Glycobiology Advance Access originally published online on July 6, 2005
Glycobiology 2005 15(11):1067-1075; doi:10.1093/glycob/cwj005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Bisecting GlcNAc mediates the binding of annexin V to Hsp47
2 Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 3 JST, Japan Science and Technology Agency, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan; 4 Department of Surgical Oncology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 5 Department of Molecular Medicine, Kochi University Medical School, 2-5-1 Akebono-cho, Kochi 780-8520, Japan; and 6 Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan
1 To whom correspondence should be addressed; e-mail: miyoshi34{at}biochem.med.osaka-u.ac.jp
Received on December 6, 2004; revised on June 25, 2005; accepted on June 26, 2005
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : ß-D-mannoside ß-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 µM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.
Key words: annexin V / bisecting GlcNAc / Hsp47
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
Oligosaccharides play a pivotal role in a multitude of biological processes such as protein folding (Yoshida et al., 2002
), stability (Bell et al., 2003), targeting (Ihrke et al., 2001), and clearance (Negroiu et al., 2003), as well as cell-matrix and cell–cell interactions (Akama et al., 2002). If specific structures of oligosaccharides have a biological function, a lectin that binds to them could be a key factor in several processes. In N-linked oligosaccharides, "bisecting N-acetylglucosamine" (GlcNAc), which is reported to be responsible for a variety of biological functions, is a unique structure (Taniguchi et al., 1996, 1999). This structure is formed by the attachment of a ß1-4 GlcNAc residue to a core ß-mannose, the reaction of which is catalyzed by UDP-N-acetylglucosamine : ß-D-mannoside ß-1,4-N-acetylglucosaminyltansferase III (GnT-III). It has been reported that increases in the level of bisecting GlcNAc sugar chains lead to some significant alterations in cells including an altered sorting of glycoproteins (Sultan et al., 1997), splenic colonization of leukemia cells in athymic mice (Yoshimura et al., 1996), changes in epidermal growth factor receptor signaling (Sato et al., 2001) and E-cadherin/ß-catenin functions (Kitada et al., 2001). These observations suggest that a particular protein molecule, a lectin that recognizes the bisecting GlcNAc sugar chain might be involved in such processes and that an alteration in the oligosaccharide structure of a specific glycoprotein leads to changes in its function. In our previous study, annexin V was identified as a lectin that binds the bisecting GlcNAc oligosaccharide structure (Gao-Uozumi et al., 2000).
Annexin V is a member of a family of structurally related proteins that bind to phospholipids in a calcium-dependent manner. Although precise biological functions in vivo have not yet been elucidated completely, many in vitro experiments indicate that annexin V exhibits membrane channel activity (Berendes et al., 1993), acts as an antiblood coagulation factor (Chap et al., 1988), plays a role in viral entry and infection by influenza and hepatitis B viruses (Huang et al., 1996), and functions in cytoskeletal–membrane interactions (Katayanagi et al., 1999). In recent years, annexins have been extensively studied on their carbohydrate binding property. Annexins have been suggested to act as a critical component in regulating secretary pathway as a result of this property (Rosales and Ernst, 1997). In addition to the binding activity toward bisecting GlcNAc structure, annexin V has also been reported to interact with glycosaminoglycans (Ishitsuka et al., 1998). Although many extracellular events are now proposed to be annexin mediated, annexin V was originally thought to be an intracellular protein. Our previous study demonstrated that bisecting GlcNAc structures accumulated in intracellular organs in forskolin-treated M31 hepatoma cells (Sultan et al., 1997). Taking into account the carbohydrate binding activity of annexin V and its localization, we postulate that annexin V may contribute to the traffic of intracellular glycoproteins through the bisecting GlcNAc structure.
The aim of this study was to isolate annexin V-binding intracellular glycoproteins by affinity chromatography using a glutathione S-transferase (GST)-annexin V-immobilized column, to better understand the mechanism by which annexin V exerts its biological function for glycoprotein sorting.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
Identification of Hsp47 as one of the annexin V-binding proteins
Since annexin V has been reported to bind to bisecting GlcNAc sugar chains, we attempted to isolate cellular glycoproteins that interact with annexin V in a oligosaccharide-dependent manner. Cell lysates were prepared by extraction of cultured GnT-III transfected rat hepatoma M31 cells. A lectin blotting was presented to show that reactivity to erythroagglutinating phytohemagglutinin (E4-PHA) increased significantly in the GnT-III-transfected cells compared with mock cells (Figure 1A). The extract was then subjected to a GST-annexin V-immobilized glutathione sepharose 4B affinity column, and the bound proteins were eluted as described in the Materials and methods. All fractions were concentrated, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in 10% polyacrylamide gels and visualized by Coomassie brilliant blue staining (Figure 1B). Two major candidates, band 1 and band 2, were successfully separated in the adsorbed fraction, which were eluted parallel to the elution pattern of GST-annexin V that was dissociated from glutathione sepharose 4B column by 1M NaCl in elution buffer (data not shown). They were not retained on a GST-immobilized glutathione sepharose 4B column (data not shown). The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane, and the amino acid sequences were determined. The N-terminal sequence, AEVKKPVEAAAPG, was successfully determined for band 1. This peptide sequence corresponds to the rat heat shock protein 47 (Hsp47) (Figure 1C). In addition, band 1 was specifically recognized using an anti-Hsp47 monoclonal antibody (Figure 1D). According to the peptide sequence analysis, band 2 appeared to be annexin II that had been reported to bind annexin V in a Ca2+-dependent manner (Brooks et al., 2002
). Since Hsp47 is a protein that has potential N-glycosylation sites and the function of its oligosaccharides has not been examined, we focused on this molecule.
