Glycobiology Advance Access originally published online on July 9, 2007
Glycobiology 2007 17(10):1061-1069; doi:10.1093/glycob/cwm074
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VIPL has sugar-binding activity specific for high-mannose-type N-glycans, and glucosylation of the 
1,2 mannotriosyl branch blocks its binding
2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience BLD 602, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
3 RIKEN, Saitama 351-0198, Japan
4 CREST, JST, Saitama 332-1102, Japan
1 To whom correspondence should be addressed; Tel: +81-4-7136-3614; Fax: +81-4-7136-3619; e-mail: yamamoto{at}k.u-tokyo.ac.jp
Received on February 9, 2007; revised on July 1, 2007; accepted on July 1, 2007
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Abstract |
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VIP36-like protein (VIPL) was identified as an endoplasmic reticulum (ER) resident protein with homology to VIP36, a cargo receptor involved in the transport of glycoproteins within cells. Although VIPL is structurally similar to VIP36, VIPL is thought not to be a lectin, because its sugar-binding activity has not been detected in several experiments. Here, recombinant soluble VIPL proteins (sVIPL) were expressed in Escherichia coli, biotinylated with biotin ligase and oligomerized with R-phycoerythrin (PE)-labeled streptavidin (SA). As measured with flow cytometry, PE-labeled sVIPL–SA bound to deoxymannojirimycin (DMJ)- or kifunensine (KIF)- but not to swainsonine (SW)-treated HeLaS3 cells in the presence of calcium. A surface plasmon resonance analysis showed that the avidity of sVIPL was enhanced after it formed a complex with SA. The binding of PE-labeled sVIPL–SA was abrogated by endo ß-N-acetylglucosaminidase H treatment of the DMJ- or KIF-treated cells. Competition with several high-mannose-type N-glycans inhibited VIPL binding, and indicated that VIPL recognizes the Man
1–2Man1–2Man sequence. Glucosylation of the outer mannose residue of this portion decreased the binding. Although the biochemical characteristics of VIPL are similar to those of VIP36, the sugar-binding activity of VIPL was stronger at neutral pH, corresponding to the pH in the lumen of the ER, than under acidic conditions. Key words: high mannose type / lectin / N-glycan / VIPL
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Introduction |
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In the endoplasmic reticulum (ER), nascent proteins are cotranslationally folded and modified with N-glycans on the Asn residues of the glycosylation consensus motif (Asn-X-Ser/Thr) (Nilsson and von Heijne 1993
). Calnexin and calreticulin, in combination with the associated thiol oxidoreductase ERp57, recognize the glucose residue of monoglucosylated high-mannose-type N-glycans and facilitate protein folding and the formation of disulfide bridges (Schrag et al. 2003; Trombetta 2003; Helenius and Aebi 2004). Glucosylated glycoproteins are deglucosylated by glucosidase II, but unfolded or misfolded glycoproteins are distinguished from properly folded glycoproteins and reglucosylated by uridine diphosphate (UDP)-glucose: glycoprotein glucosyltransferase (UGGT). Together, these processes, known as the calnexin/calreticulin cycle, guarantee the correct folding of glycoproteins (Schrag et al. 2003; Trombetta 2003; Helenius and Aebi 2004).
Correctly folded glycoproteins are then packaged into transport vesicles with the aid of cargo receptors and transported from the endoplasmic reticulum (ER) to the Golgi apparatus via the ER–Golgi intermediate compartment (ERGIC) (Ponnambalam and Banting 1996; Schrag et al. 2003; Trombetta 2003; Helenius and Aebi 2004). This transport is facilitated by intracellular lectins acting as cargo receptors. ERGIC-53, which localizes in the ER, the ERGIC, and the Golgi area, is a type I transmembrane protein whose luminal part is homologous to the leguminous lectins of plants (L-type lectin). It recognizes high-mannose-type sugar moieties on glycoproteins as a transport signal and functions in the secretion of glycoproteins including cathepsin C, cathepsin Z, and blood coagulation factors V and VIII by circulating between the ER and the Golgi apparatus (Klumperman et al. 1998; Vollenweider et al. 1998; Appenzeller et al. 1999; Moussalli et al. 1999; Zhang et al. 2005). VIP36 also has homology to leguminous lectins, recognizes high-mannose-type sugar chains, and transports glycoproteins