JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M313245200 on January 12, 2004

J. Biol. Chem., Vol. 279, Issue 13, 12943-12950, March 26, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/13/12943    most recent
M313245200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Appenzeller-Herzog, C.
Right arrow Articles by Hauri, H.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Appenzeller-Herzog, C.
Right arrow Articles by Hauri, H.-P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

pH-induced Conversion of the Transport Lectin ERGIC-53 Triggers Glycoprotein Release*

Christian Appenzeller-Herzog{ddagger}, Annie-Claude Roche§, Oliver Nufer{ddagger}, and Hans-Peter Hauri{ddagger}||

From the {ddagger}Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and §Glycobiologie, Centre de Biophysique Moleculaire, CNRS, F-45071 Orléans, France

Received for publication, December 4, 2003 , and in revised form, January 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient endoplasmic reticulum (ER) export of some secretory glycoproteins. Binding of cargo to ERGIC-53 in the ER requires Ca2+. Cargo release occurs in the ERGIC, but the molecular mechanism is unknown. Here we report efficient binding of purified ERGIC-53 to immobilized mannose at pH 7.4, the pH of the ER, but not at slightly lower pH. pH sensitivity of the lectin was more prominent when Ca2+ concentrations were low. A conserved histidine in the center of the carbohydrate recognition domain was required for lectin activity suggesting it may serve as a molecular pH/Ca2+ sensor. Acidification of cells inhibited the association of ERGIC-53 with the known cargo cathepsin Z-related protein and dissociation of this glycoprotein in the ERGIC was impaired by organelle neutralization that did not impair the transport of a control protein. The results elucidate the molecular mechanism underlying reversible lectin/cargo interaction and establish the ERGIC as the earliest low pH site of the secretory pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
After translocation into the ER,1 soluble secretory proteins, here termed cargo proteins, start their journey along the organelles of the secretory pathway. This process requires correct folding and ongoing sorting from resident proteins of the compartments through which they move. The first such separation occurs during transport from the ER to the Golgi apparatus. The molecular nature of this sorting event has been extensively studied and led to two models that may coexist and each apply for a distinct subset of cargo proteins (1). According to the bulk-flow model, ER-exit occurs by default and requires no transport signals. Retention signals would be required for keeping ER resident proteins in the ER and retrieval signals would salvage those few that inadvertently escape. Conversely, the receptor-mediated model positions the targeting information on the cargo proteins themselves that would carry positive sorting signals for ER exit. These signals are recognized by membrane spanning transport receptors that couple the cargo proteins to the cytosolic vesicle budding machinery and cycle between the ER and post-ER compartments. Several transport receptors have been identified in the past few years, including ERGIC-53 (2), Emp24p (3), and Erv29p (4), each acting as an ER export receptor for a subset of cargo proteins.

The type I transmembrane protein ERGIC-53 is ubiquitously expressed and constitutively cycles between ER and ER-Golgi intermediate compartment (ERGIC) (5). In the ERGIC the protein segregates from anterograde-directed protein traffic and returns to the ER largely bypassing the Golgi apparatus (6). ERGIC-53 is a lectin. In its luminal part it carries a carbohydrate recognition domain (CRD) with a {beta}-sandwich-fold (7) that shares significant sequence similarity and many structural details with the carbohydrate binding sites of plant L-type lectins (8-10). It preferentially binds to D-mannose (11) and recognizes protein-linked high mannose-type oligosaccharides in vivo (2). The lack of functional ERGIC-53 leads to inefficient secretion of the glycoproteins pro-cathepsin C (12) and blood coagulation factors V and VIII (13).

Cross-linking studies have established a cathepsin Z-related protein (catZr) as a model glycoprotein cargo for ERGIC-53 and documented that cargo capture starts in the ER and cargo release occurs in the ERGIC (2). Although such a differential binding of the transport receptor to its cargo is fundamental, the molecular nature of this process has remained elusive. Based on the finding that lectin activity of ERGIC-53 strictly depends on Ca2+ (2, 11), we have speculated earlier that a drop of calcium levels along the ER to ERGIC pathway may trigger glycoprotein dissociation (5). Indeed, an imaging approach that measures total Ca2+ in ultrathin cryosections revealed positive signals for both ER and Golgi, but ERGIC elements remained below detection levels (14). Nevertheless, calcium deprivation as the sole determinant for cargo release appears unlikely, as the concentration gradient of free Ca2+ from ER to ERGIC may be subtle. Apart from that, a pH-driven sorting mechanism, in analogy to the endosomal system, has been suggested based on in vitro studies (15, 16). Organellar pH is determined by the presence of active H+ v-ATPase pumps, a 106-kDa complex consisting of 13 polypeptides, and of opposed H+ leak rates (17). Whereas the progressive acidification from the ER (pH 7.1-7.4) to the trans-Golgi network (pH 5.9-6.3) has been established, it is still largely questioned, however, if significant proton pumping and organelle acidification occurs already in the early secretory pathway, i.e. the ERGIC/cis-Golgi region (for review see Ref. 18). The evidence is rather sketchy and includes the observations that the v-ATPase proteolipid subunit co-fractionates with ERGIC membranes, the v-ATPase inhibitor bafilomycin A1 produces a cell-type specific Golgi to ER retrograde transport defect, and there is minor overlap of DAMP staining with ERGIC-53 (19, 20). The notion of pre-Golgi acidification is far from being established and requires further evidence to be proven.

By studying the lectin properties of ERGIC-53 in more detail, we report here that low pH modulates the activity of ERGIC-53 in vitro and in vivo. The data suggest a molecular scenario underlying reversible lectin inactivation that involves protonation of a conserved histidine sensor residue and loss of Ca2+. In conjunction with our previous studies, the results establish the ERGIC as the earliest acid compartment of the secretory pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—The following antibodies were used: mAb 9E10.2 against a c-myc epitope (ATCC CRL 1729), mAb G1/93 against human ERGIC-53 (21), and polyclonal antibody against horse fibronectin (kindly provided by Matthias Chiquet, University of Bern). A rabbit antiserum against catZr was obtained by keyhole limpet hemocyanin-coupled immunization of the following peptide sequences: CMADRINIKRKGAWPS and CKHGIPDETCNNYQA. 4-Isothiocyanatophenyl {alpha}-D-mannopyranoside and Affi-102 beads (Bio-Rad) were used to prepare immobilized D-mannose (22). D-Mannose and chloroquine were from Sigma, endoglycosidase D (endo D) and monensin from Calbiochem, and endoglycosidase H (endo H) from Roche Diagnostics. Nigericin was kindly provided by Jean Pieters (Biozentrum, Basel).

