suppressor factor b conjugates to endophilin II andregulates phagocytosis Morihiko Nakamura and Shunsuke Shimosaki Department of Cooperative Medical Research, Collaboration Center, Shima
Trang 1suppressor factor b conjugates to endophilin II and
regulates phagocytosis
Morihiko Nakamura and Shunsuke Shimosaki
Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo, Japan
Introduction
Monoclonal nonspecific suppressor factor b (MNSFb)
is a 14.5 kDa fusion protein consisting of a protein
with 36% identity with ubiquitin and ribosomal
pro-tein S30 The ubiquitin-like segment is cleaved from
ribosomal protein S30 in the cytosol [1] MNSFb is covalently attached to certain lysines of specific target proteins, including Bcl-G, a proapoptotic protein [2] MNSFb conjugates to Bcl-G and regulates the
Keywords
endophilin; macrophage; phagocytosis;
ubiquitin-like protein
Correspondence
M Nakamura, Department of Cooperative
Medical Research, Collaboration Center,
Shimane University, Izumo 693-8501, Japan
Fax: +81 853 20 2913
Tel: +81 853 20 2916
E-mail: nkmr0515@med.shimane-u.ac.jp
(Received 13 July 2009, revised 31 July
2009, accepted 3 September 2009)
doi:10.1111/j.1742-4658.2009.07348.x
Monoclonal nonspecific suppressor factor b (MNSFb) is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions Previous studies have demonstrated that MNSFb covalently binds to the intracellular proapoptotic protein Bcl-G in cells of the macrophage cell line Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis In this study, we purified a 62 kDa MNSFb adduct from murine liver lysates by sequential chromatography
on DEAE and anti-MNSFb IgG-conjugated Sepharose MALDI-TOF MS fingerprinting revealed that this MNSFb adduct consists of an 8.5 kDa MNSFb and endophilin II, a member of the endophilin A family MNSFb may conjugate to endophilin II with a linkage between the C-terminal Gly74 and Lys294 We confirmed this result by immunoprecipita-tion⁄ western blot studies Endophilin II was ubiquitously expressed in vari-ous tissues, although a truncated form was observed in liver The 62 kDa MNSFb–endophilin II was specifically expressed in liver and macrophages Small interfering RNA-mediated knockdown of endophilin II and⁄ or MNSFb promoted phagocytosis of zymosan in Raw264.7 cells Conversely, cotransfection of endophilin II and MNSFb cDNAs inhibited the phagocy-tosis of zymosan Such inhibition was not observed in cells expressing a mutant of endophilin II in which Lys294 was replaced by arginine These results suggest that the post-translational modification of endophilin II by MNSFb might be implicated in phagocytosis by macrophages
Structured digital abstract
l MINT-7261558 , MINT-7261537 , MINT-7261546 : MNSF beta (uniprotkb: P35545 ) physically interacts ( MI:0915 ) with Endophilin-2 (uniprotkb: Q62419 ) by anti bait coimmunoprecipitation ( MI:0006 )
Abbreviations
EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MNSF, monoclonal nonspecific suppressor factor; siRNA, small interfering RNA; tEnd-II, 30 kDa truncated form of endophilin II.
