Glyco-Fonun section
dothelial cell receptor and DEC-205. Further conclusions about
evolution of binding specificities of the CRDs of these receptors must await ligand-binding studies on the endothelial cell
receptor and DEC-205.
Nomenclature
Currently, "mannose receptor family" seems to be the default
name for the group of receptors containing multiple C-type
CRDs (Wu et al, 1996). While there might be a temptation to
consider names like "polylectins," such a designation is probably inappropriate since some, perhaps most, members of the
family probably do not bind carbohydrates. A better name for
the group must await more information about the functions of
each receptor.
References
Drickamer.K. (1993) Ca2+-dependent carbohydrate-recognition domains in
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TaylOT,M.E., ConaryJ.T., Lennarz>i.R., Stahl,P.D. and DrickamerJC. (1990)
Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J. BioL Chem., 265,12156-12162.
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21323-21330.
Is human galectin-1 activity modulated
by monomer/dimer equilibrium?
Virginie Giudicelli, Didier Lutomski, Matthieu
Levi-Strauss1, Dominique Bladier, Raymonde
Joubert-Caron and Michel Caron2
Laboratoire de Biochimie et Technologic des Prot6ines, UFR Leonard de
Vinci, Universit6 Paris Nord, 74 Rue Marcel Cachin, F-93017 Bobigny
Cedex, France and 'INSERM U.I 14, College de France, 11 Place
Marcelin-Berthelot, 75005 Paris, France
^ o whom correspondence should be addressed
Key words: galectin-I/human brain/lectin/size exclusion chromatography
The galectins are a family of fi-galactoside-binding mammalian lectins characterized by a highly conserved carbohydraterecognition domain (CRD) showing a characteristic set of
highly conserved amino acid residues (Caron et al., 1990; Barondes et al, 1994; Gabius, 1994; Kasai and Hirabayashi,
1996). Both intra- (Avellana-Adalid et al., 1994; Hubert et al.,
1995; Wang et al, 1995) and extracellular functions (Li et al,
1992; Fowlis et al, 1995; Ozeki et al, 1995) have been proposed for these lectins. The most common galectin having a
single CRD is galectin-1 (GAL1), which is thought to have
growth regulatory and immunomodulatory activities (Wells
and Malluci, 1991; Lutomski et al., 1995). The principal physiological roles of this protein in human remain unknown. It has
been suggested that they differ according to whether GAL1 is
predominantly monomeric or dimeric (PeriTlo et al, 1995).
The direct demonstration of an equilibrium between monomeric and dimeric forms of GAL1 is restricted to a report
showing that recombinant GAL1 from CHO cells occurs in an
active monomeric form that can reversibly dimerize (Cho and
Cumming, 1995). This work shows clearly that dimerization is
dependent on the concentration of galectin. This demonstration
is consistent with the observation that, for many oligomers in
which there are relatively weak attractive interactions between
the subunits, the dissociation into subunits can be accomplished by simple dilution. However, not all subunits in oligomers can be separated in this way. For oligomeric proteins
containing identical polypeptide chains, such as GAL1, in most
cases the formation of monomers from the oligomer requires
dissociating agents of sufficient strength that the tertiary structure of the monomers is disrupted along with the destruction of
the quaternary structure. Only rarely are the interchain interactions sufficiently weak and different in kind compared with
the intrachain interactions, that the dissociation of oligomers
into folded monomers can be achieved (Eisenstein and
Schachman, 1989).
Therefore, whether or not a monomer-dimer equilibrium
occurs for GAL1 in all mammalian species, and serves a functional roles, remains unknown. High resolution size-exclusion
chromatography provides an excellent means for separating
monomers from dimers according to the difference in their
sizes. We used a dextran-based composite gel (Sephacryl) for
determining the quaternary structure of recombinant human
GAL1 (rGALl) and of purified tissular (human brain) GAL1
(Avellana-Adalid et al., 1990). This matrix is convenient for
size exclusion HPLC of soluble proteins (LeMaire et al,
1980); and in experiments with oligomeric plant lectins it has
vin
Glyco-Forum section
amidomethylated rGALl) or on their free amino groups (biotiny lated rGALl), than with unmodified rGALl. Identical elution profiles were also obtained with native GAL1 isolated
from the human brain. The position of the peak detected by UV
adsorption was confirmed using biotinylated rGALl by determining the amount of biotinylated protein in each fraction. The
chromatography profiles obtained by this method were similar
to those obtained by UV adsorption and showed no peak which
could be interpreted as monomeric GALL Finally, we tested
the possibility that the salt concentration might affect the quaternary structure of the lectin though ionic bonds. The behavior
of rGALl was not modified by increasing the ionic strength
(0.5-1 M NaCl).
