Polarized epithelial cells have distinct apical and basolateral membrane domains, each of which contains specific proteins and lipid components (Rodriguez-Boulan and Powell, 1992). In polarized MDCK cells, not only proteins and phospholipids but also various glycoconjugates are sorted into transport vesicles in the trans-Golgi network (TGN), delivered to the apical or basolateral cell surface (Simons and van Meer, 1988; Simons and Wandinger-Ness, 1990; Matter and Mellman, 1994), and function in the specialized domains.
It has been recently demonstrated that lectin-like proteins such as mannose-6-phosphate receptor (Dahms et al., 1989), calnexin (Ou et al., 1993; Ware et al., 1995), and calreticulin (Nauseef et al., 1995) play important roles in intracellular vesicular transport. Accordingly, we focused on an intracellular lectin which might be related to trafficking mechanism of glycoproteins. VIP36 (vesicular-integral membrane protein of 36 kDa) was isolated as a component of glycolipid-enriched detergent-insoluble complexes containing the apical marker protein and is present in carrier vesicles (Fiedler et al., 1994). The exoplasmic/luminal domain of VIP36 is homologous to the N-terminal luminal domain of ERGIC (ER-Golgi intermediate compartment)-53 (Fiedler and Simons, 1994), which is an intermediate compartment marker (Schweizer et al., 1988) and identical to MR60, a mannose-binding protein (Arar et al., 1995). Although the biological function of VIP36 is unknown as in the case of ERGIC-53, both proteins show a significant similarity to leguminous plant lectins, suggesting that they might be involved in the delivery of saccharide-bearing molecules in the secretory pathway (Fiedler and Simons, 1994). Fiedler and Simons, (1996) previously reported that VIP36 might weakly recognize N-acetylgalactosamine (GalNAc) residue and does not interact with [3H]mannose-labeled glycopeptides. However, since we obtained the contradictional results in preliminary experiments, we reinvestigated the precise carbohydrate binding specificity of VIP36.
Figure 1. Effects of temperature on specific binding of GST/Vip36 to post-nuclear supernatant (PNS) (a), and effects of pH on specific binding to secretory proteins (b) and PNS (c) prepared from metabolically [35S]-labeled MDCK cells. [35S]-labeled PNS and secretory proteins were prepared as described in Materials and methods and incubated for 1 h at 37°C(solid circles) or 4°C (open circles) in binding buffer (pH 6.0) containingthe indicated concentration of GST/Vip36 (a) or in the indicated buffer(solid circles, MES; solid triangles, MOPS; inverted solid triangles, Tris) containing 0.8 µM GST/Vip36 (b and c). The amount of specific binding was determined by subtracting the amount obtained in the same binding assay with GST. Addition of an excess amount of monosaccharides or trypsin-digested glycoproteins to the binding assay is shown as × (0.2 M [alpha]-methylmannoside), solid stars (0.2 M GalNAc), solid squares (1 mM transferrin), or open squares (1 mM thyroglobulin).
Here we show that recombinant VIP36 expressed as a fusion protein with glutathione-S-transferase (GST), GST/Vip36, binds to secretion proteins and postnuclear supernatant (PNS) proteins in MDCK cells under the condition of pH 6.0 at 37°C. This binding is independent of Ca2+ and is specifically inhibited by several nanomoles of glycopeptides with high-mannose type sugar chains containing [alpha]1->2Man residues and [alpha]-amino substituted asparagine. The binding constant between GST/Vip36 and thyroglobulin containing high-mannose type sugar chains can be measured by means of a biosensor based on surface plasmon resonance. Our results strongly suggest that VIP36 functions as an intracellular lectin recognizing high-mannose type sugar chains in MDCK cells.