Recombinant DNAs—myc/His6 (11) was mutated using the QuikChange method (Stratagene) and the following primers (coding strand): D152S, 5'-CTGTGGAATGGTGTTGGAATATTTTTTTCTTCTTTTGACAATGATGGAAAG-3'; H175Q, 5'-GGACAAATCCAGTATGACCATCAAAATGACGGGG-3'; H178Q, 5'-GGACAAATCCATTATGACCAGCAAAATGACGGGG-3'.

Cell Culture, Transfections, Drug Treatment, and in Situ Cross-linking—COS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU ml-1 penicillin, 100 µg ml-1 streptomycin, and 1 µg ml-1 amphotericin B (Sigma). Cells were transfected using the DEAE-dextran method (23) (10 µg of DNA/10-cm dish). CHO-KI cells and Lec1-derived stable clones (GM or GMAA (24)) were grown in {alpha}-minimal essential medium supplemented with 10% fetal bovine serum, 100 IU ml-1 penicillin, 100 µg ml-1 streptomycin, and 1 µgml-1 amphotericin B. Chloroquine (100 µM) was added 1 h before metabolic labeling and was present throughout the pulse-chase period. For pH-clamp experiments, cells were incubated for 15 min at 37 °C in 10 mM HEPES, 10 mM MES (pH 6.0, 6.5, 7.0), 60 mM NaCl, 60 mM KCl, 1.5 mM K2HPO4, 1 mM MgSO4, 2 mM CaCl2, 10 mM glucose, 10 mM methionine, and 10 µM each of nigericin and monensin (adapted from Ref. 25). AlF4 - was used at the following final concentrations: 30 mM NaF and 50 µM AlCl3. In situ cross-linking with dithiobis(succinimidylpropionate) (DSP) (Pierce) was performed on intact cells (2).

Mannose Binding Assay—myc/His6-ERGIC-53 was purified from transiently transfected COS-1 cells solubilized in 10 mM Tris (pH 7.4), 150 mM NaCl, 20 mM imidazole, 1% Triton X-100. Cleared lysate (100,000 x g, 1 h) from 10 10-cm dishes was incubated for 1 h at room temperature with 100 µl of Ni2+-nitrilotriacetic acid-agarose (Qiagen) under constant agitation. The beads were washed in the same buffer. Bound protein was eluted in 10 mM Tris (pH 7.4), 150 mM NaCl, 0.4 M imidazole, 0.04% Triton X-100 and dialyzed against the same buffer lacking imidazole. Purified ERGIC-53 was shock frozen and stored in aliquots at -80 °C. Mannose binding was performed for 4 h at 4 °Cin10 mM HEPES, 10 mM MES (pH 7.4) (or as indicated; pH was set at 4 °C), 150 mM NaCl, 0.5 mM CaCl2 (or as indicated), 0.04% Triton X-100 in a volume of 100 µl using 50 ng of protein and 20 µl (dry volume) of D-mannose beads. After removing the supernatant, beads were washed twice with binding buffer and bound protein was eluted in 100 µl of Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.04% Triton X-100 for 5 min at 4 °C. B- and s-fractions were analyzed by Western blotting using anti-myc and the ECL detection system (Amersham Biosciences) and quantified with a ChemImagerTM device and AlphaEaseTM software (Alpha Inotech Corporation).

Pulse-Chase Experiments, Immunoprecipitation, and Endoglycosidase Digest—Cells were labeled with [35S]methionine, chased in {alpha}-minimal essential medium containing 10 mM unlabeled methionine, and subjected to immunoprecipitation (26) using mAb G1/93 or polyclonal antibody against fibronectin and protein A-Sepharose (Amersham Biosciences). Immunoprecipitation of intracellular catZr was performed after antigen denaturation at 95 °C in 30 mM triethanoamine/HCl (pH 8.1), 100 mM NaCl, 5 mM EDTA containing 1.6% SDS followed by addition of an excess of Triton X-100 (2% final concentration) in the same buffer. Secreted catZr was methanol-chloroform precipitated (27) and then subjected to the same procedure. Where indicated, cells were treated with DSP prior to solubilization, or immunoprecipitates were digested with endo D or endo H as previously described (24). Gels were analyzed and quantified on a STORM 820 PhosphorImager (Amersham Biosciences) or by fluorography.

Immunofluorescence Microscopy—GM-Lec1 cells were cultured in 8-well multichamber Permanox slides (Milian, Plan-les-Ouates, Switzerland), fixed with 3% paraformaldehyde, permeabilized with phosphate-buffered saline, 0.1% saponin, and processed for indirect immunofluorescence using mAb G1/93 and GAM IgG(H+L)-Alexa Fluor 488 (Molecular Probes, Leiden, Netherlands). Confocal laser scanning images were acquired on a Leica microscope (TCS NT) with a 63x objective (NA 1.32).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Lectin Activity of ERGIC-53 Is Modulated by pH and Ca2+—To gain insight into the molecular mechanism of the ERGIC-53/cargo interaction, we designed an in vitro lectin assay. Full-length oligomeric ERGIC-53 carrying a NH2-terminal myc epitope and a His6 tag at the C terminus (myc/His6 (11)) was purified from COS-1 cells (Fig. 1A) and incubated with immobilized D-mannose in the presence of 0.5 mM CaCl2. Bound and unbound protein was visualized by immunoblotting using anti-myc. Binding of 50 ng of protein was complete, required Ca2+, and could be competed with 0.2 M free D-mannose (Fig. 1B), indicating specificity.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 1.
Binding of purified ERGIC-53 to D-mannose is acid sensitive. A, recombinant, myc/His6-tagged ERGIC-53 was expressed in COS-1 cells and purified on a Ni2+ resin. Purified ERGIC-53 forms disulfide-linked dimers and hexamers (Ref. 11 and data not shown). Under reducing conditions, it shows a single band of 53 kDa on a silver-stained gel. B, 50 ng of ERGIC-53 was incubated with D-mannose Affi-Gel beads in the presence of 0.5 mM CaCl2. Soluble (s) and bound protein (b) was visualized by immunoblotting using anti-myc. Note that lectin binding can be competed with free D-mannose. C, ERGIC-53 was bound to mannose beads in buffers with gradually decreased pH (mean ± S.D., n = 3). D, the supernatant of a binding assay at pH 6.0 (0.5 mM CaCl2) was split and one-half was neutralized with 30 mM Tris buffer and reincubated with mannose beads. B- and s-fractions of the first and second incubation are shown. Note that neutralization can reactivate ERGIC-53.