In summary, it is clear that the concept of an equilibrium
0.05-
0.04
O
,_ [rGALl] =
2QMM
(VI
O
O
E
c
CD
O
0.025 -
o
-
0.02
oh
0.5
sr^ri
0.75
Kav
been reported that results obtained using dextran-based media
were more reliable than those obtained using other HPLC media (Young and Jackson, 1984).
Typical elution profile obtained for rGALl and the calibration curve for molecular weight determinations are shown in
Figure 1. Size exclusion chromatography of human rGALl
yielded a fairly symmetrical peak with a calculated molecular
weight of about 26,000, consistent with those previously reported for dimeric human placenta GAL1 (Hirabayashi et at,
1987; Niambar et al., 1987). To investigate the possibility of
dimer dissociation of human GAL1, we prepared rGALl in
buffer at various concentrations. The diluted samples were kept
at 4°C for 24 h to promote equilibrium, and the HPLC profile
of each sample was examined. The galectin had similar chromatography profile independently of its concentration. Even at
low concentration (2mM) of rGALl a dimeric structure was
observed (Figure 2). Similar results were obtained with GAL1
derivatives modified either on their free thiol groups (carbox-
120
150 180
Minutes
0.04
120
150 180
Minutes
0.01
[rGALl ] - 2 \.
I
c
<~D
o
206
Fig. 1. Sephacryl S-200 size exclusion HPLC of affinity purified human
rGALl. A 100 pJ sample of rGALl containing 60 u.g of galectin was
loaded onto a 60 x 1.6 cm column equilibrated with 10 mM
Na2HPCyNaH2PO4, 150 mM NaCl, pH 7.5, 1 mM P-mercaptoethanol
buffer via injection loop and was eluted at 0.5 ml/mn. Effluent was
monitered ar 206 run. Inset calibration graph of molecular weight against
Kav. Kav, the coeficient describing the fraction of stationnary gel volume
available for diffusion of a given species, was calculated with the equation
Ka y = (Ve - VO)/(V, - Vo), where Ve is the elution volume of the protein,
V o is the void volume determined with blue dextran, and V, is the total
volume of the packed bed. Molecular weight standards: bovine serum
albumin, 67,000; ovalbumin, 43,000; chymotrypsinogen A, 25,000;
nbonuclease A, 13,700; tyrosine, 181. The arrow marks the Kav of rGALl.
-
C\J
Q
Q
O
-
\ ' '.
o
0.02 -
0.005
:•-
.1
.
120
150 180
Minutes
120
-
i
'•-._..
150 180
Minutes
Fig. 2. Size exclusion HPLC of human rGALl at different concentrations:
rGALl (50 u.M) was diluted in PBS-p mereaptoethanol to various
concentrations (50|iM - 2 jiM) and allowed to sit at 4°C for 24 h to
equilibrate. Samples (100 uJ) were injected onto a Sephacryl S-200 column
and elution was monitored by absorbance at 206 ran. Retention times were
calculated from the maximum of the peak.
IX
Glyco-Forum section
between monomers and dimers of GAL1 as a general phenomenon regulating its activity has to be questioned. As no data
shows a monomerization of human GAL1, one must be careful
before concluding that results on monomer-dimer equilibrium
obtained on a rodent model have relevance to human GALl.
Furthermore, several lines of evidence strongly support the
concept of a dimeric stable structure of human GALl. On
reducing SDS-PAGE, a faint band is often observed representing dimers of the protein that are not fully dissociated even
after heating in SDS and (3-mercaptoethanol. And in tissue and
cell extracts there is some evidences that GALl is not free but
associated with its biological partners to form high mass complexes. In light of the fact that multivalent interactions require
at least a bivalent lectin, it is likely that human GALl occurs
in the cytosol as a dimer. The same is expected to be true for
externalized galectin interacting with glycosylated cell surface
receptors. Dimeric GALl should be necessary to induce biological effects for which clustering of receptors is required
(Sharon, 1994).
Sharon.N. (1994) When lectin meets oligosaccharides. Struct. Biol, 1, 843845.
Wang.L., Inohara.H., Pienta.KJ. and Raz.A. (1995) Galectin-3 is a nuclear
matrix protein which binds RNA. Biochem, Biophys. Res. Commun., 217,
292-303.
Wells.V. and Mallucci.L. (1991) Identification of an autocrine negative growth
factor: mouse B-galactoside-binding protein is a cytostatic factor and cell
growth regulator. Cell, 64, 91-97.
