©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ERGIC-53, a Membrane Protein of the Endoplasmic Reticulum-Golgi Intermediate Compartment, Is Identical to MR60, an Intracellular Mannose-specific Lectin of Myelomonocytic Cells (*)

(Received for publication, November 2, 1994; and in revised form, December 14, 1994 )

Chantal Arar (1)(§) Valérie Carpentier (1)(§) Jean-Pierre Le Caer (2) Michel Monsigny (1) Alain Legrand (1) Annie-Claude Roche (1)(¶)

From the  (1)Laboratoire de Biochimie des Glycoconjugués et Lectines Endogènes, Université d'Orléans et Centre de Biophysique Moléculaire, CNRS, Bâtiment B, rue Charles Sadron, 45071 Orléans, Cedex 02, France and the (2)Institut Alfred H. Fessard, CNRS, 91198 Gif-sur-Yvette, Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS and DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A mannose-specific membrane lectin (MR60) isolated from human myelomonocytic HL60 cells by affinity chromatography is expressed in intracellular organelles of immature monocytes (Pimpaneau, V., Midoux, P., Monsigny, M., and Roche, A. C. (1991) Carbohydr. Res. 213, 95-108). It is not present at the cell surface and is immunochemically and structurally distinct from the M(r) 175,000 mannose receptor of mature macrophages. MR60 cDNA was isolated and characterized; on the basis of its sequence, MR60 is not related to any known mammalian lectins. Surprisingly, MR60 was found to be identical to ERGIC-53 (Schindler, R., Itin, C., Zerial, M., Lottspeich, F., and Hauri, H. P.(1993) Eur. J. Cell Biol. 61, 1-9), a type I integral membrane protein, defined as a marker of the intermediate compartment that recycles between the Golgi apparatus and endoplasmic reticulum; MR60/ERGIC-53 shares with VIP-36 significant homologies with leguminous plant lectins (Fiedler, K., and Simmons, K.(1994) Cell 77, 625-626). We extend these findings in evidencing a structural homology between MR60/ERGIC-53 and mammalian galectins (soluble beta galactose binding proteins). MR60/ERGIC-53 is the first lectin characterized as an endoplasmic reticulum-Golgi protein. Accordingly, this intracellular mannose binding protein could be involved in the traffic of glycoproteins between endoplasmic reticulum and the Golgi apparatus.


INTRODUCTION

A mannose-specific membrane lectin, MR60, has been isolated on a mannose-substituted column from membranes of monocyte/macrophage precursor cell lines, HL60 and U937 cells(1) . The isolated protein agglutinates polystyrene beads substituted with neoglycoproteins in a mannose-dependent manner and is localized uniquely in cytoplasmic organelles(2) . This mannose-specific lectin differs from that of the macrophages (3) by its molecular mass, its cell differentiation-related expression, as well as by its localization. Under denaturing and reducing conditions, MR60 migrates as a M(r) 60,000 unique band in SDS-polyacrylamide gel electrophoresis; under non reducing conditions, MR60 migrates as a M(r) 120,000 dimer. MR60 does not contain any N-glycanase-sensitive oligosaccharide(2) . To elucidate the structure and the function of MR60, the cDNA cloning was undertaken. Reverse transcription-polymerase chain reaction experiments were performed using degenerate oligonucleotides based on peptidic sequences as primers. A 550-base pair partial cDNA was amplified and was used as a specific probe to screen an HL60 cDNA library. The 5kilobase full-length cDNA was sequenced, and the peptidic sequence of MR60 was deduced. The sequence of MR60 is essentially identical to that of ERGIC-53, a protein of the endoplasmic reticulum-Golgi intermediate compartment of unknown function(4) . On the basis of a secondary structure prediction, MR60/ERGIC-53 shares with VIP-36 (5) significant homologies with leguminous plant lectins(6) . In addition, we show that the amino-terminal moiety of MR60, which is mainly made of beta-strands, is closely related with galectins, animal beta-galactose binding proteins(7) .


MATERIALS AND METHODS

Sequence of Peptides

MR60 was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to a polyvinylidene difluoride membrane allowing to determine the NH(2)-terminal sequence(8) . The internal sequences were determined upon digestion with the endoproteinase Asp-N (9) and purification on a reverse-phase column(8) .