The recombinant GST/Vip36 bound [35S]-labeled proteins from MDCK cells
VIP36 is isolated from TGN-derived vesicles of MDCK cells and its luminal domain shows homology to leguminous plant lectins and ERGIC-53 (Fiedler and Simons, 1994; Fiedler et al., 1994). As the first step to identify the functional role of VIP36, the carbohydrate binding specificity of VIP36 was studied. The luminal region of VIP36, Vip36, was expressed as a fusion protein with GST in E.coli, and the GST/Vip36 was purified by means of a glutathione Sepharose 4B column. First, we investigated whether the GST/Vip36 binds [35S]-labeled secretory proteins or [35S]-labeled PNS prepared from metabolically labeled MDCK cells under the conditions described in Materials and methods. The amount of binding observed using GST itself in the presence of glutathione-coupled Sepharose 4B, which was less than 10% of the amount using GST/Vip36, was taken as background. As shown in Figure
Figure 2. Effects of divalent cations on GST/Vip36 binding. Total membrane and cytosolic proteins were prepared as described under Materials and methods. The binding assay was carried out by incubation for 1 h at 37°Cin binding buffer (pH 6.0) containing 0.8 µM GST/Vip36, 4 ×106 d.p.m. [35S]-labeled protein and 5 mM EDTA or the indicated cation at 1 mM. GST/Vip36 bound glycoproteins
Vip36 binds secretory proteins as well as membrane-bound proteins in MDCK cells as shown in Figure Binding of GST/Vip36 was inhibited by high-mannose type glycopeptides containing [alpha]1->2Man residues
The results described so far suggested that Vip36 binds some high-mannose type sugar chains of secretory glycoproteins and membrane glycoproteins. In order to determine the further precise carbohydrate binding specificity of Vip36, various high-mannose type glycoconjugates were prepared and added to the binding assays. As summarized in Table II, upon testing of a series of high-mannose type glycopeptides, only Man6-9·GlcNAc2·NAc·Asn inhibited the binding of Vip36 effectively and, in contrast, Man5·Glc-NAc2·NAc·Asn did not inhibit the binding, consistent with the effect of glycoproteins on the binding activity as shown in Figure
Figure 3. Effects of various kinds of glycoproteins on specific binding of GST/Vip36 to PNS. The binding assay was carried out by incubationfor 1 h at 37°C in binding buffer (pH 6.0) containing 0.8 µM GST/Vip36,4 × 106 d.p.m. [35S]-labeled PNS and indicated concentrations of various glycoproteins. Open circles, thyroglobulin; solid circles, VSG; open triangles, ovalbumin; solid triangles, hCG; inverted open triangles, alkaline phosphatase; inverted solid triangles, ribonuclease B; solid squares, asialo-batroxobin glycopeptides. [alpha]-Amino substituted asparagine residue is necessary for binding of GST/Vip36
Next, in order to determine the aglycon specificity, the inhibitory effects of high-mannose type sugar chains with various reducing terminal side structures were examined. As shown in Table IIand Figure
Table I. The association constant between GST/Vip36 and porcine thyroglobulin was 2.1 × 108 M-1
Using a biosensor based on surface plasmon resonance, we analyzed the association constant between GST/Vip36 and glycoproteins. Transferrin or thyroglobulin, at 500 nM in each instance, was introduced onto the sensor chip with immobilized GST/Vip36 or GST. No specific affinity was observed between transferrin and GST/Vip36, whereas a specific interaction between thyroglobulin and GST/Vip36 was evident (Figure
Figure 4. Effects of high-mannose type sugar chains with various reducing terminal side structures on specific binding of GST/Vip36 to PNS. The experimental conditions were the same as those in the Figure 3 caption, except that various kinds of high-mannose type sugar chains were added at indicated concentrations. Open circles, M9·GlcNAc2·NAc·Asn; solid circles, M9·GlcNAc2· Asn; open triangles, M9·GlcNAc·GlcNAc; solid triangles, M9·GlcNAc.
Figure 5. The carbohydrate binding specificity of GST/Vip36.
Figure 6. Sensorgrams showing the interaction between glycoproteins and GST/Vip36. Either 500 nM transferrin (A) or 500 nM thyroglobulin (B) in binding buffer was introduced at a flow rate of 30 µl/min onto the sensor surface with immobilized GST/Vip36, and the interaction was monitored at 37°C. The specific interaction between glycoproteins and GST/Vip36 was obtained by subtracting the amount of interaction between glycoproteins and GST as background.
Table II.
