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].

– ⁄ –
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-

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

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-

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
+ ⁄ +

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

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

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

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-

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

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

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

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

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

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

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-

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.

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

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

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-

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
phore A23187 (Sigma-Aldrich, Stockholm, Sweden). Cell
fractions and conditioned media were recovered and subjec-
ted to immunoblot analysis. In addition, cytospin slides
were prepared.
Immunohistochemistry
Cytospin slides were stained with rabbit anti-mMCP-6
serum (1 : 200 dilution in NaCl ⁄ P
i
). Biotinylated goat
anti-(rabbit IgG) serum (affinity purified; Vector Laborat-
ories, Burlingame, CA) was used as secondary antibody

Tyrode’s buffer. To half of the cells, A23187 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 lLof1mm p-nitro-
phenyl-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 lLof
0.05 m Na
2
CO
3
(pH 10.0). The absorbance of each reaction
was read at 405 nm. In addition, cell pellets containing
1 · 10
6
cells recovered at six different 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.
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