 
This assay was used to further study the lectin properties of ERGIC-53. First, we tested the possibility that mannose binding was sensitive to acid. To this end, binding experiments were performed in buffers of pH 7.4, corresponding to the pH of the ER (18), down to pH 6.0. Fig. 1C shows that the fraction of active ERGIC-53 was gradually reduced to 13 ± 6% at pH 6.0. Inactivation by pH 6.0 treatment could be readily reversed by neutralization (Fig. 1D). Interestingly, the sensitivity of the lectin to low pH was linked to Ca2+ as it was completely suppressed if [Ca2+] was raised to 5 mM (Fig. 2A). In a next set of experiments the binding of ERGIC-53 was assayed at constant pH over a range of [Ca2+] (Fig. 2B). Half-maximal binding was observed in the presence of 150 µM Ca2+ at pH 7.4, 230 µM Ca2+ at pH 6.5, and 800 µM Ca2+ at pH 6.0. We conclude that pH sensitivity of ERGIC-53 is at least in part because of pH-induced changes in Ca2+ affinity raising the possibility that pH sensing and Ca2+ complexation may be mediated by the same amino acid(s).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 2.
Acid sensitivity of ERGIC-53 is modulated by Ca2+. A, binding of ERGIC-53 as described in the legend to Fig. 1C, but in the presence of 5 mM CaCl2. Under these conditions lectin activity is clearly restored at low pH. B, mannose binding at pH 7.4, 6.5, or 6.0 in the presence of different concentrations of CaCl2. A typical experiment and the data of three independent experiments are shown (mean ± S.D., n = 3).

 
Histidine 178 Has the Hallmarks of a Molecular pH Sensor—In search of the molecular mechanism underlying acid sensitivity we scanned the CRD of ERGIC-53 for potentially titratable residues. Histidine is the only basic amino acid that can be considerably protonated in moderately acidic solutions as used above. His-178 is both conserved in ERGIC-53 orthologs and positioned in a characteristic {alpha}-helix in the active site of ERGIC-53 (Fig. 3, A and B (7)). To mimic and freeze the unprotonated state, His-178 was mutated to glutamine and the mutant lectin purified. According to our expectations, H178Q would abrogate or at least reduce pH sensitivity of mannose binding. However, we found ERGIC-53(H178Q) to be inactive (Fig. 3C). In contrast, mutating the non-conserved His-175 only minimally affected the binding. Taken together with the central position in the CRD, the fact that a neutral mutation of His-178 inactivates lectin function indicates that this residue may be directly involved in mannose binding or, more likely, Ca2+ complexation (see Fig. 2B). To further support this idea, we probed the mannose binding of a Ca2+-binding mutant (D152S) that was designed on the basis of the close homology of ERGIC-53 and plant lectins (8, 28). As expected, D152S abrogated lectin activity comparable with H178Q (Fig. 3C), consistent with a role of His-178 in Ca2+ binding. These findings support the notion that His-178 serves as a molecular pH sensor of ERGIC-53. We call the {alpha}-helical loop comprising His-178 the histidine ion sensor (HIS)-loop.



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 3.
Histidine 178 is conserved and essential for lectin activity. A, sequence alignment of the CRDs of ERGIC-53 orthologs (ClustalW 1.82, using default settings). Amino acid numbering is shown for the human and rat sequence. These sequence data are available from GenBankTM/EMBL/DDBJ under accession numbers P49257 [GenBank] (human), Q9TU32 (monkey), Q62902 [GenBank] (rat), Q9D0F3 (mouse), Q91671 (frog), Q9GR90 (tunicata), P90913 (worm), and Q9V3A8 (fly). B, structural comparison of animal and plant L-type lectins. Shown are the closely related {beta}-sandwich folds of the CRD of rat ERGIC-53 (7) and E. corallodendron lectin (ECorL (37)). The inner concave {beta}-sheet oriented toward the sugar binding pocket (asterisk) is colored in yellow. The imidazole ring of His-178 in the mannose binding pocket is shown in red. Note that the {alpha}-helical loop comprising His-178 is replaced by a shorter loop in ECorL (red colored backbone), and that this space instead is filled by an extended amino acid insertion relative to the ERGIC-53 structure (orange colored backbone). Figures were designed using RasMol V2.5. C, ERGIC-53 (myc/His6) variants carrying the H175Q, H178Q, or D152S mutation were purified and probed for in vitro lectin activity in the presence of 5 mM CaCl2. Note that only ERGIC-53(H175Q) specifically binds to mannose beads.

