Heparan Sulfate 6-O-Sulfotransferase Isoform-dependent Regulatory Effects of Heparin on the Activities of Various Proteases in Mast Cells and the Biosynthesis of 6-O-Sulfated Heparin*

Background: Heparin regulates mast cell proteases. Results: Both HS6ST-1 and HS6ST-2 are involved in 6-O-sulfation of heparin. The contents of tryptase and CPA in mast cells depend on 6-O-sulfation of heparin but chymase does not. Conclusion: The 6-O-sulfation pattern regulates differently the storage of MC-specific proteases. Significance: The fine structure of heparin may be essential for MC homeostasis. Heparan sulfate 6-O-sulfotransferase (HS6ST) is an enzyme involved in heparan sulfate (HS) biosynthesis that transfers a sulfate residue to position 6 of the GlcNAc/GlcNSO3 residues of HS, and it consists of three isoforms. Heparin, the highly sulfated form of HS, resides in connective tissue mast cells and is involved in the storage of mast cell proteases (MCPs). However, it is not well understood which isoform(s) of HS6ST participates in 6-O-sulfation of heparin and how the 6-O-sulfate residues in heparin affect MCPs. To investigate these issues, we prepared fetal skin-derived mast cells (FSMCs) from wild type (WT) and HS6ST-deficient mice (HS6ST-1−/−, HS6ST-2−/−, and HS6ST-1−/−/HS6ST-2−/−) and determined the structure of heparin, the protease activity, and the mRNA expression of each MCP in cultured FSMCs. The activities of tryptase and carboxypeptidase-A were decreased in HS6ST-2−/−-FSMCs in which 6-O-sulfation of heparin was decreased at 50% of WT-FSMCs and almost lost in HS6ST-1−/−/HS6ST-2−/−-FSMCs, which lacked the 6-O-sulfation in heparin nearly completely. In contrast, chymase activity was retained even in HS6ST-1−/−/HS6ST-2−/−-FSMCs. Each MCP mRNA was not decreased in any of the mutant FSMCs. Western blot analysis showed that tryptase (mMCP-6) was almost absent from HS6ST-1−/−/HS6ST-2−/−-FSMCs indicating degradation/secretion of the enzyme protein. These observations suggest that both HS6ST-1 and HS6ST-2 are involved in 6-O-sulfation of heparin and that the proper packaging and storage of tryptase, carboxypeptidase-A, and chymase may be regulated differently by the 6-O-sulfate residues in heparin. It is thus likely that 6-O-sulfation of heparin plays important roles in regulating MCP functions.

Mast cells (MCs) are classified into connective tissue-type mast cells (CTMCs) and mucosal mast cells (22). Each mast cell type is differentiated by the types of glycosaminoglycans and MC-specific proteases present and is involved in a variety of physiological and pathological processes (23)(24)(25)(26)(27)(28)(29)(30). CTMCs mainly produce heparin, whereas mucosal mast cells primarily produce highly sulfated chondroitin sulfate (23,31,32). Biosynthesis of heparin in mast cells and the structure of heparin required for the interaction with granular proteins are largely unknown. NDST-2, C5-epimerase, and HS3ST-1 have been reported to be involved in the biosynthesis of heparin because NDST-2-null mice (33,34), C5-epimerase-null mice (35), and HS3ST-1-null mice (36) generate abnormal heparin (heparin with low sulfate or low IdoUA, low anticoagulant activity); however, it has not been studied which isoform(s) of HS6ST catalyzes the addition of the 6-O-sulfate residues in heparin in vivo. From studies of NDST-2-null mice, it is evident that heparin is required for the storage of several proteases in the granules; however, it is currently unknown as to why different proteases require 6-O-sulfate residues in heparin for their stable packaging and storage in the granules. In this study, we isolated fetal skin-derived mast cells (FSMCs) from HS6ST-deficient mice (HS6ST-1 Ϫ/Ϫ , HS6ST-2 Ϫ/Ϫ , and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ double KO), and we then investigated the role of these sulfotransferases in the biosynthesis of heparin. We also determined which MCPs were affected by deficient 6-O-sulfation of heparin. As the results, we demonstrated first that 6-O-sulfation of heparin was nearly abolished in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs and then that although some chymase activities remained, tryptase (mMCP-6) and CPA activities were almost completely absent in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs, suggesting important roles of HS6ST-2 not only in the heparin 6-O-sulfation but also in the CTMC functions.
