The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state

Violaine Carrière, Véronique Piller, Alain Legrand, Michel Monsigny and Annie-Claude Roche1

Glycobiologie, Centre de Biophysique Moléculaire, CNRS, Rue Charles Sadron, F-45071 Orléans Cedex 02, France

Received on December 1, 1998; revised on February 26, 1999; accepted on March 6, 1999

MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus-intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C-terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.

Key words: ERGIC-53/ intracellular lectin/oligomerization/mannose-binding protein

Introduction

The mannose-specific lectin, MR60, was first isolated on a mannose-substituted column from membranes of HL60 cells (Pimpaneau et al., 1991). This lectin is a type I transmembrane protein which includes a luminal N-terminal domain, a transmembrane domain, and a short C-terminal cytosolic domain. The sequence of MR60 (Arar et al., 1995) is identical (except for one amino acid) to that of ERGIC-53 (Schindler et al., 1993), a protein of the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC) which has been shown to shuttle between the endoplasmic reticulum (ER), the intermediate compartment, and the cis-Golgi apparatus. The sequence of MR60/ERGIC-53 from human cells and that of the rat homologous p58 reveals 89% identity at the amino acid level (Lahtinen et al., 1996); homologous proteins have been characterized in Caenorrhabditis elegans and Xenopus. This conservation includes the carbohydrate recognition domain (CRD) which is in the lumen of the organelles. On the basis of a secondary structure prediction, MR60/ERGIC-53 was shown to be relatively close to VIP36, a vesicular integral membrane protein presumably recycling between the plasma membrane and the Golgi apparatus (Fiedler et al., 1994); both proteins share common features with leguminous lectins (Fiedler and Simons, 1994). Peptidyl sequences as well as peculiar amino acid residues which are involved in the metal binding and the CRD of leguminous lectins (Sharon, 1993) are well conserved. The substitution of either D121/A or N156/A in the CRD abolished the lectin function (Itin et al., 1996). In addition, the amino-terminal moiety of MR60 encompassing the CRD is closely related to the galectin CRD (Arar et al., 1995; for a review, see Roche and Monsigny, 1996). Both the carbohydrate binding and the recycling properties of the MR60 protein led to the proposal that this lectin may carry newly synthesized glycoproteins in the ER and facilitate their transport to the Golgi apparatus (Arar et al., 1995; Itin et al., 1996). Recent data support a more precise proposal: MR60/ERGIC-53 may function as a chaperone for the efficient secretion of a subset of glycoproteins, including factors V and VIII (Nichols et al., 1998) and the precursor of the lysosomal enzyme cathepsin C (Vollenweider et al., 1998).

ERGIC-53 is a homo-oligomeric transmembrane protein which dimerizes immediately upon synthesis and then forms homohexamers (Schweizer et al., 1988). Similarly, p58, the MR60 rat homologous protein forms dimers and hexamers (Lahtinen et al., 1992). According to Lahtinen et al., (1996), the overexpression of the c-myc epitope-tagged p58 resulted in the accumulation of the protein in the ER and in an enlarged Golgi complex up to the plasma membrane; the c-myc epitope-tagged p58 formed homodimers and homo-oligomers as well as heterodimers and hetero-oligomers with the endogenous protein. In the present study, we investigated whether the state of MR60 oligomerization is related to the lectin activity. The MR60 lacking the last 38 amino acids at the C-terminal end formed dimers, still bound to immobilized mannosides and was revealed inside transfected cells with fluorescein-labeled mannosylated neoglycoproteins. On the contrary, a shorter protein lacking a stretch of 133 amino acids in the C-terminal region, including the two cysteine residues close to the putative transmembrane domain, did not dimerize, did not bind immobilized mannosides and was not revealed in situ by the relevant fluorescent neoglycoprotein.

Results

MR60 constructs

Knowing that the carbohydrate recognition domain (CRD) is localized in the luminal domain, we used epitope-tagged constructs of MR60 to look for any relationship between the mannose binding property and the oligomerization state of the protein. Two expression vectors, pMR60[Delta]473Myc-His and pMR60[Delta]378Myc-His (Figure 1) were constructed. Both constructs lack the putative transmembrane domain (residues 481-498, a stretch of hydrophobic residues) and the cytosolic tail (residues 499-511). Two fragments of the MR60 cDNA, encoding either a protein of 473 amino acids or a protein of 378 amino acids were subcloned into the pcDNA3.1/Myc-His expression vector. The resulting constructs encode two proteins containing 513 and 418 amino acids respectively, including the c-myc epitope followed by a His6 tag in C-terminal position.