|
Role of sugar chains in the interaction between annexin V and Hsp47
To assess whether Hsp47 is actually N-glycosylated, and whether the N-glycan mediates the binding of Hsp47 to annexin V, we treated the cell extracts with N-glycosidase F. A cell extract was prepared from GnT-III/annexin V cDNA cotransfected M31 cells. N-glycosidase F treatment was done at a final concentration of 3 U/mL at 37°C overnight, followed by immunoprecipitation using an anti-Hsp47 antibody or an anti-annexin V antibody. The western blot analysis using anti-Hsp47 antibody revealed that Hsp47 was separated into 2 groups dependent on whether it was N-glycosylated or not. Most of the Hsp47 was N-glycosylated because the band of Hsp47 after N-glycosidase F treatment had a greater mobility on SDS–PAGE than the nontreated one (Figure 2A). Only N-glycosylated Hsp47 was coprecipitated with annexin V and the binding activity of annexin V to Hsp47 was nearly abolished by the N-glycosidase F treatment (Figure 2A). A remaining band found at both lanes of anti-Hsp47 and anti-annexin V after the N-glycosidase F treatment was considered due to the incomplete digestion of N-glycosidase F.
|
To further verify the interaction of annexin V with N-glycosylated Hsp47, experiments involving the addition of free oligosaccharides were performed. Agalacto bisected biantennary or the corresponding nonbisected sugar chains were added to cell lysates in a concentration of 2 mM, before immunoprecipitation with anti-annexin V antibody. A cell lysate without exogenous oligosaccharide was used as a negative control (Figure 2B, control). In the presence of bisecting GlcNAc sugar chains, only a small amount of glycosylated Hsp47 was bound to annexin V, the majority of this protein still remained in the supernatant (Figure 2B). On the other hand, the nonbisecting GlcNAc sugar chain did not affect the interaction with anti-annexin antibody, and all of the glycosylated Hsp47 was found in the precipitation fraction (Figure 2B). These data strongly suggest that bisecting GlcNAc plays an important role in the interaction between annexin V and Hsp47.
Measurement of the binding of annexin V to Hsp47 by surface plasmon resonance
The above described results demonstrate that N-glycans, especially bisecting GlcNAc sugar chains mediate the binding of Hsp47 to annexin V. To further confirm this, we determined whether Hsp47 and annexin V are able to bind in a cell-free system using a surface plasmon resonance system. Combined with gelatin-agarose, Bio-gel P-6DG, and carboxymethyldextran (CM) ion-exchange chromatography, Hsp47 was purified from GnT-III or mock transfected M31 cells (Figure 3B, upper and middle panel). A typical purification procedure was shown in Figure 3A. An E4-PHA lectin blot analysis showed that Hsp47 from GnT-III transfectants contained more bisecting GlcNAc structures than that from M31 mock cells (Figure 3B, bottom panel).
|
To verify the changes in the core structure by GnT-III, pyridylaminated (PA) oligosaccharides of Hsp47 purified from mock and GnT-III-transfected M31 were exhaustively digested with a complete mixture of glycosidase, which contained sialidase and ß-galactosidase, followed by analysis using reversed phase high performance liquid chromatography (HPLC) (Figure 4, inset panel). The major peak of each cell was identified as the PA-agalacto-biantennary oligosaccharides for normal cell and PA-agalacto-bisected-biantennary oligosaccharides for GnT-III transfected cells using MALDI-TOF mass analysis (Figure 4). The value of 1420.37(M+N)+ corresponds to that of PA-agalacto-biantennary oligosaccharides, and the value of 1622.341(M+N)+ corresponds to that of PA-agalacto-bisected-biantennary oligosaccharides.
|
When the binding of fluid-phase Hsp47 to annexin V immobilized on a carboxymethyldextran biosensor chip was measured, it was found that Hsp47 derived from GnT-III transfectants had a much stronger binding activity to the immobilized annexin V than that derived from mock cells (Figure 3C). Next, Hsp47 purified from GnT-III transfectants was then separated into two groups according to the existence of bisecting GlcNAc sugar chain using an E4-PHA agarose column. Hsp47 bearing bisecting GlcNAc sugar chains was observed to have a much stronger binding to annexin V than Hsp47 that did not bind to the E4-PHA column (Figure 3D). The interaction pattern between Hsp47 bearing bisecting GlcNAc sugar and annexin V was very similar to that between annexin V and the bisecting GlcNAc sugar chain (Gao-Uozumi et al., 2000).
