A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components Frida Henningsson*, Sonja Hergeth*, Robert Cortelius, Magnus A ˚ brink and Gunnar Pejler Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Center, Uppsala, Sweden Mast cells (MCs) are characterized by their large con- tent of electron-dense secretory granules, and these granules are released following MC activation, a pro- cess that can be accomplished by various mechanisms, including antigen-mediated crosslinking of surface- associated IgE and exposure to neuropeptides, anaphy- latoxins or calcium ionophores [1,2]. The MC granules contain a broad array of bioactive compounds, with the exact composition being dependent on the partic- ular species and subclass of MC [1,3]. Histamine is a major constituent of all types of MC, and it is now well recognized that MC granules can contain a num- ber of different cytokines, such as tumor necrosis factor-a, interleukin (IL)-4, IL-5, IL-13, transforming growth factor-b and vascular endothelial growth factor [2]. Moreover, the MC granules contain b-hexosamini- dase and a number of MC-specific neutral proteases: chymases, tryptases and carboxypeptidase A (CPA) [4,5]. In addition, MC granules contain large amounts of highly sulfated and thereby negatively charged pro- teoglycans (PGs) of the serglycin (SG) type, and it is these PGs that give the typical metachromatic staining of MCs with cationic dyes [6]. In MCs, SG PGs can accommodate either (or both) chondroitin sulfate or heparin side chains, depending on MC subclass [7]. The processes involved in MC degranulation, in par- ticular the signal transduction pathways, have been the subject of intense investigation [8,9]. In contrast, strik- ingly little is known regarding the actual formation of MC secretory granules. For example, the factors that Keywords mast cells; proteases; proteoglycans; serglycin; sorting Correspondence G. Pejler, Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Center, Box 575, 751 23 Uppsala, Sweden Fax: +46 18 550762 Tel: +46 18 4714090 E-mail: Gunnar.Pejler@bmc.uu.se *These authors contributed equally to this work (Received 28 June 2006, revised 15 August 2006, accepted 4 September 2006) doi:10.1111/j.1742-4658.2006.05489.x In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphol- ogy of secretory granules as well as the fate of the secretory granule prote- ases in bone marrow-derived mast cells from serglycin + ⁄ + and serglycin – ⁄ – mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defect- ive in serglycin – ⁄ – mast cell granules. The low levels of mast cell proteases present in serglycin – ⁄ – cells had a granular location, as judged by immu- nohistochemistry, and were released following exposure to calcium iono- phore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degrada- tion, exocytosis or defective intracellular processing. In contrast, b-hexosa- minidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive path- way of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles. Abbreviations BMMC, bone marrow-derived mast cell; CPA, carboxypeptidase A; MC, mast cell; mMCP, mouse mast cell protease; PG, proteoglycan; SG, serglycin; TEM, transmission electron microscopy. FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS 4901 determine the sorting of granular components are lar- gely undefined, and the mechanisms that lead to the assembly of the electron-dense, metachromatically staining granules seen in mature MCs have been poorly investigated. In a recent study, we generated a mouse strain in which the SG gene was targeted [10]. We found that, in the absence of SG, mature metach- romatically staining granules were not observed. Fur- thermore, we noted that storage, but not mRNA expression, of the various MC proteases was dramatic- ally defective in SG – ⁄ – MCs. However, the underlying mechanisms behind these observations were not defined. The aim of this study was therefore to provide insights into this issue by determining the fate of SG- dependent proteases in cells with an inactivated SG gene. Our results are compatible with a model of secre- tory granule maturation in which SG PG is not involved in the transport of compounds into secretory vesicles, but is essential for retention of certain constit- uents after their entry into the granules. Results This study was undertaken to resolve the mechanism behind the previously observed severe granule defects in SG – ⁄ – MCs [10]. Given the dramatic effects of the SG knockout on granular staining properties and storage of proteases, it was first important to determine whether the lack of SG affected the actual assembly of granules and whether secretory granules were also functional in the absence of SG. To this end, bone marrow cells were recovered from SG + ⁄ + and SG – ⁄ – mice and were in vitro differentiated into mature bone marrow-derived MCs (BMMCs) by culturing in medium containing IL-3 [11,12]. Cells were recovered at various stages of cellular differentiation, and their morphology was examined after staining with May–Gru ¨ nwald ⁄ Giemsa. At day 0, as expected, no cells with an MC-like appearance were observed. Starting from day 5, cells containing ‘empty’ (May–Gru ¨ nwald ⁄ Giemsa-negative) vesicles were observed. Such vesicles were seen both in SG + ⁄ + and SG – ⁄ – cells, indicating that their formation was not dependent on SG. When the cells were cultured further, the number of May–Gru ¨ nwald ⁄ Giemsa-negative