Figure 1. Scheme of the MR60 constructs. MR60 contains a luminal domain (open rectangle), a transmembrane domain (solid rectangle) and a cytosolic domain (vertically striped rectangle). The pMR60[Delta]473Myc-His construct contains the luminal domain with 473 residues and lacks the putative transmembrane and the cytosolic domains of the MR60 protein. The pMR60[Delta]378Myc-His construct contains the 378 N-terminal residues of MR60 and lacks a part of the luminal domain and both the putative transmembrane and cytosolic domains. Both constructs carry a c-myc epitope tag (diagonally striped rectangles) and a His6 epitope tag (gray rectangles) at their C-terminal ends. Five cysteines are present in the translated protein: (*) C18 is in the signal peptide, (solid circle) C190 and (open circle) C230 are in the CRD, (open square) C466 and (solid square) C475 are close to the putative transmembrane domain.

Immunofluorescence detection of recombinant protein

To ascertain the expression of the recombinant proteins, Cos cells were transfected with the two truncated constructs as well as the full-length gene. Endogenous MR60 (Figure 2a) was visualized as vesicular elements concentrated in the Golgi region but also spread throughout the cytoplasm, as previously described for p58 (Saraste and Svensson, 1991). The full-length recombinant protein accumulated both in the ER and in an enlarged Golgi complex (Figure 2b) as previously described for ERGIC-53 (Itin et al., 1995) and for p58 (Lahtinen et al., 1996). Both the truncated proteins MR60[Delta]473 and MR60[Delta]378 were detected throughout the cells by using anti-myc antibodies (Figure 2c,d) as well as by anti-p58 antibodies (data not shown), indicating that those truncated proteins were efficiently expressed as a protein, down to the probe located in the C-terminal position. Similar expression was obtained with other cells such as HeLa (Figure 6a,b).


Figure 2. Immunofluorescence localization of endogenous MR60, r.MR60, MR60[Delta]473 and MR60[Delta]378. Cos cells (a), or transiently transfected Cos cells expressing pMR60 (b), pMR60[Delta]473Myc-His (c) or pMR60[Delta]378Myc-His (d), were fixed 24 h after transfection, permeabilized and then incubated either with anti-p58 luminal peptide antiserum (a, b) or with the anti-myc antibody (c, d) followed by FTC-goat anti-rabbit (a, b) or FTC-goat anti-mouse (c, d) to stain the intracellular endogenous MR60 lectin (a) or recombinant proteins (b-d), respectively (scale bar, 10 µm).

The transmembrane domain and the cytosolic domain are not required for oligomerization

The pMR60[Delta]473Myc-His expression vector was used to transiently transfect Cos cells. After pulse labeling with [35S] methionine and immunoprecipitation with the anti-myc epitope antibody of the truncated MR60 from the solubilized cell extracts, the immunoprecipitated proteins were separated by SDS-PAGE. The MR60[Delta]473 protein, lacking the putative transmembrane and cytosolic domains, immunoprecipitated from Cos cells transfected by the pMR60[Delta]473Myc-His vector, appeared as a single band under nonreducing conditions, corresponding to the dimer, with no hexameric form (Figure 3A, lane 1, and Figure 4, lane 3) and to the monomer (Figure 3A, lane 2) under reducing conditions. Therefore, the formation of dimers is not dependent upon the C-terminal moiety. The endogenous MR60 immunoprecipitated from nontransfected Cos cells by using the anti-p58 peptide antiserum gave, in SDS-PAGE under nonreducing conditions, two bands corresponding to the dimeric and hexameric forms (Figure 4, lane 1). By using the anti-p58 peptide antiserum, two close bands corresponding to the dimeric form were clearly seen in Cos cells transfected by the pMR60[Delta]473Myc-His vector (Figure 4, lane 2): the upper one corresponding to the recombinant protein and the lower one corresponding to the endogenous lectin. This lower band was not detected when the anti-myc was used (Figure 4, lane 3).


Figure 3. Oligomerization of recombinant proteins MR60[Delta]473 and MR60[Delta]378. Cos cells were transfected with pMR60[Delta]473Myc-His or pMR60[Delta]378Myc-His cDNAs. Twenty-four hours after transfection, cells were pulsed for 30 min with [35S] methionine and chased for 1 h 30 min. The labeled proteins were treated with the anti-myc antibody for precipitating the recombinant proteins MR60[Delta]473 and MR60[Delta]378. After immunoprecipitation, MR60[Delta]473 protein (A) and MR60[Delta]378 (B) were separated by SDS-10% PAGE under non-reducing conditions (lanes 1) and under reducing conditions (lanes 2). M and D, stand for the monomeric and the dimeric forms, respectively.