 
ERGIC-53/Glycoprotein Association Is Sensitive to Acidic pH—Does the binding of ERGIC-53 to a known glycoprotein ligand also depend on neutral pH? We studied intracellular binding of catZr to ERGIC-53 by in situ cross-linking (2) in cells that had been subjected to a pH-clamp procedure. pH clamping adapts all cellular compartments to the pH of the applied buffer, irrespective of organelle-specific ion concentrations and permeabilities (25). Because this procedure perturbs transport along the biosynthetic pathway (18),2 we made use of GMAA cells that stably express a transport-impaired ERGIC-53 mutant that is restricted to the ER (24), but still efficiently cross-links to catZr by DSP (2). This cross-linker covalently links the two proteins and after reductive cleavage slightly increases their apparent molecular mass. We chose to assess the effect of pH 6.5 and 6.0 treatment in relation to neutral pH taking into account pKa(His) of ~6.5. Because chemical coupling of amino groups by DSP works poorly below pH 7, the cells were cross-linked at pH 7.4 after metabolic labeling with [35S]methionine and pH clamping. ERGIC-53 immunoprecipitates were then analyzed by SDS-PAGE and phosphorimaging. Cross-linking efficiency, as determined by quantification of the DSP-induced gel-mobility shift of GMAA-ERGIC-53 (Fig. 4, compare lane 1 to other lanes), only minimally changed upon acid treatment indicating that incubation at neutral pH after the pH clamp allowed the cells to re-establish neutral ER pH (Fig. 4, lanes 2-4, GMAA-ERGIC-53). To confirm this, neutralization during cross-linking was imposed by inclusion of the lysosomotropic agent chloroquine that is known to raise intraorganellar pH within seconds (18). As shown in the lower panel of Fig. 4 (DSP control/chloroquine), the variations in cross-linking efficiency with or without addition of chloroquine remained statistically insignificant. The amount of the catZr-doublet (2) cross-linked to GMAA-ERGIC-53 on the other hand was significantly reduced with decreasing pH (Fig. 4, lanes 2-4, catZr). During the cross-linking procedure at neutral pH some reassociation may occur, but this process seems to be slow at 4 °C in the viscous environment of the ER lumen. Consistent with this, binding of catZr to ERGIC-53 after acidic pH clamping was only partially restored if cross-linking was performed in the presence of chloroquine (Fig. 4, lanes 5-7, catZr). Because pH-induced misfolding of catZr, a predicted lysosomal enzyme, is unlikely, we conclude that an artificially applied acidic pH in the lumen of the ER drives dissociation of catZr from ERGIC-53.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 4.
Intracellular binding of ERGIC-53 to a glycoprotein substrate is impaired by acidification of cells. Upper panel, Lec1 cells stably expressing GMAA-ERGIC-53 were labeled with [35S]methionine for 2 h, pH clamped as indicated, cross-linked by DSP in the presence or absence of 0.2 mM chloroquine, and subjected to immunoprecipitation with anti-ERGIC-53. Two different exposures of the same gel are shown for GMAA-ERGIC-53 and catZr. Lower panel, quantification of cross-linked catZr (bars) and cross-linking efficiency (lines), i.e. DSP-"cap" on ERGIC-53 bands both normalized against total ERGIC-53 (mean ± S.D., n = 3). Levels of control cells with pH clamp 7.0 were set to 100%. The decrease of catZr signals is statistically significant, variations in cross-linking efficiency are not (p < 0.05, Student's t test).

 
Neutralization of the ERGIC Inhibits Cargo Release—We wondered if pH-driven glycoprotein dissociation from ERGIC-53 also occurs in living cells without acidic manipulation. Our previous studies have localized the site of catZr cargo release from ERGIC-53 to the ERGIC (2). If indeed an endogenous acidification mechanism of the ERGIC is the driving force for the dissociation, we expect that neutralization of the intra-organellar pH would delay this process. We chose to use the dibasic compound chloroquine that elevates the pH within acidic organelles by a weak base mechanism at micromolar concentrations (minimizing osmotic swelling) and by the lack of primary amino groups does not interfere with DSP cross-linking (see above).

To verify that such organelle neutralization does not generally affect membrane traffic in the early secretory pathway, which would lead to misinterpretations, we assayed the recycling of ERGIC-53 by two different methods. First, pulse-chase experiments were performed in the presence or absence of 100 µM chloroquine using CHO-KI-derived GM-Lec1 cells (24), and immunoprecipitated ERGIC-53 was probed for sensitivity to endo D. Lec1 cells lack the Golgi enzyme N-acetylglucosamine transferase I, and as a consequence N-glycosylated proteins, upon passage through the cis-Golgi, become and remain endo D-sensitive by the action of Golgi mannosidase I. As the major recycling route of ERGIC-53 largely bypasses the cis-Golgi (6), the acquisition of endo D sensitivity is a slow process (24) and an exact measure of ERGIC-53 trafficking. Fig. 5A shows that chloroquine treatment does not slow down the recycling of glycosylated ERGIC-53. As a second read-out for recycling, we studied the localization of ERGIC-53 by immunofluorescence microscopy in GM-Lec1 cells that had been treated with . This drug accumulates recycling proteins in the ERGIC in a reversible manner (24). Very much like in control cells, in cells pretreated with chloroquine, ERGIC-53 was concentrated in a juxtanuclear area and the reticular ER fluorescence diminished after treatment (Fig. 5, C and F). After wash-out, these effects were reversed in both neutralized and control cells (Fig. 5, D and G). These results suggest that recycling kinetics of ERGIC-53 as well as morphology of the ER-Golgi interface remain undisturbed in the presence of chloroquine.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 5.
ERGIC-53 recycling is not impaired by chloroquine treatment. A, GM-Lec1 cells were pulsed for 20 min with [35S]methionine and chased as indicated. Immunoprecipitated ERGIC-53 was probed for endo D sensitivity and analyzed on 7-10% gradient gels. Where indicated, chloroquine was added to the culture medium. Asterisk, endogenous non-glycosylated ERGIC-53. Control and chloroquine kinetics are statistically indistinguishable (n = 3, p > 0.1, Student's t test). B-G, immunofluorescence localization of ERGIC-53 in GM-Lec1 cells treated with AlF4 as indicated. For organelle neutralization, chloroquine was added 1 h before AlF4 - and was present until fixation. Note that AlF4 reversibly concentrates ERGIC-53 to the Golgi area irrespective of the presence or absence of chloroquine. Bar, 10 µm.