Isolation of Fetal Skin-derived Mast Cells-FSMCs were prepared from 15.5-day-old wild type (WT), HS6ST-1 Ϫ/Ϫ , HS6ST-2 ϩ/Ϫ , HS6ST-2 Ϫ/Ϫ , and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ embryos of the C57BL/6 mouse strain according to Yamada et al. (37) with a slight modification. Briefly, internal organs, limbs, tails, and heads were removed from the embryos, and the trunks were cut into small pieces that were digested with 0.25% trypsin in PBS containing 0.53 mM EDTA for 10 min at 37°C. The residues were repeatedly digested, and then the supernatant was passed through a 100-m mesh. The cells in the filtrate were collected by centrifugation at 120 ϫ g for 10 min and were subjected to erythrolysis in ACK lysing buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM EDTA). After repeated washing in Hanks' balanced salt solution, the cell pellets were suspended at 5 ϫ 10 4 cells/ml in RPMI 1640 medium containing 10 ng/ml IL-3, 10 ng/ml stem cell factor, 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM nonessential amino acids, 10 mM sodium pyruvate, 25 mM HEPES buffer, 50 M 2-mercaptoethanol, and penicillin/streptomycin and cultured at 37°C in a CO 2 incubator for 4 weeks. After incubation, nonadherent cells were harvested and then layered on a Percoll density gradient and centrifuged. The cells that pelleted at the bottom were used as FSMCs.
Isolation and Structural Analysis of Glycosaminoglycans-The ears from 8-week-old WT and HS6ST-2 Ϫ/Ϫ mice were cut into small pieces, defatted with acetone, and dried. The FSMCs and the ear samples were suspended in 0.2 M NaOH and incubated for 16 h at room temperature, neutralized with 4 M acetic acid, and then incubated at 37°C for 2 h in 50 mM Tris-HCl (pH 7.5) containing DNase I, RNase A, and 10 mM MgCl 2 . Subsequently, the cells were subjected to proteinase (Actinase-E; Sigma) digestion at a final concentration of 1 mg/ml and incubated for 2 h at 37°C. The reaction was stopped by heating at 100°C for 5 min, and the samples were centrifuged at 14,000 ϫ g for 10 min to remove any insoluble materials. The supernatants were diluted with an equal volume of 20 mM Tris-HCl buffer (pH 7.5) and loaded onto a 0.3-ml DEAE-Sephacel column equilibrated with the same buffer. The columns were washed with 10 column volumes of buffer containing 0.2 M NaCl and then eluted with 4 column volumes of 2 M NaCl in 20 mM Tris-HCl buffer (pH 7.5). Next, 3 volumes of cold 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate and 1 mM EDTA were added to the eluates, and then the GAGs were recovered by centrifugation. An aliquot of the GAG was digested with a mixture of 0.2 milliunits of heparitinase I, 0.1 milliunits of heparitinase II, and 0.2 milliunits of heparinase in 50 l of 50 mM Tris-HCl buffer (pH 7.5), 1 mM CaCl 2 , and 5 g of bovine serum albumin at 37°C for 2 h. After filtration of the digests in an Ultrafree MC (5-kDa molecular mass cutoff filtering unit; Millipore Corp., Bedford, MA), the unsaturated disaccharide products in the filtrates were analyzed by reverse phase ion pair chromatography using a Sensyu Pak Docosil column with a fluorescence detector (Model RF-10AxL, Shimadzu Co. Kyoto, Japan) according to Toyoda et al. (38) with slightly modified elution conditions, in which unsaturated disaccharide products after separation by the chromatography were reacted with 2-cyanoacetoamide (Wako, Osaka, Japan) as a post-labeling reagent.