DNA Amplification and Cloning

Based on internal sequences, two degenerate oligonucleotides were synthesized by Eurogentec (B-Seraing) (where N is A, C, G, or T; H is A, C, or T; R is A or G; and Y is C or T): ATHTGGTAYGCNGARAA, based on the amino acid sequence IWYAEN, and TCYTCYTTYTTYTTRTC, based on the amino acid sequence DKKKEE. Reverse transcription-polymerase chain reaction was carried out on HL60 RNA using these primers. The amplified product was subcloned, sequenced, and used to screen a human HL60 cDNA library, kindly provided by Dr. M. Fukuda (San Diego, CA). Overlapping clones that represent the full-length cDNA were characterized by restriction endonuclease map and sequencing.

Secondary Structure Analysis

The protein sequence was submitted to hydrophobicity analysis (10) using the DNAstar protein program (DNAstar Inc, Madison, WI). The average hydropathy across a 9-amino acid window around the residue was calculated for each amino acid, and the secondary structure prediction was determined(11) .


RESULTS and DISCUSSION

A new mannose-specific lectin has been evidenced in membrane of myelomonocytic cells (HL60) and purified by affinity chromatography on a mannoside-substituted column(1) . Monoclonal antibodies raised against this lectin show that MR60 is localized in intracellular organelles around the nucleus(2) . To elucidate its structure and its function, the MR60 cDNA was cloned and sequenced. The MR60 amino terminus sequence (DGVGGDPAVAL) was obtained with a low yield, suggesting that the amino terminus is partly blocked. Additional sequences were obtained upon endoproteinase Asp-N fragmentation and peptide separation by reversed-phase chromatography. Based on these sequences, degenerate oligonucleotides were synthesized and used in reverse transcriptase-polymerase chain reaction experiments leading to a partial MR60 cDNA of 550 base pairs corresponding to a stretch of 180 amino acids, including the sequence of internal peptides. The 550-base pair polymerase chain reaction product was used to isolate the full-length cDNA upon screening a HL60 cDNA library. The nucleotide sequence of the isolated cDNA has been submitted to the GenBank/EMBL Data Bank.

This sequence is identical to ERGIC-53 (4) except for one nucleotide giving a seryl residue in ERGIC-53 and a threonyl residue in MR60 at position 153. ERGIC-53 is a type I membrane protein of the endoplasmic reticulum-Golgi intermediate compartment (12, 13) and recycles between cis-Golgi network and endoplasmic reticulum(14) . Fiedler and Simons (6) have recently reported a significant homology among ERGIC-53, VIP-36 (a component of glycolipid rich rafts)(5) , and three leguminous lectins. From this limited homology, they speculated that ERGIC-53 and VIP-36, which are involved in the secretory pathway, are members of a putative novel class of animal lectins. The present data demonstrate that ERGIC-53, which is identical with MR60, is a lectin. This finding identifies a resident endoplasmic reticulum-Golgi protein as a lectin for the first time. Independently, we have shown that MR60 binds beads coated with mannosylated bovine serum albumin but does not bind those coated with either sugar-free or glucosylated bovine serum albumin(2) . According to its identity with the ERGIC-53 and to its lectin activity, MR60 could be involved in the traffic of nascent glycoproteins between the endoplasmic reticulum and the cis-Golgi apparatus. As it has been recently shown, the impairment of the removal of glucose from newly synthesized glycoproteins by a glucosidase inhibitor, such as castanospermine, leads to a rapid degradation of these glycoproteins(15, 16) . A lectin, such as MR60/ERGIC-53, which does not contain itself any glycan, could bind nascent glycoproteins as soon as their glucose residues have been stripped off. Therefore, this lectin could protect the early processed glycoproteins and/or convey them in a shuttling process between endoplasmic reticulum and Golgi apparatus.