Different concentration of Hsp47 bearing a bisecting GlcNAc sugar chain was used to measure the interaction with the immobilized annexin V and Kd was calculated as 5.5 µM. These analyzes support the view that a bisecting GlcNAc is important in the binding of annexin V to glycoproteins.
Colocalization of annexin V with Hsp47
Since annexin V has been reported to be present in various cellular locations (Spreca et al., 1992; Sun et al., 1992), we examined whether annexin V was colocated with bisected sugar chains or Hsp47 in M31 GnT-III and annexin V cotransfected cells. An immunofluorescence study was performed as described in the Experimental procedures. Cells were stained with anti-annexin V full-length polycolonal antibody (Figure 5A and D), fluorescein isothiocyanate (FITC)-labeled-E4-PHA (Figure 5B) and anti-Hsp47 monoclonal antibody (Figure 5E). Although that reactivity to E4-PHA increased significantly in the GnT-III-transfected cells compared with that of mock cells (Figure 1A), the bisecting GlcNAc structure, represented by FITC-E4-PHA positive spots, was found to have accumulated around the Golgi compartment although no staining was detected at the cell surface. Although Hsp47 was detected mainly in the endoplasmic reticulum and Golgi complex, annexin V was found to be colocated with the FITC-labeled-E4-PHA positive spots (Figure 5C) and with Hsp47 at Golgi, but not endoplasmic reticulum (ER) on merged images (Figure 5F). The lectin blotting of whole cell lysate of mock and GnT-III transfected M31 cells with E4-PHA showed the difference of the amount of glycoproteins that are positive for the E4-PHA staining. These observations as well as the result of surface plasmon resonance support the conclusion that annexin V recognizes Hsp47 in a bisecting-GlcNAc-sugar-chain-dependent manner in vivo.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
Hsp47 is reported to be a heat-shock protein that interacts transiently with procollagen during its folding, assembly, and transport from the ER in mammalian cells (Satoh et al., 1996
; Tasab et al., 2000). In this study, Hsp47 was identified as one of the proteins that binds to annexin V. In addition, the binding of annexin V to Hsp47 was characterized by immunoprecipitation with pretreatment with N-glycosidase F or E4-PHA precipitation, surface plasmon resonance analysis, and immunofluorescence microscopy analysis. The findings showed that the interaction between Hsp47 and annexin V was dependent on the presence of oligosaccharides, bisecting GlcNAc in particular.
Potential N-glycosylation sites have been deduced, based on the protein sequence of human, rat, mouse and chicken Hsp47 (Clarke et al., 1991, 1992) and the number of potential N-glycosylation sites in these sequences is two for the human and chicken Hsp47, three in the rat and the mouse Hsp47, respectively. Possible functions of the carbohydrate structure of Hsp47 were investigated for the first time in this study. Treatment of rat Hsp47 with N-glycosidase F reduced its molecular mass from approximately 47 to 38kDa, which corresponds to the expected sizes of three N-linked oligosaccharides, suggesting that all three of the N-glycosylation sites are glycosylated. A remaining band after the N-glycosidase F treatment was found at both lanes of immunoprecipitation with anti-Hsp47 antibody and that with anti-annexin V antibody. For the remaining band had the same molecular mass as the nontreated lane, it was considered that 3U/mL of N-glycosidase F was not enough to release all the N-glycans of Hsp47.
Besides E4-PHA lectin blot, we assessed the changes in the core structure of PA-oligosaccharides purified from Hsp47 of mock cells or that of GnT-III-transfected cells using reversed phase HPLC and MALDI-TOF mass analysis. The major peak on the HPLC profile of GnT-III-transfected cells was certificated as a core structure with bisecting GlcNAc [GlcNAcß1-2Man
1-6(GlcNAcß1-2Man1-3)(GlcNAcß1-4)Manß1-4GlcNAcß1-4GlcNAc (Gn(Gn)Gn)].
Phospholipids, calcium, and glycosaminoglycans have been reported to be ligands for annexin V according to biochemistry and structure information (Huber et al., 1990; Kojima et al., 1992; Swairjo et al., 1995; Campos et al., 1998; Capila et al., 1999). Contrary to the proposal that annexin V has carbohydrate-binding activity specific to sialoglycoproteins, Hsp47 purified from both M31 cells and M31 GnT-III transfectants does not contain 2-3- or 2-6-linked sialic acid residues, as evidenced by lectin blot analysis of Maackia amurensis lectin (MAM) and Sambucus sieboldiana agglutinin (SSA; data not shown). This absence of sialic acid residues did not affect the binding between Hsp47-annexin V consistent with our previous report that annexin V recognizes bisecting GlcNAc, suggesting that bisecting GlcNAc plays a critical role in this carbohydrate-mediated interaction. For annexins are known as a family of homologous proteins, it needs more studies to show if other annexins, besides annexin V, have interactions with Hsp47 mediated by bisecting GlcNAc.