Mono Q Column Chromatography-The crude GAGs from the ears of WT and HS6ST-2 Ϫ/Ϫ mice, prepared as described above, were digested with 100 milliunits of chondroitinase ABC at 37°C for 1 h to digest the chondroitin sulfate (CS). The chondroitinase ABC-resistant GAGs were precipitated with 3 volumes of cold 95% ethanol containing 1.3% potassium acetate and 1 mM EDTA and recovered by centrifugation at 14,000 ϫ g for 30 min. The precipitates were dissolved in 0.5 ml of 0.1 M NaCl in 50 mM Tris-HCl (pH 7.2) (Buffer A) and were applied to a Mono Q column (GE Healthcare) equilibrated with Buffer A. The column was developed with Buffer A for 5 min (0 -5 min), then with a linear gradient from 0. precipitated by the addition of 3 volumes of cold 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate and 1 mM EDTA, followed by centrifugation. The disaccharide composition of each fraction was analyzed as described above. The yield of each fraction was calculated from the total amount of disaccharides obtained from HPLC analysis.
Western Blot Analysis-FSMC proteins were extracted in PBS containing 2 M NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor mixture (1 tablet per 10 ml, complete Mini; Roche Applied Science) for 30 min on a rotary shaker, and the extracts were clarified by centrifugation (11,000 ϫ g for 30 min). Protein concentration was determined using a Dc protein assay kit (Bio-Rad), and 60 g of protein were separated by 10% SDS-PAGE. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were immunoblotted with anti-mouse tryptase (MCP-6) antibody. The blot was developed using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Western Lightning Plus; PerkinElmer Life Sciences). The bands were detected using luminescent image analyzer (Fuji Film, Tokyo, Japan).
Expression Levels of MCPs and HS6STs mRNAs by RT-PCR-Total RNA from WT, HS6ST-2 Ϫ/Ϫ , and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs was isolated using TRIzol (Invitrogen) and purified (PureLink RNA mini kit; Invitrogen). The reverse-transcription reaction was performed using the high capacity cDNA archive kit (Applied Biosystems, Foster City, CA) and 1.0 g of total RNA as the template. The expression levels of the genes were semi-quantitatively determined by RT-PCR using the primer pairs shown in Table 1. The data were obtained from two independent experiments. Quantitative RT-PCR was performed using specific primer pairs and SYBR Green (Takara, Shiga, Japan) according to the manufacturer's protocol. Each transcript was normalized to reactions using ␤-actin-specific primer pairs (Takara, Shiga, Japan). The PCR products were analyzed in real time using the ABI Prism 7700 system. The values were obtained in two independent experiments performed in triplicate.

Sequence Forward
Reverse Assay of the Activities of Tryptase, Chymase, and Carboxypeptidase A-FSMCs were solubilized in lysis buffer (PBS containing 2 M NaCl and 0.5% Triton X-100), with 100 l of lysis buffer, 1-2 ϫ 10 5 FSMCs. In a final volume of 120 l, the reaction mixtures contained 10-l aliquot of the extracts containing 1 g of protein, 20 l (S-2586 and S-2288) or 40 l (M-2245) of a 1.8 mM aqueous solution of chromogenic substrates. S-2586 (Chromogenix, Molndal, Sweden), S-2288 (Chromogenix, Milano, Italy), and M-2245 (Bachem AG, Bubendorf, Switzerland) were used to determine the activity of chymotrypsinlike proteases (chymase), trypsin-like proteases (tryptase), and CPA, respectively. The reaction mixtures were incubated at 37°C. The activities were monitored as the absorbance at 405 nm. To examine the effects of exogenous heparin on protease activity, heparin was added to the reaction mixtures at three different concentrations (0.08, 0.8, and 8 g/ml).
Histocytochemistry of Cultured Fetal Skin Mast Cells-Fiveweek-old cultured skin mast cells were placed into a 4-well slide glass and centrifuged at 30 ϫ g for 5 min. The medium was carefully aspirated, and the cells were fixed with methanol, acetic acid, 10% formaldehyde (85:5:10) for 30 min at 4°C (39). The cells were double-stained with Alcian blue and safranin O and reacted with naphthol AS-D chloroacetate to detect the chloroacetate esterase activity (34).