vesi- cles gradually decreased in both SG + ⁄ + and SG – ⁄ – cells. This was accompanied by the appearance, from about day 12, of May–Gru ¨ nwald ⁄ Giemsa-positive granular structures in SG + ⁄ + cells. A gradual increase in May–Gru ¨ nwald ⁄ Giemsa staining was seen over time in SG + ⁄ + cells. In contrast, May–Gru ¨ nwald ⁄ Giemsa-pos- itive vesicles were not seen in SG – ⁄ – cells at any stage of differentiation (not shown). These results are in agree- ment with those of a previous study [10]. One potential explanation for the lack of May– Gru ¨ nwald ⁄ Giemsa-negative vesicles in SG – ⁄ – cells at later stages of differentiation could be that immature granules are generated in the absence of SG, but that the lack of SG causes their disruption. Alternatively, secretory granules could be present at late stages of maturation also in SG – ⁄ – cells, but not be visible by conventional microscopy. To provide further insights into this issue, we examined the cells at the ultrastruc- tural level by transmission electron microscopy (TEM). The TEM analysis indeed revealed the existence of secretory granule-like organelles in SG – ⁄ – cells, and these organelles were found in approximately equal numbers as in SG + ⁄ + cells (Fig. 1; upper panels). However, the morphology of the granules was differ- ent; whereas dense core formation was seen in SG + ⁄ + granules, the contents of the SG – ⁄ – granule were of more amorphous character, without defined electron- dense cores (Fig. 1; lower panels). To address whether the secretory granules were functional, we measured the ability of the MCs to release b-hexosaminidase, a granule component, upon exposure to calcium ionophore A23187. As shown in Fig. 2A, equal amounts of b-hexosaminidase were released by SG + ⁄ + and SG – ⁄ – cells after calcium iono- phore stimulation, and the kinetics of release were sim- ilar. Furthermore, the levels of b-hexosaminidase in conditioned medium from nonstimulated cells were similar in cultures of both genotypes (Fig. 2A), indica- ting that the lack of SG PG did not result in increased spontaneous release of b-hexosaminidase. These find- ings indicate that the general ability of MCs to degran- ulate is not dependent on SG. Experiments were also undertaken to investigate whether the level of stored b-hexosaminidase is influenced by SG. Although b-hexosaminidase activity was already detected at day 0, the intracellular content of this enzyme increased markedly after 6 days of culture, and reached a plat- eau from about day 12 (Fig. 2B). Both the kinetics of accumulation and the level of maximal storage were virtually identical in SG + ⁄ + and SG – ⁄ – cells, indica- ting that the storage of b-hexosaminidase is independ- ent of SG. The results above indicate that functional MC secre- tory granules are formed independently of SG PG. Hence, the defective storage of MC proteases in the absence of SG [10] is not due to defects in the forma- tion or functionality of granular compartments. In order to understand the mechanism by which SG PG promotes storage of these compounds, the strategy in the next set of experiments was to follow the fates of the SG-dependent proteases when SG was absent. To address these issues, we examined the expression, Role of serglycin in secretory granule assembly F. Henningsson et al. 4902 FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS cellular storage, processing and secretion of SG and of various MC proteases at different stages of MC differ- entiation, as described below. SG core protein transcript was already clearly detectable at day 0, but the level of transcript appeared to increase after 5 days of culture (Fig. 3A). In con- trast, no detectable mRNA for mouse MC protease 5 (mMCP-5; a chymase), the tryptase mMCP-6 or CPA was detected at day 0. Starting from day 5, however, mMCP-6 and CPA transcripts were clearly detected, and they appeared to increase further after 12 days of culture. The onset of mMCP-5 mRNA expression was somewhat delayed, with clearly detectable transcript being seen from day 12. mMCP-5, mMCP-6 and CPA transcripts were detected in both SG + ⁄ + and SG – ⁄ – cells, and the kinetics as regards onset of mRNA expression were similar in both genotypes, indicating that the knockout of SG does not affect cellular differ- entiation into MCs, as judged by the transcription of the MC protease genes. Further experiments were carried out to examine how the mRNA expression profiles of SG + ⁄ + and SG – ⁄ – cells were reflected at the protein level (Fig. 3B). Immunoblot analysis of SG + ⁄ + cell extracts showed that neither of the MC proteases were present at day 0. mMCP-5 protein was detected starting from day 12, i.e. at the same time as when gene transcription was first seen. mMCP-5 protein accumulated over time, with a plateau of maximal storage seen after 26 days of culture. mMCP-6 storage showed similar kinetics as for mMCP-5. In contrast, CPA protein was detected as early as after 5 days of culture, and a maximal plat- eau of storage was already seen at day 12. Both pro- CPA and mature CPA were detected in SG + ⁄ + cells. A dramatically different pattern was seen in SG – ⁄ – cells. mMCP-5 protein was not detected at any time point, and mMCP-6 protein, although being detect- able, was present at markedly lower levels than in SG + ⁄ + cells. Notably, however, mMCP-6 accumula- tion in SG – ⁄ – cells showed similar kinetics as in SG + ⁄ + cells. In contrast, the