Figure 7. Identification of GST/Vip36 binding glycoproteins derived from MDCK cells. [35S]-labeled secretory proteins (lanes 1-5) and PNS (lanes 6-10) were prepared from MDCK cells as described in Materialsand methods. The binding assay was carried out in the presence or absence of excess amounts of thyroglobulin, with or without treatment of Endo H for 16 h at 37°C. The bound glycoproteins to GST/Vip36 (or GST) were subjected to SDS-PAGE (12%) followed by autoradiography. Lanes 1 and 6, total secretory proteins and PNS, respectively; lanes 2 and 7, GST bound proteins; lanes 3 and 8, GST/Vip36 bound proteins; lanes 4 and 9, GST/Vip36 bound proteins in the presence of thyroglobulin; lanes 5 and 10, GST/Vip36 bound proteins with treatment of Endo H for 16 h at 37°C.
This paper clearly demonstrated that VIP36 recognizes high-mannose type glycans containing two [alpha]1->2mannose residues and [alpha]-amino-substituted asparagine, and the aglycon specificity is unique as follows: VIP36 does not bind (Man[alpha]1->2)1~4-Man5·GlcNAc2·Asn, or (Man[alpha]1->2)1~4Man5·GlcNAc2 but binds (Man[alpha]1->2)1~4Man5·GlcNAc2·NAc·Asn. The binding character indicates that the positively charged primary amino residue of asparagine disturbs the lectin activity of VIP36, which requires not only carbohydrate moieties but also [alpha]-amino substituted asparagine. Such a binding character of VIP36 might allow it to discriminate between glycoproteins to be transported and luminal degradation products such as free oligosaccharides and asparagine-linked oligosaccharides. Although the importance of reducing terminal side structures in interactions with lectins has been reported for several lectins including lentil lectin (Kornfeld et al., 1981), pea lectin (Kornfeld et al., 1981), and E4-PHA (Kobata and Yamashita, 1989), the carbohydrate binding specificity of VIP36 including [alpha]-amino substituted asparagine is demonstrated for the first time in this paper.
Fiedler and Simons, (1996) previously reported that the interaction between MDCK cells and VIP36 is inhibited by high concentrations of GalNAc, and 0.1% of [3H]galactose-labeled glycopeptides in MDCK cells binds to VIP36-agarose column and eluted by 0.1 M GalNAc; by contrast, [3H]mannose-labeled glycopeptides pass through the column. However, we obtained the different results by using [35S]-labeled membrane/secretory proteins in MDCK cells for VIP36-binding assay. The binding activity between GST/Vip36 and [35S]-labeled membrane/secretory proteins in MDCK cells was not inhibited by 0.2 M GalNAc or 1 mM batroxobin glycopeptides containing [beta]-GalNAc residues on the nonreducing terminal (Figures
ERGIC-53 recycling between ER and cis-Golgi is one of the candidates possessing lectin activity which may function in relation to intracellular transport. ERGIC-53 is identical to the mannose-binding protein MR60 (Arar et al., 1995) and shows high homology to VIP36 in the regions of the carbohydrate binding sites and Ca2+ binding site; however, these two proteins are quite different from each other in terms of lectin character. ERGIC-53 recognizes a mannose in a Ca2+-dependent manner (Itin et al., 1996), while VIP36 neither recognizes monosaccharides nor requires any divalent cations for binding to high-mannose type sugar chains. Because VIP36 was shown to bind Ca2+ as reported by Fiedler and Simons, (1996), there remains the possibility that VIP36 binds other components in a Ca2+-dependent manner. This point has to be resolved by analyzing point mutants of VIP36 in the calcium binding site in subsequent studies.
In relation to the vectorial transport in the epithelial cells, Wandinger-Ness et al., (1990) isolated distinct transport vesicles mediating the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. The apical membrane has an unusually high glycosphingolipid content, leading to the hypothesis that glycolipids and apical proteins are co-sorted in the TGN into the vesicular carriers that are formed by glycolipid self-association into microdomains or "rafts" onto which apically destined proteins attach. Because VIP36 is one of the membrane proteins present in TGN-derived transport vesicles isolated from the MDCK cells and its coding cDNA sequence is highly homologous to that of legume lectin (Fiedler and Simons, 1994; Fiedler et al., 1994), the lectin activity of VIP36 might be somewhat related to the sorting machinery in TGN, although we do not have any evidence at this time.