The C-terminal moiety of the luminal domain is required for the dimerization of MR60

Knowing that the transmembrane and cytosolic domains are not involved in the dimerization of the protein, a shorter construct pMR60[Delta]378Myc-His was prepared to investigate the putative involvement of the C-terminal moiety of the luminal domain in the dimerization of the MR60. The electrophoresis pattern of the MR60[Delta]378 protein obtained by immunoprecipitation was identical under both nonreducing (Figure 3B, lane 1) and reducing (Figure 3B, lane 2) conditions, appearing as monomers, with no dimeric form. Therefore, a part of the luminal domain is mandatory for the dimerization of the protein. More precisely, the fragment 379-473 (95 amino acids) is involved in the formation of the dimeric protein. Again, because MR60[Delta]473 is dimeric in the presence of SDS under nonreducing conditions, and because MR60[Delta]378 is monomeric under similar conditions, it appears that the cysteine residue in this moiety (379-473), precisely that in position C466 is mandatory to stabilize MR60[Delta]473 in its dimeric form. In addition, partially degraded proteins bearing the myc motif were visualized after immunoprecipitation with the anti-myc antibody; indeed, two bands of lower molecular weight were evidenced (Figure 3, lanes B1 and B2).


Figure 4. Immunoprecipitation of the endogenous MR60 and the truncated MR60[Delta]473 proteins with anti-p58 peptide antiserum. Cos cells were transfected with pMR60[Delta]473Myc-His. Twenty-four hours after transfection, cells were pulsed for 30 min with [35S] methionine and chased for 1 h 30 min. Untransfected cells, as controls, were labeled under the same conditions. The labeled proteins were treated with anti-p58 antiserum for precipitating both the endogenous and the recombinant proteins (lanes 1 and 2) or by anti-myc antibody for precipitating only the recombinant MR60[Delta]473 protein (lane 3). Proteins were separated on SDS-6%PAGE under reducing conditions. In lane 1, from non-transfected cells only the endogenous MR60 could be visualized, in lane 2 from transfected cells both endogenous MR60 and recombinant truncated MR60[Delta]473 were visualized, while in lane 3 only the recombinant protein was visualized. D and H stand for the dimeric and hexameric forms, respectively.

Mannoside binding property of the truncated proteins

MR60 is a mannoside-specific lectin (Pimpaneau et al., 1991; Itin et al., 1996) present in the cell in an oligomeric form. The role of the oligomerization on the sugar binding capability of the protein is not yet known. To shed light on this matter the sugar binding properties of the truncated proteins leading to either the dimeric or monomeric forms were investigated. After pulse-chase metabolic labeling, proteins were extracted and incubated with mannoside-substituted beads according to a procedure modified from Pimpaneau et al., (1991). The proteins adsorbed on the mannoside-substituted gel were selectively eluted with 0.2 M d-mannose and immunoprecipitated either with anti p58-peptide antiserum or with anti-myc antibodies. As a control to detect the presence of the protein of interest, independently of its ability to bind immobilized mannoside, proteins extracted from the cell lysates were directly immunoprecipitated and analyzed: the endogenous MR60 protein is shown in Figure 5A (lane 1) and the recombinants proteins MR60[Delta]473 and MR60[Delta]378 in Figure 5B,C (lanes 1). The MR60[Delta]473 protein was adsorbed on and selectively eluted from the mannoside immobilized on gel (Figure 5B, lanes 2-4) showing a behavior similar to that of the genuine MR60 protein (Figure 5A, lanes 2-4). Conversely, the shorter protein MR60[Delta]378 was not adsorbed on the mannoside substituted gel (Figure 5C). Therefore, it appears that MR60[Delta]378 protein which lacks the C-terminal fragment of the luminal moiety and which contains the putative carbohydrate recognition domain has lost its lectin function.