 
Therefore, we used chloroquine to study the role of luminal pH in the kinetics of catZr release from ERGIC-53. It has been shown that cargo binding by ERGIC-53 persists as far as to the ERGIC, where dissociation takes place (2). To measure the rate of catZr dissociation, GM-Lec1 cells were pulse-labeled with [35S]methionine and chased for the indicated times with or without addition of chloroquine. After cross-linking the amount of catZr co-immunoprecipitated with anti-ERGIC-53 was analyzed. Chloroquine indeed delayed the release of catZr (Fig. 6A). We reasoned that this observation could reflect: (i) enhanced cross-linking in the presence of chloroquine; (ii) an unspecific block in secretion that would slow down transport of catZr at the level of ER exit, possibly followed by its degradation; or (iii) impaired dissociation from ERGIC-53 in the ERGIC. An effect of chloroquine on the efficiency of DSP treatment can be excluded (Fig. 4 and 30 min chase in Fig. 6A). To test the second possibility we performed a pulse-chase analysis in GM-Lec1 cells with an antiserum that specifically recognizes catZr.2 As shown in Fig. 6B, in untreated cells, catZr gradually converted from the proform to the mature enzyme indicating its targeting to the lysosomal pathway. In contrast, upon treatment with chloroquine, catZr remained unprocessed and the proform persisted slightly longer, which is consistent with its slower transit through the early secretory pathway. Furthermore, we analyzed the appearance of the metabolically labeled, unprocessed catZr-proenzyme in the culture medium from chloroquine-treated or control cells. Regardless of neutralization the cells secreted catZr (Fig. 6C). In chloroquine-treated cells the inhibition of intracellular maturation (Fig. 6B), which may arise from the impaired function of acid hydrolases and/or from transport defects, caused hypersecretion of catZr, a phenomenon known for lysosomal proteins. This renders it impossible to quantitatively compare the rate of secretion with or without neutralization and most likely accounts for the seeming paradox of retention in the ERGIC versus enhanced secretion of catZr. Collectively, these findings demonstrate that chloroquine neither initiates the degradation nor blocks the secretion of catZr, but causes some setback in its early transport through the secretory pathway. To discriminate whether under these conditions secretory transport is generally affected or if this delay is specific for catZr, we sought to measure the transport of an independent secretory marker. To this end, we examined glycosylation modifications on fibronectin, a ubiquitous component of the extracellular matrix. Because we failed to detect the accumulation of endo D-sensitive fibronectin in GM-Lec1 cells (data not shown), we used endo H and wt CHO-KI cells. Contrary to Lec1 cells, fibronectin secreted from this cell line was resistant to endo H as expected (data not shown). To study ER to medial-Golgi transport of fibronectin we performed pulse-chase/endo H experiments and assessed the acquisition of endo H resistance. Fig. 6D shows that intracellular fibronectin was readily lost with or without chloroquine and that this drug did not slow down fibronectin trafficking from the ER to the medial-Golgi. We conclude that chloroquine treatment specifically retards the dissociation of catZr from ERGIC-53 indicating that pH-induced conversion of the lectin domain of ERGIC-53 in the ERGIC contributes to efficient release of its glycoprotein cargo.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 6.
Dissociation of glycoprotein cargo from ERGIC-53 is delayed upon organelle neutralization. A, pulse-chase analysis of catZr release. GM cells were pulsed for 10 min and chased as indicated with or without addition of chloroquine. After DSP treatment, ERGIC-53 was immunoprecipitated and co-precipitated catZr was analyzed by phosphorimaging. The diagram shows the decrease of catZr signals normalized against ERGIC-53 and expressed as percentage of the value after 30 min of chase (mean ± S.D., n = 3; Student's t test for chloroquine versus control at time points 60, 90, and 120: p < 0.02, p < 0.01, and p < 0.02). B, maturation of catZr in the presence or absence of chloroquine as seen with a pulse (15 min)-chase experiment. P, proform; m, mature form. C, secretion time course of pulse-labeled (15 min) catZr from control- and chloroquine-treated GM cells. D, CHO-KI cells were metabolically labeled for 20 min and chased for the indicated times. Fibronectin (FN) immunoprecipitates were treated with or without endo H and resolved on a 4-10% gradient gel. Quantification of three independent experiments is shown (mean ± S.D.).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have uncovered the molecular mechanism of reversible lectin binding of ERGIC-53 to its substrates. Subtle pH changes inactivate and reactivate the CRD of ERGIC-53 in vitro and in vivo. We propose a model for cargo release by pH-induced loss of Ca2+ (Fig. 7). Ca2+-complexed ERGIC-53 binds cargo in the ER at neutral pH. Upon arrival in the ERGIC, His-178 is protonated because of lowered luminal pH and Ca2+ is lost. Because Ca2+ is required for the lectin activity of ERGIC-53 (Refs. 2 and 11, and this study), its loss leads to the inactivation of the mannose binding pocket resulting in the release of glycoprotein cargo. Subsequently, the transport receptor is recycled back to the ER, where, with a deprotonated and reactivated CRD, it can start a new round of cargo capture. Interestingly, a similar mechanism has been reported for C-type lectins, such as asialoglycoprotein receptor, and their glycoprotein cargo upon endocytosis from the plasma membrane (29, 30). In that case, however, the acidification ranges from extracellular pH 7.3 to endosomal pH 5.4 and, as a consequence, pH sensing by the asialoglycoprotein receptor can be achieved by a cluster of amino acids that includes an aspartic acid and an arginine residue (31). It is important to note that the [Ca2+] range we found to allow pH-sensitive mannose binding of ERGIC-53 (Fig. 2B) corresponds well to physiological Ca2+ levels in the ER (32). Accordingly, a mechanism that maintains lower levels of free Ca2+ in the ERGIC would further promote pH-induced loss of Ca2+ on ERGIC-53 and thereby contribute to efficient cargo release in a moderately acidic milieu. In quantitative terms, still 89 ± 4% binding would occur in the presence of 0.5 mM Ca2+ assuming a pHERGIC of 6.5. A more prominent decrease in the fraction of active ERGIC-53 at that pH (25 ± 12%), however, would be seen, if [Ca2+]ERGIC came to 0.2 mM (Figs. 1 and 2). Such thinking does not only demonstrate how the potentiation of these two parameters, pH and [Ca2+], can fully describe the molecular process of cargo dissociation, but also provides the theoretical basis for our observation, that elevating solely the pH of the ERGIC leads to a relatively moderate defect (80 ± 2% binding at pH 7.4, 0.2 mM [Ca2+]) in glycoprotein release (Fig. 6A). Although high concentrations of free Ca2+ have been measured within both the ER (~0.4 mM) and the Golgi (~0.3 mM) (32-34), we are still lacking quantitative records of [Ca2+]ERGIC. The in vivo demonstration of differential Ca2+ regulation along the secretory pathway will be one of our challenges in the future.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 7.
Model for cargo release by pH-induced loss of Ca2+. Upon arrival in the ERGIC, His-178 in the HIS-loop of ERGIC-53 is protonated because of lowered luminal pH (1) leading to the loss of one (or more) Ca2+ (2). Loss of Ca2+ as a cofactor inactivates the mannose binding pocket (3) and triggers cargo release. MBP, mannose binding pocket.

