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J. Biol. Chem., Vol. 282, Issue 38, 28246-28255, September 21, 2007

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Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VIP36^*

Tadashi Satoh, Nathan P. Cowieson¹, Wataru Hakamata^¶, Hiroko Ideo^||**, Keiko Fukushima^||**, Masaaki Kurihara^¶, Ryuichi Kato, Katsuko Yamashita^||**, and Soichi Wakatsuki²

From the Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia, ^¶Division of Organic Chemistry, National Institute of Health Sciences (NIHS), Tokyo 158-8501, Japan, ^||Innovative Research Initiatives, Tokyo Institute of Technology, Yokohama 226-8503, Japan, and ^**Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo 101-0062, Japan

Received for publication, April 11, 2007 , and in revised form, June 25, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca²⁺ and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel -sandwich and binds one Ca²⁺ adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca²⁺ ion orients the side chains of Asp¹³¹, Asn¹⁶⁶, and His¹⁹⁰ for carbohydrate binding. This result explains the Ca²⁺-dependent carbohydrate binding of this protein. The Man--1,2-Man--1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca²⁺-dependent and D1 arm-specific manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In eukaryotic cells, post-translational modification of secreted proteins and intracellular protein transport between organelles are ubiquitous features. One of the most studied systems is the N-linked glycosylation pathway in the synthesis of secreted glycoproteins (1-3). The N-linked glycoproteins are subjected to diverse modifications and are transported through the endoplasmic reticulum (ER)³ via the Golgi apparatus to their final destinations inside and outside of the cell. Incorporation of the cargo glycoproteins into the transport vesicles is mediated by transmembrane cargo receptors, which have been identified as intracellular lectins. For example, mannose 6-phosphate receptor (4) functions as a cargo receptor for lysosomal proteins in the trans-Golgi network, whereas ER-Golgi intermediate compartment (ERGIC)-53 (5, 6) and its yeast orthologs Emp46/47p (7) are transport lectins for glycoproteins that are transported out of the ER.

VIP36, vesicular-integral protein of 36 kDa, was originally isolated from Madin-Darby canine kidney cells as a component of detergent-insoluble, glycolipid-enriched complexes containing apical marker (8). Confocal and immunoelectron microscopic experiments have suggested that VIP36 is distributed by either the pre-Golgi secretory pathway (9-11) or post-Golgi pathway (8, 12). Furthermore we showed that VIP36 is involved in intracellular transport, in the secretion of glycoproteins (e.g. clusterin) from polarized Madin-Darby canine kidney cells (13), and in the secretion of -amylase from rat parotid glands (14). Taken together, VIP36 appears to play significant roles not only in vesicular transport from the ER to the Golgi complex but also from the trans-Golgi network to the plasma membrane.