between the Golgi and the ER or the cell surface (Fiedler et al. 1994; Fiedler and Simons 1996; Hara-Kuge et al. 1999; Schrag et al. 2003; Hara-Kuge et al. 2004). VIPL (VIP36-like protein), a novel member of this family, was identified in database screenings as a protein with 43% similarity to ERGIC-53 and 68% similarity to VIP36 (Neve et al. 2003; Nufer et al. 2003). Its orthologues are broadly distributed in many eukaryotes from human to fission yeast, whereas VIP36 is restricted to higher organisms. This phylogenetic finding suggests that VIP36 evolved from VIPL by gene duplication (Nufer et al. 2003). VIPL is also predicted to have type I transmembrane topology with a putative carbohydrate-recognition domain homologous to L-type lectins (Neve et al. 2003; Nufer et al. 2003). Although the structural similarity between VIP36 and VIPL is very high, their characteristics are quite different. VIP36 and VIPL have the same cytoplasmic ER exit motif (KRFY) on their C-termini, but VIPL has an additional arginine at the fifth position from the C-terminus, creating the ER localization motif RKR. As a result, VIPL is a resident of the ER (Neve et al. 2003; Nufer et al. 2003), unlike ERGIC-53 and VIP36, which cycles in the secretory pathway. The sugar-binding activity of VIPL is also thought to be different from those of ERGIC-53 and VIP36; for example, HA-tagged VIPL does not bind to immobilized Man or fluorescein isothiocyanate–bovine serum albumin (BSA) conjugated to Glc, Man, or GlcNAc as ERGIC-53 does (Nufer et al. 2003). Moreover, VIPL does not compete with ERGIC-53 for binding to immobilized Man (Nufer et al. 2003). Based on these observations, VIPL is thought to be an L-type lectin without sugar-binding activity. On the other hand, another group reported that knockdown of VIPL mRNA using short interfering RNA in HeLa cells significantly slowed down the secretion of two glycoproteins (35 and 250 kDa) into the medium (Neve et al. 2003), suggesting that VIPL does function in the secretion of glycoproteins, possibly by serving as an ER export receptor, similarly to ERGIC-53.
To clarify the characteristics of VIPL and understand its function, we analyzed the sugar-binding activity and specificity of recombinant soluble VIPL (sVIPL) using cells with modified cell surface carbohydrates as ligands. The inhibition of cell surface binding of sVIPL–SA by chemically synthesized glycans demonstrated that VIPL preferentially binds to the Man1–2Man1–2Man sequence on the A arm of high-mannose-type N-glycans.
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Results |
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Preparation of soluble VIPL–SA complexes
We prepared a cDNA encoding a recombinant sVIPL corresponding to the luminal part of VIPL, which includes a putative lectin domain, and expressed it in Escherichia coli. The sVIPL had a C-terminal enzymatic biotinylation signal and was biotinylated with the biotin ligase BirA to allow oligomerization with SA, which possesses four binding sites for biotin. A gel-shift assay with an excess amount of SA indicated that almost all of the sVIPL was biotinylated (data not shown). For flow cytometric analysis, we mixed sVIPL and SA-PE at a ratio of 4:1, allowed the mixture to stand for 1 h at 4°C, and used them as probes without purification. To identify the oligomeric forms of the sVIPL–SA complex mixed at ratios of 4:1 and 10:1 that were present in the probes, we performed gel-filtration chromatography. The mixture of sVIPL and SA at a 10:1 molar ratio was eluted from a Superdex-200 HR column in two peaks corresponding to molecular weights of 188 and 32 kDa, respectively (Figure 1A). Based on the relative molecular weight of the peaks, these are thought to be sVIPL tetramers complexed with SA and the corresponding sVIPL monomers, respectively. The elution profile indicated that the complex of sVIPL and SA mixed at a ratio of 4:1 contained sVIPL tetramers complexed with SA, trimers complexed with SA, and sVIPL monomers at a ratio of approximately 4:5:2 (Figure 1B). Incubation time of sVIPL and SA mixture was prolonged to 2 or 4 h, and then, complex formation of sVIPL tetramer by gel filtration was analyzed under the same condition. Elution profiles of 2-h and 4-h incubation samples showed the same elution profiles as shown in Figure 1B (data not shown), indicating that some other factors (steric hindrance, etc.) might interfere with complete tetramer formation.