Trang 2mitogen-activated protein kinase (MAP kinase)
path-way by inhibiting the activation of extracellular
signal-regulated kinase (ERK) [3] Several ubiquitin-like
proteins are implicated in interferon signaling
MNSFb, ISG15, FAT10 and NUB1L are induced by
interferon-c [4–7] Among them, MNSFb and FAT10
are closely related to apoptosis [2,8]
The endophilins are a family of proteins identified in
a search for SH3 domain-containing proteins The
most extensively studied isoform of the mammalian
endophilins, brain-specific endophilin I [9,10], is
necessary for synaptic vesicle endocytosis [11–13]
Endophilin II, which is ubiquitously expressed [14], is
a member of the endocytic endophilin family, but its
function in regulating endocytosis remains unclear In
this study, we observed that the authentic 45 kDa
endophilin II is expressed in various mouse tissues,
including brain, testis, and spleen Interestingly,
heter-ogeneous species with molecular masses of about 62,
45 and 25 kDa were observed in liver lysates We
dem-onstrated that MNSFb covalently conjugates to
endo-philin II in liver and cells of the macrophage cell line
Raw264.7, and that MNSFb–endophilin II complex
formation might be implicated in phagocytosis in
macrophages
Results
Purification of MNSFb adducts from murine liver
In previous studies, we have shown that MNSFb
cova-lently conjugates to various target proteins [2,4]
Dur-ing the studies, we observed that several MNSFb
adducts were found in liver extracts of mice To
fur-ther study the mechanism of action of MNSFb, we
tried to isolate the MNSFb conjugates When an
extract from fresh murine liver was chromatographed
through a DEAE–Sepharose column, and various
frac-tions were subjected to western blotting by using
antibody against MNSFb, several MNSFb
antibody-reactive conjugates were eluted from the column by
100 mm NaCl (Fig 1, lane 5) We further purified
these conjugates by MNSFb antibody affinity
chroma-tography The final preparation gave two silver-stained
bands on SDS⁄ PAGE with mobilities corresponding to
62 and 95 kDa under reducing conditions (Fig 1, lane 3)
We observed that antibody against MNSFb recognized
these two bands (Fig 1, lane 6) These MNSFb
anti-body-reactive proteins were first subjected to
N-termi-nal sequence aN-termi-nalysis after electroblotting Despite
repeated attempts, ambiguous signals were obtained
from each protein (about 100 pmol) For internal
sequencing, the protein bands were digested in-gel with
trypsin Selected peptides from the 62 kDa band were subjected to sequence analysis, and the results showed the amino acid sequences of MNSFb and an irrelevant protein (data not shown) No clear results were obtained from the 95 kDa band
MALDI-TOF MS analysis of MNSFb adducts
To identify the target molecule of MNSFb, MALDI-TOF MS was performed by separating the 62 kDa MNSFb adduct by SDS⁄ PAGE under reducing condi-tions Bands corresponding to the 62 kDa MNSFb adduct were excised and subjected to in-gel digestion with trypsin as described Then, the resulting mixtures
of peptides were analyzed by MALDI-TOF MS Table 1 shows the peptide masses observed by MALDI-TOF MS of the 62 kDa MNSFb adduct puri-fied from murine liver The resulting sets of peptide masses were then used to search the NCBI database for potential matches Seven of the peptides in the MALDI spectrum matched endophilin II protein, pro-viding a sequence coverage of 32% This result indicates that the 62 kDa MNSFb adduct is an MNSFb–endophilin II protein complex Endophilin II
is a member of the endophilin A family, and is ubiqui-tously expressed in various tissues Endophilin II possesses the SH3 domain, which is critical for associa-tion with synaptojanin-1 and dynamin A signal was
-95 kDa MNSF adduct -62 kDa MNSF adduct
1 2 3 4 5 6
97
65
45
31
kDa-CBB Silver WB: anti-MNSF
Fig 1 Purified fractions of MNSFb adducts analyzed by SDS ⁄ PAGE (12% polyacrylamide gel) and immunostained for pro-tein Lanes 1 and 4 contain an aliquot (100 lg of total protein) from liver lysates prepared from Balb ⁄ c mice; lanes 2 and 5 contain an aliquot from the DEAE chromatography purification step; lanes 3 and 6 contain an aliquot from the anti-MNSFb affinity chromatogra-phy purification step; lanes 1 and 2, CBB (Coomassie brilliant blue)-stained; lane 3, silver-blue)-stained; lanes 4–6, immunostained with antibodies against MNSFb Mobilities of the 95 and 62 kDa MNSFb adducts and the molecular mass standards (kDa) are indicated to the right and left of the figure, respectively.