Figure 5. MR60 and MR60[Delta]473 but not MR60[Delta]378 proteins bind to a mannoside-substituted gel. Recombinant proteins were expressed in Cos cells. Non transfected cells were used as a control for endogenous MR60. Cells were harvested 24 h after transfection, washed, and lysed. The solubilized material was diluted in binding buffer containing 15 mM CaCl2, 1mM MgCl2 and adsorbed onto the mannoside immobilized on gel overnight in an end-over-end shaker. The gel (500 µl) was packed in a column, washed with 50 ml binding buffer, and eluted with 3 ml 0.2 M d-mannose in the binding buffer. One milliliter fractions were collected and the MR60 related proteins were immunoprecipitated from each fraction either with anti-p58 luminal peptide antiserum for the endogenous MR60 (A, lanes 2-4) or with the anti-myc antibody for the recombinant proteins MR60[Delta]473 (B, lanes 2-4) and MR60[Delta]378 (C, lanes 2-4). Precipitated proteins were separated by SDS-PAGE under non reducing conditions. As a control to detect the presence of a protein of interest independently of its ability to bind immobilized mannoside, a part of the solubilized material was directly immunoprecipitated either with the anti-p58 luminal peptide antiserum for the endogenous MR60 protein (A, lane 1) or with the anti-myc antibody for the recombinant proteins MR60[Delta]473 (B, lane 1) and MR60[Delta]378 (C, lane 1). M, D, and H indicate the positions of the monomeric, dimeric and hexameric forms, respectively.


Figure 6. Binding of fluorescein-labeled mannosylated bovine serum albumin. HeLa cells were transfected with pMR60[Delta]473 (a, c) or pMR60[Delta]378 (b, d) cDNAs. Twenty-four hours after transfection the cells were fixed, permeabilized, and incubated with F-ManBSA. Subsequently, the cells were incubated with anti-myc antibody followed by LRSC-goat anti-mouse. MR60[Delta]473 and MR60[Delta]378 were visualized in the rhodamine channel (a and b), and the F-neoglycoprotein in the fluorescein channel (c and d), respectively (scale bar, 10 µm).

The difference in the sugar binding ability of the two truncated MR60 proteins described in this paper was confirmed by in situ labeling with fluoresceinylated neoglycoproteins. The labeling of MR60[Delta]473 expressed in HeLa cells by the anti-myc antibody and by fluoresceinylated mannosylated serum albumin (F-ManBSA) was quite similar, indicating that the truncated protein was able to bind mannosides (Figure 6a,c). Under the same conditions, fluoresceinylated neoglycoproteins bearing other sugars, such as F-LacBSA, F-GlcNAcBSA, failed to label cells expressing the truncated MR60 protein (data not shown). Cells expressing this smaller MR60[Delta]378 protein were not labeled with F-ManBSA, they were only labeled with the anti-myc antibody (Figure 6b,d); a limited area of transfected cells was slightly labeled by F-ManBSA but not by anti-myc antibody, indicating that the F-ManBSA labeling was due to the endogenous MR60 protein.

Discussion

MR60, which was first isolated by affinity chromatography on a column containing mannoside-substituted gel, is a calcium-dependent mannose-specific lectin (Pimpaneau et al., 1991); its cDNA was isolated and characterized (Arar et al., 1995) showing that MR60 sequence is not related to any known mammalian lectin but is identical to ERGIC-53, a marker of the intermediate compartment (Schweizer et al., 1988; Schindler et al., 1993). The amino acid sequence deduced from the cDNA together with a panel of biochemical data show that this lectin is a type I transmembrane protein with a predicted cleavable signal sequence in N-terminal position, has a large luminal domain, a single hydrophobic (putatively transmembrane) domain and a short C-terminal cytosolic tail. Newly synthesized human ERGIC-53 and rat p58 have been shown in vitro to rapidly dimerize and subsequently assemble into hexamers, which at steady state are in equilibrium with the dimers. The high molecular mass proteins resulting from oligomerization have been suggested to be stabilized by disulfide bonds (Schweizer et al., 1988, Lahtinen et al., 1992).

