Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor*

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of ∼6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugarbinding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of ϳ6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.
N-linked oligosaccharides contribute to the folding, transport, and degradation of glycoproteins via interactions with a variety of intracellular lectins (1)(2)(3)(4). The processing of the N-linked oligosaccharides is initiated in the endoplasmic reticulum (ER) 2 by the removal of the glucose residues from the Glc 3 Man 9 GlcNAc 2 oligosaccharide structure. Mannose trimming is subsequently initiated in the ER and continues in the Golgi complex by a series of mannosidases prior to the branching and extension of the oligosaccharides by Golgi glycosyltransferases (5)(6)(7). Calnexin and calreticulin, which are ER chaperones with lectin activities, specifically bind monoglucosylated high mannose-type oligosaccharides, i.e. Glc 1 Man 9 GlcNAc 2 , expressed on unfolded proteins and assist their folding (8 -10). Degradation of glycoproteins is governed by the trimming of the middle branch of their carbohydrate moieties by ER mannosidase I and by association with ER degradationenhancing ␣-mannosidase-like protein (EDEM) (11)(12)(13).
Secretory and membrane glycoproteins from ER-resident proteins that are correctly folded and sorted leave the ER and are exported to the Golgi complex via the ER-Golgi intermediate compartment (ERGIC). ERGIC-53, which is a 53-kDa type I transmembrane protein used as a popular marker for the ERGIC, constitutively cycles between the ER and the ERGIC, serving as a cargo receptor for some glycoproteins (14). The crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, revealed a striking structural similarity to Ca 2ϩ -dependent leguminous lectins and calnexin (15,16).
Cargo glycoproteins are further sorted in the Golgi, where the vesicular integral protein of 36 kDa (VIP36), the type I membrane glycoprotein with a CRD similar to that of ERGIC-53, is thought to act as a cargo receptor for quality control (17,18). Although VIP36 is highly localized in the cis-Golgi network, the carbohydrate moieties of VIP36 are modified by Golgi enzymes, suggesting that this glycoprotein recycles between the Golgi and the ER (19).
To gain deeper insight into the mechanisms underlying the molecular actions of VIP36 and ERGIC-53, it is essential to reveal the sugar-binding properties of these cargo receptors in detail. Although both of the cargo receptors interact with glycoproteins carrying high mannose-type oligosaccharides (20 -23), detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. Although ERGIC-53 has been shown to bind glycoproteins in a Ca 2ϩ -and pH-dependent manner (24), a Ca 2ϩ requirement for the sugar binding of VIP36 remains controversial (25,26).
In the present study, we analyzed the sugar-binding properties of a recombinant CRD domain of VIP36 (VIP36-CRD) by the frontal affinity chromatography (FAC) method, because this method is suitable for analyzing weak interactions (27)(28)(29). For FAC analyses, we prepared a pyridylaminated (PA) sugar library consisting of 21 kinds of oligosaccharides, including isomeric structures. On inspection of the FAC data, the mannose residues predominantly involved in association with VIP36-CRD had been identified in the high mannose-type oligosaccha-* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation. 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. 1 To whom correspondence should be addressed: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe ride structures. The sugar branch specificity of VIP36 thus revealed formed a basis for understanding the molecular function of this cargo receptor in transport of glycoproteins.
Protein Expression-The DNA fragment encoding amino acids residues 45-296 of human VIP36, which was designated VIP36-CRD, was cloned into the pET-3c plasmid vector with a C-terminal poly(H) tag moiety. The protein was expressed in Escherichia coli BL21 (DE3) pLysS strain (Stratagene) in LB medium. Production of recombinant proteins was induced by the addition of 0.08 mM isopropyl ␤-D-thiogalactopyranoside. The poly(H) fusion protein was purified from cell lysates with Ni 2ϩ -nitrilotriacetic acid column (Amersham Biosciences). The fusion protein was cleaved by incubation with PreScission protease (Amersham Biosciences), and the poly(H) tag was removed by dialysis. The protein was further purified using a Superose 12 gel filtration column (Amersham Biosciences).
FAC Analysis-0.4 mM VIP36-CRD was dissolved in 1 ml of 0.2 M NaHCO 3 , pH 8.3, containing 0.1 M D-mannose and coupled to the HiTrap NHS-activated column following the manufacturer's instruc-tions (Amersham Biosciences). After immobilization, the agarose beads were taken out from the cartridge and packed into a stainless steel column (4.0 ϫ 10 mm) (GL Sciences).