 
The presented model implies a molecular link between pH sensing and Ca2+ complexation. Indeed, our in vitro mannose binding studies indicated a modulation in the affinity of ERGIC-53 to Ca2+ ions in a slightly acidified environment (Fig. 2B). Furthermore, we identified His-178 to be essential for lectin activity, presumably in its deprotonated state. Glutamine cannot mimic the function of His-178, suggesting that this amino acid may be part of a precise ligand binding pocket. These findings led us to propose that the pH sensor residue His-178, directly or indirectly, is involved in Ca2+ binding, and that its protonation would trigger loss of the cation by a repulsive interaction (Fig. 7). Whether such a mechanism would involve one or more Ca2+ ions is not known. It is worth noting that the closely related lectin Vip36 has been shown to bind two Ca2+ ions (35). Co-crystallization of ERGIC-53 with Ca2+ and a sugar ligand will help to address this issue and verify our prediction. In the calcium-free crystal structure of ERGIC-53 (7) His-178 is located in an {alpha}-helical loop next to the mannose binding site that we termed the HIS-loop. It must be emphasized that in the crystal, notably the central metal/sugar binding loop C (36), was disordered in the absence of Ca2+ (7). Hence, the structure presented in Fig. 3B might resemble the inactive form of the CRD with protonated His-178 and the conformation of the HIS-loop in the presence of Ca2+ may significantly differ. Moreover, we illustrate in Fig. 3B that the HIS-loop is characteristic for the animal L-type lectin ERGIC-53 and may functionally replace the B loop (Ref. 36, shown in orange) of the plant L-type lectin family (represented here by Erythrina corallodendron lectin (37), for alignments see Ref. 28).3 Consequently, although His-178 is conserved among animal L-type lectins, leguminous lectins as well as yeast homologues of ERGIC-53 lack a central histidine sensor suggesting that these proteins perform functions unrelated to those known for ERGIC-53.

By which mechanism and in which compartment of the secretory pathway v-ATPase starts H+ pumping and, as a consequence, organelle acidification occurs, is still a matter of debate (18). Herein, we have used the weak base chloroquine that elevates the pH within acidic compartments. This treatment delays the dissociation of catZr from ERGIC-53 indicating that the site of cargo release, the ERGIC, is acidified and serves as a target for neutralizing agents. Importantly, we have found that catZr is not nonspecifically trapped in the ER in chloroquine-treated cells. Additionally, we demonstrated that this drug neither generally affects the secretory transport from the ER to the medial-Golgi nor retards the recycling pathway of ERGIC-53. The latter observation stands in sharp contrast to a study that concluded that pre-Golgi acidification by v-ATPase is required for retrograde transport to the ER (20). Using the v-ATPase inhibitor bafilomycin A1 the authors showed tubulation of the ERGIC, redistribution of {beta}-COP, and some retardation of brefeldin A-stimulated retrograde transport of Golgi mannosidase II that seems to be highly cell type-specific (18). In the light of our quantitative recycling studies in the presence of chloroquine (Fig. 5A), however, it appears likely that these observations were not a result of organelle neutralization but rather because of other effects caused by bafilomycin A1 treatment (18). Although the staining pattern of ERGIC-53 showed some minor overlap with acidic compartments that were trapped (and neutralized) with the weak base DAMP, consistent with our chloroquine experiments (Fig. 5B), no tubulation phenotype was seen in these cells and the obvious negative control using bafilomycin A1 was omitted (20). Other suggestions in the literature for an acidification mechanism of the ERGIC or early Golgi were deduced from pH-dependent in vitro processes such as the binding of ADP-ribosylation factor to microsomes (38), the dissociation of RAP from LDL receptor-related protein (15), and the binding of KDEL peptides to KDEL receptor (16). None of these pH dependences, however, could be (i) demonstrated in situ or (ii) attributed to the action of titratable sensor amino acids. Finally, a proteolytic pre-Golgi event that remained unclassified appeared to depend on organelle acidification (39, 40). The data presented here, however, combine the in vitro characterization of a pH-driven molecular mechanism with the documentation of its in vivo relevance and thus, to our knowledge, add the highest level of evidence for pre-Golgi acidification. As targeting of a pH probe (25, 41) exclusively to the ERGIC is impossible because of the lack of a marker protein that does not recycle through the ER, a reliable measurement of pHERGIC has never been reported and may hardly be expected. Even so, the conclusions presented herein are of particular biomedical interest given the fact that multiple diseases including cystic fibrosis (42) and Alzheimer's disease (43) have been associated with molecular processes in the ERGIC.