The exoplasmic/luminal domain of VIP36 as well as the luminal domain of ERGIC-53 and Emp46/47p share homology with L (leguminous)-type lectins and are thus identified as carbohydrate recognition domains (CRDs). It has been shown that ERGIC-53 interacts with glycoproteins carrying high mannose type glycan by endo--N-acetylglucosaminidase H treatment (15-17) and binds glycoproteins in a Ca²⁺- and pH-dependent manner (18). We have previously found that VIP36 has high affinity for high mannose type glycans containing Man--1,2-Man residues in Man_7-9(GlcNAc)₂-Asn peptides (19). Kamiya et al. (20) recently reported in detail the carbohydrate binding properties of VIP36 assayed by frontal affinity chromatography. This work suggested the Ca²⁺ dependence of carbohydrate binding and the specificity for the D1 arm, Man--1,2-Man--1,2-Man residues, of high mannose type glycans (corresponding to Man(D1)-Man(C)-Man(4); Fig. 1). In addition, using a flow cytometry-based method, it was also demonstrated that VIP36 binds glycoproteins carrying high mannose type glycans (21). These observations suggested that VIP36 is involved in the transport of glycoproteins via high mannose type glycans.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Chemical structures of Man9(GlcNAc)2. The individual carbohydrate residues of Man9(GlcNAc)2 are labeled. The D1 arm of Man9(GlcNAc)2 is colored in green.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Synthesis of Man--1,2-Man--1,2-Man, Man--1,2-Man- -1,3-Man, and Man--1,2-Man--1,6-Man—Couplings of phenyl 3,4,6-tri-O-benzyl--D-thiomannopyranoside (i), phenyl 2,4,6-tri-O-benzyl--D-thiomannopyranoside (ii), and phenyl 2,3,4-tri-O-benzyl--D-thiomannopyranoside (iii) having hydroxyl groups at the C-2, C-3, and C-6 positions (25) and 1,2-di-O-acetyl-3,4,6-tri-O-benzyl--D-mannopyranose (iv) (26) were performed under conditions well established for -mannosidation (trimethylsilyl trifluoromethanesulfonate/CH₂Cl₂) to give phenyl 2-O-acetyl-3,4,6-tri-O-benzyl--D-mannopyranosyl-(12)-3,4,6-tri-O-benzyl--D-thiomannopyranoside (v), phenyl 2-O-acetyl-3,4,6-tri-O-benzyl--D-mannopyranosyl-(13)-2,4,6-tri-O-benzyl--D-thiomannopyranoside (vi), and phenyl 2-O-acetyl-3,4,6-tri-O-benzyl--D-mannopyranosyl-(16)-2,3,4-tri-O-benzyl--D-thiomannopyranoside (vii) (27), respectively. Subsequent deacetylation of the mannobioses (v, vi, and vii) gave phenyl 3,4,6-tri-O-benzyl--D-mannopyranosyl-(12)-3,4,6-tri-O-benzyl--D-thiomannopyranoside (viii), phenyl 3,4,6-tri-O-benzyl--D-mannopyranosyl-(13)-2,4,6-tri-O-benzyl--D-thiomannopyranoside (ix), and phenyl 3,4,6-tri-O-benzyl--D-mannopyranosyl-(16)-2,3,4-tri-O-benzyl--D-thiomannopyranoside (x) (27), respectively. Introduction of the non-reducing end of the mannose residue to the mannobioses (viii, ix, and x) using 1-O-acetyl-2,3,4,6-tetra-O-benzyl--D-mannopyranose (xi) (28) was performed using the same -mannosidation method to give phenyl 2,3,4,6-tetra-O-benzyl--D-mannopyranosyl-(12)-3,4,6-tri-O-benzyl--D-mannopyranosyl-(12)-3,4,6-tri-O-benzyl--D-thiomannopyranoside (xii), phenyl 2,3,4,6-tetra-O-benzyl--D-mannopyranosyl-(12)-3,4,6-tri-O-benzyl--D-mannopyranosyl-(13)-2,4,6-tri-O-benzyl--D-thiomannopyranoside (xiii), and phenyl 2,3,4,6-tetra-O-benzyl--D-mannopyranosyl-(12)-3,4,6-tri-O-benzyl--D-mannopyranosyl-(16)-2,3,4-tri-O-benzyl--D-thiomannopyranoside (xiv), respectively. Finally complete deprotection of synthesized mannotriose derivatives (xii, xiii, and xiv) afforded -D-mannopyranosyl-(12)--D-mannopyranosyl-(12)--D-mannopyranose, -D-mannopyranosyl-(12)--D-mannopyranosyl-(13)--D-mannopyranose, and -D-mannopyranosyl-(12)--D-mannopyranosyl-(16)--D-mannopyranose, respectively. These mannotrioses were isolated on a COSMOSIL Sugar-D column (Nacalai Tesque) using an isocratic solvent composed of 65% MeCN and 35% H₂O. NMR and MS spectra of these compounds were in good agreement with those reported for closely related compounds (29, 30).

Preparation of Man₃GlcNAc and Man₆(GlcNAc)₂-Asn—Man₃GlcNAc was prepared from urine of a mannosidosis patient as described previously (31). Briefly 10 ml of urine containing 10 mg of creatinine was concentrated to 1 ml and centrifuged for 20 min at 3,000 rpm. The supernatant was subjected to Bio-Gel P-4 (200-400 mesh) column chromatography (2.6 x 97 cm). The column was eluted with water containing 0.002% phenylmercuric nitrate, and the hexose content in each fraction was analyzed with phenol-sulfuric acid reagent. Fractions between Man₂GlcNAc and Man₄GlcNAc were pooled, sequentially subjected to Bio-Gel P-4 (under 400 mesh) column chromatography (2 x 100 cm) at 55 °C, and eluted with distilled water by monitoring with a refractometer. The fraction corresponding to Man₃GlcNAc was collected, and the structure of Man₃GlcNAc was identified as Man--1,2-Man--1,3-Man--1,4-GlcNAc by methylation analysis and sequential exoglycosidase digestion using Man12-specific Aspergillus saitoi -mannosidase, jack bean -mannosidase, and snail -mannosidase. The yield was 1100 nmol. Man₆(GlcNAc)₂-Asn was prepared from ovalbumin as described previously (32).