FAC analysis was carried out as described previously (27)(28)(29). PA oligosaccharides were dissolved at a concentration of 10 nM in 10 mM Tris-HCl (pH 8.0), 10 mM HEPES (pH 7.0 -7.5), or 10 mM MES (pH 5.5-6.5) containing 1 mM CaCl 2 or 5 mM EDTA and applied onto the VIP36-CRD column at a flow late of 0.25 ml/min at 20°C. The elution profile was monitored by the fluorescence intensity at 400 nm (excitation at 320 nm). The retardation compared with the control oligosaccharide was computed using the difference of each elution volume, V f . The dissociation constant, K d , of VIP36-CRD for Man 9 GlcNAc 2 -PA (M9.1) was determined using equation 1 (28), where [A] 0 , V 0 , and B t are initial concentration of the PA oligosaccharide, the elution volume of the control sugar, and the total amount of immobilized VIP36-CRD in the column, respectively. The elution profile was monitored by UV light absorption at 300 nm to avoid possible quenching caused by the relatively high concentration of the PA sugar. For the determination of V 0 , 50 M p-nitrophenyl-␤-D-galactopyranoside was used. K d (ϭ 1/K a ) was calculated based on the retardation V f Ϫ V 0 measured at concentrations of 150, 100, 50, 25, and 10 mM M9.1.
The relative affinity of each oligosaccharide was calculated under conditions where [A] 0 is negligibly small compared with K d , using In this case, the heptasaccharide Neu5Ac␣2-3Gal␤1-3GalNAc␤1-4(Neu5Ac␣2-8Neu5Ac␣2-3)Gal␤1-4Glc-PA was used as a control sugar to determine V 0 . K a values are mean Ϯ S.D. of three independent experiments.

Characterization of Sugar-binding Specificity of VIP36 by the FAC
Analysis-In this study, we constructed a PA sugar library for the FAC analyses to determine the sugar-binding specificity of VIP36-CRD. Fig.  1 shows typical elution profiles of the PA oligosaccharides overlaid with that of the control sugar. Based on the retardation volume V f Ϫ V 0 , we could estimate relative affinities of the individual PA oligosaccharides for VIP36-CRD. The K a of VIP36-CRD for M9.1 was determined as 0.97 (Ϯ0.02) ϫ 10 4 M Ϫ1 on the basis of dependence of retardation on the ligand concentration. The obtained data were treated according to Equation 1 (Fig. 2). The value of K d was calculated from the slope, which corresponds to ϪK d . The B t value, which was obtained from the intercept on the ordinate, was 2.1 ϫ 10 Ϫ8 mol for the immobilized VIP36-CRD column. Inspection of these data allows us to calculate K a values for all other PA oligosaccharides using Equation 2 (TABLE ONE). The PA sugars with higher affinities for VIP36-CRD are all high mannosetype possessing the Man␣1-2Man␣1-2Man branch (D1 branch), i.e. M9.1, M8.1, M8.2, and M7.1. Glucosylation or trimming of the D1 arm resulted in significant reduction in affinity for VIP36-CRD. The complex-and hybrid-type oligosaccharides were shown to exhibit lower affinities, which is consistent with the previous analysis (26).
Ca 2ϩ and pH Dependence of Sugar Binding of VIP36-CRD-Next, we analyzed the Ca 2ϩ and pH dependence of the sugar-binding affinity of VIP36-CRD by the FAC method. Fig. 3 shows the elution profiles of M9.1 on the VIP36-CRD affinity column in the presence of 1 mM CaCl 2 or 5 mM EDTA. No significant retardation of elution of M9.1 was observed in the presence of EDTA, indicating that Ca 2ϩ is required for sugar binding of VIP36-CRD.
Both ERGIC-53 and VIP36 possess two Ca 2ϩ -binding sites in their CRDs. The x-ray crystallographic study of the CRD of p58/ERGIC-53 has revealed that one Ca 2ϩ -binding site consists of Asp-160, Phe-162, Asn-164, and Asp-189, and the other is composed of Asp-163, Asp-165, Asn-169, Asn-170, and Asp-189 (16). The amino acid residues, forming the former Ca 2ϩ -binding site are all conserved in VIP36-CRD. There exists one conserved histidine residue (His-178 and His-190 in ERGIC-53 and VIP36, respectively) in close proximity in this Ca 2ϩ -binding site. A neutral mutation of His-178 inactivated the lectin function of ERGIC-53 (24).
We assessed the effect of a mutation of His-190 on the sugar binding of VIP36-CRD by the FAC method. A His3 Tyr substitution at position 190 resulted in the disappearance of the retardation of the elution of M9.1, indicating that His-190 plays an essential role in the sugar binding of VIP36-CRD (data not shown).
Finally, we analyzed the pH dependence of the sugar binding of VIP36-CRD. Fig. 4 shows the K a values under varying pH conditions of VIP36-CRD for M9.1, which exhibits a bell-shaped pH dependence with a maximum near pH 6.5.

DISCUSSION
VIP36 has been reported to recognize high mannose-type oligosaccharides containing Man␣1-2Man residues (26). In this study, we attempted to estimate the contribution to VIP36 binding of the individual mannosyl branches of high mannose-type oligosaccharides by use of    the sugar library, including isomeric PA oligosaccharides. The present study exemplifies that the FAC method is extremely powerful in analyzing weak interactions in a quantitative manner. The K a values of VIP36-CRD for the PA oligosaccharides assessed in the present study were in the range 0.07-1.16 ϫ 10 4 M Ϫ1 (TABLE ONE). Such a low affinity binding is widely observed for lectin-carbohydrate interaction systems, which often adopt affinity enhancement by multivalent interaction (35). It is conceivable that VIP36 clusters on the luminal membrane and thereby achieves affinity enhancement due to multiple interactions with its cargos.