Since E4-PHA is a plant lectin that is known to have binding specificity for bisecting GlcNAc sugar chains (Narasimhan et al., 1986), the E4-PHA-staining-spots are mainly considered to be intracellular proteins that contain bisecting GlcNAc. Our present study indicated that bisecting GlcNAc accumulated in intracellular organelles in the case of GnT-III and annexin V cotransfected M31 cells. A fractionation study of the major organelles of GnT-III and annexin V cotransfected M31 cells (data not shown) and immunofluorescence microscopy (Figure 5) suggests that annexin V, as well as E4-PHA-staining-spots, accumulates mainly in Golgi apparatus or the ER-Golgi intermediate compartment. Considering the subcellular colocalization of annexin V and its bisecting-GlcNAc-binding activity, we hypothesize that annexin V may play an important role in the transport of cellular glycoproteins.
Although elevations in GnT-III expression levels lead to some significant cellular alterations (Yoshimura et al., 1996; Sultan et al., 1997; Kitada et al., 2001; Sato et al., 2001), the population of glycoproteins that can be modified by bisecting GlcNAc is not large (Ekuni et al., 2002). The limited number of GnT-III-target glycoproteins and bisecting GlcNAc-recognizing lectins appear to play an important role in a variety of biological functions of bisecting GlcNAc. This study shows, for the first time, that Hsp47 is a target protein of GnT-III. Since Hsp47 was reported to interact with collagen, we investigated the effect of bisecting GlcNAc of Hsp47 on their interaction. The interaction of Hsp47 and type 1 collagen was examined using a solid phase assay as reported (Vaillancourt and Cates, 1991). However, no obvious differences were detected in this preliminary experiment (data not shown). The immunofluorescence microscopy findings indicated that annexin V seemed to be colocated with Hsp47 in the Golgi apparatus or the ER-Golgi intermediate compartment. These data suggested that the binding of annexin V to Hsp47 was much more likely to affect the ER retrieval of Hsp47 than to affect its interaction with procollagen. We hypothesize that, in addition to the ER-retention signal on its C-terminal (Satoh et al., 1996), bisecting GlcNAc can also act as a sorting signal for Hsp47. It has been suggested that some glycoproteins may contain both N- or O-glycans and some other motifs as sorting signals (Ihrke et al., 2001). As a result, it has been postulated that Hsp47, bearing bisecting GlcNAc, might follow a complex cellular route as a result of two targeting motifs that compete or in combination with one another in various sorting situations. Further investigations will be required to prove the hypothesis relating to the oligosaccharide functions of Hsp47 as well as for Hsp47 itself.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
Materials
GST-annexin V fusion protein was expressed in Escherichia coli strain BL21 using the plasmid pGEX2T. Glutathione sepharoseTM 4B, protein G sepharoseTM 4 fast flow, and Gelatin sepharoseTM 4B were purchased from Amersham Pharmacia (Uppsala, Sweden). Dulbecco’s modified Eagle’s medium (D-MEM), fetal calf serum, G418, and streptomycin were obtained from Sigma Chemical (St. Louis, MO). Penicillin G potassium was purchased from Banyu (Tokyo, Japan). The effectence transfection reagent was obtained from Qiagen (Germany). A mouse anti-annexin V monoclonal antibody and a rabbit anti-annexin V full-length antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse anti-Hsp47 monoclonal antibody was purchased from Stressgen (San Diego, CA). N-glycosidase F was obtained from Roche (Basel, Switzerland). Polyvinylidene difluoride membrane was obtained from Millipore (Bedford, MA). Biotinylated E4-PHA and MAM were obtained from Seikagaku (Tokyo, Japan). A detection kit for horseradish peroxidase-avidin complex (ABC kit) was obtained from Vector Laboratories (Burlingame, CA). Bio-Gel P-6DG was bought from Bio-Rad (Hercules, CA) and CM-Toyopearl 650 was obtained from Tosoh (Tokyo, Japan). GlycoTAGTM Reagent kit was a product of Takara (Kyoto, Japan). Enhanced chemiluminescence (ECLTM ) reagents were purchased from Amersham International (Buckinghamshire, UK).
Cell culture and cell lines
A rat hepatoma cell line mRLN-31 (M31) was obtained from the Japanese Cancer Resources Bank (Tokyo, Japan). Cells were grown in D-MEM supplemented with 10% fetal bovine serum, 50 units/mL penicillin sulfate and 50 µg/mL streptomycin at 37°C under 5% CO2, 95% air. GnT-III-cDNA-transfected cells, annexin V-cDNA-transfected cells, and GnT-III/annexin V-cDNA-double-transfected cells were established by transfecting rat GnT-III cDNA that was subcloned into a pHOOKTM -2 and/or human annexin V cDNA that was inserted into a mammalian expression vector, pCXN2 (Niwa et al., 1991). Mock transfectants were prepared by introduction of each vector alone. All the transfectants were selected with 1 mg/mL of geneticin. The transfection was carried out using the Effectence Transfection Reagent according to the manufacturer’s protocol. The expression of each protein was checked by western blot.