[alpha]-Amino substituted Asn-linked high-mannose type glycans are recognized in a Ca2+-independent manner by VIP36, and VIP36 binds sugar chains most effectively at pH 6.0, which is very close to the pH condition in TGN. Such characters may be useful to resolve the functional role of VIP36. It is the subsequent interest whether VIP36 functions to sort glycoproteins having high-mannose type glycans in TGN and to transport them to the apical surface in MDCK cells. There have already been some reports concerning the roles of N-linked glycans in intracellular transport. Scheiffele et al., (1995) showed that growth hormone, which is nonglycosylated and secreted from both sides in MDCK cells, is secreted from the apical side when N-glycosylated. Gut et al., (1998) demonstrated that N-linked glycans are recognized in apical sorting of membrane proteins.
In addition to VIP36, VIP21 (Kurzchalia et al., 1992) which is identical to caveolin (Glenney, 1992), and annexin XIIIb (Fiedler et al., 1995) were identified as vesicular integral membrane proteins. The functional roles of each factor and the correlation among such factors including VIP36 remain unclear and further studies are needed. These studies might lead to elucidation of the roles of glycoconjugates in the mechanisms of sorting and vesicle formation in intracellular transport. Materials and chemicals
MDCK cells, strain II were donated by Dr. M. Tashiro (National Institute of Infectious Diseases, Tokyo, Japan) and maintained in MEM supplemented with 5% FBS in plastic dishes. Expre35S35S (37.0 TBq (1000 Ci)/mmol) and d-[2-3H(N)]-mannose (17.6 Ci/mmol) were purchased from DuPont NEN (Boston, MA), endo-[beta]-N-acetylglucosaminidase H (Endo H) from Seikagaku Kogyo Co., LTD. (Tokyo, Japan). Mannose-6-phosphate, transferrin, hCG, thyroglobulin, and ribonuclease B were purchased from Sigma Chemical Co. Human placental alkaline phosphatase was kindly provided by Dr. Y.Ikehara (Fukuoka University, School of Medicine, Japan). Batroxobin was from American Diagnostical Inc. (Greenwich, CT). VSG of Trypanosoma brucei were prepared as described previously (Fukushima et al., 1997). Man5-9·GlcNAc were obtained from Man5-9·GlcNAc2 by Endo H digestion. Man5-6·GlcNAc2 and Man7-9·GlcNAc2 were prepared by hydrazinolysis of ovalbumin (Tai et al., 1975) and porcine thyroglobulin (Ito et al., 1977), respectively. Metabolic labeling and cell fractionation
Cells at confluence in 100 mm dishes were incubated for 16 h in medium containing 1.5 mg/l l-methionine, 3.1 mg/l l-cysteine·2HCl, and 25 µCi/ml Expre35S35S. [35S]-labeled cells were washed twice in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2, and lysed by incubation on ice for 30 min in lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris/HCl, pH 7.0) containing 1 mM PMSF, 100 kallikrein U/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin (protease inhibitor cocktail). The lysate was centrifuged for 10 min at 3000 r.p.m., and the supernatant, hereafter called PNS, was used for binding assay. The total MDCK cell membrane fraction was prepared from cell homogenate according to Kurzchalia et al., (1992). Briefly, 2 ml of the homogenate adjusted to 1.5 M sucrose by adding sucrose was overlaid with 5 ml of 1.2 M sucrose followed by 3 ml of 0.8 M sucrose. Both sucrose solutions were prepared in 3 mM imidazole, pH 7.4, and 1 mM DTT. After centrifugation in RPS40T rotor at 27,000 r.p.m. for 20 h, fractions corresponding to the interface between 0.8 M and 1.2 M sucrose were pooled and used as the cellular membrane fraction. The supernatant after centrifuging cell homogenate at 100,000 × g for 30 min was used as the cytosol protein fraction. Preparation of [35S]-labeled secretory proteins
The confluent cells cultured in plastic dishes were incubated for 30 min in starvation medium lacking methionine and cysteine, and labeled for 2 h with 250 µCi/ml Expre35S35S. After removing the labeling medium, the cells were washed three times with normal culture medium and incubated for another 2 h in the culture medium. The medium was collected and applied to a PD-10 (Pharmacia Biotech) column in each instance to separate the labeled glycoproteins from free [35S]methionine. Preparation of GST/Vip36