So far, it was not clear whether or not the capacity of MR60 to specifically bind mannosides, was dependent on its oligomerization state. A close relation between the oligomerization and the lectin activity has already been demonstrated for several animal or plant lectins. One well characterized lectin, the human hepatic asialoglycoprotein receptor (ASGP-R) is a noncovalent hetero-oligomer (mainly trimer) composed of two closely related subunits, H1 and H2. In agreement with the requirement of a coexpression of H1 together with H2 to achieve a high affinity binding to asialoglycoprotein ligands at the cell surface (McPhaul and Berg, 1986; Shia and Lodish, 1989), it was concluded that three galactoses born by N-linked oligosaccharides interact simultaneously with two sites on two different H1 subunits and with one site on H2 subunit. Recent results suggested that the stalk segments of the receptor subunits oligomerize to constitute an [alpha]-helical coiled coil stalk on top of which the carbohydrate recognition domain is exposed for ligand binding (Bider et al., 1996). An other example is given by the native mannan-binding protein (MBP), a calcium-dependent mammalian serum lectin, with a collagen-like domain characterized by an NH2-terminal cysteine-rich domain, a neck domain and a carbohydrate recognition domain; this protein forms several disulfide-dependent oligomeric structures (hexamers of trimers) (for a review, see Epstein et al., 1996). The truncated recombinant protein with a short collagen domain is able to form trimers by noncovalent association of the neck domain and binds sugar with a specificity similar to that of the native form (Eda et al., 1998). Usually, lectins have one sugar binding site per subunit, but wheat germ agglutinin (WGA) is an unusually dimeric plant lectin bearing four carbohydrate binding sites (Privat et al., 1974); the two binding sites are noncooperative, spatially distinct and formed by amino acid residues of both protomers (Wright et al., 1984). Conversely, in some instances, the high affinity of a lectin for its ligands derives from the presence of multiple carbohydrate binding recognition domains on one subunit as in the case of the macrophage mannose specific receptor (Stahl et al., 1984). Here, we show that the lectin capacity of MR60 is correlated with its oligomerization state. The full-length protein which properly oligomerizes in dimers and hexamers as well as the pMR60[Delta]473 which only forms dimers, both bind to mannoside substituted gel and are eluted with high mannoside concentration (0.2 M D-mannose); d-mannose started to elute MR60/ERGIC-53 at a concentration of 0.1 M (Itin et al., 1996). In contrast, the shorter recombinant protein which does not form stable dimers, was neither retained nor even retarded on a gel containing immobilized mannoside. This result shows that the affinity for the gel containing immobilized mannoside was below a low threshold (about 103 l × mol-1); indeed, usually proteins with a low affinity for their substrate or their ligand are able to bind such a gel, as in the case of hydrolases (Cuatrecasas and Anfisen, 1971; Jakoby and Wilchek, 1974). The ability of MR60[Delta]473 to bind mannoside was corroborated by the fact that cells expressing this truncated protein were labeled in a similar manner with anti-myc antibody and with the mannoside bearing neoglycoprotein, while the inability of MR60[Delta]378 to bind mannoside was also evidenced in the case of cells transfected with the smaller protein MR60[Delta]378 (Figure 6).

As shown by MR60[Delta]378 gel electrophoresis (Figure 3B, lanes 1 and 2), a large part of the truncated protein appeared upon immunoprecipitation, as a major band migrating as a monomer. Two minor bands of low molecular weight were also visualized by the anti-myc antibody. This partial degradation is reminiscent of that of the unfolded H2b asialoglycoprotein receptor subunit expressed alone in the HepG2 cells which was partially degraded in the ER (Wikström and Lodish, 1993). Conversely, when a mutated ERGIC-53 (the two C-terminal phenylalanines were replaced by two alanines) was expressed in HeLa cells, the protein, which was not able to leave the ER, was however acting as a lectin being able to bind fluoresceinylated and mannosylated serum albumin (F-ManBSA) (Vollenweider et al., 1998). This result is probably related to a stabilization of MR60/ERGIC-53 due to the presence of the cytosolic part and the membrane domain.

The oligomerization state of MR60/ERGIC-53 and p58 proteins has been suggested to be stabilized by disulfide bonds (Schweizer et al., 1988; Lahtinen et al., 1992), without excluding the role of a different part of the protein. Here, we show that a truncated MR60 protein lacking both the transmembrane and the cytosolic domains gives a dimer. In addition, we show that while the cytosolic and transmembrane domains are not involved in the dimerization of the protein, a part of the luminal domain is essential to form dimers and hexamers. Indeed, the C-terminal moiety of the luminal domain is essential for the dimer formation, since the recombinant pMR60[Delta]473 protein is capable of forming a dimer while the shorter protein pMR60[Delta]378 is not. The amino acid sequence of MR60/ERGIC-53 carries four cysteine residues in its luminal domain (Schindler et al., 1993; Arar et al., 1995). The two first cysteines, residues 190 and 230, which are part of the carbohydrate recognition domain (according to Fiedler and Simons, 1994; Arar et al., 1995; Itin et al., 1996) are clearly not involved in the stabilization of the dimers since the truncated protein MR60[Delta]378 which possesses both of them, does not appear as a dimer in the absence of reducing agent. The two other cysteines which are close to the putative transmembrane domain are involved in the stabilization of the oligomerization state. The cysteine 466 stabilizes the dimeric form since the MR60[Delta]473 appears as a dimer in the absence of a reducing agent and as a monomer in the presence of a reducing agent; the cysteine 475 seems to be required to stabilize higher oligomeric forms since its absence in the MR60[Delta]473 is correlated with a total lack of hexamers while the full recombinant protein, which contains both the cysteines 466 and 475, appears as dimers and hexamers.