SDS–PAGE, western blot analysis, and lectin blot analysis
Ten percent SDS–PAGE was carried out by the method of Laemmli (1970). For western blot and lectin blot analyses, the electrophoresed proteins were transferred onto a PVDF membrane. After blocking with 3% bovine serum albumin, the membrane was incubated with biotinylated lectin or horseradish peroxidase (HRP) conjugated antibody. After washing three times, the membranes were then incubated with peroxidase-conjugated antibody to bind HRP-conjugated primary antibody or HRP-conjugated biotin-avidin complex for detecting the biotinylated lectin. Detection was performed with an ECLTM kit according to the manufacturer’s recommended protocol.
Purification of ligand proteins of annexin V
GnT-III-transfected M31 cell cultures (>80% confluent) were rinsed twice with ice-cold phosphate-buffered saline (PBS) and harvested in lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (w/v) Nonidet P-40, and 1:1000 protease inhibitor cocktail). Cell lysates were centrifuged at 15,000 x g for 15 min at 4°C, the supernatants were collected, desalted, and then applied to a GST-annexin V-fusion-protein-immobilized glutathione sepharose 4B column, which had been equilibrated with 50 mM Tris–HCl, pH 7.4, 5 mM CaCl2, 100 mM NaCl, and 0.01% Triton X-100. This affinity column was prepared depended on the GST-glutathione binding activity. A GST-immobilized glutathione sepharose 4B column was used as a negative control. The columns were incubated with the supernatant at 4°C for 2h, followed by washing with 10 bed volumes of equilibrating buffer. Proteins that bound to the columns were eluted with 1 M NaCl in equilibrating buffer.
Preparation of oligosaccharides
In this study, two different oligosaccharides, agalacto bisected oligosaccharides Gn(Gn)Gn and agalacto nonbisected oligosaccharides GlcNAcß1-2Man1-6(GlcNAcß1-2Man1-3)Manß1-4GlcNAcß1-4GlcNAc (GnGn) were prepared as previously reported (Gao-Uozumi et al., 2000). Briefly, N-linked oligosaccharides were prepared from bovine 
-globulin and digested with sialidase and ß-galactosidase. The agalacto nonbisected oligosaccharides were purified from the digested oligosaccharides by HPLC using an TSK-gel ODS-80 column (4.6 x 150 mm). Aliquots of the purified oligosaccharides were pyridylaminated using GlycoTagTM reagent kit and then subjected to structural analysis. A bisected sugar chain, Gn(Gn)Gn, was prepared from the attachment of ß1,4 GlcNAc to the purified GnGn sugar chain, the reaction of which is catalyzed by GnT-III. The structures were confirmed by nuclear magnetic resonance (NMR) and reversed phase HPLC, after pyridylamination. These PA-oligosaccharides were also used as the standards in structure analysis involving HPLC.
Immunoprecipitation
Cells were lysed as described above. Nonspecific binding proteins were cleared by preincubation with protein G-sepharose 4FF beads for 1 h at 4°C. After pelleting the protein G-sepharose 4FF beads by centrifugation at 3000 rpm, 5 min, 5 µg/mL of mouse anti-Hsp47 monoclonal antibody or 5 µg/mL of mouse anti-annexin V monoclonal antibody was incubated with the supernatant overnight at 4°C. The immune complexes were collected with protein G-sepharose 4FF beads. When the effect of free sugar chains on Hsp47-annexin V complex was investigated, agalacto bisected biantennary sugar chain or its corresponding nonbisected sugar chain was added to the cell lysate to a final concentration as 2mM, before the start of immunoprecipitation.
Purification of Hsp47 from M31 cells
Cells from f 150 mm x 50 culture plates of confluent M31 parent cells or GnT-III-transfected cells were used in a typical isolation of Hsp47 as described (Vaillancourt and Cates, 1991). Gelatin-agarose affinity chromatography, Bio gel P-6DG column, and CM-sepharose chromatography were included in this purification (Figure 3A). About 18 µg of Hsp47 was purified from cells described above and the yield was about 0.1%.
Enzyme treatments
In the digestion of cellular glycoproteins with N-glycosidase, the cell lysate was treated overnight with 3 U/mL of N-glycosidase at 37°C. The enzyme-treated cell lysate was then used for immunoprecipitation, to check the interaction between annexin V and Hsp47.