Trang 3detected at 2096.3 Da that corresponds to amino acids
7–25 of MNSFb Importantly, a pair of signals was
detected at 1161.4 and 1605.0 Da (Table 2) These
sig-nals correspond to digestion fragments in which amino
acids 291–297 of endophilin II are covalently linked by
an isopeptide bond to amino acids 71–74 of MNSFb,
and amino acids 291–301 are linked by an isopeptide
bond to amino acids 71–74 Collectively, MNSFb may
conjugate to endophilin II with a linkage between the
C-terminal Gly74 and Lys294 Lys294 is specific for
endophilin II among all members of the endophilin
family (Fig 2)
Immunoblot and immunoprecipitation analyses
To confirm that MNSFb covalently conjugates to
endophilin II, we performed immunoblotting of
samples purified by MNSFb antibody affinity
chroma-tography The results of the blotting revealed that anti-body against endophilin II specifically recognized the
62 kDa, but not the 95 kDa, MNSFb adduct (Fig 3A), indicating that the 62 kDa band is a com-plex of MNSFb with endophilin II
To further study these interactions, we performed immunoprecipitation experiments using antibodies against endophilin Cell lysates were prepared from mouse liver and immunoprecipitated with antibodies directed against MNSFb, and associated proteins were analyzed by western blot analysis by using antibodies against endophilin As shown in Fig 3B, MNSFb associated with endophilin II Immunoprecipitation of cell lysates with normal IgG followed by western blot analysis revealed no detectable association of endophi-lin II, indicating the specificity of MNSFb for endo-philin II In addition, converse immunoprecipitation with antibody against endophilin II and immunoblot analysis with antibody against MNSFb confirmed the association between endophilin II and the MNSFb adduct Brain extracts were also examined by immuno-precipitation⁄ western blot As depicted in Fig 3C, neither antibody against endophilin I nor antibody against endophilin II recognized the MNSFb adduct Thus, we concluded that the 62 kDa MNSFb adduct
is an MNSFb–endophilin II complex and is specifically expressed in liver Collectively, the results of immuno-blotting, together with the internal peptide sequences
in Table 1 and MALDI-TOF MS analysis, show that MNSFb covalently binds to endophilin II via an isopeptide bond
We next performed immunoblotting analysis to mea-sure endophilin II levels in various organs of mice As shown in Fig 4, the 45 kDa endophilin II is expressed
Table 1 Assignment for peptide fragments from a trypsin digest of the 62 kDa MNSFb adduct The 62 kDa MNSFb adduct was digested
by trypsin and subjected to MALDI-TOF MS analysis The data in the second column are the mass values obtained experimentally, whereas the results in the third column are those calculated from the trypsin fragmentation of the gene products of endophilin II and MNSFb The fourth column indicates the numbers of the first and last amino acid of the indicated endophilin II and MNSFb peptides, whereas the fifth shows the corresponding amino acid sequences.
Mass (MH+)
Endo-II
MNSFb
Table 2 Isopeptide bonds between the C-terminal glycine of
MNSFb and the lysine of endophilin II The 62 kDa MNSFb adduct
was digested by trypsin and subjected to MALDI-TOF MS analysis.
The data in the first column are the mass values obtained
experi-mentally, whereas the results in the second column are those
calculated from the trypsin-fragmented peptide complexes The
third column shows the corresponding amino acid sequences of
MNSFb and endophilin II (in bold).