Structures of PA oligosaccharides from N-glycans used in this study and the obtained K a values for VIP36-CRD immobilized column
We have revealed that VIP36 preferentially binds the D1 branch of high mannose-type oligosaccharides. Principally, the FAC data has shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. A cycle of glucose trimming by glucosidase II and reglucosylation by UDP-glucose:glycoprotein glucosyltransferase coupled with the correct folding of some glycoproteins in the ER, which is assisted by calnexin and/or calreticulin (1,36). The D1 arm is trimmed by cis-Golgi mannosidase I, giving rise to the Man 5 GlcNAc 2 structure, which also has a lower affinity for VIP36-CRD (6,38). These data indicate that VIP36 exhibits optimal binding affinities for the glycoproteins that leave the calnexin/calreticulin cycle in the ER and yet do not undergo trimming of the D1 mannosyl branch in the cis-Golgi (Fig. 5). The present FAC data also suggest that the affinity for cargos of VIP36 is nominally affected by mannose trimming by ER mannosidases I and II, which gives rise to Man8 and Man7 structures (5).
Sugar binding of ERGIC-53 has shown to be Ca 2ϩ -dependent (24,39). Appenzeller et al. (22) have reported that the efficiency of crosslinking of ERGIC-53 and its cargo was reduced in Lec1 cells treated with castanospermine, indicating that sugar binding of ERGIC-53 is affected by untrimmed glucose residues. Taken together, our data suggest that ERGIC-53 and VIP36 share common sugar-binding specificities. However, there exist significant differences in the pH dependence of sugar binding between them. ERGIC-53 efficiently binds immobilized man-nose at pH 7.4 but not at slightly lower pH, whereas VIP36 exhibits a bell-shaped pH dependence of binding to M9.1, with optimal pH values of ϳ6.5. Similar pH dependence has been reported for the binding of VIP36 to secretory and postnuclear supernatant proteins (26).
In ERGIC-53, on the basis of the mutagenesis data, ionization of His-178, which is located in the ␣-helix participating in the Ca 2ϩ coordination, has been suggested to lead to the loss of Ca 2ϩ in the sugarbinding pocket, thereby resulting in a reduction of its lectin activity upon acidification (24). Although the data presented here suggest that ionization of His-190 of VIP36, which corresponds to His-178 of ERGIC-53, is similarly responsible for the decrease in its sugar-binding affinity to below pH 6.5, the bell-shaped pH dependence cannot be attributed to a single titratable group. There exist several titratable groups, e.g. His-129, in the proximity of the putative sugar-binding site of VIP36-CRD, which was predicted based on the crystal structure of p58-CRD (15,16). These amino acid residues are candidates for pH sensing. Further structural and mutational analyses of VIP36-CRD will help to identify the amino acid residue(s) responsible for the pH dependence of sugar binding.
There is progressive acidification from the ER through Golgi to the trans-Golgi network because of H ϩ pumping by v-ATPase (41). The typical pH values of the ER, cis-Golgi, and trans-Golgi network have been reported to be 7.2, 6.4, and 5.4, respectively (41,42). It has been reported that ERGIC-53 binds to the glycoprotein cargo in the ER and dissociates before reaching the cis-Golgi (22), which is consistent with its pH dependence of sugar binding (24). By inspection of the specificity and optimal pH of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins retaining the intact D1 mannosyl branch in the cis-Golgi network and recycles to the ER, where it releases the cargos due to higher pH. It is possible that VIP36 catches and retrieves glycoproteins that have escaped the trimming of the D1 branch, and glycoprotein carrying the correctly trimmed high mannose-type oligosaccharides or more mature hybrid-or complex-type oligosaccharides can no longer interact with VIP36 and move in the secretory pathway.
It has been shown that imperfectly folded or partially assembled proteins that exit the ER fail to escape the early secretory system entirely, because they are retrogradely transported from post-ER compartments to the ER (43,44). This retrieval mechanism is mediated at least partially by interactions between proteins possessing a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence, such as immunoglobulin heavy chain-binding protein (BiP) and the KDEL receptor (40). A cross-linking experiment has demonstrated that VIP36 interacts with BiP in HEK293 cells (37). One intriguing possibility is that VIP36 contributes to the retrograde transport of some misfolded or partially assembled glycoproteins in cooperation with BiP and the KDEL receptor, protecting the D1 branch of the cargos, which is subjected to quality control in the ER afterward, against the attack of cis-Golgi mannosidase I.
In summary, our study provides direct evidence that VIP36 binds the D1 mannosyl branch of its target glycoproteins, which is tightly associated with quality control at the ER.