Effect of HS6ST-2 Null Mutations on 6-O-Sulfation of Ear
Heparin-We previously showed that the IdoUA(2SO 4 )-GlcNSO 3 (6SO 4 ) contents in HS/heparin from mouse ears of WT mice and HS6ST-1-deficient mice were nearly identical and that only the GlcNAc-6SO 4 residues were significantly reduced in the ears of HS6ST-1 Ϫ/Ϫ mice (11). Considering that CTMCs are abundant in the ear, these observations suggest that HS6ST isoform(s) other than HS6ST-1 may be involved in 6-O-sulfation of heparin. Alternatively, both HS6ST-1 and other isoform(s) may be involved in 6-O-sulfation of heparin in WT mice, and the other isoform(s) may compensate for the deletion of HS6ST-1. In this study, we examined whether or not HS6ST-2 contributes to the 6-O-sulfation of HS/heparin in the ear using HS6ST-2 Ϫ/Ϫ mice. Disaccharide composition analysis of HS/heparin obtained from the ear showed that ⌬Di-(N,6,2)triS derived from IdoUA(2SO 4 )-GlcNSO 3 (6SO 4 ) and ⌬Di-(N,6)diS derived from HexA-GlcNSO 3 (6SO 4 ) were both markedly reduced in HS/heparin from HS6ST-2 Ϫ/Ϫ mice, compared with those in wild type mice. In contrast, ⌬Di-(N,2)diS derived from HexA(2SO 4 )-GlcNSO 3 was increased in HS/heparin from HS6ST-2 Ϫ/Ϫ mice (Fig. 1A). These data suggest that although HS6ST-2 is involved in 6-O-sulfation of heparin, other isoforms also contribute to 6-O-sulfation of heparin because IdoUA(2SO 4 )-GlcNSO 3 (6SO 4 ) from HS/heparin of HS6ST-2 Ϫ/Ϫ mice was about 50% of that from HS/heparin of WT mice. To further clarify the involvement of HS6ST-2 in the 6-O-sulfation of heparin, we tried to separate HS and heparin from each other by anion exchange chromatography (Mono Q column). Under the conditions described under "Experimental Procedures," HS (bovine kidney), shark cartilage, and heparin standards eluted from the column at 36.7, 42.5, and 45.6 min, respectively. HS/heparin obtained from the ear of WT or HS6ST-2 Ϫ/Ϫ mice were separated into six fractions by this column as follows: fraction I (30.5-33.5 min), fraction II (34 -36.5 min), fraction III (37-41 min), fraction IV (41.5-44 min), fraction V (44.5-46 min), and fraction VI (46.5-49 min). Fractions I to VI were then subjected to disaccharide analysis. The disaccharide compositions of these fractions are shown in Fig. 1C. In fraction VI from WT mice, ⌬Di-(N,6,2)triS and ⌬Di-0S derived from the GlcUA-GlcNAc unit were 64% and less than 5%, respectively, of the total disaccharides. Such a disaccharide composition is characteristic of a typical heparin. In contrast, the ⌬Di-(N,6,2)triS of fraction VI from HS6ST-2 Ϫ/Ϫ mice was 39% of the total disaccharides. The decrease in the proportion of ⌬Di-(N,6,2)triS was accompanied by an increase in the proportion of ⌬Di-(N,2)diS. The total amounts of HS/heparin in these fractions were calculated from the yield of each disaccharide. As shown in Fig. 1B, fraction VI from WT mice accounted for 58% of the total HS/heparin, whereas fraction VI from HS6ST-2 Ϫ/Ϫ mice accounted for less than 9% of the total HS/heparin. In addition, the proportions of fraction IV and V from HS6ST-2 Ϫ/Ϫ mice were higher than those from WT mice. These observations support the idea that HS6ST-2 is mainly involved in 6-O-sulfation of heparin.
Heparan Sulfate 6-O-Sulfotransferases-1 and -2 Are Involved in 6-O-Sulfation of Heparin-We first confirmed that the FSMCs prepared according to Yamada et al. (37) possessed the characteristics of CTMCs; WT-FSMCs were double-stained with safranin O and Alcian blue ( Fig. 2A) and reacted with AS-D to detect chloroacetate esterase activity (Fig. 2B). Safranin O stains (in red) heparin, a marker of connective tissue-type mast cells, but it does not stain chondroitin sulfate. In contrast, Alcian blue stains both heparin and chondroitin sulfate. Most WT-FSMCs were strongly stained with safranin O, indicating that heparin is abundant in these cells. Chloroacetate esterase activity is a mast-cell marker. WT-FSMCs had high esterase activity. These results indicated that most FSMCs appear to be CTMCs, as described by Yamada et al. (37).