total amounts of CPA antigen (pro-CPA + mature CPA) were approximately equal in SG – ⁄ – and SG + ⁄ + cells. An interesting observation was that only the pro-form of CPA was Fig. 1. Transmission electron micrographs. The upper panels show representative mature (5 weeks of culture) bone marrow-derived mast cells (BMMCs) from serglycin (SG) + ⁄ + and SG – ⁄ – mice (original magnification 5000·). The lower panels show enlarged granules (original magnification · 40 000). F. Henningsson et al. Role of serglycin in secretory granule assembly FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS 4903 detected in SG – ⁄ – cells, indicating that pro-CPA processing into mature protease is strongly dependent on SG. A plausible explanation for this finding is that the protease(s) that are responsible for the pro-CPA processing is dependent on SG. In agreement with such a notion, we showed recently that pro-CPA processing was defective in cathepsin E – ⁄ – MCs and that the storage of cathepsin E in MCs is dependent on heparin PG [13]. Thus, a likely explanation for the defective pro-CPA processing in SG – ⁄ – MCs is that the lack of SG also results in defective cathepsin E storage and that this, in turn, results in defective pro-CPA processing, leading to an accumulation of pro-CPA. Next, we investigated the possibility that the pro- teases were constitutively secreted in the absence of SG PG. Conditioned media were collected at different stages of MC differentiation, and were analyzed for the presence of secreted MC proteases. As shown in Fig. 3C, mMCP-5 protein was present at low levels in conditioned medium from SG + ⁄ + cells after prolonged culture, but was absent in medium from SG – ⁄ – cells. In contrast, mMCP-6 protein was clearly detected, starting from about day 14, in conditioned medium from both SG + ⁄ + and SG – ⁄ – cells. CPA, pre- dominantly in its mature form, was secreted into the medium by SG + ⁄ + cells, starting at about day 5. In contrast, only the pro-form of CPA was secreted by SG – ⁄ – cells. The total level of secreted CPA (pro-CPA and mature CPA) was somewhat higher in medium from SG – ⁄ – than in that from SG + ⁄ + MCs, in partic- ular at early time points (Fig. 3C). Note that, at early time points, pro-CPA dominated over mature protease, both intracellularly (Fig. 3B; day 5) and in conditioned medium from SG + ⁄ + cells (Fig. 3C; days 6 and 12), indicating that efficient processing of pro-CPA is dependent on the degree of MC maturation. In accord- ance with this notion, only the mature form of CPA is detected in fully mature connective tissue-type MCs recovered in vivo [13], and only the pro-form of CPA is detected in poorly differentiated transformed cell lines of MC origin (M. Grujic & G. Pejler, unpub- lished results). The results above indicate that mMCP-6 and pro- CPA are secreted by SG – ⁄ – MCs. A possible explan- ation for these findings would be that the absence of SG causes missorting of mMCP-6 and pro-CPA into the constitutive rather than into the regulated pathway of secretion. If indeed this were the case, mMCP-6 and pro-CPA would not be present in the secretory gran- ules, and exposure of SG – ⁄ – cells to MC-degranulating agents would not cause increased release of mMCP-6 and pro-CPA. If, on the other hand, mMCP-6 and pro-CPA are in fact located in secretory granules also in SG – ⁄ – cells, MC degranulation would be expected to induce their release. In order to address these possibil- ities, SG + ⁄ + and SG – ⁄ – MCs were exposed to the cal- cium ionophore A23187, a compound that is regularly used as an MC secretagogue [14]. As shown in Fig. 4A, exposure of SG + ⁄ + cells to A23187 resulted in clearly detectable mMCP-6 and CPA in conditioned medium. Strikingly, calcium ionophore stimulation also resulted in the release of mMCP-6 and pro-CPA by SG – ⁄ – cells (Fig. 4A). The implication of these find- ings is that mMCP-6 and pro-CPA are sorted into releasable secretory vesicles despite the absence of SG. To obtain further evidence for this, SG – ⁄ – cells were stained for mMCP-6 antigen, before and after expo- sure to calcium ionophore. In resting SG – ⁄ – cells, 0 12345 0 10 20 30 40 50 60 70 80 90 100 Hours +/+ non-stim +/+ A23187 -/- non-stim -/- A23187 Hexosaminidase release (% of total) A 0 010203040 20 40 60 80 100 120 Days of culture +/+ -/- Hexosaminidase content (% of maximal) B Fig. 2. b-Hexosaminidase content and release. (A) Conditioned media from mature (5 weeks of culture) serglycin (SG) + ⁄ + (filled symbols) and SG – ⁄ – (open symbols) cells were analyzed for b-hex- osaminidase activity, without stimulation (squares) or after addition of A23187 (circles). (B) SG + ⁄ + (filled squares) and SG – ⁄ – (open squares) cells taken at different stages (days 0–33) of differenti- ation in interleukin (IL)-3-containing medium were analyzed for total intracellular b-hexosaminidase activity. Results are expressed as percentages, where the b-hexosaminidase content in SG + ⁄ + cells at day 33 is set as 100%. Results are expressed as means of tripli- cate determinations ± SD. Role of serglycin in secretory granule assembly F. Henningsson et al. 4904 FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS mMCP-6 was found in granule-like compartments close to the plasma membrane (Fig. 4C), indeed sup- porting the idea that mMCP-6 is transported into secretory granules even in the absence of SG PG. Fur- thermore, after exposure to A23187, SG – ⁄ – cells showed signs of degranulation and it was also evident that the released granules stained positively for mMCP-6 (Fig. 4C). Preimmune serum gave only dif- fuse overall staining of SG – ⁄ – cells and a total absence of granular staining of either unstimulated or A23187- stimulated cells (Fig. 4C). Next, we investigated the possibility that the MC proteases are subjected to degradation by lysosomal proteases when SG PG is absent. For this purpose, cells were incubated with NH 4 Cl in order to raise the pH of acidic intracellular compartments, including lysosomes and secretory granules, and thereby inacti- vate lysosomal proteases. Incubation of SG – ⁄ – MCs with NH 4 Cl did not affect the level of intracellular mMCP-6, indicating that degradation by lysosomal mechanisms is not a primary fate of mMCP-6 when SG is absent (Fig. 5). In contrast, NH 4 Cl caused an SG mMCP-6 mMCP-5 CPA HPRT 0512 19 26 33 0512 19 26 33 SG -/- SG +/+ A Days: 0512 19 26 33 05 12 19 26 33Days: 0614 20 26 33 0614 20 26 33Days: mMCP-6 mMCP-5 pro-CPA CPA B mMCP-6 mMCP-5 pro-CPA CPA C Fig. 3. mRNA expression and protein analysis. (A) Total RNA was prepared from serglycin (SG) + ⁄ + and SG – ⁄ – bone marrow-derived cells after different durations (days 0–33) of culture in medium containing interleukin (IL)-3. The RNA was used for analysis of the expression of mouse mast cell protease (mMCP)-5, mMCP-6, carboxypeptidase A (CPA) and SG by RT-PCR. Expression of hypoxanthine–guanine phopho- ribosyltransferase was used as housekeeping control. (B) Cell extracts were prepared from cells taken at various stages (days 0–33) of differ- entiation and were subjected to immunoblot analysis using antisera towards mMCP-5, mMCP-6 and CPA. (C) Secretion of MC proteases from SG + ⁄ + and SG – ⁄ – cells. Conditioned media were recovered from SG + ⁄ + and SG – ⁄ – cells at various stages (days 0–33) of differentiation in IL-3-containing medium. The media were concentrated and subjected to immunoblot analysis for mMCP-5, mMCP-6 and CPA. The results shown are representative of three independent experiments. F. Henningsson et al. Role of serglycin in secretory granule assembly FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS 4905 accumulation of mMCP-5 protein in SG – ⁄ – cells, whereas mMCP-5 levels were not affected in SG + ⁄ + MCs (Fig. 5; upper panel). This indicates that mMCP- 5, in the absence of SG, is degraded by proteases with low pH optima, possibly in lysosomal compartments. Interestingly, several ‘lysosomal’ proteases, e.g. cathep- sin C, cathepsin D and cathepsin E, have been found not only in lysosomes but also in MC secretory gran- ules [13,15,16]. Thus, the degradation of mMCP-5 does not necessarily have to involve transport to lysosomes, but could in fact occur within the secretory granule compartment. NH 4 Cl did not cause any noticeable accumulation of pro-CPA or mature CPA in either SG + ⁄ + or SG – ⁄ – MCs. However, the NH 4 Cl treat- ment resulted in the accumulation of an intermediate form of CPA, of somewhat lower molecular weight than pro-CPA (Fig. 5; lower panel). Most likely, this compound represents an intermediate in processing. These findings indicate that the processing of pro-CPA occurs in (at least) two steps, and that the processing of the intermediate form of CPA to mature protease is dependent on a (lysosomal?) protease with an acidic pH optimum. Control experiments showed that NH 4 Cl did not affect cellular viability (not shown). Degradation by the proteasome pathway could con- stitute an alternative degradative pathway in the absence of SG. However, incubation of cells with lac- tacystin, an inhibitor of proteasome function, did not cause any accumulation of MC proteases in SG – ⁄ – MCs (not shown). AB C Fig. 4. Protease release after mast cell (MC) degranulation. Serglycin (SG) + ⁄ + and SG – ⁄ – MCs (after 5 weeks of culture) were treated with calcium ionophore A23187. (A) Medium fractions from SG + ⁄ + and SG – ⁄ – cells were subjected to immunoblot analysis using anti- sera towards carboxypeptidase A (CPA) and mouse mast cell protease (mMCP)-6. Note the increase in extracellular mMCP-6 and CPA antigen, in both SG + ⁄ + and SG – ⁄ – cul- tures, after stimulation with A23187. (B) Cell fractions from SG + ⁄ + and SG – ⁄ – cells were analyzed for mMCP-6 and CPA by immuno- blotting. (C) Cytospin slides were prepared from nontreated and A23187-treated SG – ⁄ – cells and were immunohistochemically stained for the presence of mMCP-6 anti- gen. Note the granular staining for mMCP-6, both before and after A23187 stimulation. The results shown are representative of three independent experiments. Role of serglycin in secretory granule assembly F. Henningsson et al. 4906 FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS As shown in Fig. 3A, SG core protein mRNA was already expressed at day 0. However, maximal MC protease accumulation was not obtained until about day 26 (Fig. 3B), a finding that may appear contradict- ory, considering the strong dependence of the MC pro- teases on SG for storage. This indicates that the levels of stored proteases are not directly related to the amount of SG core protein mRNA being expressed. One potential explanation could be that the amount of actual sulfated PGs is not directly correlated with the level of SG mRNA, and it was therefore of interest to also follow the levels of sulfated PGs during the course of MC differentiation. To this end, SG + ⁄ + MCs at different stages of differentiation were biosynthetically labeled with 35 SO 4 2– . 