mMCP-5, mMCP-6, and CPA3 affects the gran- GAGs from both WT and multiprotease-deficient cells were ules at the ultrastructural level. depolymerized by nitrous acid (pH 1.5) (Fig. 6A). This shows that the sulfated GAGs belong to the heparin/HS family rather than The absence of multiple MC proteases results in defective being of CS type (CS is not degraded by nitrous acid [pH 1.5]). storage of heparin in cultured MCs Furthermore, the GAG chains from WT and multiprotease- A potential explanation for the reduced metachromatic staining deficient cells were of similar size (Fig. 6A) and had approxi- (see Fig. 4) is that multiple-protease deficiency causes a reduction mately equal anionic charge density (Fig. 6B). Notably, the GAGs in proteoglycan content of MCs. To assess this possibility we first from both WT and multiprotease-deficient cells eluted at similar labeled PCMCs biosynthetically with [35S]sulfate, which will be positions on anion exchange chromatography as did standard incorporated as either O-sulfate- or N-sulfate groups in the GAG heparin (Fig. 6B). This indicates that the GAGs produced are of side chains of the corresponding proteoglycans. After a 24-h la- high negative charge density, which is a characteristic feature of beling period, proteoglycans were purified. As judged by total heparin as opposed to HS (7). Hence, the sensitivity to depoly- [35S]sulfate incorporation, there was a trend toward a decrease in merization by nitrous acid in combination with the high negative the amount of cell-associated proteoglycans in multiprotease- charge density classifies the [35S]-labeled GAGs produced by deficient MCs accompanied by a trend toward increased proteo- PCMCs as heparin (rather than HS). The Journal of Immunology 3935

FIGURE 5. Effect of multiprotease deficiency on granule ultrastructure. Peritoneal cells were recovered from WT and multiprotease-deficient (KO) mice and were analyzed by TEM. The lower panels represent enlarged

views of MC granules. Note that the absence of multiple MC proteases Downloaded from causes an altered granule ultrastructure.

Importantly, note that the biosynthetic labeling procedure will only detect those proteoglycans that are synthesized during the time frame of the labeling period, but will not account for those that have accumulated in MCs over time. Hence, there is a possibility that the http://www.jimmunol.org/ biosynthetic labeling approach will not reflect the total content of stored proteoglycans in MCs. To provide a more complete picture of the stored proteoglycans of WT versus multiprotease-deficient MCs, we therefore employed an alternative method based on enzymatic digestion of unlabeled GAGs with either heparin FIGURE 6. Characterization of GAGs synthesized by WT and multi- or chondroitinase ABC, followed by RPIP-HPLC analysis for protease-deficient MCs. PCMCs from WT and multiprotease-deficient 35 detection and quantification of formed disaccharides (18). In ad- (KO) mice were biosynthetically labeled with [ S]sulfate, followed by dition to providing a measurement of total GAG content, the purification of labeled proteoglycans and release of GAG chains from the corresponding proteoglycans. (A) GAGs were subjected to size separation by guest on October 1, 2021 enzymatic/RPIP-HPLC method also gives detailed information of using a Superdex 200 column either before (filled symbols) or after (open the disaccharide composition of the respective GAG chains. In symbols) treatment with nitrous acid (HNO [pH 1.5]). (B) GAG chains 35 2 agreement with the [ S]sulfate labeling approach, the enzymatic/ from WT and multiprotease-deficient (KO) PCMCs were subjected to RPIP-HPLC approach revealed that heparin/HS is a dominating anion exchange chromatography. The filled symbols represent the [35S]- GAG synthesized by both WT and multiprotease-deficient PCMCs labeled GAGs from WT and KO cells. The open symbols indicate the as shown by the high content of disaccharide species characteristic elution positions of unlabeled standard CS and heparin, as indicated re- for GAGs of heparin/HS type (Fig. 7A). Notably, among the various spectively (assayed by the carbazole method; monitored at A530 nm). The heparin/HS disaccharide species, there was a striking dominance solid line indicates the concentration of the eluent (LiCl). 