We then confirmed that the defect in HS6ST-1 did not alter the 6-O-sulfation of heparin/HS synthesized in FSMCs as was observed in the HS/heparin obtained from the ear. We isolated glycosaminoglycans from WT and HS6ST-1 Ϫ/Ϫ -FSMCs and analyzed their disaccharide compositions by digestion with a heparanase-I, heparanase-II, and heparinase mixture (Fig. 3A). In the digested products from the glycosaminoglycans obtained from WT-FSMCs, the proportions of ⌬Di-(N,6,2)triS, ⌬Di-0S, and ⌬Di-6S were 51-60%, 2-9%, and less than 2%, respectively, of total disaccharides. Undigested glycosaminoglycans, mainly consisting of chondroitin sulfate, accounted for only 4.7 Ϯ 1.2% of the heparin/HS in the glycosaminoglycans, again indicating that the FSMCs were CTMCs (37,39). The disaccharide composition of the glycosaminoglycans isolated from HS6ST-1 Ϫ/Ϫ -FSMCs was nearly the same as that of the glycosaminoglycans obtained from WT-FSMCs, although the proportion of ⌬Di-(N,6,2)triS was slightly lower. These results also indicated that, as was observed in the ear, either HS6ST-1 barely contributed to the 6-O-sulfation of heparin in the FSMCs or the absence of HS6ST-1 was compensated by other HS6ST isoforms.

Effect of 6-O-Sulfation in Heparin on MCP Activities
To determine whether the defective 6-O-sulfation of heparin/HS affected CS biosynthesis in the FSMCs, we digested the glycosaminoglycans obtained from WT, HS6ST-2 ϩ/Ϫ , HS6ST-2 Ϫ/Ϫ , and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs with chondroitinase ABC, and we analyzed the disaccharide composition of CS (Fig. 3C). In CS from WT and all three mutant FSMCs, the total ⌬Di-4S, which was derived from GlcUA-GalNAc(4SO 4 ), and ⌬Di-diS E , which was derived from GlcUA-GalNAc(4,6-SO 4 ), was nearly constant (92-94% of total disaccharides) among these glycosaminoglycans, indicating that the major parts of the repeating unit in these CSs bear a 4-O-sulfate group and that the degree of 4-O-sulfation was not altered in the three mutant FSMCs. In contrast, the ratio of ⌬Di-diS E /(⌬Di-4S ϩ⌬Di-diS E ) varied slightly and was 0.18 for WT-CS and 0.13 for HS6ST-1 Ϫ/ Ϫ /HS6ST-2 Ϫ/Ϫ -CS. These values are much lower than those observed in BMMCs (0.28) (40), which are thought to be immature mast cells. These results indicated that 6-O-sulfation of the GalNAc(4SO 4 ) residues of chondroitin sulfate was only slightly affected by the defects in the 6-O-sulfation of heparin/HS.