These data suggest that these two cysteines are involved in the formation of links between two proximal subunits:

Indeed, a recent report by Lahtinen et al., (1999) using site-directed mutagenesis of the two cysteine residues close to the transmembrane domain of p58 is in agreement with this scheme, by showing that only one of those cysteines is required to stabilize a dimeric form.

The lack of sugar binding capacity of the truncated MR60[Delta]378 brings a new light on the molecular mechanism related to the mutations observed in ERGIC-53 in the case of the combined factors V and VIII deficiency patients (Nichols et al., 1998). Indeed, in one patient the frameshift mutation associated with a G insertion at the level of codons 29-30 led to a peptide containing 101 amino acids, 71 of which do not belong to the normal MR60/ERGIC-53 protein. This protein has little chance to be recognized by anti-MR60/ERGIC-53 antibodies and does not contain a CRD. Conversely, in another patient the splice donor mutation after the codon 383 allowed the synthesis of a truncated protein slightly longer than MR60[Delta]378. In our hands, such a short protein was clearly evidenced by using either anti-p58 polyclonal antibodies or anti-myc antibodies. In the Nichols paper, the short protein was not visualized inside the cells because the authors were using a monoclonal antibody (G1/93) which is probably directed against an epitope not present in that short protein; this short protein was also not revealed on a Western blot which was designed to show proteins with a molecular mass close to that of the full-length ERGIC-53 protein. Moreover, the short protein corresponding to the splice donor mutation lacks the cysteine residue close to the membrane and therefore can presumably neither form a dimer nor act as a lectin, i.e., a sugar binding protein. Thus, in the case of the splice donor mutation, the truncated protein even if it is present, cannot act as a sugar dependent chaperone.

In conclusion, both the dimeric and hexameric forms are able to bind mannosides while the monomeric form is not.

Materials and methods

Reagents

[35S] Methionine and Thermo Sequenase radiolabeled terminator cycle sequencing kit were from Amersham (Buckinghamshire, UK); cell culture media (DMEM, and DMEM without l-methionine), fetal bovine serum (FBS) and methionine from Gibco (Grand Island, NY); dithiothreitol (DTT), protease inhibitor cocktail for mammalian cell extracts [4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, transepoxysuccinyl-l-leucylamido-(4-guanido)-butane, bestatin, leupeptin, aprotinin], as well as phenylmethylsulfonyl fluoride (PMSF), Triton X-100, paraformaldehyde (PFA), saponin, bovine serum albumin (BSA), glycine and d-mannose were from Sigma (Saint-Quentin-Fallavier, France). Affi-102 agarose beads were from Bio-Rad (Richmond, CA). The rabbit polyclonal anti-luminal p58 peptide antibodies raised against a peptide corresponding to amino acids 158-170 from the rat protein (Lahtinen et al., 1996) was kindly provided by U. Lahtinen (Ludwig Institute for Cancer Research, Stockholm, Sweden); mouse monoclonal anti-myc epitope antibody was from Invitrogen (Leek, The Netherlands). FTC-goat anti-mouse IgG (H+L), FTC goat anti-rabbit immunoglobulins and lissamine rhodamine (LRSC)- F(ab[prime])2 fragment goat anti-mouse IgG (H+L) were from Jackson ImmunoResearch Laboratories. Rabbit anti-mouse immunoglobulin antibody, rabbit normal immune serum and fixed Staphylococcus aureus cell suspension (Pansorbin) were from Calbiochem-Novabiochem (La Jolla, CA). Enlightning was from Du Pont de Nemours (Brussels, Belgium).

Neoglycoproteins were prepared by coupling isothiocyanatophenyl-glycosides to bovine serum albumin and fluoresceinylated neoglycoproteins were obtained by using fluorescein isothiocyanate as previously described (Roche et al., 1983; Monsigny et al., 1984); fluorescent neoglycoproteins were purified by gel filtration on Ultrogel GF05 (Biosepra, Villeneuve la Garenne, France) and precipitated from a 1mg/ml solution in water by adding 9 volumes of absolute ethanol.