2 2 of the trisulfated disaccharide HexA(2-O-SO3 )-GlcNSO3 (6-O- 2 SO3 ) (denoted NS6S2S). A high content of this highly sulfated Although heparin was identified as the major GAG produced by disaccharide is a characteristic feature of heparin as opposed to the PCMCs, a smaller portion of CS was also recovered (∼16% of HS (7). Importantly, the high content of the NS6S2S disaccharide total GAG content) (Fig. 7C). However, there was no reduction species is in agreement with the high overall negative charge den- in the amount of CS when comparing WT and multiprotease- sity as judged by anion exchange chromatography (see Fig. 6B). deficient MCs (Fig. 7C). The CS disaccharide composition was 2 2 Significant amounts of the disulfated HexA-GlcNSO3 (6-O-SO3 ) similar in CS from WT and multiprotease-deficient MCs (Fig. 7C), species (NS6S) were also recovered, whereas low or undetectable being strongly dominated by the monosulfated HexA(4S)-GalNAc 2 2 2 levels of HexA(2-O-SO3 )-GlcNSO3 (NS2S) and of various mono- (4S) and disulfated HexA-GalNAc(4,6-di-O-SO3 ) (6S4S) disac- or nonsulfated species were seen (Fig. 7A). charide species. When comparing the disaccharide profiles of WT versus multiprotease-deficient MCs, it was evident that the multiple- The absence of multiple MC proteases results in defective protease deficiency caused a reduction in the total heparin con- storage of MC heparin in vivo tent, as reflected by a profound reduction of the NS6S2S and NS6S Next, we investigated whether the multiprotease deficiency causes disaccharide species (Fig. 7A). However, there were no significant similar effects on the storage of MC heparin in vivo as those differences in the relative distribution of the various disaccharide observed in cultured PCMCs. For this purpose we prepared extracts variants when comparing heparin from WT versus multiprotease- of whole ear skin tissue from WT and multiprotease-deficient deficient MCs (Fig. 7B), indicating that the multiple protease animals and analyzed their contents and structure of heparin/HS deficiency did not affect the structural properties of the heparin. and CS using the enzymatic/RPIP-HPLC method. Importantly, Hence, these findings indicate that the multiprotease deficiency in note that this approach accounts for all of the GAGs present in the MCs causes a profound reduction in the amount, but not structure, ear skin, that is, not only GAGs produced by MCs. As seen in Fig. of heparin stored within the secretory granules. 7F, CS constituted the main type of GAG in whole ear skin tissue 3936 PROTEASE-DEFICIENT MAST CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. Effect of multi-MC protease deficiency on content and structure of GAGs. GAGs were isolated from PCMCs (A–C) and ear skin tissue (D–F) from WT and multiprotease-deficient mice. GAGs were depolymerized to disaccharide size by treatment with chondroitinase ABC and heparinase I, II, and III, respectively. Disaccharides were separated by RPIP-HPLC. (A and D) The disaccharide composition of heparin/HS recovered from PCMCs (A) and ear skin (D) of WT and multiprotease-deficient (KO) mice as indicated. Disaccharide composition is given as nanomoles per 106 cells (PCMCs) or per milligram wet tissue (ear skin). (B and E) Percentage of disaccharide variants in heparin/HS from PCMCs (B) and ear skin (E). (C and F) The disaccharide composition of CS recovered from PCMCs (C) and ear skin (F) of WT and multiprotease-deficient (KO) mice as indicated. The disaccharide composition is given as nanomoles per 106 cells (PCMCs) or per milligram wet tissue (ear skin). Heparin/HS disaccharides (A–D): NAc, HexA-GlcNAc; NS, HexA- 2 2 2 2 2 2 2 GlcNSO3 ; 6S, HexA-GlcNAc(6-O-SO3 ); 2S, HexA(2-O-SO3 )-GlcNAc; NS6S, HexA-GlcNSO3 (6-O-SO3 ); NS2S, HexA(2-O-SO3 )-GlcNSO3 ; - 2 2 2 2 2S6S, HexA(2-O-SO3 )-GlcNAc(6-O-SO3 ); NS6S2S, HexA(2-O-SO3 )-GlcNSO3 (6-O-SO3 ). CS disaccharides (C, F): OS CS/Hya, nonsulfated di- 2 2 2 saccharides of CS or hyaluronan origin; 4S, HexA-GalNAc(4-O-SO3 ); 6S, HexA-GalNAc(6-O-SO3 ); 2S, HexA(2-O-SO3 )-GalNAc; 6S4S, HexA- 2 2 2 2 2 2 GalNAc(4,6-di-O-SO3 ); 4S2S, HexA(2-O-SO3 )-GalNAc(4-O-SO3 ); 6S2S, HexA(2-O-SO3 )-GalNAc(6-O-SO3 ); 6S4S2S, HexA(2-O-SO3 )-Gal- 2 NAc(4,6-di-O-SO3 ). Mean 6 SEM; n =3.*p # 0.05, **p # 0.01, ***p # 0.001. from both WT and multiprotease-deficient mice, accounting for and NS6S (83% reduction) (Fig. 7D), whereas the levels of NS2S ∼91 and ∼97% of the total GAG content, respectively. However, and various non- or monosulfated disaccharides were unaffected significant amounts of heparin/HS disaccharides were also detected (Fig. 7D). These findings indicate that the absence of multiple MC (Fig. 7D, 7E). Among these, similar to PCMCs, the trisulfated proteases causes a profound reduction in the amount of stored NS6S2S was the dominating species and NS6S was also present in heparin in MCs in vivo. appreciable amounts (although considerably lower than NS6S2S) (Fig. 7D). However, differing from PCMCs, significant amounts of Discussion various non- and monosulfated disaccharide species and NS2S In this study, to our knowledge we describe for the first time the were observed (Fig. 7D). generation of a mouse strain simultaneously lacking multiple A comparison of the GAG content of ear