Protein Expression and Purification—The DNA fragment for residues 51-301, which correspond to the CRD and part of the stalk domain of canine VIP36, was cloned into the BamHI and EcoRI sites of pGEX4T-1 plasmid (GE Healthcare). The recombinant VIP36 was expressed in Escherichia coli BL21(DE3). Cells were harvested after induction with 0.1 mM isopropyl -D-thiogalactoside (Wako) for 15 h at 20 °C and lysed by sonication in phosphate buffered saline buffer containing 2 mM CaCl₂. The cell lysate was loaded on a glutathione-Sepharose 4B column (GE Healthcare). The glutathione S-transferase (GST) fusion protein was eluted by glutathione (Wako) and cleaved by thrombin protease (GE Healthcare). The cleaved proteins were passed through a glutathione-Sepharose 4B column to remove GST protein and further purified by a benzamidine-Sepharose 4FF column (GE Healthcare) to remove the thrombin protease. The purified protein was dialyzed against 10 mM MES (pH 6.5) and 2 mM CaCl₂.

Crystallization and X-ray Data Collection—Initial crystallization conditions were screened using the Large Scale Protein Crystallization and Monitoring System (PXS) (33). The crystallization conditions of VIP36 in its Ca²⁺-bound form were obtained in a buffer containing 18 mg ml^-1 protein, 15% (w/v) polyethylene glycol 4000, 1.5 M NaCl, and 0.1 M MES (pH 6.5) with incubation at 277 K for 4 days. For the metal free-form, the Ca²⁺-bound crystal was soaked with this buffer containing 10 mM EDTA to remove Ca²⁺. The crystal of Ca²⁺.Man-bound VIP36 was obtained by soaking the Ca²⁺-bound crystal with the buffer containing 50 mMD-mannose (Sigma). The Ca²⁺.Man₂-bound VIP36 was co-crystallized in a buffer containing 10 mg ml^-1 protein, 3.4 mM 2-mannobiose (Sigma), 5% (w/v) polyethylene glycol 4000, 0.3 M MgCl₂, and 0.1 M MES (pH 6.5) with incubation at 277 K for 1 week. On the other hand, the Ca²⁺.Man₃GlcNAc-bound VIP36 was co-crystallized in a buffer containing 10 mg ml^-1 protein, 3.4 mM Man₃GlcNAc, 10% (w/v) polyethylene glycol 4000, and 0.4 M imidazole malate (pH 6.0) with incubation at 277 K for 3 weeks. Despite the extensive co-crystallization with Man₃GlcNAc the electron density map did not show any interaction with the GlcNAc moiety; only the Man₃ portion was recognized by VIP36. Data sets of the metal-free and Ca²⁺.Man₂ - and Ca²⁺. Man₃GlcNAc-bound forms were collected under cryogenic conditions with crystals soaked with a cryoprotectant buffer containing 20% (v/v) glycerol. The Ca²⁺-, Ca²⁺.Man-bound crystals were soaked with a buffer containing 2.5 M LiCl instead of 1.5 M NaCl for data collection under cryogenic conditions. The diffraction data were processed using HKL2000 (34). The metal-free and Ca²⁺-, Ca²⁺.Man-, and Ca²⁺.Man₂-bound crystals belong to space group C2 with two molecules per asymmetric unit. In contrast, the Ca²⁺.Man₃GlcNAc-bound crystal belongs to space group P2₁2₁2₁ with five molecules per asymmetric unit. The crystallographic parameters of VIP36 are shown in Table 1.