Mass (MH + )
MLGG(71–74)
MLGG(71–74)
Trang 4in different mouse tissues, including brain (cerebrum
and cerebellum) and testis It should be pointed out
that the 30 kDa truncated form of endophilin II
(tEnd-II) was consistently observed in liver (Fig 4A,
lane 4) The N-terminal region of endophilin II might
be cleaved, because we employed antibodies against
the middle (in this study) and C-terminal (not shown)
regions of endophilin II We examined the expression
of endophilin II in the murine Raw264.7
macrophage-like cell line and peritoneal macrophages, because
endophilin II is a member of the endocytic endophilin
family We observed two bands (62 kDa MNSFb–
endophilin II and 45 kDa endophilin II) in the western
blot of the macrophages (Fig 4B, lanes 1 and 2)
Interestingly, heterogeneous bands, including the
62 kDa band (MNSFb–endophilin II), were
reproduc-ibly observed in liver lysates but not in brain lysates
(Fig 4B, lanes 3 and 4: dialyzed against NaCl⁄ Pi in
the presence of protease inhibitors) This observation
is consistent with the results for the formation of the
62 kDa complex of MNSFb and endophilin II in liver
(Fig 3)
Regulation of phagocytosis by endophilin II
Endophilin II is one of three members of the subgroup
endophilin A, but its function in regulating endocytosis
remains unclear It has been reported that endophilin I
and endophilin III are implicated in receptor-mediated
endocytosis [15] Thus, we assessed the possible roles
of endophilin II in the phagocytosis of zymosan in
macrophages We employed Raw264.7 cells, which
have been studied in our laboratory [3] The 62 kDa
MNSFb adduct and endophilin II are expressed in
untreated Raw264.7 cells (Figs 4B and 5A)
Endophi-lin II small interfering RNA (siRNA), but not control
scramble siRNA, specifically reduced the expression of
endophilin II but not of a-tubulin (Fig 5A) It should
be noted that the expression of 62 kDa MNSFb–endo-philin II was also decreased As can be seen in Fig 5B, Raw264.7 cells phagocytized zymosan particles (30.3% ± 3.6%) Cells capturing more than two ops-onized zymosan particles were judged as positive We examined the effect of endophilin II siRNA on the phagocytosis of opsonized zymosan in Raw264.7 cells The treatment of Raw264.7 cells with endophilin II siRNA significantly enhanced phagocytosis (1.9-fold) (Fig 5C) Interestingly, MNSFb siRNA, which has been used in the previous studies [3], showed similar effects on phagocytosis, although the effect was less potent (1.4-fold) (Fig 5C) To determine whether MNSFb–endophilin II formation in Raw264.7 cells is involved in the phagocytosis of zymosan, immunoblot-ting was performed with the use of antibodies against endophilin II We did not observe 62 kDa MNSFb– endophilin II in the cells treated with MNSFb siRNA (Fig 5A), suggesting that MNSFb siRNA was effec-tive in reducing MNSFb protein levels Importantly, Raw264.7 cells transfected with endophilin II and MNSFb siRNA (double knockdown) enhanced phago-cytosis to a similar extent as observed for cells treated with endophilin II siRNA alone Thus, it is unlikely that free MNSFb plays a central role in the phagocy-tosis The facilitatory effect of MNSFb siRNA was lower than that of endophilin II siRNA In addition, the expression of 62 kDa MNSFb–endophilin II was much lower than that of endophilin II Together, these results strongly indicate that MNSFb–endophilin II has a much higher inhibitory activity than endophilin
II alone Transient DNA transfection experiments were performed to confirm this idea As can be seen in Fig 5D, transfection with pcDNA3.1–endophilin II resulted in significant inhibition of the phagocytosis
of zymosan (71.1%) in Raw264.7 cells, consistent with the data from siRNA knockdown experiments (Fig 5C) No such inhibition was observed in cells
Fig 2 Primary structure of mouse endophi-lins End-I, endophilin I; End-II, endophilin II; End-III, endophilin III The SH3 domain is boxed In End-II, the Lys294 residue respon-sible for isopeptide formation is in bold The lengths and numbers of the endophilins are indicated.
Trang 5expressing a mutant endophilin II that has an arginine
in place of the lysine at position 294 that is responsible
for MNSFb conjugation Cotransfection of the
expres-sion vectors encoding endophilin II and MNSFb
caused stronger inhibition of the phagocytosis of
zymosan (75.1%) We observed weak but significant
inhibition of zymosan phagocytosis in cells transfected
with pcDNA3.1–MNSFb Immunoblotting analysis
showed that the expression of MNSFb–endophilin II
was increased in cotransfected cells (Fig 5E) Collec-tively, these observations demonstrate that Lys294 of endophilin II is the only site of MNSFb conjugation
A
B
C
65
kDa-A
B
45
31
65
45
31
kDa-8
-End-II
-tEnd-II
-End-II
- tEnd-II -MNSF/End-II
Fig 4 Tissue distribution of endophilin II (A) Tissue homogenates (50 lg of protein each) obtained from the indicated organs were subjected to immunoblotting analysis using antibody against endo-philin II Lane 1: cerebrum Lane 2: cerebellum Lane 3: brain stem Lane 4: liver Lane 5: testis Lane 6: epididymis Lane 7: prostate gland Lane 8: seminal vesicle (B) Lane 1: peritoneal macrophage lysates Lane 2: Raw264.7 cells Lane 3: liver lysates dialyzed against NaCl ⁄ P i in the presence of protease inhibitors Lane 4: brain lysates dialyzed as liver in lane 3.