Young,N.M. and Jackson.G.E.D. (1984) Anomalous behavior of lectins in
size-exclusion high-performance liquid chromatography and gel electrophoresis. J. Chromat., 336, 397-402.
Albert Neuberger (1908-96): founder of
modern glycoprotein research
References
Avellana-Adalid.V., Joubert.R., Bladier.D. and Caron.M. (1990) Biotinylated
derivative of a human brain lectin: synthesis and use in affinoblotting for
endogenous ligand studies. Anal. Biochem., 190, 26—31.
Avellana-Adalid,V., Rebel.G., Caron,M., Cornillot.J.D., Bladier.D. and
Joubert-Caron.R. (1994) Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differenciation. Glycoconjugate J., 11, 286-291.
Barondes.S.H., Cooper.D.N.W., Gitt,M.A. and Leffler.H. (1994) Galectins.
Structure and function of a large family of animal lectins. J. Biol. Chem.,
269, 20807-20810.
Caron.M., Bladier.D. and Joubert.R. (1990) Soluble galactoside-binding vertebrate lectins: a protein family with common properties. Int. J. Biochem.,
22, 1379-1385.
Cho.M. and Cummings.R.D. (1995) Galectin-1, a B-galactoside-binding lectin
in Chinese hamster ovary cells. 1. Physical and chemical characterization. J.
Biol. Chem., 270, 5198-5206.
Eisenstein.E. and Schachman.H.K. (1989) Determining the roles of subunits
in protein function. In Creighton.T.E. (ed.), Protein Function. IRL Press,
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Fowlis.D., Colnot.C, Ripoche.M.A. and Poirier.F. (1995) Galectin-3 is expressed in the notochord, developing bones, and skin of the postimplantation
mouse embryo. Dev. Dynam., 203, 241—251.
Gabius.H.J. (1994) Non-carbohydrate binding partners/domains of animal
lectins. Int. J. Biochem., 26, 469-477.
Hirabayashi,J., Kawasaki,H., Suzuki.K and Kasai,K.I. (1987) Further characterization and structural studies on human placenta lectin. J. Biochem., 101,
987-995.
Hubert,M., Wang.S.Y., Wang.J.L., Seve.A.P. and Hubert.J. (1995) Intranuclear distribution of galectin-3 in mouse 3T3 fibroblasts: comparative
analysis by immunofluorescence and immunodetection microscopy. Exp.
Cell Res., 220, 397^06.
Kasai.K.I. and HirabayashiJ. (1996) Galectins: a family of animal lectins that
decipher glycocodes. J. Biochem., 119, 1-8.
LeMaire.M., Rivas.E. and MollerJ.V. (1980) Use of gel chromatography for
determination of size and molecular weight of proteins: further cautions.
Anal. Biochem., 106, 12-21.
Li.W.X., Joubert-Caron.R., Eloumami.H., Bladier.D., Caron.M. and Baumann,N. (1992) Regulation of a beta-galactoside-binding lectin and potential ligands during postnatal maturation of rat brain. Dev. Neurosci., 14,
290-295.
Lutomski.D., Caron.M., Bourin.P., Lefebure.C, Bladier.D. and JoubertCaron.M. (1995) Purification and characterization of natural antibodies that
recognize a human brain lectin. J. Neuroimmunol., 57, 9—15.
Niambar.M.P., Basu.D. and Appukuttan,P.S. (1987) Physical properties and
binding-site amino acid residues of galactoside-binding protein of human
placenta. J. Biosci., 11, 331-338.
Ozeki.Y., Matsui.T., Yamamoto.Y., FunahashiJ., HamakoJ. and TitaniJC.
(1995) Tissue fibronectin is an endogenous ligand for galectin-1. Glycobiology, 5,255-261.
Perillo.N.L., Pace.K.E., SeilhamerJ.J. and Baum,L.G. (1995) Apoptosis of T
cells mediated by galectin-1. Nature, 378, 736-739.
Albert Neuberger, who passed away in London on August 14,
1996, was an outstanding scientist of enormous intellectual
stature, a distinguished biochemist, and an inspiring teacher.
He also rendered many services to the scientific community
and to society in his country, in Israel and elsewhere.
Sixty years ago Neuberger proved for the first time the existence of glycoproteins, by demonstrating that carbohydrate is
an integral part of a protein. Some 25 years later he identified,
together with his coworkers, the first carbohydrate-peptide
linking group. He thus laid the foundation for modern glycoprotein research. Neuberger also made seminal contributions to
other areas of biochemistry, primarily porphyrins and lectins.
Ovalbumin—the first identified glycoprotein
Most proteins are now known to be glycosylated, and the detection, purification, and characterization of glycoproteins pose
hardly any problems, even when available only in microgram