Recombinant cDNAs

The sequence extending from the HindIII site (nucleotide 6) to the PmeI site (nucleotide 1673) of MR60 cDNA (GenBank U09716) was introduced in the HindIII and EcoRV sites of the pcDNA3 expression vector (Invitrogen) and a BglII restriction nuclease site was inserted 9 nucleotides upstream the ATG codon using polymerase chain reaction (PCR) to produce the plasmid pMR60. This expression vector contains the complete coding sequence of MR60. The pMR60 vector was used as a template to construct the two partially deleted genes as follows. First, an intermediate plasmid (pMR[Delta]Myc-His) was constructed by insertion of a BglII/EcoRI fragment from pMR60 (representing the first 918 nucleotides of the MR60 cDNA) into the expression vector pcDNA3.1/Myc-His (Invitrogen) opened by BamHI/EcoRI enzymes. Secondly, the two different deleted fragments representing the C-terminal part of the recombinant protein were obtained by PCR and used to construct the plasmid pMR60[Delta]473Myc-His carrying the MR60 cDNA deleted from nucleotide 1419 (amino acid 474) to the nucleotide 1553 (amino acid 511) preceding the stop codon; PCR was carried out with the following antisense oligonucleotide carrying an EcoRV site 5[prime]-TGCTGGATATCTTGGAAATGGTGGTAGTTC-3[prime] and a sense oligonucleotide 5[prime]-CTGCACAAGGGCATTTTG-3[prime]. For the second vector pMR60[Delta]378Myc-His, carrying a cDNA deleted from the nucleotide 1152 (amino acid 379) to the nucleotide 1553 preceding the stop codon, as above, the same sense oligonucleotide was used and the antisense oligonucleotide carried an EcoRV site: 5[prime]-TGCTGGATATCTCATTCCTGCTCCTCTTTT-3[prime]. The PCR products were digested by EcoRI/EcoRV enzymes and the digestion fragments were inserted into the intermediate plasmid pMR[Delta]Myc-His to generate expression vectors pMR60[Delta]473Myc-His and pMR60[Delta]378Myc-His, respectively. All PCR products were sequenced using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham, Buckinghamshire, UK), confirming that no error has been introduced by PCR.

Cell culture and transfections

Cos-7 and HeLa cells (ATCC, Rockville, MD) were grown in DMEM supplemented with 10% FBS, 100 I.U./ml penicillin, 100 µg/ml streptomycin. For radiolabeling, cells (106) were plated 1 day before the transfection in 10 cm dishes. For immunofluorescence microscopy, cells (4 × 104) were plated 1 day before transfection on glass slides in four-well multichambers. Cells were transiently transfected with 5µg DNA for 106 cells using polyethylenimine (PEI) (Boussif et al., 1995) or ExGen (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions. Biosynthetic labeling and immunofluorescence detection were performed 24 h after transfection.

Metabolic labeling and immunoprecipitation

For radiolabeling, cells at 50% confluency were incubated (30 min at 37°C) in methionine-free medium containing 10% dialyzed FBS before adding [35S] methionine (9.25 × 106 Bq per 10 cm dish) for 30 min at 37°C. Upon labeling, cells were chased (1 h 30 at 37°C) in a complete culture medium containing 2 mM cold methionine. The cells were then washed twice in PBS, harvested, and lysed in lysis buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4 containing 1% Triton X-100, the protease inhibitor cocktail (100 µl/107 cells) and 1 mM PMSF). The lysates were cleared by centrifugation (15,000 × g for 15 min in a microfuge), and the supernatants were diluted in dilution buffer (5 mM EDTA, 250 mM NaCl, 20 mM Tris-HCl at pH 8 containing 1% Triton X-100 and 2.5 mg/ml BSA). Before immunoprecipitation of the endogenous MR60, the diluted Cos cell lysate was cleared by an overnight incubation at 4°C with 10 µl normal rabbit serum followed by an addition of a 10% suspension of fixed Staphylococcus aureus cells (30 min at 4°C) as described by Carlsson and Fukuda, (1986). After centrifugation (30 s at 10,000 × g in a microfuge), the immunoprecipitation of the endogenous MR60 was performed by adding 5 µl of the anti-luminal p58 peptide antiserum to the supernatant followed by an overnight incubation at 4°C. For immunoprecipitation of the c-myc-tagged recombinant proteins, 3 µg of the mouse monoclonal anti-myc antibody were added to the transfected Cos cell lysates. After about 5 h at 4°C, a rabbit anti-mouse antibody was added and the solution was incubated overnight at 4°C. Recovering of immune complexes from both endogenous and recombinant proteins was performed as above by addition of fixed S.aureus cells. The immune complexes were then washed once with dilution buffer, three times with a high salt buffer (5 mM EDTA, 400 mM NaCl, 20 mM Tris-HCl at pH 8 containing 1% Triton X-100) and once with 10 mM Tris-HCl at pH 7.4. The immunoprecipitated proteins were solubilized at 95°C in loading buffer (60 mg/ml SDS, 375 mM Tris-HCl, at pH 6.8 and 30% glycerol (v/v)) with or without a reducing agent (100 mM DTT). Proteins were separated on SDS (1 mg/ml) -10% polyacrylamide slab gels under reducing conditions or on SDS (1mg/ml)-6 % polyacrylamide slab gels under nonreducing conditions. The gels were fixed in a mixture of isopropanol, acetic acid and water (25/10/65, per volume) and treated for fluorography with Enlightning according to the manufacturer's instructions.