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Binding of PE-labeled sVIPL–SA complex to HeLaS3 cells treated with DMJ, KIF, or SW
We prepared membrane-based high-mannose-type glycans by the treating HeLaS3 cells with the
-mannosidase inhibitors, deoxymannojirimycin (DMJ), kifunensine (KIF), and swainsonine (SW). DMJ inhibits ER -mannosidase I and II, and causes accumulation of Man9GlcNAc2 (Elbein 1991). KIF strongly inhibits ER -mannosidase I but not ER -mannosidase II, and causes accumulation of the Man9GlcNAc2 and Man8GlcNAc2 isomer C (Elbein 1991; Weng and Spiro 1993). SW, which inhibits Golgi -mannosidase II, causes hybrid-type glycans to be expressed on the cell surface (Tulsiani and Touster 1983). When we treated HeLaS3 cells with 1 mM DMJ or 2 µg/mL KIF, the binding of GNA, which is specific for high-mannose- or hybrid-type N-glycans, increased about sixfold, and the binding of DSA, which is specific for complex-type N-glycans, decreased by half. The effect of treating with DMJ or KIF was almost the same. When we treated HeLaS3 cells with 2 µg/mL SW, the binding of GNA increased about 4.5-fold, and the binding of DSA mildly decreased. The effect of SW was slightly different from that of DMJ or KIF (Figure 2A). These results indicate that the treatment with DMJ, KIF, and SW worked as expected. Carbohydrate structures on the surface of intact HeLaS3 cells and those of HeLaS3 cells after treatment of DMJ, KIF, and SW has already been analyzed by pyridylamino (PA) derivatization, two-dimensional high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization–time-of-flight–mass spectrometry (MALDI-TOF-MS), as described previously (Kawasaki et al. 2007). Of all the sugar chains expressed in intact HeLaS3 cells, 36.2% are complex-type N-glycans, 42.2% of high-mannose-type N-glycans, and the remaining other types of sugar chains are present on the cell surfaces. Following treatment of HeLaS3 cells with 1 mM DMJ for 24 h, complex-type glycans decreased from 36.2% to 2.7% whereas the percentage of high-mannose-type glycans, particularly Man7–9GlcNAc2, increased from 30.7% to 69.6%. The same result was obtained after HeLaS3 cells were treated with 2 µg/mL KIF for 24 h.
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Next we measured the binding of PE-labeled sVIPL–SA complexes to DMJ-, KIF-, and SW-treated HeLaS3 cells. Although sVIPL–SA complexes did not bind to HeLaS3 cells, they bound strongly to DMJ- and KIF-treated cells; mean fluorescence intensity (MFI) values were two orders of magnitude higher (Figure 2A). In contrast, the binding of sVIPL–SA complexes to HeLaS3 cells did not change after SW treatment. Because SW enhanced the expression of hybrid-type but not high-mannose-type glycans, sVIPL may specifically bind to high-mannose-type glycans on cell surfaces.
Surface plasmon resonance (SPR) analysis of the binding of sVIPL–SA complexes to immobilized glycoproteins
We confirmed the sugar-binding activity of sVIPL by surface plasmon resonance (SPR) using BIAcore. Porcine thyroglobulin (PTG), bovine ribonuclease B (RNase B), and chicken immunoglobulin Y (IgY) were immobilized on sensor chips, and the binding of sVIPL–SA complexes to these immobilized glycoproteins was measured. sVIPL–SA complexes at 100 µg/mL bound to PTG but did not bind to either RNase B or IgY (Figure 2B). Because PTG has several kinds of high-mannose-type glycans (Tsuji et al. 1981), these observations are in good agreement with the previous data obtained with flow cytometry. The binding of sVIPL–SA complexes to PTG was in a dose-dependent manner (Figure 2C). We calculated the Ka value of sVIPL–SA complex binding to PTG to be 1.5 x 106 M–1. The affinity of the binding of sVIPL monomer to glycoproteins was too weak to be demonstrated and sVIPL did not bind to PTG even at 100 µg/mL (Figure 2D), making it difficult to determine the biological relevance of the carbohydrate-binding activity of VIPL. Oligomerization of sVIPL increases its avidity, making it possible to monitor the binding of sVIPL to ligands.
Sugar-binding specificity of sVIPL–SA complexes
To verify that the binding of sVIPL–SA complexes to DMJ-treated cells depends on high-mannose-type glycans, we examined the binding of PE-labeled sVIPL–SA complexes to DMJ-treated cells after a 2-h incubation with endo H at 37°C. The endo H treatment, which removes high-mannose-type glycans, was effective because it decreased the binding of GNA, which is specific for high-mannose-type glycans, but did not change the binding of DSA, which is specific for complex-type sugar chains (Figure 3A). The endo H treatment of DMJ-treated HeLaS3 cells almost completely abolished the binding of PE-labeled sVIPL–SA complexes to the cells (Figure 3A), indicating that sVIPL binds to high-mannose-type glycans on DMJ-treated cells.