skin from WT versus proteases stored within MCs of the connective tissue type, that is, multiprotease-deficient animals showed that the CS content and mMCP-4, mMCP-6 and CPA3. Previous studies have shown that disaccharide composition was indistinguishable (Fig. 7F). In CPA3 and mMCP-5 show a strong interdependence for storage contrast, the multiprotease deficiency caused a profound reduction within MCs; that is, the knockout of CPA3 causes a profound in the amounts of heparin/HS-derived disaccharides (Fig. 7D, 7E). impairment of mMCP-5 storage (at the protein level) and vice In particular, the multiprotease deficiency caused a dramatic re- versa (13, 19). Consequently, MCs of the multiprotease-deficient duction in those disaccharides that were shown to be characteristic mouse strain additionally lack mMCP-5 at the protein level. Al- for MCs (see data for PCMCs), that is, NS6S2S (82% reduction) together, the generated mouse strain thus lacks a panel of stored The Journal of Immunology 3937 proteases, as a result of both genetic targeting and posttransla- as a contradiction. Importantly, however, note that the biosynthetic tional effects. Importantly, note that all of the genetically targeted labeling approach only accounts for those GAGs that are produced proteases (i.e., mMCP-4, mMCP-6, and CPA3) show an MC- within the limited time frame of the labeling experiment, whereas specific expression pattern (4, 22). Hence, any effects of the the enzymatic/RPIP-HPLC approach accounts for all of the GAGs multiprotease deficiency can be attributed to effects related to that have accumulated in MCs as they mature during several MCs. We anticipate that the multiprotease-deficient strain gener- weeks. An important conclusion is thus that the actual synthesis of ated in this study will be an important tool to assess the global heparin is not influenced by the multiple protease absence, as effects of the MC proteases. reflected by the short time frame [35S]sulfate labeling approach. Previous studies have shown that the storage of mMCP-4, Rather, the reduced total heparin content, as shown by the mMCP-5, mMCP-6, and CPA3 is strongly dependent on sergly- enzymatic/RPIP-HPLC approach, is likely due to long-term ef- cin, whereas the storage of mMCP-7 and of the mucosal MC fects of the multiprotease deficiency on the ability of MCs to store protease mMCP-1 is serglycin-independent (9, 10, 20, 21). The heparin. mice generated in this study thus lack all of the MC-specific Interestingly, although CS synthesis in cultured MCs was un- proteases that are known to be dependent on serglycin for storage. detectable using the [35S]sulfate labeling approach, CS disac- Most likely, the dependence of these proteases on serglycin is charides in appreciable amounts were detected when applying the explained by electrostatic interactions between the GAG chains of enzymatic/RPIP-HPLC methodology. A likely explanation for this serglycin and the respective proteases. In support of this notion, seeming discrepancy is that CS might accumulate in MCs at early serglycin expressed in a MC context has a remarkably high an- stages of maturation, that is, at stages preceding the time point ionic charge density and, conversely, the serglycin-dependent MC when the [35S]sulfate labeling was performed. Another potential proteases display high positive surface charges and bind strongly explanation would be that CS synthesis in MCs occurs at a slower Downloaded from to GAGs, in particular to heparin, in purified systems (23–26). rate than does the synthesis of heparin. Hence, the short time Moreover, MCs lacking N-deacetylase/N-sulfotransferase 2, an frame of the [35S]sulfate labeling procedure would predominantly required for sulfation of the heparin chains attached to the detect biosynthetically labeled heparin rather than CS. serglycin core protein, have similar defects in protease storage as When comparing the heparin/HS disaccharide profiles of PCMCs those seen in serglycin2/2 MCs (21, 27, 28). In further support for versus skin tissue, some notable differences were observed. In an electrostatic mechanism, there is a strong positive correlation the heparin/HS from PCMCs, there was a clear dominance of http://www.jimmunol.org/ between the net positive charge of various MC chymases and their NS6S2S and NS6S, suggesting that these two species are signature extent of dependence on serglycin for storage (22). disaccharides characteristic for mature MCs. In skin tissue, NS6S2S A major finding in this study was that the simultaneous absence and NS6S were also found in high amounts but, in contrast to of serglycin-dependent proteases caused a major distortion of the PCMCs, appreciable amounts of various non- and monosulfated secretory granule integrity. This was indicated by the