35 S-labeled PGs were recovered both from conditioned medium (secreted PGs) and from cell extracts, and were quantified. As shown in Fig. 6A, the levels of secreted PGs were similar at all time points tested. In contrast, the levels of intracellu- lar PGs increased markedly over time. Notably, the level of intracellular PGs did not reach a plateau, as observed for the proteases (Fig. 3B). Rather, the level of cell-associated PGs showed a continuous increase over time (Fig. 6A). Notably, the latter is in good agreement with the relatively late appearance of May– Gru ¨ nwald ⁄ Giemsa-positive granules as compared to the onset of SG mRNA expression (see above). Next, the possibility that the different abilities of MCs to store proteases at different stages of differenti- ation could be due to differences in the electrostatic charge of the PGs was addressed. For this purpose, MC PGs recovered at different time points during the course of MC development were examined by anion exchange chromatography. At early time points (day 10), there was a distinction between two separate PG populations, one with low anionic charge density [coeluting with standard chondroitin sulphate A (CS-A)] and one population with a markedly higher charge (coeluting with standard pig mucosal heparin) (Fig. 6B). Similar elution profiles were seen for PGs recovered from the cell layer and from conditioned medium. In contrast, only highly charged PGs were seen at day 23 (Fig. 6B) and day 34 (not shown), again with similar charge densities being displayed by cell- associated and extracellular PGs. Together, these results indicate that the MC maturation process is associated with both increased total synthesis of sulfated PGs and increased charge density of the synthesized PGs. Discussion Although the knockout of both SG [10] and N-de- acetylase ⁄ N-sulfotransferase-2 [17,18], the latter being an enzyme involved in the sulfation of heparin chains attached to the SG core protein, has provided strong evidence for a crucial role of PGs in mediating the storage of secretory granule compounds in MCs, the mechanism behind these observations has not been established. One potential mechanism would be that SG is important for the formation of the secretory granule. However, we show here that SG – ⁄ – MCs also displayed clearly discernible secretory vesicle-like struc- tures. By conventional microscopy, such vesicles were May–Gru ¨ nwald ⁄ Giemsa-negative and, interestingly, May–Gru ¨ nwald ⁄ Giemsa-negative vesicles were also seen in SG + ⁄ + cells at early stages of differentiation. Most likely, these structures represent immature secre- tory granules in which the PG content is too low to stain with May–Gru ¨ nwald ⁄ Giemsa. In accordance with this, it was observed that the intracellular content of highly sulfated PGs was relatively low at the corres- ponding (early) time point. At later stages of differenti- ation, in contrast, SG + ⁄ + MCs showed staining with May–Gru ¨ nwald ⁄ Giemsa, and this correlated well with a marked increase in the recovery of highly sulfated intracellular PGs. The presence of secretory vesicle-like structures in SG – ⁄ – cells was also supported at the ultrastructural level. TEM analysis showed the presence of highly elec- tron-dense granules in SG + ⁄ + cells, but also showed Fig. 5. Inhibition of lysosomal proteases. Serglycin (SG) + ⁄ + and SG – ⁄ – cells (after 5 weeks of culture) were incubated for 6 h with 20 m M NH 4 Cl, or for 20 h with 5 mM NH 4 Cl. Cell extracts were subsequently subjected to immunoblot analysis using antisera towards carboxypeptidase A (CPA) and mouse mast cell protease (mMCP)-6. The arrow indicates a ‘semiprocessed’ form of CPA. The results shown are representative of three independent experi- ments. F. Henningsson et al. Role of serglycin in secretory granule assembly FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS 4907 an abundance of granule-like organelles in SG – ⁄ – cells. Importantly, however, the granule matrix in SG – ⁄ – cells was more amorphous than in SG + ⁄ + cells, and showed less defined dense core formation. Our results also pro- vide evidence that SG – ⁄ – cells retain the full capability to undergo stimulus-induced degranulation, as deter- mined by the ability to release b-hexosaminidase in response to calcium ionophore. Together, our data thus indicate that SG PG is not necessary for the formation of MC secretory granules, and nor is SG involved in mechanisms of degranulation. Another possible explanation for the storage defects seen in SG – ⁄ – MCs would be that SG PG is needed for correct intracellular sorting of the MC proteases into the secretory granules, the alternative fate being secre- tion by the constitutive pathway. If this was the case, it would be expected that SG-binding compounds such as the MC proteases would be preferentially released into the extracellular space by SG – ⁄ – MCs. Such mis- sorting would result in excessive accumulation of granule compounds in conditioned medium from SG – ⁄ – cells. We here provide evidence that CPA, in its pro-form, is secreted at higher levels by SG – ⁄ – cells than by their SG + ⁄ + counterparts, indeed indicating that the lack of SG PG causes increased constitutive release. However, rerouting into the constitutive path- way of secretion does not seem to be a general effect on all MC proteases when SG PG is lacking, as shown 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 20 40 60 0 100 200 300 400 500 0 100 200 300 400 500 20 40 60 0 100 200 300 0 100 200 300 20 40 60 20 40 60 A 530 ; LiCl concentration (M -1 ) Fraction number Fraction number Cell fraction Medium fraction B Day 23 Day 10 Day 23 Day 10 hep CS 0 2000 4000 6000 8000 10000 Day 10 Day 23 Day 34 A cell fractions medium fractions Fig. 6. Analysis of sulfated proteoglycans. Serglycin (SG) + ⁄ + bone marrow cells were cultured for different times (10, 23 34 days) in medium containing interleukin (IL)-3 and were biosynthetically labeled with 35 SO 4 2– (A) Total recovery of 35 S-labeled proteogly- cans per 1 · 10 6 cells into cell (filled bars) and medium (hatched bars) fractions. (B) Charge density of sulfated proteoglycans. 