Loss of ERGIC-53 expression in humans causes combined deficiency of coagulation factors V and VIII (13). A recent study has revealed a second protein implicated in this disease, MCFD2, an EF-hand protein that co-purifies with ERGIC-53 in a Ca2+-dependent way (44). Our data show that ERGIC-53 purified in the absence of Ca2+ (see "Experimental Procedures") or from Ca2+-depleted cells (data not shown) still efficiently binds mannose upon readdition of Ca2+. Thus, this activity appears to be independent on complex formation with accessory proteins. The requirement for MCFD2 may be restricted to specific substrates such as coagulation factors V and VIII and the molecular role of this protein remains to be characterized. In summary, our study provides evidence that the function of ERGIC-53 as a transport lectin is determined by molecular switches in its substrate binding site itself that are triggered by changes in the ion composition between subcellular compartments involving pH and, presumably, calcium.


   FOOTNOTES
 
* This work was supported by the Swiss National Science Foundation and the University of Basel. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Research Director of Inserm. Back

|| To whom correspondence should be addressed. Tel.: 41-61-267-2222; Fax: 41-61-267-2208; E-mail: Hans-Peter.Hauri{at}unibas.ch.

1 The abbreviations used are: ER, endoplasmic reticulum; catZr, cathepsin Z-related protein; CRD, carbohydrate recognition domain; DSP, dithiobis(succinimidylpropionate); endo D, endoglycosidase D; endo H, endoglycosidase H; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; HIS, histidine ion sensor; mAb, monoclonal antibody; CHO, Chinese hamster ovary; MES, 4-morpholineethanesulfonic acid. Back

2 C. Appenzeller-Herzog and H.-P. Hauri, unpublished observations. Back

3 After submission of this manuscript, Velloso and colleagues (45) published the crystal structure of ERGIC-53 in complex with Ca2+. His-178 complexes one of the two calcium ions by its N{delta}-1 atom via a water molecule, whereas N{epsilon}-2 may be directly involved in ligand binding. The side chain of Asp-152 directly coordinates the same Ca2+. Back