Fig 3 Western blot (WB) and immunoprecipitation (IP) analysis of MNSFb adducts (A) Samples purified by anti-MNSFb affinity chro-matography from mouse liver cell lysates were analyzed by wes-tern blot Lane 1: control rabbit IgG Lane 2: antibodies against MNSFb Lane 3: antibodies against endophilin II (B) Liver cell extracts (300 lg) were immunoprecipitated with antibodies against MNSFb (lane 2) or normal IgG (lane 1) Immunoprecipitates were analyzed by western blot with antibodies against endophilin II (lanes 1 and 2) Conversely, the extracts (300 lg) were immunopre-cipitated with antibodies against endophilin II (lane 4) or normal IgG (lane 3) Immunoprecipitates were analyzed by western blot with antibodies against MNSFb (lanes 3 and 4) (C) Brain cell extracts (300 lg) were immunoprecipitated with antibodies against MNSFb (lanes 1 and 2) Immunoprecipitates were analyzed by western blot with antibodies against endophilin II (lane 1) or endophilin I (lane 2) The cell extracts (5 lg) were analyzed by western blot with anti-bodies against endophilin I (lane 3) In (A) and (B), mobilities of the
62 kDa MNSFb adduct (arrowhead) and the molecular mass stan-dards (kDa) are indicated to the right and left of the figure, respec-tively In (C), the mobility of 40 kDa endophilin I (End-I) is indicated
to the right of the figure.
Trang 6and that this post-translational modification may
nega-tively regulate phagocytosis in macrophages
Discussion
We have previously demonstrated that MNSFb forms
an isopeptide bond with Bcl-G, a proapoptotic protein
MNSFb becomes isopeptide-linked via its C-terminal
diglycine motif, by analogy with ubiquitin and other
ubiquitin-like modifiers, such as SUMO MNSFb–Bcl-G
directly binds to ERKs, and inhibits ERK activation by
MAP kinase kinase 1 [3] Similarly, in this study, we
showed that MNSFb covalently binds to endophilin II in
liver and Raw264.7 cells We isolated a 62 kDa MNSFb
adduct from mouse liver lysates (Fig 1) MALDI-TOF
MS analysis of the 62 kDa MNSFb adduct showed that
MNSFb conjugates to endophilin II with a linkage
between the C-terminal Gly74 and Lys294 The
molecu-lar mass of 62 kDa was somewhat molecu-larger than that of
endophilin II conjugated by a single molecule of MNSFb (calculated molecular mass, 49.242 Da) One may specu-late that MNSFb might conjugate to at least one lysine
in addition to Lys294 in endophilin II However, we did not observe any candidate fragments with theoretical masses by MALDI-TOF MS Additionally, experiments with mutants indicate that Lys294 of endophilin II is the only site of MNSFb conjugation (Fig 5E)
Endophilins are SH3 domain-containing proteins Three isoforms of endophilin have been identified: endophilin I, which functions during synaptic vesicle recycling; endophilin II, which is ubiquitously distrib-uted throughout many tissue types; and endophilin III, which is expressed in brain and testis [16] Although many studies have been focused on endophilin I, the underlying mechanism of action of endophilin II remains unclear Lua and Low have reported [17] that overexpression of endophilin II enhances epidermal growth factor (EGF)-stimulated receptor endocytosis
1
A
B
MNSF/
End-II-
-tubulin-Untreated cells
Opsonized zymosan
Control End-II siRNA End-II cDNA
MNSF – – + + – +
End-II mEnd-II – + – + – – – –
1
0
– + – +
**
**
*
3
0
** **
*
2
1
End-II siRNA
MNSF siRNA
– + – + – – + +
MNSF/End-II-MNSF – – + + – +
End-II
mEnd-II – + – + – – – –
– + – +
End-II-