35 S-labeled proteoglycans isolated from cell and medium fractions, both derived from SG + ⁄ + cells, were mixed with internal stand- ards of heparin (hep) and chondroitin sulfate (CS) and were subjected to anion exchange chromatography on a DEAE–Sephacel col- umn. The column was eluted with a linear gradient of LiCl. Fractions were analyzed for radioactivity (filled symbols) and for uronic acid in order to detect the internal standards (open symbols; A 530 ). The results shown are representative of two independent experi- ments. Role of serglycin in secretory granule assembly F. Henningsson et al. 4908 FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS by the fact that mMCP-6 and mMCP-5 were found at similar or higher levels in medium from SG + ⁄ + cells than in medium from their SG – ⁄ – counterparts. More- over, our data provide evidence that the SG-dependent MC proteases are in fact present in functional secre- tory granules even in the absence of SG PG. This indi- cates that transport of MC proteases into the secretory granule compartments can occur independently of SG PG. Hence, SG does not appear to function as general sorting vehicle for MC proteases. From the data presented here and previously [10], it is clear that knockout of the SG gene results in a major reduction of mMCP-5, mMCP-6 and CPA storage. However, the blockade is not complete, indicating that the proteases can actually be stored to some extent in MC granules even in the absence of SG PGs to which they can bind. In turn, this may be explained by low lev- els of granular storage in the absence of any partner PG. An alternative explanation could be that there are low levels of PGs other than SG in the MC granule, and that such PG species can provide some compensation for the lack of SG in terms of promoting MC protease storage. However, the presence of non-SG PG species within MC granules remains to be demonstrated. So, how does the lack of SG cause such a dramatic reduction of stored MC proteases? Although general mechanisms involved in the intracellular sorting of granule components are still relatively poorly defined, two major hypotheses have emerged: ‘sorting by entry’ and ‘sorting by retention’ [19]. In the sorting by entry hypothesis (e.g. the mannose 6-phosphate system [20]), each secretory granule compound has a unique sorting signal that interacts with a cognate receptor on the luminal side of specific regions in the trans-Golgi network, leading to budding from the trans-Golgi network of vesicles containing only molecules with the corresponding specific sorting signals. In the sorting by retention hypothesis, certain compounds entering the immature granules may carry sorting motifs that inter- act with the limiting membrane, but luminal proteins that are not associated with the trans-Golgi network membrane may also be included in the budding vesicle. According to this hypothesis, the contents of the imma- ture granule are subsequently refined, both by conden- sation of selected compounds and by removal of others by vesicular transport, e.g. to lysosomes for destruction, or to the extracellular space by ‘constitutive-like’ or ‘piecemeal’ exocytosis [19,21]. This process will eventu- ally result in secretory granule maturation. Although we cannot at this stage with certainty define the role of SG PG in this process, our results are clearly compat- ible with a model in which SG organizes secretory gran- ule maturation according to the sorting by retention hypothesis. In support of this, all of the MC proteases that have been shown to be SG-dependent for storage, i.e. mMCP-4, mMCP-5, mMCP-6 and CPA (this study and [10]), display high affinity for sulfated glycosami- noglycans [22–25]. It is therefore possible that mMCP- 4, mMCP-5, mMCP-6 and CPA are transported into immature granules independently of SG, but that their retention within the granules is dependent on their tight electrostatic interaction with SG PG. However, our data indicate that interaction with SG PG is not an absolute prerequisite for retention of all granule com- pounds within the granule, as shown by the lack of SG dependence for the storage of b-hexosaminidase. The sorting for retention model of granule matur- ation implies that compounds not selected for retent- ion are expelled from the maturing granule by vesicular transport. In line with this model, our results suggest that mMCP-5 is targeted to degradation if not retained by SG. We also see a marked secretion of pro-CPA by SG – ⁄ – cells, possibly as a consequence of defective retention. However, there is also secretion of CPA, albeit in its mature form, from SG + ⁄ + cells. mMCP-6 is also secreted by SG – ⁄ – cells, but in con- trast to pro-CPA and mature CPA, mMCP-6 secretion was somewhat higher in SG + ⁄ + cells than in their SG – ⁄ – counterparts. However, the level of mMCP-6 protein in the conditioned medium from SG – ⁄ – cells was considerably higher than the intracellular level, indicating that secretion rather than storage is the dominating pathway for mMCP-6 in the absence of SG. One possible explanation for these findings is that there is continuous low-level release of secretory gran- ule compounds in normal MCs, a process often referred to as ‘piecemeal’ degranulation [21]. In the absence of SG as a retention vehicle, this slow release may constitute the dominating pathway. In summary, this study has provided the first insights into the mechanism by which SG PG regulates MC secretory granule homeostasis. Experimental procedures Cell culture Experiments were performed on SG + ⁄ + and SG – ⁄ – mice, back-crossed to C57BL ⁄ 6J for 10 generations. The experi- ments were approved by the local ethical committee. BMMCs were obtained by culturing femural and tibial bone marrow cells in DMEM (SVA, Uppsala, Sweden), supplemented with 10% heat-inactivated fetal bovine serum (Biotech line AS), 50 lgÆmL )1 gentamicin sulfate (SVA), 2mml-glutamine (SVA) and 50% WEHI-3B conditioned medium (which contains IL-3) for 3 weeks. The cells were F. Henningsson et al. Role of serglycin in secretory granule assembly FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS 4909 kept at a concentration of 500 000 cellsÆmL )1 , and the med- ium was changed every third day. Staining Three hundred thousand cells were collected on cytospin slides (700 r.p.m., 5 min) and stained with May–Gru ¨ nw- ald ⁄ Giemsa. The slides were first fixed in methanol for 3 min, and then stained with May–Gru ¨ nwald (Merck, Sol- lentuna, Sweden) for 15 min. After being washed with water, the slides were stained with 5% Giemsa (Merck) in water for 10 min. TEM Cells were fixed for 6 h in 2% glutaraldehyde in a 0.1 m sodium cacodylate buffer supplemented with 0.1 m sucrose, and this was followed by 1.5 h of postfixation in 1% osmium tetroxide dissolved in the same cacodylate buffer. After dehydration in ethanol, the cells were embedded in the epoxy resin Agar 100 (Agar Scientific, Stansted, UK). Ultrathin sections were placed on copper grids covered with a film of polyvinyl formal plastic (Formvar; Agar Scientific) and contrasted with uranyl acetate and lead citrate. Elec- tron micrographs were taken with a Hitachi electron micro- scope (Hitachi Ltd, Tokyo, Japan). RT-PCR Total RNA was isolated using the NucleoSpin RNA II kit (Macherey Nagel, Du ¨ ren, Germany). Total RNA was used for first-strand cDNA synthesis using SuperScriptII for RT-PCR using primers specific for the MC proteases and SG. Hypoxanthine–guanine phosphoribosyltransferase was used as a positive control for the RT-PCR. The PCR prim- ers used were as specified elsewhere [10]. Immunoblotting At each studied time point, 2 · 10 6 cells were taken from the main cultures, centrifuged at 300 g, (Megafuge 1.0R; Heraeus; equipped with a swing out rotor), resuspended in 1 mL of serum-free medium (as described above), and further cultured overnight. The cells were thereafter pelleted by centrifugation at 300 g (10 min, Megafuge 1.0R; Heraeus; equipped with a swing out rotor), and both the pellet and the medium fraction were recovered and stored at ) 20 °C until analysis. Before analysis, the recovered media were concentrated 50 times using Amicon Ultra-4 centrifugal filter device (Millipore, Solna, Sweden) and then mixed with 20 lL of SDS ⁄ PAGE sample buffer containing 5% b-mercaptoethanol. Cell pellets (1 · 10 6 cells) were dissolved in 300 lL of SDS ⁄ PAGE sample buffer containing 5% b-mercaptoethanol. Immunoblotting was carried out as previously described [10]. Proteoglycan isolation and analysis SG + ⁄ + cells (20 · 10 6 ) from days 9, 22 and 33 of culture were biosynthetically labeled overnight with 0.32 mCi of carrier-free 35 SO 4 2– (GE Healthcare, Uppsala, Sweden). Cells were pelleted by centrifugation for 10 min at 300 g (Megafuge 1.0R; Heraeus; equipped with a swing out rotor). Cells and conditioned media were stored at ) 20 °C until further analysis. For isolation of cell fraction glycos- aminoglycans, cell pellets were solubilized in 500 lLof NaCl ⁄ P i ⁄ 2 m NaCl ⁄ 0.5% Triton X-100 at 4 °C for 30 min. Then, the solubilisates were diluted with 9.5 mL of H 2 O ⁄ 0.5% Triton X-100 and applied to columns contain- ing 0.4 mL of DEAE–Sephacel, equilibrated with 50 mm Tris ⁄ 0.1 m NaCl ⁄ 0.1% Triton X-100 (pH 8.0). Conditioned media were loaded directly onto the columns. After wash- ing with 10 mL of 50 mm Tris ⁄ HCl (pH 8.0) ⁄ 0.1 m NaCl ⁄ 0.1% Triton X-100 and 10 mL of 50 mm sodium acetate ⁄ 0.15 m NaCl ⁄ 0.1% Triton X-100 (pH 4.0), samples were eluted with 50 mm sodium acetate ⁄ 2 m NaCl (pH 5.5). Four fractions of 1100 lL each were collected and analyzed for radioactivity by scintillation counting. Fractions containing radioactive material were pooled and diluted with H 2 O to yield 0.05 m NaCl, and subjected to anion exchange chromatography on a 5 mL column of DEAE–Sephacel connected to an FPLC system. The col- umn was eluted with a gradient of increasing concentra- tions of LiCl, from 0.05 m to 2 m in 50 mm sodium acetate (pH 4.0) at a flow rate of 0.5 mLÆmin )1 . Fractions (0.5 mL) were collected and