Lectin affinity chromatography
E4-PHA agarose was used as a ligand in lectin affinity chromatography (Shinkawa et al., 2003) for separating Hsp47 purified from GnT-III-transfected M31 cells based on the content of bisecting GlcNAc. Purified Hsp47 was applied to the column that had been previously equilibrated with 5 mM HEPES/NaOH (pH 8.0). The column was eluted with the buffer containing 0.1 M K2B4O7. Chromatography was performed at 4°C. The column was washed using the buffer containing 0.5 M NaCl before the next purification. From 18 µg of Hsp47 purified from GnT-III-transfected cells, 4.5 µg of Hsp47 bearing bisected GlcNAc sugar chain was recovered.
Preparation and structure analysis of PA oligosaccharides from purified Hsp47
N-linked oligosaccharides of Hsp47 was liberated by N-glycosidase F, at 37°C for 24 h. The released oligosaccharides were labeled with 2-aminopyridine, using GlycoTag reagent kit. Excess reagent was removed by gel filtration and the resulting PA oligosaccharides were further purified using a cellulose cartridge Glycan preparation kit (Takara, Japan) according to the procedures recommended. To digest the PA-oligosaccharides into their corresponding core structures, a complete glycosidase mixture that consists of sialidase and ß-galactosidase was used. The oligosaccharides were incubated in 60 µL of 0.1 M citrate-phosphate buffer (pH 5.0) with 50 milliunits of Arthrobacter ureafaciens sialidase (Nacalai Tesque, Osaka, Japan) and 50 milliunits of jack bean ß-galactosidase at 37°C for 24 h. To analyze the core structure of cell N-glycans, the digested PA-oligosaccharides were subjected to an HPLC system (Shimazu, Japan) equipped with an TSK-gel 80TM column (4.6 x 150mm, Tosoh) as described before (Koyota et al., 2001).
MALDI-TOF MS analysis
MALDI-TOF MS was performed with a Bruker Daltonics’ ultraflex instrument (Bruker Daltonics, Billerica, MA). The mass spectra were acquired in the reflection mode under a 20-kV accelerating voltage with positive detection. 2,5-Dihydroxybenzoic acid (10 mg/mL) was used as the matrix.
Measurement of the binding of Hsp47 to annexin V by surface plasmon resonance assay
To determine whether Hsp47 bound to annexin V in a cell-free system, we assessed the interaction between these proteins by a surface plasmon resonance assay using a Biacore 2000 instrument (Biacore, Piscataway, NJ). Annexin V was immobilized on a carboxymethyldextran (CM5) biosensor chip using amine coupling. Hsp47, purified from cells or eluted from the E4-PHA agarose column, were dissolved in 10 mM HEPES-Na, pH7.4, 140 mM NaCl, and 2.5 mM CaCl2. The binding of increasing concentrations of Hsp47, delivered at a flow rate of 5 µL/min, was then measured in real time. The sensor surface was regenerated with a 10-µL pulse of 10 mM glycine–HCl, pH2.0. To minimize bulk effect, the flow cell, to which nothing was immobilized, was used to subtract the contribution of nonspecific interactions. Kinetic parameters were calculated using the BIA evaluation 2.1 program according to the manufacturer’s recommendations.
Immunofluorescence microscopy
GnT-III/annexin V cotransfected M31 cells were washed once in PBS(-) and fixed with 4% paraformaldehyde in PBS for 20 min at 4°C, followed by washing with PBS(-), and quenching for 20 min with 1% saponin in PBS(-) that contains 1% bovine serum albumin to block and permeabilize the cells. The cells were then incubated for 1 h at room temperature with the mouse anti-Hsp47 antibody (1 µg/mL), rabbit anti-annexin V antibody (1 µg/mL), and FITC-conjugated E4-PHA (5 µL/100 µL). Primary antibody binding was detected with a FITC-labeled goat antibody to mouse IgG or rhodamine-B conjugated goat antibody to rabbit IgG. Both of the secondary antibodies were 50-time-diluted. Finally the cells were washed six times for 5 min with PBS (-). Confocal microscopy was done on a LSM410 Zeiss confocal microscope (Carl Zeiss, Oberkochen, Germany).
|
Supplementary data |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
Supplementary data are available at Glycobiology online (http://glycob.oupjournals.org/).
|
Acknowledgments |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
This study was supported by a grant-in-aid for Scientific Research (S) (13854010) from the Japan Society for the Promotion of Science; the 21st Century COE program and grant-in-aid for Cancer Research and Scientific Research of Priority Areas (15025238) from the Ministry of Education, Science, Culture, Sports and Technology, and Japan Science and Technology Agency (JST).
|
Abbreviations |
|---|
ECL, enhanced chemiluminescence; E4-PHA, erythroagglutinating phytohemagglutinin; FITC, fluorescein isothiocyanate; GlcNAc, N-acetylglucosamine; Gn(Gn)Gn, GlcNAc- ß1-2Man
1-6(GlcNAcß1-2Man1-3) (GlcNAcß1-4)Manß14- GlcNAcß1-4GlcNAc; GnGn, GlcNAc ß1-2Man1-6(Glc- NAcß1-2Man1-3)Manß1-4GlcNAc ß1-4GlcNAc; GnT-III, UDP-N-acetylglucosamine : ß-D-mannoside ß-1,4-N-acetylglucosaminyltansferase III; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; MALDI-TOF, matrix assisted laser desorption/ionization-time of flight; MAM, Maackia amurensis lectin; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SSA, Sambucus sieboldiana agglutinin |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
|---|
Akama, T.O., Nakagawa, H., Sugihara, K., Narisawa, S., Ohyama, C., Nishimura, S., O’Brien, D.A., Moremen, K.W., Millan, J.L., and Fukuda, M.N. (2002) Germ cell survival through carbohydrate-mediated interaction with Sertoli cells. Science, 295, 124–127.