Total RNA was extracted from MDCK cells using ISOGEN (Nippon Gene, Tokyo, Japan) and cDNA synthesis was carried out using Ready-to-Go T-Primed First-Strand Kit (Pharmacia Biotech). The target cDNA encoding the extracellular domain of VIP36 (Vip36, amino acids 76-304) was amplified by PCR performed by 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The primers used were 5[prime]-GGGGGATCCGATATAACTGACGGT-3[prime] as sense primer and 5[prime]-GGGGAATTCCCCCACTTCGGAAGTTTC-3[prime] as antisense primer. After the sequence of the amplified fragment was confirmed the identical one shown by Fiedler et al., (1994), the fragment was inserted into downstream of GST using the EcoRI and BamHI sites in pGEX-2TK (Pharmacia Biotech). Vip36 was expressed as a fusion protein, i.e., fused with GST in E.coli and GST/Vip36 was purified by affinity chromatography using glutathione Sepharose 4B according to the manufacturer[prime]s instructions. GST was expressed and purified by the same method. The purified GST/Vip36 and GST were dialyzed against 25 mM MES, pH 6.0, 150 mM NaCl, and 1 mM PMSF, and the dialysates were concentrated with an Amicon Centricon-30. Assay of binding of GST/Vip36 to [35S]-labeled proteins of MDCK cells
The binding of GST/Vip36 to [35S]-labeled proteins was assayed in a solution containing 0.8 µM GST/Vip36 or GST, 4 × 106 d.p.m. [35S]-labeled proteins derived from MDCK cells, 25 mM MES and 150 mM NaCl, pH 6.0, in a total volume of 100 µl, with incubation for 60 min at 37°C. After incubation, 50 µl of a 50% slurry of glutathione Sepharose 4B equilibrated with the same binding buffer above was added to each assay sample and mixed gently for 30 min. The Sepharose beads were then washed four times with binding buffer by centrifugation at 5000 r.p.m. for 10 s at 4°C. Scintillation fluid containing Triton X-100 was added, the samples were mixed and centrifuged. Then the radioactivity of the supernatant was determined. The radioactivity obtained in control samples with GST was taken as background. In another case, the samples were subjected to SDS-PAGE and the proteins bound to GST/Vip36 were visualized by autoradiography. Preparation of high-mannose type aspartyloligosaccharides from porcine thyroglobulin
High-mannose type oligosaccharides, Man7-9·GlcNAc2, were prepared from 3 g of porcine thyroglobulin as described in a previous paper (Ito et al., 1977). Each oligosaccharide was converted to aspartyloligosaccharides by treatment with Arthrobacter protophormiae endo-[beta]-N-acetylglucosaminidase (Endo A) (Fan et al., 1995). Aspartyl-[beta]-N-acetylglucosamine (Asn-GlcNAc) was prepared from Fmoc-conjugated Asn-GlcNAc (kindly provided by Dr. T.Inazu, Noguchi Institute, Tokyo, Japan) by treatment with 20% piperidine. Five hundred microliters of reaction mixture containing 0.1 M Asn-GlcNAc, 30% acetone, each oligosaccharide at 1 mM, and 40 mU of Endo A (kindly provided by Dr. K.Takegawa, Faculty of Agriculture, Kagawa University, Japan) in 50 mM ammonium acetate (pH 6.0) was incubated at 37°C for 15 min and boiled to stop the reaction. After Sephadex G-25 gel filtration to remove excess Asn-GlcNAc, oligosaccharide fractions were applied to a AG 50W-X2 cation exchange column (0.7 × 5.2 cm, H+ form; Bio-Rad). Unreacted oligosaccharides passed through the column, whereas aspartyl oligosaccharides bound to the column and were eluted with 50 mM CaCl2. Fractions were immediately neutralized and desalted. The yield of aspartyl oligosaccharides in the endo A reaction was 40%. Man5·GlcNAc2·Asn and Man6·GlcNAc2·Asn were prepared from ovalbumin as described in a previous paper (Tai et al., 1975). Solid phase binding assay based on surface plasmon resonance
The affinity between VIP36 and human transferrin or porcine thyroglobulin was measured using a GST/Vip36- or GST-immobilized assay system in a BIAcore 2000 instrument (BIACORE AB., Uppsala, Sweden). GST/Vip36 or GST was covalently immobilized on the sensor surface by amine coupling. The amount of GST/Vip36 immobilized was 12,759.0 RU and the amount in the case of GST was 11,838.3 RU. Either 500 nM transferrin or 500 nM thyroglobulin in binding buffer was introduced onto the surface at a flow rate of 30 µl/min. The interaction was monitored at 37°C as the change in the surface plasmon resonance response. The apparent rate constants for binding were determined by the methods of Fägerstam et al., (1992).