Binding to the mannoside-substituted gel

The mannoside immobilized on gel was prepared by coupling 4-isothiocyanatophenyl [alpha]-d-mannopyranoside with activated agarose beads bearing amino terminal groups (Affi-102) as described by Pimpaneau et al., (1991). [35S] methionine-labeled Cos cell lysates were diluted in the binding buffer (15 mM CaCl2, 1 mM MgCl2, 150 mM NaCl, 10 mM Tris-HCl at pH 7.4 containing 0.15 % Triton X-100), and incubated under gentle stirring overnight at 4°C with the mannoside-substituted gel (108 cells/100µl). The beads were washed with 100 volumes of the binding buffer. Endogenous and recombinants MR60 were eluted with 6 gel volumes of binding buffer containing 0.2 M d-mannose. The endogenous and recombinant proteins were immunoprecipitated from the eluted fractions and analyzed as described above.

Immunofluorescence microscopy

Twenty-four hours after transfection the cells were fixed and permeabilized. All incubations were conducted at room temperature. Cos cells and HeLa cells transiently transfected or untransfected cells used as controls, were washed twice with phosphate-buffered saline (PBS) pH 7.4, fixed for 30 min in PBS containing 20 mg/ml PFA, washed twice with PBS containing 20 mM glycine, and permeabilized 20 min in PBS containing 1 mg/ml saponin and 20 mM glycine. The anti-myc antibody was used at a 1/400 dilution in PBS containing 1 mg/ml saponin. Before adding the anti-luminal p58 peptide antiserum the cells were incubated for 30 min in PBS containing 2% per volume goat serum in order to prevent unspecific binding (Lahtinen et al.,1996) and then incubated with the anti-luminal p58 peptide antiserum (1/300 in PBS containing 1 mg/ml saponin). Cells were then washed four times with PBS containing 1 mg/ml saponin and incubated 30 min with either FTC-goat anti-mouse or LRSC-goat anti-mouse or FTC-goat anti-rabbit antisera (1/200). For the double immunofluorescence, permeabilized cells were first incubated for 30 min with 20 µg/ml of the fluorescein-labeled mannosylated bovine serum albumin (F-ManBSA). After extensive washes, cells were mounted in a PBS-glycerol mixture (1:1) per volume, containing 10 mg/ml DABCO (1,4-diaza bicyclo[2,2,2]octane) as an antifading agent (Johnson et al., 1982).

Confocal microscopy analysis

Cells were analyzed with a confocal imaging system MRC-1024 (Bio-Rad) equipped with a Nikon microscope (Nikon, Tokyo, Japan), a 60× Planapo objective (numerical aperture 1.4) and a krypton/argon laser tuned to produce both 488 nm fluorescein excitation and 564 nm rhodamine excitation wavelength beams allowing simultaneous reading of both fluorescent signals. Pictures were recorded with a Kalman filter (average of 10 to 15 images). Images were treated using Adobe Photoshop software (Adobe Systems Inc., Mountain View, CA).

Acknowledgments

We thank Françoise Fargette for her valuable help in producing the plasmids and Marie-Thérèse Bousser for her expert technical assistance in cell experiments. We are grateful to Dr. Ulla Lahtinen (Ludwig Institute for Cancer Research, Stockholm) for providing us with the p58 anti-luminal peptide antiserum. V.C received a fellowship from Ministère de l'Enseignement Supérieur et de la Recherche Scientifique et Technique. This work was supported by grants from the Agence Nationale de Recherche sur le SIDA and from the Association pour la Recherche sur le Cancer (ARC 6132). V.P. is full-time investigator at the Centre National de la Recherche Scientifique; M.M. and A.L. are Professor and Assistant-Professor, respectively, at the University of Orléans; and A.C.R. is Research Director at the Institut National de la Santé et de la Recherche Médicale.

Abbreviations

BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum-Golgi-intermediate compartment; FBS, fetal bovine serum; F-ManBSA, fluoresceinylated and mannosylated bovine serum albumin; PFA, paraformaldehyde; PMSF, phenylmethylsulfonyl fluoride; r.MR60, full-length recombinant protein.

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