Bell, S.L., Xu, G., Khatri, I.A., Wang, R., Rahman, S., and Forstner, J.F. (2003) N-linked oligosaccharides play a role in disulphide-dependent dimerization of intestinal mucin Muc2. Biochem. J., 373, 893–900.[CrossRef][Web of Science][Medline]
Berendes, R., Voges, D., Demange, P., Huber, R., and Burger, A. (1993) Structure-function analysis of the ion channel selectivity filter in human annexin V. Science, 262, 427–430.
Brooks, N.D., Grundy, J.E., Lavigne, N., Derry, M.C., Restall, C.M., MacKenzie, C.R., Waisman, D.M., and Pryzdial, E.L. (2002) Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5. Biochem. J., 367, 895–900.[CrossRef][Web of Science][Medline]
Campos, B., Mo, Y.D., Mealy, T.R., Li, C.W., Swairjo, M.A., Balch, C., Head, J.F., Retzinger, G., Dedman, J.R., and Seaton, B.A. (1998) Mutational and crystallographic analyses of interfacial residues in annexin V suggest direct interactions with phospholipid membrane components. Biochemistry, 37, 8004–8010.[CrossRef][Medline]
Capila, I., VanderNoot, V.A., Mealy, T.R., Seaton, B.A., and Linhardt, R.J. (1999) Interaction of heparin with annexin V. FEBS. Lett., 446, 327–330.[CrossRef][Web of Science][Medline]
Chap, H., Comfurius, P., Bevers, E.M., Fauvel, J., Vicendo, P., Douste-Blazy, L., and Zwaal, R.F. (1988) Potential anticoagulant activity of lipocortins and other calcium/phospholipid binding proteins. Biochem. Biophys. Res. Commun., 150, 972–978.[CrossRef][Web of Science][Medline]
Clarke, E.P. and Sanwal, B.D. (1992) Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins. Biochim. Biophys. Acta, 1129, 246–248.[Medline]
Clarke, E.P., Cates, G.A., Ball, E.H., and Sanwal, B.D. (1991) A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors. J. Biol. Chem., 266, 17230–17235.
Ekuni, A., Miyoshi, E., Ko, J.H., Noda, K., Kitada, T., Ihara, S., Endo, T., Hino, A., Honke, K., and Taniguchi, N. (2002) A glycomic approach to hepatic tumors in UDP-N-acetylglucosamine : ß-D-mannoside ß–1,4-N-acetylglucosaminyltansferase III (GnT-III) transgenic mice induced by diethylnitrosamine (DEN): identification of haptoglobin as a target molecule of GnT-III. Free Radic. Res., 36, 827–833.[CrossRef][Web of Science][Medline]
Gao-Uozumi, C.X., Uozumi, N., Miyoshi, E., Nagai, K., Ikeda, Y., Teshima, T., Noda, K., Shiba, T., Honke, K., and Taniguchi, N. (2000) A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine. Glycobiology, 10, 1209–1216.
Huang, R.T., Lichtenberg, B., and Rick, O. (1996) Involvement of annexin V in the entry of influenza viruses and role of phospholipids in infection. FEBS. Lett., 392, 59–62.[CrossRef][Web of Science][Medline]
Huber, R., Romisch, J., and Paques, E.P. (1990) The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes. EMBO J., 9, 3867–3874.[Web of Science][Medline]
Ihrke, G., Bruns, J.R., Luzio, J.P., and Weisz, O.A. (2001) Competing sorting signals guide endolyn along a novel route to lysosomes in MDCK cells. EMBO J., 20, 6256–6264.[CrossRef][Web of Science][Medline]
Ishitsuka, R., Kojima, K., Utsumi, H., Ogawa, H., and Matsumoto, I. (1998) Glycosaminoglycan binding properties of annexin IV, V, and VI. J. Biol. Chem., 273, 9935–9941.
Katayanagi, K., Van de Water, J., Kenny, T., Nakanuma, Y., Ansari, A.A., Coppel, R., and Gershwin, M.E. (1999) Generation of monoclonal antibodies to murine bile duct epithelial cells: identification of annexin V as a new marker of small intrahepatic bile ducts. Hepatology, 29, 1019–1025.[CrossRef][Web of Science][Medline]
Kitada, T., Miyoshi, E., Noda, K., Higashiyama, S., Ihara, H., Matsuura, N., Hayashi, N., Kawata, S., Matsuzawa, Y., and Taniguchi, N. (2001) The addition of bisecting N-acetylglucosamine residues to E-cadherin down-regulates the tyrosine phosphorylation of beta-catenin. J. Biol. Chem., 276, 11956–11962.