Computer-aided Model Building—The model of VIP36·Man₈(GlcNAc)₂-Asn complex was built using coordinates of well ordered high mannose type glycans on glycoprotein crystal structures (human pancreatic -amylase (Protein Data Bank code 1BSI), Erythrina corallodendron lectin (Protein Data Bank code 1LTE), and exo-(1,3)--glucanase (Protein Data Bank code 1H4P [PDB] )) and mannosyl ligand-bound VIP36 structures. The corresponding glycan residues were superimposed on each other, and appropriate coordinates were used as follows: Asn-GlcNAc(1), Protein Data Bank code 1BSI; GlcNAc(2), Protein Data Bank code 1LTE; Man(3)-Man(4)-Man(C), Man₃GlcNAc-bound VIP36; Man(D1), Man₂-bound VIP36; Man(4')-(Man(A))-Man(B)-Man(D3), Protein Data Bank code 1H4P [PDB] ; VIP36, Man₂-bound form. Based on the above model and human salivary -amylase (Protein Data Bank code 1SMD) structures, the complex model of VIP36·rat salivary -amylase carrying Man₈(GlcNAc)₂ was built. The salivary -amylase was docked onto the VIP36·Man₈(GlcNAc)₂-Asn complex model through super-imposition with an N-glycosylation site (Asn⁴⁶¹) of the salivary -amylase and the asparagine residue of the high mannose type glycan bound with VIP36.

Surface Plasmon Resonance (SPR) Analysis—SPR measurements were carried out at 25 °C using a Biacore 2000 (Biacore) equipped with a CM5 sensor chip. GST-VIP36 (residues 51-322) was purified by affinity chromatography using glutathione-Sepharose 4B and benzamidine-Sepharose 4FF columns. The purified protein was immobilized on the flow cell using the amine coupling method. Various concentrations of mannotrioses (Man--1,2-Man--1,2-Man, Man--1,2-Man--1,3-Man, and Man--1,2-Man--1,6-Man) and Man₆(GlcNAc)₂-Asn in sample buffer (50 mM HEPES (pH 6.0) and 1 mM CaCl₂) were injected over the flow cells at a flow rate of 20 µl/min using HBS-P buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% surfactant P20) as the running buffer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Crystallization and Overall Structure of Exoplasmic/Luminal Domain of VIP36—The exoplasmic/luminal domain (residues 51-301) of VIP36, corresponding to the CRD and part of the short stalk domain, was crystallized. Despite extensive crystallization screening, diffraction quality crystals of the CRD (residues 51-278) alone could not be produced. The crystal structure of the exoplasmic/luminal domain of Ca²⁺-bound VIP36 was solved by the molecular replacement method using the Ca²⁺-bound ERGIC-53 CRD (Protein Data Bank code 1R1Z) (23) as a search model. The crystal belongs to space group C2 with two molecules (A and B) per asymmetric unit. Both VIP36 molecules are related by 2-fold symmetry, forming a pseudo-homodimer. However, gel filtration data demonstrate that this protein is monomeric in solution (supplemental Fig. 1).

To obtain crystals of mannose-bound VIP36, the Ca²⁺-bound crystals were soaked in a solution containing mannose in molar excess. The Man₂-bound form was obtained by co-crystallization. To obtain the metal-free form, the Ca²⁺-bound crystal was soaked with buffer containing 10 mM EDTA. Crystallization of VIP36 in the absence of Ca²⁺ was not successful. Following treatment with EDTA the Ca²⁺ is completely absent from molecule B, whereas approximately half of the Ca²⁺ ions are removed from molecule A. In the C2 crystal form, crystallization of VIP36 in complex with longer oligomannoses was not successful due to the crystal packing around the ligand-binding site. To find other crystal forms, we therefore carried out crystallization screening in the presence of Ca²⁺ and longer oligomannoses, Man--1,2-Man--1,2-Man, Man--1,2-Man--1,3-Man, Man₃GlcNAc, and Man₆(GlcNAc)₂-Asn. Diffraction quality crystals were obtained from co-crystallization with the Man₃GlcNAc alone. The crystal belongs to space group P2₁2₁2₁ with five molecules (A, B, C, D, and E) per asymmetric unit. The structure has the Man--1,2-Man--1,3-Man moiety in molecules A and B, whereas the GlcNAc moiety is disordered. In contrast, only one mannose residue is ordered in molecule C, and all the carbohydrate residues are disordered in molecules D and E. The dimer interfaces of the P2₁2₁2₁ crystal form are different from that of the C2 crystal form, suggesting that VIP36 is monomeric.

