(Received for publication, November 2, 1994; and in revised form, December 14, 1994 )
From the
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 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
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.
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 60,000 unique band in
SDS-polyacrylamide gel electrophoresis; under non reducing conditions,
MR60 migrates as a M
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
-strands, is closely related with galectins, animal
-galactose binding proteins(7) .
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 -sheets, the
strands of which have some conserved or homologous amino acids, while
the links between the
-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
-strands and
-turns, while the COOH-terminal moiety
contains mainly
-helices and
-turns (Fig. 1). The
presence of short
-strands in the amino-terminal moiety of MR60 is
reminiscent of the structure of the galectins and of the pea seed
lectin. The MR60
-strands were aligned with those of pea seed
lectin (PSL) (
)(Fig. 2A). Inside the related
-strands, some amino acids are invariant and some are conserved.
By comparing the sequences of
-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
-strands of MR60
correspond to the 5 and 6
-strand sheets of
galectin-2(17) . One
-strand called S`3 is located between
galectin S3 and S4
-strands. The sequence corresponding to the
very short galectin F5
-strand could also be helicoidal in the
case of MR60. Inside the identified MR60
-strands, several amino
acids are identical with those of corresponding
-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
-strands of galectin-2, Asp-157
corresponds to Glu-52 between S4 and S5
-strands, and Lys-232
corresponds to Arg-108 between F5 and S2
-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 -strands are identified by a line under the corresponding amino acids tracks. The
-strands in the five- and six-stranded
-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
(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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09716[GenBank].