In a parallel experiment, we analyzed the expression profiles of Hs6st-1, Hs6st-2, and Hs6st-3 mRNA using semi-quantitative RT-PCR (Fig. 4). It is evident that Hs6st-1 and Hs6st-2 are the predominant isoforms in WT-FSMCs. We previously showed that HS6ST-2 is present as the long form and the one spliced form (HS6ST-2S) (8,9). In the RT-PCR used in this study, the transcript sizes for the long form and the short form of HS6ST should be 507 and 387 bp, respectively. The expression profiles of Hs6st-2 mRNA in WT-FSMCs suggested that more than 85% of the mRNA was a short form (see the major band corresponding to the short form and a faint band above the major one corresponding to the long form in Fig. 4A). The profiles also showed no expression of either form of Hs6st-2 mRNA in HS6ST-2 Ϫ/Ϫ and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs (Fig. 4B). The expression of Hs6st-3 mRNA was barely detected in WT and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs, indicating that the expression level of heparan sulfate 6-O-sulfotransferase-3 gene (Hs6st-3) is very low in CTMCs. The expression level of 3-Osulfotransferase-1 (Hs3st-1) did not differ among WT-, HS6ST-2 Ϫ/Ϫ -, and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs. In addition, N-deacetylase/N-sulfotransferase-2 (Ndst-2), another important heparin biosynthetic enzyme, which when disrupted leads to the production of abnormal heparin (33,34), was expressed in mutant FSMCs nearly at the same level as in the WT-FSMCs. These data appear to be consistent with the observation that the N-sulfate residue content in the HS/heparin chains in the WT and mutant FSMCs was nearly equal. Serglycin transcript, to which heparin is attached, was also expressed in the mutant FSMCs at the same level as in the WT-FSMCs. Taken together, these results suggest that N-sulfation of heparin and the expression of serglycin core protein were not affected by deletion of HS6ST-1 and/or HS6ST-2. Because structural differences in heparin were barely detected between HS6ST-1 Ϫ/Ϫ -FSMCs and WT-FSMCs, and the expression of serglycin was not altered by deletion of HS6ST-1 compared with WT, 3 it is likely that the interaction of mast cell proteases with serglycin in the granules of HS6ST-1 Ϫ/Ϫ -FSMCs is similar to that in the granules of WT-FSMCs. Therefore, the effects of deletion of HS6ST-1 on the expression and storage of mast cell proteases were not analyzed in this study.

Activities of Various Mast Cell Proteases Are Regulated Differently by the 6-O-Sulfate Residues in Heparin-Previous in
vivo studies using Ndst-2-deficient mice (33,34) showed that the MC proteases interacted with heparin and that the mutant mice had very low sulfated heparin, suggesting the sequential 3 H. Habuchi, unpublished data. reaction of heparin modification, namely that the N-deacetylation/N-sulfation of GlcNAc residues catalyzed by NDST-2 is the first step in the modification, followed by 2-O-sulfation of HexA residues and 6-O-sulfation of GlcNSO 3 residues. Therefore, those studies using NDST-2-deficient mice suggested that the sulfation of heparin is important for the interaction between heparin and mast cell proteases but provided no evidence for the roles of O-sulfate residues in heparin. As indicated above, we found that deletion of HS6ST-2 or deletion of both HS6ST-1 and HS6ST-2 caused a decrease or disappearance of 6-O-sulfated disaccharide units in heparin, whereas deletion of HS6ST-1 alone did not. Therefore, we expected that deletion of HS6ST-2 or deletion of both HS6ST-1/HS6ST-2 would reveal the role of 6-O-sulfated residues of heparin in the protease-heparin interactions in mast cell granules.
However, there are alternative explanations, for example, heparin with the altered structure synthesized in the mutant FSMCs might not be sufficient to support full protease activity. To determine such a possibility, we investigated the effects of normal heparin on the protease activities. Increasing concentrations of heparin (0.08, 0.8, and 8 g/ml) were added to the reaction mixture containing the extracts from HS6ST-1 Ϫ/Ϫ / HS6ST-2 Ϫ/Ϫ -FSMCs, and no increase in the activities of tryptase, CPA, or chymase were observed (Fig. 5), suggesting that the abnormal structure of heparin did not cause a reduction of the protease activities in the extract of mutant FSMCs.
Considering these results together, it is likely that the 6-Osulfate residues in heparin are essential for the proper packaging and/or storage of active tryptase (mMCP-6) and CPA in CTMCs and that different structures in heparin are required for the interaction with chymases.  D). A and C, expression of each mRNA was measured by semi-quantitative RT-PCR using primer pairs as described under "Experimental Procedures." The expression level of ␤-actin was used as a control. The mRNA expression was compared between WT and HS6ST-2 Ϫ/Ϫ FSMCs (A) or between HS6ST-2 Ϫ/Ϫ and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs (C). B and D, mRNA expression was quantitatively measured using SYBR Green according to the manufacturer's protocol, and expression levels were normalized to ␤-actin expression as described under "Experimental Procedures." The mRNA expression was compared between WT and HS6ST-2 Ϫ/Ϫ -FSMCs (B) or between HS6ST-2 Ϫ/Ϫ and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs (D). Relative mRNA expression values were obtained from two independent experiments that were performed in triplicate, and error bars represent the standard deviation. FIGURE 7. Immunoblot analysis of tryptase (mMCP-6) from HS6ST-1 ؊/؊ / HS6ST-2 ؊/؊ -FSMC. Proteins were extracted with a high salt buffer containing Triton X-100, EDTA, and a mixture of protease inhibitors. Sixty micrograms of protein was subjected to 10% SDS-PAGE, and the immunoblots were probed using an antibody against mMCP-6 as described under "Experimental Procedures." The extraction buffer was loaded in the left lane as a control.