The CRD of VIP36 has an overall globular shape composed of a -sandwich of two antiparallel -sheets and is composed of 17 -strands and three 3₁₀ helices, each with a single turn (Fig. 2A). The -sandwich comprises a seven-stranded (2-5-14-7-8-9-10) concave -sheet and a six-stranded (1a,b-15-6-11-12-13a) convex -sheet in a variation of the jelly roll fold. The -strands are numbered according to the secondary structure numbering scheme of ERGIC-53 (22). A -hairpin (strands 3 and 4) is inserted between 2 and 5. Residues Cys²⁰² (strand 10) and Cys²³⁹ (strand 13a) form a disulfide bond. The stalk domain is composed of a -strand (16), a short -helix (H4), and one turn of a 3₁₀ helix (H5). The 16 forms a -sheet with 13b on the face of the protein opposite to the ligand-binding site between the concave and convex -sheets. The stalk domain (residues 289-301) contributes to an extensive crystal contact (Fig. 2A and supplemental Fig. 2) that explains the successful crystallization of this construct.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Overall structure of the exoplasmic/luminal domain of VIP36. Ribbon models of the VIP36 (Man2-bound form, molecule A) are shown in A. The secondary structures are highlighted (-strands belonging to the concave -sheets, yellow; -strands belonging to convex -sheets, blue; -strands belonging to -hairpin, cyan; -strands belonging to the short -sheet formed between the stalk domain and one of the loops of the CRD, magenta; helices, red), and the loops of the CRD and stalk domain are colored gray and green, respectively. The bound Ca2+ is shown as a pink sphere. The bound oligomannoses are superimposed from the VIP36 complex structures with Man--1,2-Man and Man--1,2-Man--1,3-Man and are shown as a green stick model. The reducing-end mannose residue in the Man2-bound form is omitted because its position is almost the same as that of the Man3GlcNAc-bound form. Positions of Loops 1, 2, and 3, which are bound to the oligomannose, are indicated. The surface models (B) are shown in the same orientations as in A and colored according to the electrostatic surface potential (blue, positive; red, negative; scale from -10 to +10 kT/e).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Ca2+-binding site and its conformational changes upon Ca2+ binding of VIP36. The Ca2+-bound (molecule A) and metal-free (molecule B) structures are shown in stereo and colored in yellow and green, respectively. Residues coordinating Ca2+ and those with notable conformational changes are shown in ball- and-stick models. Water molecules are labeled W1 and W2.Ca2+-coordinating bonds are solid lines, and hydrogen bonds are dotted lines.

Specific Binding of VIP36 to D1 Arm of High Mannose Type Glycan—Previous studies have suggested that VIP36 CRD recognizes D1 arm, Man--1,2-Man--1,2-Man residues of high mannose type glycans (19, 20). In fact, we observed using SPR analysis that VIP36 has higher affinity for Man--1,2-Man--1,2-Man oligosaccharide (corresponding to Man(D1)-Man(C)-Man(4) of the D1 arm) than for Man--1,2-Man--1,3-Man (Fig. 4). No interaction was observed between VIP36 and Man--1,2-Man--1,6-Man. In addition, we observed that Ca²⁺ ion is required for the interaction between VIP36 and Man₆(GlcNAc)₂-Asn (supplemental Fig. 3). The calculated dissociation constant between VIP36 and Man₆(GlcNAc)₂-Asn in the presence of 1 mM Ca²⁺ was 70 µM.On the other hand, no interaction was observed in the presence of 5 mM EDTA.

Structure of VIP36 in Complex with Ca²⁺ and Man—In the structure of VIP36 in complex with Ca²⁺ and mannose, the mannose is located in a pocket neighboring the Ca²⁺-binding site on the concave -sheet and has well defined electron density (Fig. 5A). The mannose-binding site (also called the "primary binding site" hereafter) comprises 7 and Loops 1, 2, and 3. A number of specific contacts can be seen between the protein and the ligand. The mannose is bound by Asp¹³¹ (O-1 and O-2), Asn¹⁶⁶ (N-2), His¹⁹⁰ (N-2), Gly²⁶⁰ (N), Asp²⁶¹ (N), and Leu²⁶² (N) through hydrogen bonds in the complex (Fig. 5D). Incidentally the side-chain positions of Asp¹³¹, Asn¹⁶⁶, and His¹⁹⁰ in the primary binding site are stabilized by Ca²⁺.