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To clarify the precise sugar-binding specificity of sVIPL, we next performed an inhibition assay using a series of synthesized oligosaccharides (Figure 3B), as previously described (Matsuo and Ito 2003
; Matsuo et al. 2003); Man9GlcNAc2 (M9), Man8GlcNAc2 isomer B (M8B), Man8GlcNAc2 isomer C (M8C), Man7GlcNAc2 (M7), Glc1Man9GlcNAc2 (G1M9), Glc1Man8GlcNAc2 isomer B (G1M8B), Glc1Man8GlcNAc2 isomer C (G1M8C), and Glc1Man7GlcNAc2 (G1M7). sVIPL–SA complexes were preincubated with each oligosaccharide for 1 h, and their bindings to the DMJ-treated HeLaS3 cells were measured in the presence of each oligosaccharide. M9, M8B, M8C, and M7 at the concentration of 5 mg/mL blocked the binding of sVIPL–SA complexes to the cells by approximately 60–80%, and at 0.5 mg/mL, it blocked the binding by 40% (Figure 3C). G1M9 and G1M8B partially inhibited the binding only at 5 mg/mL (Figure 3C), and G1M8C and G1M7 did not block the binding of PE-labeled sVIPL–SA complexes to the cells even at 5 mg/mL (Figure 3C). High-mannose-type oligosaccharides M9, M8B, M8C, and M7 inhibited the binding of PE-labeled sVIPL–SA complexes to DMJ-treated cells more efficiently than monoglucosylated oligosaccharides G1M9, G1M8B, G1M8C, and G1M7, respectively, indicating that the Man1–2Man1–2Man arm (the A arm in Figure 3B, M9) may be recognized by sVIPL. Glucosylation of nonreducing terminal mannose decreases the binding of sVIPL to sugar chains. Mannose trimming of the B and/or C arms of M9 did not affect the inhibition, demonstrating that sVIPL primarily recognizes the Man1–2Man1–2Man sequence of A arm.
Soluble VIPL binds to sugars via a putative sugar-binding site
VIPL shares significant similarity with leguminous lectins, and the conserved aspartic acid corresponding to Asp128 of VIPL is essential for the sugar-binding activity of other leguminous lectins (Figure 4A). When this conserved aspartic acid was substituted with asparagine, sugar-binding activities of other cargo receptors were abrogated as described previously (Kawasaki et al. 2007). To test whether Asp128 is essential for VIPL binding, we mutated sVIPL Asp128 to Asn to form sVIPLD128N and then assayed the binding of PE-labeled sVIPLD128N–SA complexes to DMJ- or KIF-treated HeLaS3 cells. The PE-labeled sVIPLD128N–SA complexes did not bind to the cells at any concentration examined (from 0.01 to 10 µg/mL), although PE-labeled sVIPL–SA complexes bound to the cells in a dose-dependent manner (Figure 4B, C). The substitution of Asp128 to Asn also prevented the binding of SA complexes to immobilized PTG at 100 µg/mL in an SPR analysis (Figure 4D). These findings suggest that the binding of sVIPL to sugars is mediated by a putative sugar-binding site conserved among leguminous lectins.
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Characterization of sVIPL–SA complex binding
The binding analyses of PE-labeled sVIPL–SA complexes to HeLaS3 cells described above were performed in the presence of 1 mM calcium chloride. Because leguminous lectins, which are homologous to VIPL, have metal-binding sites and require calcium and manganese ions to bind to sugars, we examined the effects of calcium and manganese ion on the binding of sVIPL–SA complexes. To test whether calcium ions are required for the sugar-binding activity of sVIPL, we performed a binding assay in the presence of 5 mM EDTA instead of 1 mM CaCl2 in every step. The presence of EDTA completely prevented the binding of PE-labeled sVIPL–SA complexes to KIF-treated HeLaS3 cells (Figure 5A). To test whether manganese ions could substitute for calcium ions, the binding of PE-labeled sVIPL–SA complexes was performed in the presence of both 1 mM manganese chloride and 5 mM EGTA, which is a Ca2+-selective chelator. PE-labeled sVIPL–SA complexes did not bind to the cells even in the presence of manganese ions (Figure 5A), indicating that Ca2+, but not Mn2+, is required for the sugar-binding activity of VIPL.