analyzed for 35 S radioactivity. As an internal standard, 200 lL of a mixture of unlabeled heparin (4 mgÆmL )1 ) and CS-A (5 mgÆmL )1 ; Sigma, Stockholm, Sweden) was added to each sample before anion exchange chromatography analysis. Internal standards were detected by the carbazole method: 25 lL of each fraction was mixed with 300 lL of concentrated H 2 SO 4 ⁄ 25 mm K 2 B 4 O 7 and 10 lL of carbazole reagent (0.125% carbazole in 96% eth- anol). The samples were boiled for 10 min and cooled, and the absorbance at 530 nm was measured. Inhibition of proteasome and lysosome function Mature BMMCs (1 · 10 6 ) were cultured in 5 lm lactacystin (Affiniti Research Products, Exeter, UK). After incubation overnight, cells were pelleted, solubilized and subjected to immunoblotting for mMCP-5, mMCP6 and CPA. Lyso- somal function was inhibited by incubation of cells with 5 or 20 mm NH 4 Cl. After 6–20 h, cells were pelleted, solubi- lized and subjected to immunoblotting. Degranulation To induce MC degranulation, 2 · 10 6 cells were incubated for 120 min in the presence of 2 lm of the calcium iono- Role of serglycin in secretory granule assembly F. Henningsson et al. 4910 FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... CA) was used as secondary antibody (1 : 100 dilution in NaCl ⁄ Pi) Staining was performed with a standard protocol using the biotin–avidin-based Vectastain Elite kit (Vector Laboratories) and diaminobenzadine (DAB) for detection of the secondary antibody, according to the protocol supplied by the manufacturer As a negative control, preimmune serum was used After staining, slides were dehydrated in increasing... (2004) Serglycin is essential for maturation of mast cell secretory granule J Biol Chem 279, 40897–40905 11 Tsai M, Takeishi T, Thompson H, Langley KE, Zsebo KM, Metcalfe DD, Geissler EN & Galli SJ (1991) Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor Proc Natl Acad Sci USA 88, 6382–6386 12 Razin E, Ihle JN, Seldin D, Mencia-Huerta JM, Katz... Katz HR, LeBlanc PA, Hein A, Caulfield JP, Austen KF & Stevens RL (1984) Interleukin 3: a differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan J Immunol 132, 1479–1486 13 Henningsson F, Yamamoto K, Saftig P, Reinheckel T, Peters C, Knight SD & Pejler G (2005) A role for cathepsin E in the processing of mast- cell carboxypeptidase A J Cell Sci 118,... Diamant B & Patkar SA (1975) Stimulation and inhibition of histamine release from isolated rat mast cells Dual effects of the ionophore A2 3187 Int Arch Allergy Appl Immunol 49, 183–207 15 Dragonetti A, Baldassarre M, Castino R, Demoz M, Luini A, Buccione R & Isidoro C (2000) The lysosomal FEBS Journal 273 (2006) 4901–4912 ª 2006 The Authors Journal compilation ª 2006 FEBS 4911 Role of serglycin in secretory. .. p-nitrophenyl-N-acetyl-b-d-glucosaminide (Sigma-Aldrich) in 0.05 m citrate buffer (pH 4.5) at 37 °C for 1 h As a control for total b-hexosaminidase content, cells were lysed with Tyrode’s buffer ⁄ 1% Triton X-100 and incubated as above All reactions were quenched by addition of 100 lL of 0.05 m Na2CO3 (pH 10.0) The absorbance of each reaction was read at 405 nm In addition, cell pellets containing 1 · 106 cells recovered at six... of Tyrode’s buffer To half of the cells, A2 3187 was added (final concentration 2 lm), whereas the other half was left without additions Cells were incubated for 1, 2 or 4 h Samples (50 lL) were taken at each time point, and cells were centrifuged at 300 g for 10 min (Megafuge 1.0R; Heraeus; equipped with a swing out rotor) The remaining supernatant was incubated with 100 lL of 1 mm p-nitrophenyl-N-acetyl-b-d-glucosaminide... secretory granule assembly 16 17 18 19 20 F Henningsson et al protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic ⁄ mast cell line RBL J Cell Sci 113, 3289–3298 Wolters PJ, Laig-Webster M & Caughey GH (2000) Dipeptidyl peptidase I cleaves matrix-associated proteins and is expressed mainly by mast cells in normal dog airways Am J Respir Cell. .. P & Castle D (1998) Sorting and storage during secretory granule biogenesis: looking backward and looking forward Biochem J 332, 593–610 Ghosh P, Dahms NM & Kornfeld S (2003) Mannose 6-phosphate receptors: new twists in the tale Nat Rev Mol Cell Biol 4, 202–212 4912 21 Dvorak AM (2005) Piecemeal degranulation of basophils and mast cells is effected by vesicular transport of stored secretory granule. .. chondroitin sulfate E proteoglycans, and [3H]diisopropyl fluorophosphatebinding proteins are exocytosed from activated mouse bone marrow-derived mast cells J Biol Chem 261, 15017–15021 25 Springman EB, Dikov MM & Serafin WE (1995) Mast cell procarboxypeptidase A Molecular modeling and biochemical characterization of its processing within secretory granules J Biol Chem 270, 1300–1307 FEBS Journal 273 (2006)... time points (days 0, 6, 12, 20, 26 and 33) were lysed with 1% Triton X-100 in Tyrode’s buffer and assayed for total b-hexosaminidase content as described above Acknowledgements This work was supported by grants from the Swedish Research Council, Formas, Mizutani Foundation for Glycoscience and King Gustaf V’s 80th Anniversary Fund Role of serglycin in secretory granule assembly References 1 Metcalfe . A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components Frida Henningsson*, Sonja Hergeth*,. Staining was performed with a standard protocol using the biotin–avidin-based Vectastain Elite kit (Vector Laboratories) and diamino- benzadine (DAB) for