FSMCs. These data suggest that, like tryptase, CPA also requires the 6-O-sulfate residues of heparin for packaging and storage into granules. However, other possibilities could not be excluded. The loss of CPA activity in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs may be due to defective processing of pro-CPA, because CPA processing is defective in cathepsin-E KO mice, and the activity of cathepsin-E in mast cells is strongly dependent on fully sulfated heparin, possibly because the physical colocalization of cathepsin-E and CPA is mediated by heparin (52). Therefore, it is possible that such colocalization might be functionally hampered by the 6-O-sulfate-deficient heparin in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs. However, such a possibility appears to be insufficient to explain the substantial loss of CPA in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs because partial CPA activity was detected in cathepsin-E KO mice (52), and other proteases might be involved in the processing of pro-CPA.
Because HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs synthesize abnormal heparin almost lacking in 6-O-sulfate residues, those heparin chains are supposed to be lower in total sulfation than those in WT-FSMCs (Table 3). Even HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs still had heparin with about 30% reduction in total sulfation. In addition, the total amount of heparin produced by HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs did not differ from those of WT-FSMCs (data not shown). Considering those together, the net negative charge (total sulfation) may have unlikely observed effects on the storage of mast cell proteases. However, the possibility that the effects are due in part to a decrease in the net negative charge can hardly be excluded.
The mRNA levels of the mast cell proteases were largely unaffected by the loss of 6-O-sulfation of heparin. Therefore, the most likely explanation for the differences observed in the activities of tryptases (mMCP6) and CPA between WT-FSMCs and HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs is that the post-translational processes that enable the active proteases to be packaged and stored in secretory granules are highly dependent on heparin. Interestingly, substantial chymase activity remained in the HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs. In WT and mutant FSMCs, the transcripts of two chymases (mMCP-4 and mMCP-5) were expressed strongly, whereas the expression of mMCP-1 and mMCP-2 was much lower than that of mMCP-4 and mMCP-5 (Fig. 6), which was consistent with previous studies showing that CTMCs express mMCP-4 and mMCP-5 but not mMCP-1 and mMCP-2 (47,58,59). It has been shown that mMCP-5-null mice lack CPA (60) and CPA-null mice lack mMCP-5 (61). Because CPA activity was considerably abolished in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs, the partial loss of chymase activity in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs that was observed in this study might be due to the loss of mMCP-5. If this were the case, the retained chymase activity in HS6ST-1 Ϫ/Ϫ /HS6ST-2 Ϫ/Ϫ -FSMCs would likely be from mMCP-4. Although there are several possible explanations for less effect of the defect of the 6-O-sulfate residues in heparin on chymase activity, we did show that the heparin structures required for chymase binding are different from those required for binding to tryptase (mMCP-6) and CPA.
Mast cell proteases possess a number of pivotal and individually different roles in the immune system and in diseases. Chymase plays a role in Trichinella spiralis clearance (62) and bacterial infection (63) and has been implicated as a biomarker for cardiovascular diseases (64). CPA regulates sepsis (65) and functions in degrading certain snake venom toxins (65,66). Tryptase (mMCP6) also contributes to the innate immune response toward T. spiralis (67) and Klebsiella pneumoniae infection (68). MC tryptase was also found to have attenuated arthritic response via tryptase-heparin complexes in tryptase-KO (mMCP-6 Ϫ/Ϫ ) animals that developed lower inflammation and bone/cartilage erosion than did WT mice (69). Therefore, it is possible that HS6ST activity might be involved in the specific regulation of some protease activities in CTMCs. For example, a possible up-regulation of HS6ST activity in patients during arthritic inflammation might affect the treatment of this disease. Therefore, some reagents that are able to regulate HS-6-O-sulfation might have therapeutic potential in diseases involving mast cell proteases.