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We also examined the pH dependency of sVIPL–SA complex binding in the presence of 0.5 mM calcium. The binding of PE-labeled sVIPL–SA complexes at neutral pH was higher than in acidic conditions (Figure 5B). This is consistent with the localization of VIPL in the ER (Neve et al. 2003
; Nufer et al. 2003), where the pH value in the ER is around 7.4 (Wu et al. 2000, 2001). |
Discussion |
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The novel VIPL was identified based on consensus profiles of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 (Neve et al. 2003
; Nufer et al. 2003). VIPL is a type I membrane glycoprotein with the domain organization very similar to that of VIP36, consisting (from the N-terminus) of an L-type lectin domain, stalk domain, transmembrane domain, and short cytoplasmic domain. Nufer et al. reported that HA-tagged VIPL failed to bind to immobilized mannose monosaccharide, or fluorescein-isothiocyanate-labeled BSA conjugated to Man, Glc, or GlcNAc under the conditions that had been used to determine the mannose binding of ERGIC-53 (Nufer et al. 2003). In contrast, we were able to demonstrate that VIPL has sugar-binding activity by using sVIPL–SA complexes as a probe, possibly because we used only the luminal part of the molecule containing the L-type lectin and stalk domain. It has suggested that the stalk domain in VIP36 may function to prevent steric hindrance between the lectin domain and the other domain (Kawasaki et al. 2007). Our binding experiment using SPR showed that the sVIPL–SA complex has the affinity for PTG, with an association constant (Ka) of about 1.5 x 106 M–1. In the case of leguminous Griffonia simplicifolia isolectins, the Ka values of monovalent, divalnent, trivalent, and tetravalent isolectins for human type A erythrocytes are 7.5 x 105, 2.9 x 106, 1.4 x 107, and 1.2 x 107 M–1, respectively (Knibbs et al. 1998). Furthermore, plant lectins bind to cells with an affinity several orders of magnitude higher than to the haptenic sugars. Similarly, the oligomerization of sVIPL greatly enhanced its avidity to ligands and enabled us to detect its binding to sugars.
The sugar-binding activity of VIPL was calcium-dependent; 0.1 mM calcium was enough to detect the sugar-binding activity (data not shown), and the binding of the sVIPL–SA complexes to DMJ-treated HeLaS3 cells was completely inhibited in the presence of ethylenediamine tetraacetic acid (EDTA). The binding of sVIPL–SA complexes was stronger at pH 7.5–8.0 than at acidic pH. This contrasts with the optimum pH of 6.0–6.5 reported for the sugar binding of VIP36, which localizes in the cis-Golgi (Kamiya et al. 2005). Because the pH in the ER is almost neutral, and calcium concentration in the ER ranges from approximately 0.3 to 0.5 mM (Alvarez and Montero 2002; Demaurex and Frieden 2003), it is likely that VIPL can bind to sugars in the ER.
VIPL bound to PTG, but did not bind to either bovine RNase B or chicken IgY. Bovine RNase B has a high-mannose-type sugar chain, and more than 70% of oligosaccharides are smaller molecular weight ones consisting of five mannose residues (Liang et al. 1980). Chicken IgY contains 27% of glucosylated high-mannose-type oligosaccharides, 10% of high-mannose-type oligosaccharides, and 63% of complex-type oligosaccharides (Suzuki et al. 2004). The sugar chains that strongly bound to VIPL (M9, M8B, M8C, and M7 in Figure 3C) are not present on both proteins, and further, the carbohydrate content of both proteins is less than 10%. These findings support the observation that RNase B and IgY did not bind to sVIPL–SA complexes. In the inhibition assay, VIPL specifically bound to high-mannose-type N-glycans and not to monoglucosylated N-glycans. High-mannose-type N-glycans, especially M9, M8B, M8C, and M7, strongly inhibited the binding of sVIPL to DMJ-treated cells, whereas monoglucosylated high-mannose-type glycans did not. Monoglucosylated M9 and M8B (G1M9 and G1M8B) partially inhibited the binding of sVIPL only at a higher concentration of 5 mg/mL; monoglucosylated M8C and M7 (G1M8c and G1M7) did not inhibit even at the concentration of 5 mg/mL. This result suggests that VIPL preferentially binds to the Man1–2Man1–2Man sequence on the A arm of high-mannose-type N-glycans, but glucosylation at the nonreducing terminal of this sequence abrogates the binding. This pattern of sugar-binding specificity is opposite to that of calnexin and calreticulin, which chaperone the folding of newly synthesized glycoproteins and bind to monoglucosylated high-mannnose-type N-glycans but not deglucosylated ones (Ware et al. 1995; Vassilakos et al. 1998). Because of this difference in the sugar-binding specificity of VIPL and calnexin or calreticulin, we propose that newly synthesized glycoproteins released from calnexin or calreticulin are then trapped by VIPL in the ER. To show a more quantitative data, we carried out SPR analysis. First, high-mannose-type oligosaccharides with propyl groups could not be immobilized efficiently to hydrophobic surface of sensor chip L1 to detect their interactions with sVIPL–SA. Next we carried out the immobilization of methotrexate (MTX)-derivatized high-mannose-type oligosaccharides because they could bind to dihydrofolate reductase (DHFR) with high affinity. Although MTX-derivatized oligosaccharides were successfully captured on DHFR-immobilized sensor chip CM5 at a concentration of 
45 pg/mm2, binding of sVIPL–SA on the surface could not be detected (data not shown). Density of immobilized oligosaccharides on the sensor chip might be lower to detect their interaction with sVIPL–SA.