   ACKNOWLEDGMENTS
 
We thank Pete Burkhard and Markus Meier for help in lectin structure analysis, Käthy Bucher for technical assistance, and Matthias Chiquet for kindly providing us with antibodies to fibronectin.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Warren, G., and Mellman, I. (1999) Cell 98, 125-127[CrossRef][Medline] [Order article via Infotrieve]
  2. Appenzeller, C., Andersson, H., Kappeler, F., and Hauri, H. P. (1999) Nat. Cell Biol. 1, 330-334[CrossRef][Medline] [Order article via Infotrieve]
  3. Muniz, M., Nuoffer, C., Hauri, H. P., and Riezman, H. (2000) J. Cell Biol. 148, 925-930[Abstract/Free Full Text]
  4. Belden, W. J., and Barlowe, C. (2001) Science 294, 1528-1531[Abstract/Free Full Text]
  5. Hauri, H. P., Kappeler, F., Andersson, H., and Appenzeller, C. (2000) J. Cell Sci. 113, 587-596[Abstract]
  6. Klumperman, J., Schweizer, A., Clausen, H., Tang, B. L., Hong, W., Oorschot, V., and Hauri, H. P. (1998) J. Cell Sci. 111, 3411-3425[Abstract]
  7. Velloso, L. M., Svensson, K., Schneider, G., Pettersson, R. F., and Lindqvist, Y. (2002) J. Biol. Chem. 277, 15979-15984[Abstract/Free Full Text]
  8. Loris, R., Hamelryck, T., Bouckaert, J., and Wyns, L. (1998) Biochim. Biophys. Acta 1383, 9-36[CrossRef][Medline] [Order article via Infotrieve]
  9. Schrag, J. D., Procopio, D. O., Cygler, M., Thomas, D. Y., and Bergeron, J. J. (2003) Trends Biochem. Sci. 28, 49-57[CrossRef][Medline] [Order article via Infotrieve]
  10. Loris, R. (2002) Biochim. Biophys. Acta 1572, 198-208[Medline] [Order article via Infotrieve]
  11. Itin, C., Roche, A. C., Monsigny, M., and Hauri, H. P. (1996) Mol. Biol. Cell 7, 483-493[Abstract]
  12. Vollenweider, F., Kappeler, F., Itin, C., and Hauri, H. P. (1998) J. Cell Biol. 142, 377-389[Abstract/Free Full Text]
  13. Nichols, W. C., Seligsohn, U., Zivelin, A., Terry, V. H., Hertel, C. E., Wheatley, M. A., Moussalli, M. J., Hauri, H. P., Ciavarella, N., Kaufman, R. J., and Ginsburg, D. (1998) Cell 93, 61-70[CrossRef][Medline] [Order article via Infotrieve]
  14. Pezzati, R., Bossi, M., Podini, P., Meldolesi, J., and Grohovaz, F. (1997) Mol. Biol. Cell 8, 1501-1512[Abstract]
  15. Bu, G., Geuze, H. J., Strous, G. J., and Schwartz, A. L. (1995) EMBO J. 14, 2269-2280[Medline] [Order article via Infotrieve]
  16. Scheel, A. A., and Pelham, H. R. (1996) Biochemistry 35, 10203-10209[CrossRef][Medline] [Order article via Infotrieve]
  17. Wu, M. M., Grabe, M., Adams, S., Tsien, R. Y., Moore, H. P., and Machen, T. E. (2001) J. Biol. Chem. 276, 33027-33035[Abstract/Free Full Text]
  18. Weisz, O. A. (2003) Int. Rev. Cytol. 226, 259-319[Medline] [Order article via Infotrieve]
  19. Ying, M., Flatmark, T., and Saraste, J. (2000) J. Cell Sci. 113, 3623-3638[Abstract]
  20. Palokangas, H., Ying, M., Vaananen, K., and Saraste, J. (1998) Mol. Biol. Cell 9, 3561-3578[Abstract/Free Full Text]
  21. Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L., and Hauri, H. P. (1988) J. Cell Biol. 107, 1643-1653[Abstract/Free Full Text]
  22. Pimpaneau, V., Midoux, P., Monsigny, M., and Roche, A. C. (1991) Carbohydr. Res. 213, 95-108[CrossRef][Medline] [Order article via Infotrieve]
  23. Cullen, B. R. (1987) Methods Enzymol. 152, 684-704[Medline] [Order article via Infotrieve]
  24. Kappeler, F., Klopfenstein, D. R., Foguet, M., Paccaud, J. P., and Hauri, H. P. (1997) J. Biol. Chem. 272, 31801-31808[Abstract/Free Full Text]
  25. Wu, M. M., Llopis, J., Adams, S. R., McCaffery, J. M., Teter, K., Kulomaa, M. S., Machen, T. E., Moore, H. P., and Tsien, R. Y. (2000) Methods Enzymol. 327, 546-564[Medline] [Order article via Infotrieve]
  26. Itin, C., Schindler, R., and Hauri, H. P. (1995) J. Cell Biol. 131, 57-67[Abstract/Free Full Text]
  27. Wessel, D., and Flugge, U. I. (1984) Anal. Biochem. 138, 141-143[CrossRef][Medline] [Order article via Infotrieve]
  28. Nufer, O., Mitrovic, S., and Hauri, H. P. (2003) J. Biol. Chem. 278, 15886-15896[Abstract/Free Full Text]
  29. Feinberg, H., Torgersen, D., Drickamer, K., and Weis, W. I. (2000) J. Biol. Chem. 275, 35176-35184[Abstract/Free Full Text]
  30. Feinberg, H., Park-Snyder, S., Kolatkar, A. R., Heise, C. T., Taylor, M. E., and Weis, W. I. (2000) J. Biol. Chem. 275, 21539-21548[Abstract/Free Full Text]
  31. Wragg, S., and Drickamer, K. (1999) J. Biol. Chem. 274, 35400-35406[Abstract/Free Full Text]
  32. Solovyova, N., and Verkhratsky, A. (2002) J. Neurosci. Methods 122, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  33. Pinton, P., Pozzan, T., and Rizzuto, R. (1998) EMBO J. 17, 5298-5308[CrossRef][Medline] [Order article via Infotrieve]
  34. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., and Tsien, R. Y. (2001) J. Biol. Chem. 276, 29188-29194[Abstract/Free Full Text]
  35. Fiedler, K., and Simons, K. (1996) J. Cell Sci. 109, 271-276[Abstract]
  36. Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445[CrossRef][Medline] [Order article via Infotrieve]
  37. Elgavish, S., and Shaanan, B. (1998) J. Mol. Biol. 277, 917-932[CrossRef][Medline] [Order article via Infotrieve]
  38. Zeuzem, S., Feick, P., Zimmermann, P., Haase, W., Kahn, R. A., and Schulz, I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6619-6623[Abstract/Free Full Text]
  39. Bonifacino, J. S., Lippincott-Schwartz, J., Chen, C., Antusch, D., Samelson, L. E., and Klausner, R. D. (1988) J. Biol. Chem. 263, 8965-8971[Abstract/Free Full Text]
  40. Antusch, D., Bonifacino, J. S., Burgess, W. H., and Klausner, R. D. (1990) J. Immunol. 145, 885-890[Abstract]
  41. Miesenbock, G., De Angelis, D. A., and Rothman, J. E. (1998) Nature 394, 192-195[CrossRef][Medline] [Order article via Infotrieve]
  42. Gilbert, A., Jadot, M., Leontieva, E., Wattiaux-De Coninck, S., and Wattiaux, R. (1998) Exp. Cell Res. 242, 144-152[CrossRef][Medline] [Order article via Infotrieve]
  43. Annaert, W. G., Levesque, L., Craessaerts, K., Dierinck, I., Snellings, G., Westaway, D., George-Hyslop, P. S., Cordell, B., Fraser, P., and De Strooper, B. (1999) J. Cell Biol. 147, 277-294[Abstract/Free Full Text]
  44. Zhang, B., Cunningham, M. A., Nichols, W. C., Bernat, J. A., Seligsohn, U., Pipe, S. W., McVey, J. H., Schulte-Overberg, U., de Bosch, N. B., Ruiz-Saez, A., White, G. C., Tuddenham, E. G., Kaufman, R. J., and Ginsburg, D. (2003) Nat. Genet. 34, 220-225[CrossRef][Medline] [Order article via Infotrieve]
  45. Velloso, L. M., Svensson, K., Pettersson, R. F., and Lindqvist, Y. (2003) J. Mol. Biol. 334, 845-851[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 BloodHome page
N. Kawasaki, Y. Ichikawa, I. Matsuo, K. Totani, N. Matsumoto, Y. Ito, and K. Yamamoto
The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2
Blood, February 15, 2008; 111(4): 1972 - 1979.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Kamiya, D. Kamiya, K. Yamamoto, B. Nyfeler, H.-P. Hauri, and K. Kato
Molecular Basis of Sugar Recognition by the Human L-type Lectins ERGIC-53, VIPL, and VIP36
J. Biol. Chem., January 25, 2008; 283(4): 1857 - 1861.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
K. Kato and Y. Kamiya
Structural views of glycoprotein-fate determination in cells
Glycobiology, October 1, 2007; 17(10): 1031 - 1044.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Satoh, N. P. Cowieson, W. Hakamata, H. Ideo, K. Fukushima, M. Kurihara, R. Kato, K. Yamashita, and S. Wakatsuki
Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VIP36
J. Biol. Chem., September 21, 2007; 282(38): 28246 - 28255.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Ivanov, A.-M. Dragoi, X. Wang, C. Dallacosta, J. Louten, G. Musco, G. Sitia, G. S. Yap, Y. Wan, C. A. Biron, et al.
A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA
Blood, September 15, 2007; 110(6): 1970 - 1981.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Banerjee, P. Vishwanath, J. Cui, D. J. Kelleher, R. Gilmore, P. W. Robbins, and J. Samuelson
The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation
PNAS, July 10, 2007; 104(28): 11676 - 11681.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Nonaka, B. Y. Ma, M. Ohtani, A. Yamamoto, M. Murata, K. Totani, Y. Ito, K. Miwa, W. Nogami, N. Kawasaki, et al.
Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein
J. Biol. Chem., June 15, 2007; 282(24): 17908 - 17920.
[Abstract] [Full Text] [PDF]