Structure of VIP36 in Complex with Ca²⁺ and Man₂—The Man--1,2-Man residues have extremely well defined electron density in the structure of VIP36·Man₂ complex (Fig. 5B). The 4-OH and 6-OH groups of the non-reducing mannose residue make hydrogen bonds with Ser⁹⁶ (O) and Asp²⁶¹ (O-1), respectively (Fig. 5E). In contrast, the hydrogen bond between the 6-OH group and Asp²⁶¹ (O-1) is not observed in molecule B (supplemental Fig. 4A). The binding site of the reducing-end mannose residue of Man₂ is almost the same as the primary binding site (Fig. 5, D and E).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Specific binding of GST-VIP36 to the D1 arm (Man--1,2-Man- -1,2-Man) revealed by SPR analysis. Three different mannotrioses were added over flow cells at the indicated concentrations. Specific binding of mannotrioses was obtained by subtracting the resonance unit (RU) value of the GST immobilized sensor chip from the values of GST-VIP36 immobilized sensor chips. The plots were obtained by subtracting the values measured using the sample buffer without carbohydrates. The dose binding curves were obtained from the resonance unit value at 200 s. Solid circle, Man--1,2-Man--1,2-Man; open circle, Man--1,2-Man--1,3-Man; solid triangle, Man--1,2-Man--1,6-Man.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Carbohydrate ligand-binding site of VIP36. 2Fo - Fc electron density map of mannose of the Man-bound form (A), Man--1,2-Man of the Man2-bound form (B), and Man--1,2-Man--1,3-Man of the Man3GlcNAc-bound form (C) contoured at 1.2. Secondary structures are shown as in Fig. 2A. D, structure of mannose and Ca2+-binding site of VIP36 (molecule A). E, structure of Man--1,2-Man and Ca2+-binding site of VIP36 (molecule A). F, structure of Man--1,2-Man--1,3-Man and Ca2+-binding site of VIP36 (molecule A). The bound carbohydrate residues are shown as green stick models. Residues of VIP36 binding to the ligands are shown in ball-and-stick models.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (54K): [in this window] [in a new window]   FIGURE 6. A, overall structure of ConA monomer. A purple ribbon model of ConA (molecule A) is shown. The bound Mn2+ (S1) and Ca2+ (S2) are shown as large gray and black spheres, respectively. B, comparison between VIP36 and ConA metal-binding site structures. The VIP36 (molecule A) and ConA structures are colored yellow and purple, respectively. Residues of VIP36 and ConA are labeled in black and purple, respectively. The Ca2+ in VIP36 is shown as a large pink sphere. Because the position of Ca2+ at the S2 site in ConA is almost the same as in VIP36, it is not shown. Water molecules found in the S1 site of ConA are shown as small white spheres and are labeled W3 and W4. C, overall structure of ERGIC-53 CRD. A cyan ribbon model of ERGIC-53 (molecule A) is shown. The bound Ca2+ is shown as large magenta spheres (M1 and M2). D, comparison between VIP36 (yellow) and ERGIC-53 (cyan) Ca2+-binding site structures. Because the position of Ca2+ at the M2 site in ERGIC-53 is almost the same as in VIP36, it is not shown. Water molecules found in the M1 site of ERGIC-53 are shown as small orange spheres and are labeled W5 and W6. Residues involved in the metal binding are shown as ball- and-stick models.