Kojima, K., Ogawa, H.K., Seno, N., Yamamoto, K., Irimura, T., Osawa, T., and Matsumoto, I. (1992) Carbohydrate-binding proteins in bovine kidney have consensus amino acid sequences of annexin family proteins. J. Biol. Chem., 267, 20536–20539.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[CrossRef][Medline]
Narasimhan, S., Freed, J.C., and Schachter, H. (1986) The effect of a "bisecting" N-acetylglucosaminyl group on the binding of biantennary, complex oligosaccharides to concanavalin A, Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin (RCA-120) immobilized on agarose. Carbohydr. Res., 149, 65–83.[CrossRef][Web of Science][Medline]
Negroiu, G., Dwek, R.A., and Petrescu, S.M. (2003) The inhibition of early N-glycan processing targets TRP-2 to degradation in B16 melanoma cells. J. Biol. Chem., 278, 27035–27042.
Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193–199.[CrossRef][Web of Science][Medline]
Rosales, J.L. and Ernst, J.D. (1997) Calcium-dependent neutrophil secretion: characterization and regulation by annexins. J. Immunol., 159, 6195–6202.[Abstract]
Sato, Y., Takahashi, M., Shibukawa, Y., Jain, S.K., Hamaoka, R., Miyagawa, J., Yaginuma, Y., Honke, K., Ishikawa, M., and Taniguchi, N. (2001) Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors. J. Biol. Chem., 276, 11956–11962.
Satoh, M., Hirayoshi, K., Yokota, S., Hosokawa, N., and Nagata, K. (1996) Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen. J. Cell. Biol., 133, 469–483.
Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki, M., and others. (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem., 278, 3466–3473.
Spreca, A., Rambotti, M.G., Giambanco, I., Pula, G., Bianchi, R., Ceccarelli, P., and Donato, R. (1992) Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart. J. Cell. Physiol., 152, 587–598.[CrossRef][Web of Science][Medline]
Sultan, A.S., Miyoshi, E., Ihara, Y., Nishikawa, A., Tsukada, Y., and Taniguchi, N. (1997) Bisecting GlcNAc structures act as negative sorting signals for cell surface glycoproteins in forskolin-treated rat hepatoma cells. J. Biol. Chem., 272, 2866–2872.
Sun, J., Salem, H.H., and Bird, P. (1992) Nucleolar and cytoplasmic localization of annexin V. FEBS Lett., 314, 425–429.[CrossRef][Web of Science][Medline]
Swairjo, M.A., Concha, N.O., Kaetzel, M.A., Dedman, J.R., and Seaton, B.A. (1995) Ca (2+)-bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin V. Nat. Struct. Biol., 2, 968–974.[CrossRef][Web of Science][Medline]
Taniguchi, N., Yoshimura, M., Miyoshi, E., Ihara, Y., Nishikawa, A., and Fujii, S. (1996) Remodeling of cell surface glycoproteins by N-acetylglucosaminyltransferase III gene transfection: modulation of metastatic potentials and down regulation of hepatitis B virus replication. Glycobiology, 6, 691–694.
Taniguchi, N., Miyoshi, E., Ko, J.H., Ikeda, Y., Ihara, Y., Yoshimura, M., Nishikawa, A., and Fujii, S. (1999) Implication of N-acetylglucosaminyltransferases III and V in cancer: gene regulation and signaling mechanism. Biochim. Biophys. Acta, 1455, 287–300.[Medline]
Tasab, M., Batten, M.R., and Bulleid, N.J. (2000) Hsp47: a molecular chaperone that interacts with and stabilizes correctly-folded procollagen. EMBO J., 19, 2204–2211.[CrossRef][Web of Science][Medline]
Vaillancourt, J.P. and Cates, G.A. (1991) Purification and reconstitution of a collagen-binding heat-shock glycoprotein from L6 myoblasts. Biochem. J., 274, 793–798.[Medline]
Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and others. (2002) E3 ubiquitin ligase that recognizes sugar chains. Nature, 418, 438–442.[CrossRef][Medline]
Yoshimura, M., Ihara, Y., Ohnishi, A., Ijuhin, N., Nishiura, T., Kanakura, Y., Matsuzawa, Y., and Taniguchi, N. (1996) Bisecting N-acetylglucosamine on K562 cells suppresses natural killer cytotoxicity and promotes spleen colonization. Cancer Res., 56, 412–418.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Nishioka, J.-i. Aikawa, M. Ida, I. Matsumoto, M. Street, M. Tsujimoto, and K. Kojima-Aikawa Ligand-binding Activity and Expression Profile of Annexins in Caenorhabditis elegans J. Biochem., January 1, 2007; 141(1): 47 - 55. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||