What is the function of VIPL? Unlike VIP36 and ERGIC-53, which are predominantly associated with post-ER membrane and cycles in the early secretory pathway, HA-tagged VIPL expressed in HepG2 cells is a noncycling resident protein of the ER. The localization of VIPL in the ER requires the arginine-lysine-arginine (RKR) motif in the cytoplasmic tail, and the substitution of the RKR motif by serine-serine-serine (SSS) changes the distribution of VIPL at both the cell surface and intracellular membranes (Nufer et al. 2003). VIPL(N163D)-HA mutant, which lacks sugar-binding activity, was also localized to the ER in transfected COS cells (Nufer et al. 2003). Interestingly, overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL receptor or the overall morphology of the early secretory pathway (Nufer et al. 2003). Overexpression of VIPL(N163D)-HA also caused the redistribution of ERGIC-53 in the ER; this redistribution does not involve the lectin activity of ERGIC-53 since the lectin-impaired N156A mutant of ERGIC-53 was also redistributed (Nufer et al. 2003). Such observations suggest that VIPL directly interacts with the transport receptor ERGIC-53 without bound ligands, and further, VIPL might transfer the newly synthesized glycoproteins from calnexin (or calreticulin) to ERGIC-53 in the ER during the secretory pathway. This may explain the previous observation that knockdown of VIPL mRNA significantly slows the secretion of glycoproteins to the medium as reported (Neve et al. 2003).
It is proposed that the biological function of VIPL may be evolutionally conserved in eukaryotes, because cDNAs encoding previously identified L-type lectins of Drosophila, and Caenorhabditis elegans are orthologues of VIPL rather than of VIP36, and that VIPL appeared earlier in evolution than did VIP36 (Nufer et al. 2003). Further, calnexin, calreticulin, EDEM (ER-degradation enhancing mannosidase-like proteins), and UGGT, which participate in the quality control of glycoproteins in the ER, are conserved in yeast, Drosophila, C. elegans, and vertebrates. In contrast, the machinery for transport and sorting, especially ERGIC-53 and VIP36, differ among species. Orthologues of VIP36 are present in vertebrates, and the orthologue of homodimeric ERGIC-53 in yeast is a heterooligomer of Emp46p and Emp47p (Sato and Nakano 2002). These findings may indicate that evolutionally conserved VIPL plays a part in the quality control of glycoproteins rather than in the transport or sorting of such ligands.
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Materials and methods |
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Preparation of wild-type and mutant soluble VIPL (sVIPL) proteins
A full-length cDNA coding human VIPL (accession no. NM_030805) was amplified from cDNA of human lymphoma U937 cells by PCR using the primers 5'-GGGAAAGATGGCGGCGAC-3' and 5'-GGAGGGCTCAG TAGAAGCG-3'. The cDNA encoding the soluble luminal part of VIPL (sVIPL; corresponding to amino acids 49–311) was further amplified by PCR using the primers 5'-TTTTTTTTCATATGGAGTACTTG-3' and 5'-AACCCGGGGCCACTCAGG-3', which contain restriction sites for Nde I and Sma I at the 5' and 3' ends, respectively. The amplified DNA was inserted between the Nde I and Sma I sites of the pET3c-Cbio expression vector to fuse the recombinant protein to an enzymatic biotinylation tag at the C-terminus (Wada et al. 2004
). The protein was then expressed from E. coli. To generate the sVIPL mutant VIPLD128N, Asp128 was replaced with Asn by PCR-based mutagenesis using a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) and mutated primers, 5'-GAATCTGCATGGGAATGGCTTGGCAATCTGG-3' and 5'-CCAGATTGCCAAGCCATTCCCATGCAGATTC-3', according to the manufacturer's protocol. The recombinant sVIPL and sVIPLD128N were expressed in the BL21(DE3)pLysS strain of E. coli (Novagen, Madison, WI) in the presence of 1 mM isopropyl ß-thiogalactopyranoside and recovered as inclusion bodies. The inclusion bodies were solubilized in solubilization buffer (50 mM Tris-HCl, pH 8.0, containing 6 M guanidine, 1 mM DTT, and 0.1 mM EDTA), diluted with refolding buffer (100 mM Tris-HCl, pH 8.0, containing 0.4 M arginine, 1 mM CaCl2, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, and 0.5 mM phenylmethanesulphonylfluoride [PMSF]) to a protein concentration of 6 µM and refolded in vitro by dialysis against 20 mM Tris-HCl, pH 