It was shown that ERGIC-53 contains two Ca²⁺ ions termed M1 and M2 (Fig. 6, C and D) and that the M1 ion does not lie at the same site as the S1 ion, although M2 is located at the corresponding S2 site (23). The Ca²⁺ of VIP36 corresponds to the M2 site of ERGIC-53. When the VIP36 and ERGIC-53 structures are superimposed, the M2 metal ions overlay with a separation that is less than 0.2 Å. Although the Ca²⁺ of VIP36 is equivalent to the M2 site of ERGIC-53, the electron density maps of VIP36 show no peak that could be assigned as a metal ion either at the corresponding M1 site or at any other sites within the structure. In the Ca²⁺-binding site (M2 and S2), the Ca²⁺-coordinating residues are structurally very well conserved except for Asp¹⁹³ in VIP36 (Fig. 6, B and D). The corresponding Asp¹⁹ (ConA) and Asp¹⁸⁹ (ERGIC-53) residues are coordinated by the two metal ions. In VIP36, the O-1 of Asp¹⁹³ forms a hydrogen bond with the main-chain nitrogen atom of Asp¹⁶⁷ to stabilize the Ca²⁺-binding site. As a result, only one Ca²⁺ ion fixes the ligand binding residues in VIP36. Our crystallographic studies also suggest that VIP36 did not bind other divalent cations, neither Mn²⁺ nor Mg²⁺ (data not shown). Loop 1 of VIP36 is two residues longer than that of ERGIC-53. Likewise the residues coordinating the M1 ion in ERGIC-53 (Asp¹⁶³, Asp¹⁶⁵, Asn¹⁶⁹, Asn¹⁷⁰, and Asp¹⁸⁹; shown in green in supplemental Fig. 2) are poorly conserved in VIP36. Taken together, we conclude that VIP36 binds only one Ca²⁺ ion and that the single Ca²⁺ fixes the positions of residues involved in carbohydrate ligand binding.

View larger version (52K): [in this window] [in a new window]   FIGURE 7. A, superimposition of Man-, Man2-, and Man3GlcNAc-bound forms of VIP36. The Man-, Man2-, and Man3GlcNAc-bound forms of VIP36 (molecule A) are colored in marine blue, white, and yellow, respectively. The bound Man, Man2, and Man3 are shown as magenta, white, and green stick models, respectively. B, ligand binding diagram. The model of Man--1,2-Man--1,2-Man--1,3-Man (Man4) is designated as Man(D1)-Man(C)-Man(4)-Man(3), respectively. Hydrogen bonds are indicated with dotted arrows pointing in the direction from donor to acceptor. C, comparison between the VIP36 (yellow) and ConA (purple) carbohydrate ligand-binding site. Residues binding to the ligands are shown in ball-and-stick models. D, comparison between the VIP36 (yellow) and ERGIC-53 (cyan) carbohydrate ligand-binding site. The model of Man4 is shown as a green stick model. Residues of VIP36 binding to the ligands and the corresponding ones in ERGIC-53 are shown in ball-and-stick models.

Next the carbohydrate-binding site of VIP36 was compared with that of ConA in complex with Man--1,3-(Man--1,6-)Man (corresponding to Man(3)-(Man(4'))-Man(4)) (49). Although the carbohydrate binding specificity of VIP36 and ConA is essentially different, the structural conservation of ligand-binding sites between them is observed in not only the Man(C)-binding site but also the Man(D1)- and Man(4)-binding sites of VIP36 (Fig. 7C). In the Man(C)-binding site of VIP36, the ligand binding residues are structurally very well conserved except for His¹⁹⁰ in VIP36 (Fig. 7C). The corresponding main-chain nitrogen atom of Arg²²⁸ in ConA is bound to only the 3-OH group of the mannose residue. In contrast, N-2 of His¹⁹⁰ in VIP36 is bound to the 3-OH and 4-OH groups and acts simultaneously as hydrogen bond donor and acceptor (Fig. 7B). In the Man(4)-binding site, although the carbohydrate binding loop conformation of VIP36 is largely different than that of ConA, the side-chain position of Tyr¹⁶⁴ is very similar to that of Tyr¹² in ConA.