-tubulin-Fig 5 Effect of endophilin II on formation of phagosomes in Raw264.7 cells (A) Expression of endophilin II and MNSFb–endophilin II were analyzed by western blot after treatment with siRNAs for 72 h Lanes 1, 3 and 4: transfected with scramble (control) siRNA Lane 2: endo-philin II siRNA Lane 5: MNSFb siRNA Lanes 1, 2, 4 and 5: immunostained with antibody against endoendo-philin II Lane 3: control rabbit IgG Mobilities of 62 kDa MNSFb ⁄ endophilin II (open arrowhead) and 45 kDa endophilin II (closed arrowhead) are indicated to the left and right of the figure (B) Raw264.7 cells were untreated or incubated with IgG-opsonized zymosan particles (Alexa488-labeled) for 30 min Fluo-rescence was viewed with confocal microscopy Cells were transfected with siRNA or cDNA for endophilin II as described in Experimental procedures (C) Raw264.7 cells were treated with siRNAs for 72 h before addition of zymosan particles The phagocytosis assay was performed as described in Experimental procedures Cells capturing more than two zymosan particles were regarded as positive cells Values are shown as the mean ± standard deviation (n = 5) *P < 0.05 versus untreated; **P < 0.01, one-way ANOVA test (D) Raw264.7 cells were transiently cotransfected with expression vectors encoding either the endophilin II or endophilin II mutant, along with expression vector for MNSFb The phagocytosis assay was performed after 72 h of transfection as described in Experimental procedures Values are shown as the mean ± standard deviation (n = 3) *P < 0.05 versus empty vector; **P < 0.01, one-way ANOVA test (E) Western blot analysis of extracts of cells transfected with the wild-type or mutant endophilin II cDNA The results of immunoblotting using antibodies against endophilin II and a-tubulin are shown.
Trang 7and ERK1⁄ 2 phosphorylation Angers et al [18] have
reported that the E3 ubiquitin ligase Itch ubiquitinates
endophilin I and localizes to the endosomal system
fol-lowing EGF stimulation They also mentioned that
endophilin I binds to germinal center kinase-like
kinase, suggesting a role for endophilin I in the c-Jun
N-terminal kinase signaling pathway [19] Together
with our previous studies on the regulation of ERK
activity by MNSFb, this indicates that endophilins,
ubiquitin and ubiquitin-like protein(s) may be closely
involved in MAP kinase pathways Experiments are
underway to isolate E3 ubiquitin ligase-like enzyme(s)
involved in MNSFb conjugations
Sugiura et al [15] mentioned that the variable region
of endophilin III is important in regulating transferrin
endocytosis In this study, we showed that MNSFb
covalently modifies the Lys294 in the variable region
of endophilin II and that this modification is involved
in the phagocytosis of zymosan in Raw264.7 cells
Thus, as in the case of endophilin III, the variable
region of endophilin II may be important in regulating
endocytosis in macrophages Interestingly, both
endo-philin II and endoendo-philin III negatively regulate
recep-tor-mediated internalization [15] It has been reported
that endophilin family members bind to synaptojanin
and dynamin via a Grb2-like Src homology 3 domain
(constant region) [9,20] In this context, MNSFb may
not affect the binding of endophilin II to these
part-ners It should be noted that the residue responsible
for MNSFb conjugation (Lys294) is only found in
endophilin II (Fig 2) Indeed, we did not observe
MNSFb–endophilin I in brain extracts (Fig 3C)
In preliminary experiments, we observed that
dectin-1 (b-glucan receptor) involves the mechanism of action
of endophilin II on phagocytosis in macrophages
Dec-tin-1-mediated intracellular signaling pathways
regulat-ing phagocytosis in macrophages remain largely
unknown Although Syk involves most of the functions
of dectin-1, this kinase is not required for particle
uptake in macrophages [21] Investigations are
under-way to clarify whether MNSFb–endophilin II is a
mediator in dectin-1-mediated signaling
It is interesting that MNSFb–endophilin II was
detected in liver cells in terms of endocytosis The