The secondary structure prediction of VIP-36 led Fiedler et al.(5) to hypothesize that VIP-36 is related to a leguminous plant lectin; this finding was extented to other plant lectins and to ERGIC-53(6) . More strikingly, Lobsanov et al.(17) , based on x-ray data, reported a structural homologous organization between galectin-2 and the mannose-specific lectin from pea seeds(18, 19) . It is interesting to notice that the primary structure of these two lectins is only sketchily related; they share a domain with two beta-sheets, the strands of which have some conserved or homologous amino acids, while the links between the beta-strands of pea lectin are usually much longer than those of the galectin-2. In addition, those two lectins have not the same sugar specificity; the pea seed lectin binds glycoconjugates containing mannose while galectins bind glycoconjugates containing galactose. The secondary structure prediction of MR60 shows that the amino-terminal moiety between amino acid 1 and 250 contains mainly beta-strands and beta-turns, while the COOH-terminal moiety contains mainly alpha-helices and beta-turns (Fig. 1). The presence of short beta-strands in the amino-terminal moiety of MR60 is reminiscent of the structure of the galectins and of the pea seed lectin. The MR60 beta-strands were aligned with those of pea seed lectin (PSL) (^1)(Fig. 2A). Inside the related beta-strands, some amino acids are invariant and some are conserved. By comparing the sequences of beta-strands of the human lectin L14-I (galectin-1) (20) and of the human lectin L14-II (galectin-2)(17, 21) , with the sequence of the human lectin MR60 between amino acids 81 and 269, it appeared (Fig. 2B) that 11 of the 12 beta-strands of MR60 correspond to the 5 and 6 beta-strand sheets of galectin-2(17) . One beta-strand called S`3 is located between galectin S3 and S4 beta-strands. The sequence corresponding to the very short galectin F5 beta-strand could also be helicoidal in the case of MR60. Inside the identified MR60 beta-strands, several amino acids are identical with those of corresponding beta-strands in galectin-1, galectin-2, both galectin-1 and -2, or even in galectin-1, -2, -3, and -4. Moreover, the galectin amino acids identified by Lobsanov et al.(17) as residues stabilizing side chains of the amino acids involved in the binding of the sugar ligand are also found in MR60; more precisely, Tyr-127 and Asn-130 correspond to Phe-31 and Asn-34 in S3 beta-strands of galectin-2, Asp-157 corresponds to Glu-52 between S4 and S5 beta-strands, and Lys-232 corresponds to Arg-108 between F5 and S2 beta-strands.


Figure 1: Secondary structure prediction according to Garnier et al.(10) .




Figure 2: Alignments of the MR60 sequence with those of PSL (A) and those of galectin-1 and galectin-2 (L14-I, L14-II) (B). Amino acid positions are indicated on the rightside of the alignments; a dash indicates a gap. The beta-strands are identified by a line under the corresponding amino acids tracks. The beta-strands in the five- and six-stranded beta-sheets are labeled F1-F5 and S1-S6, respectively, according to Lobsanov et al.(17) . A, a conserved residue in PSL and MR60 is indicated by a filled square, and a homologous residue is indicated by an open circle. A residue conserved in PSL, galectin-1 and -2, and MR60 is indicated by a filled circle. The peptide that is absent in the mature PSL is in boldface. B, a residue conserved in one galectin and MR60 is indicated by an open square, a residue conserved in galectin-1 and galectin-2 and MR60 is indicated by a filled square, and a residue conserved among all galectins (galectin-1, -2, -3, and -4) is indicated by a filled circle. Homologous amino acids are indicated by open circles. Residues stabilizing side chain of the residues interacting with lactose in galectin-2 are marked with an times(17) . Accession numbers for the GenBank/EMBL data bases and references are as follows: MR60, U09716; L14-I, X11829(20) ; L14-II, M87010(21) ; PSL, J01254(18) .



In conclusion, the mannose-specific membrane lectin MR60, characterized in intracellular organelles of immature monocytes, is identical with ERGIC-53, a membrane protein that shuttles between endoplasmic reticulum and cis-Golgi apparatus(12) ; the sequence homology among ERGIC-53, VIP-36, and leguminous plant lectins (6) is extended to that of a structural homology between MR60/ERGIC-53 and galectins; and MR60/ERGIC-53 appeared to be a new type of animal membrane lectin, which, on the basis of its cell localization and of its selective affinity for mannosides but not for glucosides, could be involved in the protection and/or in the traffic of early processed glycoproteins.


FOOTNOTES

*
This work was supported in part by Association de Recherche sur le Cancer Grants ARC 6132 (to A.-C. R.) and ARC 6231 (to A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09716[GenBank].