This work was supported in part by grants-in-aid from the Uehara Memorial Foundation, and by Grants-in-aid 10680592 and 10178104 (for Scientific Research on Priority Area) from the Ministry of Education, Science, Sports and Culture of Japan.
TGN, trans-Golgi network; Man, mannose; GlcNAc, N-acetyl-glucosamine; GalNAc, N-acetylgalactosamine; Asn, l-asparagine; PNS, post nuclear supernatant; Endo H, endo[beta]-N-acetylglucos-aminidase H; hCG, human chorionic gonadotropin; VSG, variant surface glycoproteins; GPI, glycosylphosphatidylinositol; Endo A, endo-[beta]-N-acetylglucosaminidase A; Asn-GlcNAc, aspartyl-[beta]-N-acetylglucosamine; TBS, Tris-buffered saline (25 mM Tris base, 137 mM NaCl, 2.7 mM KCl, pH 7.4); ERGIC, ER-Golgi intermediate compartment; VIP36, vesicular-integral membrane protein of 36kDa; GST, glutathione-S-transferase; NAc-Asn, N[alpha]-acetyl-l-asparagine.
Glycoprotein
N-Glycans/mol
Structure of glycan
Reference
Thyroglobulin
NDa
Bi- to tetra-antennary
Kondo et al., 1977
High-mannose type
Ito et al., 1977
VSG from Trypanosoma
5
Bi-antennary
High-mannose type
Zamze et al., 1991
GPI anchor
Ferguson et al., 1988
Alkaline phosphatase
1
Bi-antennary
Endo et al., 1988
GPI anchor
Redman et al., 1994
Ribonuclease B
1
High-mannose type
Liang et al., 1980
Ovalbumin
1
High-mannose type
Tai et al., 1975
Hybrid type
Transferrin
2
[alpha]2->6Sialylated bi-antennary
Spik et al., 1975
hCG
4
[alpha]2->3Sialylated mono- and bi-antennary mucin type
Endo et al., 1979
Asialo-batroxobin
2
[beta]1->4N-Acetyl-galactosaminylated bi-antennary
Tanaka et al., 1992
Compounds
Concentration for 50% inhibition
Man5 · GlcNAc2
-*
Man5 · GlcNAc2[beta]1->Asn
-
Man5 · GlcNAc2[beta]1->NAc· Asn
-
Man[alpha]1->2Man5 · GlcNAc2
-
Man[alpha]1->2Man5 · GlcNAc2[beta]1->Asn
-
Man[alpha]1->2Man5 · GlcNAc2[beta]1->NAc· Asn
1 × 10-7M
(Man[alpha]1->2)2Man5 · GlcNAc2
-
(Man[alpha]1->2)2Man5 · GlcNAc2[beta]1->Asn
-
(Man[alpha]1->2)2Man5 · GlcNAc2[beta]1->NAc· Asn
1 × 10-9M
(Man[alpha]1->2)3Man5 · GlcNAc2
-
(Man[alpha]1->2)3Man5 · GlcNAc2[beta]1->Asn
-
(Man[alpha]1->2)3Man5 · GlcNAc2[beta]1->NAc· Asn
1 × 10-9M
(Man[alpha]1->2)4Man5 · GlcNAc2
-
(Man[alpha]1->2)4Man5 · GlcNAc2[beta]1->Asn
-
(Man[alpha]1->2)4Man5 · GlcNAc2[beta]1->NAc· Asn
1 × 10-9M
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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