truncated 30 kDa form may be a consequence of the
MNSFb adduct It is possible that the N-terminal
region may be deleted from endophilin II in these
tis-sues, because specific antibodies against C-terminal
(Fig 4) and central (not shown) regions detected the
truncated form Investigations are underway to clarify
the mechanism of action of endophilin II in liver cells
Monoubiquitination is thought to regulate receptor
internalization and endosomal sorting [22] It is evident
that monoubiquitination is involved in endocytosis of EGF receptor [23] In this study, we presented data showing that endophilin II may act in concert with MNSFb to inhibit the phagocytosis of zymosan (Fig 5) Thus, ubiquitin and ubiquitin-like protein(s) may regu-late endocytosis by modification of target proteins Taken as a whole, the present study demonstrates that endophilin II may negatively regulate phagocyto-sis, and that MNSFb–endophilin II formation might
be important for potent regulation of phagocytosis in macrophages
Experimental procedures
Materials
Rabbit polyclonal antibodies against MNSFb were prepared
as previously described [4] Rabbit polyclonal antibodies against endophilin I and endophilin II were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Alexa488–zymosan A was purchased from Molecular Probes The zymosan particles were opsonized by using Opsonizing Reagents (Molecular Probes, Eugene, OR, USA) derived from purified rabbit polyclonal IgG antibodies that are specific for zymosan Rabbit TrueBlot was obtained from eBioscience (San Diego, CA, USA) DEAE cellulose was purchased from Sigma-Aldrich (Tokyo, Japan)
Purification of the MNSFb adduct
Mouse liver was obtained from BALB⁄ c mice All purifica-tion steps were performed at 4C A portion of a mouse liver (8 g) was homogenized in buffer A (20 mm Tris⁄ HCl, pH 7.5,
50 mm NaCl, 1 mm dithiothreitol) containing a protease inhibitor mixture [1 mm 4-(2-aminoethyl)-benzenesulfonyl
1 mgÆL)1pepstatin A], using a tissue blender (3· 30 s) The homogenate was centrifuged at 13 000 g for 20 min, filtered through gauze, and centrifuged at 13 000 g for another
20 min The resulting supernatant was further centrifuged at
120 000 g for 60 min to obtain a cytosolic extract, which was incubated overnight with DEAE (Sigma, Tokyo, Japan) pre-equilibrated with buffer A Following extensive washing with buffer A, bound proteins were eluted in 20 mm Tris⁄ HCl (pH 7.5) and 100 mm NaCl, and the protease inhibitor mix-ture Additional purification was achieved by anti-MNSFb affinity chromatography, as previously described [1]
Immunoblotting
Cell extracts in SDS sample buffer were subjected to 12%
membranes The membrane was incubated overnight at
Trang 8block nonspecific binding sites Subsequently, the
mem-branes were incubated with anti-MNSFb rabbit IgG in
the blocking buffer, after which they were incubated with
performed according to the enhanced chemiluminescence
detection system (Amersham Biosciences, Chalfont St Giles,
UK) We have previously demonstrated that antibody
against MNSFb does not cross-react with ubiquitin [4]
In-gel digestion and MALDI-TOF MS
All experimental procedures were described in the previous
study [2] Briefly, silver-stained spots were cut out of the
gels for in-gel digestion and digested with trypsin (Sigma)
The peptides were extracted from the gel matrix by vortexing
for 30 min, and then concentrated using Zip Tips (Millipore
Corp., Bedford, MA, USA) Peptide mass fingerprinting
was performed using a PerkinElmer⁄ PerSeptive Biosystems
(Framingham, MA, USA) Voyager-DE-RP MALDI-TOF
mass spectrometer The peptide samples were cocrystallized
with matrix on a gold-coated sample plate using 0.5 lL of
matrix (a-cyano-4-hydroxytranscinnamic acid) and 0.5 lL of