§
Received a fellowship from the Ministère de la Recherche et de la Technologie.

Directeur de Recherche INSERM. To whom correspondence should be addressed. Tel.: 33-38-51-55-37; Fax: 33-38-69-00-94; roche{at}cnrs-orleans.fr.

(^1)
The abbreviation used is: PSL, pea seed lectin.


ACKNOWLEDGEMENTS

We are grateful to Dr. R. C. Hughes (Medical Research Council, London) for critical reading of the manuscript and to Dr. M. Fukuda (La Jolla Cancer Research Foundation, La Jolla, CA) for the kind gift of the HL60 cDNA library. We thank Violaine Carrière for help with the 5`-end cDNA cloning and Martine Dubois for technical assistance.


REFERENCES

  1. Pimpaneau, V., Midoux, P., Monsigny, M., and Roche, A. C. (1991) Carbohydr. Res. 213, 95-108 [CrossRef][Medline] [Order article via Infotrieve]
  2. Carpentier, V., Vassard, C., Plessis, C., Motta, G., Monsigny, M., and Roche, A. C. (1994) Glycoconjugate J. 11, 333-338 [Medline] [Order article via Infotrieve]
  3. Stahl, P. D., and Gordon, S. (1982) J. Cell Biol. 93, 49-56 [Abstract]
  4. Schindler, R., Itin, C., Zerial, M., Lottspeich, F., and Hauri, H. P. (1993) Eur. J. Cell Biol. 61, 1-9 [Medline] [Order article via Infotrieve]
  5. Fiedler, K., Parton, R. G., Kellner, R., Etzold, T., and Simons, K. (1994) EMBO J. 13, 1729-1740 [Abstract]
  6. Fiedler, K., and Simons, K. (1994) Cell 77, 625-626 [Medline] [Order article via Infotrieve]
  7. Barondes, S. H., Castronovo, V., Cooper, D. N. W., Cummings, R. D., Drickamer, K., Feizi, T., Gitt, M. A., Hirabayashi, J., Hughes, C., Kasai, K., Leffler, H., Liu, F. T., Lotan, R., Mercurio, A. M., Monsigny, M., Pillai, S., Poirer, F., Raz, A., Rigby, P. W., Rini, J. M., and Wang, J. L. (1994) Cell 76, 597-598 [Medline] [Order article via Infotrieve]
  8. Carpentier, V. (1992) Caractérisation de Protéines Membranaires, Affines d'un Glycovariant de la Glycoprotéine Acide alpha 1 et du Mannose des Cellules de la Lignée Monocytique, Ph.D. thesis. Université d'Orléans
  9. Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent, S. B. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6970-6974 [Abstract]
  10. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  11. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120 [Medline] [Order article via Infotrieve]
  12. Schweizer, A., Fransen, J. A. M., Bachi, T., Ginsel, L., and Hauri, H. P. (1988) J. Cell Biol. 107, 1643-1653 [Abstract]
  13. Hauri, H. P., and Schweizer, A. (1992) Curr. Opin. Cell Biol. 4, 600-608 [Medline] [Order article via Infotrieve]
  14. Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C., and Klausner, R. D. (1990) Cell 60, 821-836 [Medline] [Order article via Infotrieve]
  15. Moore, S. E. H., and Spiro, R. G. (1993) J. Biol. Chem. 268, 3809-3812 [Abstract/Free Full Text]
  16. Kearse, K. P., Williams, D. B., and Singer, A. (1994) EMBO J. 13, 3678-3686 [Abstract]
  17. Lobsanov, Y. D., Gitt, M. A., Leffler, H., Barondes, S. H., and Rini, J. M. (1993) J. Biol. Chem. 268, 27034-27038 [Abstract/Free Full Text]
  18. Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549 [Abstract/Free Full Text]
  19. Einspahr, H. (1986) J. Biol. Chem. 261, 16518-16527 [Abstract/Free Full Text]
  20. Gitt, M. A., and Barondes, S. H. (1991) Biochemistry 30, 82-89 [Medline] [Order article via Infotrieve]
  21. Gitt, M. A., Massa, S. M., Leffler, H., and Barondes, S. H. (1992) J. Biol. Chem. 267, 10601-10606 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.