8.0, containing 25 mM NaCl and 1 mM CaCl2 at 4°C for 24 h. The dialyzed fraction was first applied to a UNO Q-6 column (12 mm x 53 mm; Bio-Rad, Hercules, CA) equilibrated with 20 mM Tris-HCl, pH 8.0, containing 25 mM NaCl and 1 mM CaCl2. After washes with the same buffer, the column was eluted with 18 mL of a linear gradient of NaCl from 25 to 500 mM in the same buffer. Elution was performed at a flow rate of 1 mL/min and fractions of 1.0 mL were collected. The fractions containing correctly folded sVIPL were monitored by enzyme-linked immunosorbent assay using anti-human VIPL monoclonal antibodies (VL1 and VL2, manuscript in preparation), and the purity of each fraction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). To further purify sVIPL, the fractions corresponding to correctly folded sVIPL were collected, applied to a column of Superdex-75 10/300 GL (10 x 300 mm; GE Healthcare Bio-Sciences Corp., Piscataway, NJ), and eluted with 20 mM Tris-HCl, pH 8.0, containing 30 mM NaCl and 1 mM CaCl2 at a flow rate of 0.5 mL/min at 25°C. Chromatography was performed using an AKTAexplorer HPLC system with UNICORN control software (GE Healthcare Bio-Sciences). Soluble VIPLD128N was purified by the same procedure. The purified sVIPL and sVIPLD128N proteins fused to a C-terminal biotinylation tag were biotinylated with the biotin ligase BirA (Avidity, Denver, CO) according to the manufacturer's protocol. After the remaining free biotin was removed by gel filtration using a Superdex-75 10/300 GL column and an elution buffer of 20 mM HEPES-NaOH, pH 7.4, containing 150 mM NaCl and 1 mM CaCl2, biotinylation was confirmed with a gel-shift assay in polyacrylamide gels as follows. One microgram of sVIPL was mixed with SA at a molar ratio of 1:2 at 4°C for 1 h in 10 µL of 10 mM sodium phosphate, pH 7.4, containing 137 mM NaCl and 2.68 mM KCl [PBS(–)]. The sample was added to 2 µL of 6x solubilizing buffer (375 mM Tris-HCl, pH 6.8, containing 12% SDS, 0.6% bromophenol blue, and 60% glycerol) and electrophoresed in 12.5% polyacrylamide gels under nonreducing conditions without boiling. Complexes of sVIPL and SA were analyzed by gel-filtration chromatography on a column of Superdex-200 HR 10/30 (10 x 300 mm; GE Healthcare Bio-Sciences) equilibrated with PBS(–); elution was performed with the same buffer.
Binding analysis of PE-labeled sVIPL–SA and sVIPLD128N–SA using flow cytometry
To prepare R-phycoerythrin (PE)-labeled sVIPL–SA complexes or sVIPLD128N–SA complexes, the biotinylated sVIPL or sVIPLD128N was mixed with SA conjugated to PE (SA-PE; BD Biosciences Pharmingen, San Jose, CA) at a molar ratio of 4:1 for 1 h on ice. To prepare the PE-labeled lectins, each biotinylated plant lectin, DSA (Seikagaku Kogyo, Tokyo, Japan) or GNA (EY laboratories, San Mateo, CA), was mixed with SA-PE at a molar ratio of 10:1 for 1 h on ice. The human cervical adenocarcinoma cell line HeLaS3 (Cell Resource Center for Biomedical Research, Tohoku University, Miyagi, Japan) was maintained in RPMI1640 (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (Intergen, New York), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-mercaptoethanol. Twenty microliters of HeLaS3 cells (2.0 x 105) were stained with 3 µg/mL sVIPL–SA in HEPES-buffered saline (HBS: 42 mM HEPES–NaOH, pH 7.4, containing 274 mM NaCl, 1 mM KCl, 1 mM CaCl2, and 0.2% glucose) containing 0.1% NaN3 and 0.1% BSA for 30 min at 25°C. After the cells were washed once with HBS, the fluorescence intensity was measured with a FACSCalibur Flow Cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA). The fluorescence at 575 nm corresponding to bound PE on the cell surface was recorded and converted into an MFI. In total, 104 live cells gated by forward and side scattering and exclusion of PI were acquired for analysis. To examine the binding of sVIPL–SA to the cells in the absence of calcium, 5 mM EDTA was added instead of 1 mM CaCl2 in every step. To examine whether Mn2+ is required for the sugar-binding activity of VIPL, the binding of sVIPL–SA was monitored in the presence of 1 mM MnCl2 and 5 mM EGTA to compete out the intrinsic Ca2+ ions in the protein aliquots. To test binding under several pH conditions, we used 20 mM MES–NaOH (at pH 5.5, 6.0, and 6.5) or 20 mM HEPES-NaOH (at pH 7.0, 7.5, and 8.0) containing 150 mM NaCl, 1 mM KCl, 0.5 mM CaCl2, and 0.2% glucose instead of HBS in every step.