When the carbohydrate-binding site of VIP36 was compared with the corresponding site of ERGIC-53 in complex with Ca²⁺ (23), the Man₄ binding residues of VIP36 are largely identical to the corresponding residues of ERGIC-53 (Fig. 7D and supplemental Fig. 2). The structural conservation suggests that ERGIC-53 also binds the D1 arm moiety of high mannose type glycoproteins, which is consistent with the function of ERGIC-53 as a transport lectin for high mannose type glycoproteins. These contain Man₈(GlcNAc)₂ with an intact D1 arm and are transported from the ER by the ER quality control mechanism (2, 3). However, there are some structural differences in the 5, 7, and Loops 1 and 3 regions: (i) in 5 the side-chain orientation of Ser⁹⁶ of ERGIC-53 is dissimilar to that of VIP36, (ii) the side-chain orientation of Asp¹²⁹ in 7 of ERGIC-53 is different than that of the corresponding Asp¹³¹ of VIP36, (iii) Phe¹⁶² of ERGIC-53 is replaced by Tyr¹⁶⁴ in VIP36, and (iv) the Loop 3 of ERGIC-53 is positioned further away from the ligand when compared with VIP36, and Asp²⁶¹ of VIP36 is replaced by Gly²⁶⁰ in ERGIC-53. Most significantly, Tyr¹⁶⁴ and Asp²⁶¹ in VIP36 are better suited than Phe¹⁶² and Gly²⁶⁰ in ERGIC-53 for binding to the Man₄. In ERGIC-53, the presumptive weak interactions between ERGIC-53 and carbohydrate ligands might be compensated by the oligomerization of the CRDs.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Model for binding between VIP36 and high mannose type glycan (Man8(GlcNAc)2-Asn). A, the high mannose type glycan is indicated by a stick model. In the oligosaccharide, the part determined in this study is colored in green. The modeled D2 and D3 arms and N-linked chitobiose moiety of the high mannose type glycan are shown in purple. The types of glycosidic linkages are also indicated. The individual carbohydrate residues of Man8(GlcNAc)2-Asn are shown as in Fig. 1. Residues involved in the ligand binding are shown as ball-and-stick models. B, model for binding between VIP36 and salivary -amylase carrying Man8(GlcNAc)2 in rat secretory vesicles.

It has been shown that VIP36 recycles between the ER and the Golgi complex (9-11). To date, however, there is no obvious evidence that VIP36 is involved in retrograde transport of glycoproteins from the Golgi complex to the ER. On the other hand, we revealed that VIP36 localizes in the trans-Golgi network (12) and is involved in secretion of high mannose type glycoproteins clusterin and -amylase (13, 14). It has been generally known that the D1 arm is trimmed by cis-Golgi mannosidase I to form Man₅(GlcNAc)₂ in the cis-Golgi. The carbohydrate structure has a lower affinity for VIP36 (19, 20). In this study, we have shown that VIP36 specifically binds the Man--1,2-Man--1,2-Man residues of the D1 arm of high mannose type glycan. Taken together, VIP36 might be involved in anterograde transport of certain glycoproteins carrying high mannose type glycan with the D1 arm from the ERGIC via the Golgi complex to the plasma membrane by protecting the D1 arm against trimming by cis-Golgi mannosidase I. Although it is not known whether or not high mannose type glycan of rat salivary -amylase has the D1 arm, a possible model for binding between VIP36 and salivary -amylase carrying high mannose type glycan (Man₈(GlcNAc)₂) in rat parotid acinar cells is shown (Fig. 8B).

In summary, we determined the first complex structure of the exoplasmic/luminal domain of the transport lectin VIP36 and Ca²⁺ and Man, Man₂, and Man₃GlcNAc, which are part of the D1 arm of high mannose type glycans. Our results provide structural insights into the mechanism of recognition of high mannose type glycoproteins by VIP36 in a Ca²⁺-dependent and D1 arm-specific manner. Further biochemical analysis such as subcellular localization of VIP36 on a wide variety of cells and identification of its cargo glycoproteins together with the detailed carbohydrate structures will provide further insight into the mechanism of high mannose type glycoprotein transport by VIP36.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2DUO, 2DUP, 2DUQ, 2DUR, and 2E6V) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by the Protein 3000 project, by Grant-in-aid for Young Scientists (B) 17790097 from The Ministry of Education, Culture, Sports, Science and Technology of Japan, and by research grants for research on human immunodeficiency virus/AIDS from The Ministry of Health and Labor Sciences of Japan. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.

¹ Supported by a fellowship from the Australian Synchrotron Research Program.

² To whom correspondence should be addressed. Tel.: 81-29-864-5631; Fax: 81-29-879-6179; E-mail: soichi.wakatsuki{at}kek.jp.

³ The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; CRD, carbohydrate recognition domain; GST, glutathione S-transferase; SPR, surface plasmon resonance; MES, 4-morpholineethanesulfonic acid; ConA, concanavalin A.

	ACKNOWLEDGMENTS

 
We thank Drs. L. M. G. Chavas and K. Ihara for helpful discussion and the beamline staff of BL-5A, BL-6A, and AR-NW12A at the Photon Factory, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan (Proposals 2003S2002, 2005G070, and 2006S2006) for providing the data collection facilities and support.

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