sample Cysteines were treated with iodoacetamide to form
carboxyamidomethyl cysteine, and methionine was
consid-ered to be oxidized
Immunoprecipitation
Immunoprecipitation was performed with a horseradish
rabbit IgG (TrueBlot), according to the manufacturer’s
instructions RIPA buffer (50 mm Tris, 1% Nonidet P-40,
0.25% deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm
1 mgÆmL)1 each of the protease inhibitors aprotinin,
leu-peptin, and pepstatin) extracts of Raw264.7 cells were
pre-cleared with 50 mL of anti-rabbit IgG beads for 1 h on ice
Subsequently, 5 mg of primary antibody against MNSFb
or endophilin II was added to precleared lysates and
bated on ice for an additional 1 h Samples were then
beads The beads were washed five times with RIPA buffer,
and immunoprecipitates were released from the beads by
10 min of boiling in NuPAGE LDS sample buffer
(Invitro-gen) Immunoblotting was performed with antibody against
MNSFb or antibody against endophilin II A rabbit IgG
TrueBlot was employed as a second antibody
Cell culture, the siRNAs, and transfection of cells
Cells of the Raw264.7 macrophage-like cell line (ATCC
TIB-71) was cultured routinely in DMEM with 10% fetal
bovine serum and penicillin⁄ streptomycin at 37 C and 5%
CO2 Small interfering RNA duplexes were synthesized and
purified by Qiagen, Inc (Chatsworth, CA, USA) The
5¢-CCACCCTGCCATGCTAATAAA-3¢ [3]; and endophi-lin II siRNA, 5¢-AAGGTGCTCTAGAAACACTAA-3¢ Scramble siRNA directed against 5¢-GGACTCGACGC AATGGCGTCA-3¢ was the negative control Cells were treated with siRNA according to the instructions provided with the RNAiFect transfection reagent (Qiagen, Inc.) Raw264.7 cells (1.2· 105) were treated with 3 lg of siRNA
in RPMI-1640 medium supplemented with 10% fetal boo-vine serum in the presence of the RNAiFect transfection reagent After a 48 h incubation at 37C, the medium con-taining the mixture of RNAiFect and siRNA was replaced
by DMEM containing 10% fetal bovine serum, and cells were incubated for a further 24 h Mutant endophilin II (K294R) was generated by replacing the codon for Lys294 with the codon for arginine by utilizing the LA PCR
some experiments, Raw264.7 cells were transiently cotrans-fected with expression vectors encoding either the endophi-lin II or endophiendophi-lin II mutant along with expression vector for MNSFb, as previously described [3]
Collection of peritoneal macrophages
Peritoneal macrophages were obtained using mice injected
4 days previously with 2 mL of a sterile 3% brewer thio-glycolate broth (Difco, Detroit, MI, USA) The cells were collected by centrifugation (at 400 g for 5 min), washed, and resuspended in DMEM containing 10% fetal bovine serum Lysates of the collected macrophages were used for immunoblotting
Phagocytosis assays
Raw264.7 cells were treated with siRNAs as described above Cells were seeded at 5· 104cells per well in four-well cham-ber plates (Nunc, Roskilde, Denmark) Cells were washed, and Alexa488–zymosan A (Molecular Probes) was added (10 particles per cell) and allowed to bind for 1 h at 4C Following this incubation, unbound zymosan was removed
by washing, and the cells were incubated at 37C for 30 min
to allow particle uptake To determine the internalization of zymosan, cells capturing zymosan were treated with 0.05% Trypan blue in saline for a few minutes before observations
by fluorescence microscopy To determine the phagocytosis index, we identified > 200 Alexa-positive cells in randomly chosen fields of view, and the percentage of cells capturing more than two zymosan particles was determined
Acknowledgement
This study was supported in part by Grants-in-Aid for Scientific Research (19570131 to M Nakamura)
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