Deoxymannojirimycin, kifunensine, swainsonine, and endo H treatment on HeLaS3 cells
For the modification of cell surface glycans, HeLaS3 cells were cultured in the presence of 1 mM DMJ (Sigma), 2 µg/mL KIF (Calbiochem, Darmstadt, Germany), or 2 µg/mL SW (Calbiochem) for 20 h. For the digestion of cell surface high-mannose-type N-glycans, the DMJ- or KIF-treated cells were harvested and then digested with 1.0 x 104 U/mL of endo H (New England Biolabs, Berverly, MA) in HBS containing 0.1% NaN3 and 0.1% BSA for 2 h at 37°C with gentle rotation.
Inhibition of sVIPL–SA binding by high-mannose-type glycans
The high-mannose-type oligosaccharides (propyl derivatives) were synthesized as previously described (Matsuo and Ito 2003; Matsuo et al. 2003). Ten microliters of the propyl derivative of each high-mannose-type oligosaccharide (10 mg/mL or 1 mg/mL) was mixed with 10 µL of PE-labeled sVIPL–SA (3 µg/mL) at 25°C for 1 h. Each mixture was then added to 10 µL of DMJ-treated HeLaS3 cells (2.0 x 105 cells) and incubated for 30 min at 4°C. After washing the cells with HBS containing 0.1% NaN3 and 0.1% BSA, we measured fluorescence intensity by flow cytometry as described above.
SPR analysis of the binding of sVIPL–SA complexes to immobilized glycoproteins
SPR experiments were performed with the BIAcore 3000 system (Biacore International AB, Uppsala, Sweden). PTG (purified as described previously; Tsuji et al. 1981; Yamamoto et al. 1981), bovine RNase B (Wako, Osaka, Japan), or chicken IgY (Sigma) were immobilized on the surface of sensor chips using an amine coupling kit according to the manufacturer's protocol until the resonance units (RUs) reached at approximately 7000, 2000, and 7000, respectively. In the coupling reactions, 1 RU corresponds to an immobilized protein concentration of 1 pg/mm2. Free amino groups were blocked with ethanolamine chloride. The binding of sVIPL monomers and sVIPL–SA complexes to these glycoproteins was measured in 20 mM HEPES–NaOH, pH 7.4, containing 150 mM NaCl and 1 mM CaCl2 at 25°C with a flow rate of 10 µL/min. The sVIPL monomers or sVIPL–SA complexes were dialyzed against the same buffer used in the measurements and then applied to the sensor chip. The regeneration of the chip surface was carried out with 20 mM HEPES–NaOH, pH 7.4, containing 150 mM NaCl and 10 mM EDTA. To calculate the association constant (Ka) of sVIPL–SA to PTG, sVIPL–SA at concentrations of 0, 20, 40, 60, 80, and 100 µg/mL was applied to the sensor chip. The Ka value for the binding of the sVIPL–SA complexes to PTG was obtained from the SPR binding data and calculated using biospecific interaction analysis (BIA) evaluation software (Biacore International AB) fitted with a 1:1 binding model and a molecular mass for the sVIPL–SA complex of 145 kDa.
An alignment of carbohydrate-recognition domain of leguminous lectins and L-type lectins
The following sequences of leguminous lectins and L-type lectins were obtained from databases: soybean agglutinin (SBA; P05046
[GenBank]
), peanut agglutinin (PNA; P02872
[GenBank]
), Lotus tetragonolobus agglutinin (LTA; P19664
[GenBank]
), Pisum sativum lectin (PSL; CAA47011
[GenBank]
), VIPL (NP_110432
[GenBank]
), VIP36 (NP_006807
[GenBank]
), and ERGIC-53 (NP_005561
[GenBank]
). These sequences were aligned by using T-COFFEE program (www.ch.embnet.org/software/TCoffee.html), shaded with BOXSHADE program (www.ch.embnet.org/software/BOX_form.html), and edited with Adobe Illustrator CS.
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency; Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16390019).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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