profound species as well as NS2S were also found. Because the GAGs isolated reduction in toluidine blue staining of multiprotease-deficient MCs from skin extract represent a mixture of GAGs from all of different and through the defective granule ultrastructure as seen by TEM. cell types found in the skin, we cannot with certainty establish the In contrast to the multiprotease-deficient MCs, MCs with single cellular sources of the respective disaccharide species. However, by guest on October 1, 2021 protease deficiencies exhibited normal granule morphology. This because NS6S2S and NS6S were profoundly reduced in skin from suggests that effects on granule integrity due to the lack of indi- the multiprotease-deficient mice, and considering that high ex- vidual proteases can be rescued by compensation provided by other pression of these two disaccharide units is characteristic for MCs, it proteases, whereas no compensatory mechanism is available that appears reasonable that NS6S2S and NS6S found in skin tissue are can rescue granule integrity when all of the serglycin-binding pro- MC derived. In contrast, the levels of the various non-/monosulfated teases are absent simultaneously. species and NS2S were not affected by the multiprotease deficiency, Because toluidine blue is known to stain anionic compounds and it thereby appears conceivable that these latter disaccharide such as proteoglycans, a likely explanation for the reduced tolu- species arise from low-sulfated HS proteoglycans produced by non- idine blue staining was that the protease deficiency results in effects MCs (e.g., fibroblasts). on the secretory granule proteoglycans. In agreement with this A likely explanation for the reduction in MC heparin due to the notion, we show that the simultaneous absence of serglycin-binding multiple protease absence is that the storage of heparin is dependent proteases in MCs causes a profound reduction in stored heparin, as on electrostatic interactions with the positively charged proteases. seen both in cultured MCs and in vivo. Importantly, the reduction in According to such a scenario, a reduction in positive electric charge heparin was not a consequence of a gross effect on total GAG (displayed by the MC proteases) would thus disturb the electric synthesis, as there was no reduction in the amounts of GAGs of CS charge balance within granules, leading to impaired storage of type in multiprotease-deficient MCs. Hence, the deletion of the heparin and thereby a distortion of the granule composition. Con- various serglycin-dependent proteases causes a specific defect in versely, previous studies have demonstrated that a reduction in heparin storage. Although we at present cannot explain the dif- negative charge, either due to the lack of serglycin (9, 10) or sulfated ferential effect on heparin versus CS, one possibility would be that heparin (20, 21, 27), causes severe defects in protease storage. In the CS recovered from the cultured MCs may be derived from line with these findings is also a previous report showing that MCs proteoglycans located in cellular compartments distinct from lacking histamine exhibit reduced storage of granule proteases and granules, for example, at the cell surface. Alternatively, as sup- proteoglycans (29). Although not experimentally proven, a likely ported by experimental data (21, 23, 27, 28), the MC proteases explanation for the latter findings may be that a reduction in his- may interact preferentially with heparin versus CS. A reduction in tamine (positively charged) has a downstream adverse effect on the the levels of proteases may therefore have a more limited effect on storage of negatively charged serglycin proteoglycans, and that the CS as compared with the effects on heparin storage. Intriguingly, reduced serglycin storage in turn results in impaired storage of whereas the enzymatic/RPIP-HPLC method revealed a large re- the positively charged proteases. duction of stored heparin in multiprotease-deficient versus WT Taken together, the present findings together with previous MCs, such an effect was not apparent when biosynthetically la- observations suggest that granule composition in MCs is dependent beling the cells with [35S]sulfate. This may at first glance appear on a dynamic interaction betweenvarious differently charged granule 3938 PROTEASE-DEFICIENT MAST CELLS compounds. According to this model, a reduction in either nega- 2005. Loss of histochemical identity in mast cells lacking . Mol. Cell. Biol. 25: 6199–6210. tively or positively charged granule constituents would cause im- 14. Ghildyal, N., D. S. Friend, R. Freelund, K. F. Austen, H. P. McNeil, V. Schiller, paired storage of compounds of opposite charge. and R. L. Stevens. 1994. 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