THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 15, Issue of April 9, pp.

15579 –15590, 2004

© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Molecular Recognition between Glyconectins as an Adhesion

Self-assembly Pathway to Multicellularity*
Received for publication, August 12, 2003, and in revised form, December 30, 2003
Published, JBC Papers in Press, December 30, 2003, DOI 10.1074/jbc.M308927200

Gradimir N. Misevic‡§, Yann Guerardel¶, Lazar T. Sumanovski储, Marie-Christine Slomianny ¶,

Maurice Demarty‡, Camille Ripoll‡, Yannis Karamanos**, Emmanuel Maes¶,
Octavian Popescu‡‡, and Gerard Strecker ¶
From the ‡Laboratoire des Processus Intégratifs Cellulaires, UMR 6037 CNRS, Faculté des Sciences et Techniques de
Rouen, 76821 Mont St Aignan Cedex, France, ¶Unité de Glycobiologie Structurale et Fonctionnelle, Université des
Sciences et Technologies de Lille, UMR 8576 CNRS, 59655 Villeneuve D’Ascq, France, 储Department of Research,
University Hospital of Basel, CH-4058 Basel, Switzerland, **Laboratoire de Biochimie Moléculaire et Cellulaire,
Université d’Artois, Faculté J Perrin, rue J. Souvraz, SP18, 62307 Lens, France, and ‡‡Molecular Biology Center and
Institute for Interdisciplinary Experimental Research, Babes-Bolyai University, 400006 Cluj-Napoca, Rumania

The appearance of multicellular forms of life has been The emergence of multicellularity required the simultaneous
tightly coupled to the ability of an organism to retain its development of cell adhesion and recognition properties. In
own anatomical integrity and to distinguish self from addressing the unresolved question of what may have been the

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non-self. Large glycoconjugates, which make up the out-
ermost cell surface layer of all Metazoans, are the primary
candidates for the primordial adhesion and recognition
functions in biological self-assembly systems. Atomic
force microscopy experiments demonstrated that the
binding strength between a single pair of Porifera cell
surface glyconectin 1 glycoconjugates from Microciona
prolifera can hold the weight of 1600 cells, proving their
adhesion functions. Here, measurement of molecular self-
recognition of glyconectins (GNs) purified from three
Porifera species was used as an experimental model for
primordial xenogeneic self/non-self discrimination. Phys-
icochemical and biochemical characterization of the
three glyconectins, their glycans, and peptides using gel
electrophoresis, ultracentrifugation, NMR, mass spec-
trometry, glycosaminoglycan-degrading enzyme treat-
ment, amino acid and carbohydrate analyses, and peptide
mapping showed that GNs define a new family of proteo-
glycan-like molecules exhibiting species-specific struc-
tures with complex and repetitive acidic carbohydrate
motives different from the classical proteoglycans and
mucins. In functional self-assembly color-coded bead, cell,
and blotting assays, glyconectins displayed species-
specific recognition and adhesion. Affinity-purified
monospecific polyclonal antibodies prepared against
GN1, -2, and -3 glycans selectively inhibited cell adhesion
of the respective sponge species. These results together
with species-specific coaggregation of GN carbohydrate-
coated beads with cells showed that GN glycans are func-
tional in cell recognition and adhesion. The specificity of
carbohydrate-mediated homophilic GN interactions in
Porifera approaches the binding selectivity of the evolu-
tionarily advanced immunoglobulin superfamily. Xeno-
selectivity of primordial glyconectin to glyconectin recog-
nition may be a new paradigm in the self-assembly and
non-self discrimination pathway of cellular adhesion
leading to multicellularity.
* This work was supported mainly by private funds (from G. N. M.)
and in part by the Conseil Régional Nord-Pas de Calais, CNRS, and the
University of Rouen and University of Lille. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 332-35-14-69-
08; Fax: 332-35-14-70-20; E-mail: gradimir@gradimir.com.
molecular basis for primordial self-recognition and non-self-
discrimination we focused our attention on the role of proteo-
glycan-like glyconectins in Porifera xenogeneic cellular inter-
actions, as the evolutionary most compatible model system for
ancestors of Metazoans. Electron microscopic, x-ray diffraction,
and biochemical analyses showed that large glycoconjugates
such as glyconectins, classical type proteoglycans and mucins,
are the largest multi-million molecular weight macromole-
cules, extending at least 10 times further from the cell surface
than any other cell adhesion glycoprotein (1–3). Based on this
evidence, here we put forward the hypothesis that Porifera
glyconectins, as the most peripheral cell surface environment
sensors, may have provided the initial key recognition and
adhesion functions during the emergence of Metazoan ances-
tors. This would imply that Porifera, as the simplest Metazoans
alive today, should have preserved, at least in part, a glyconec-
tin adhesion and recognition mechanism to guide the initial
phase of xenogeneic selectivity of cellular interactions. In fact,
in recent atomic force microscopy experiments we demon-
strated with purified molecules that the strength of glyconectin
1 to glyconectin 1 binding is fundamental in the cohesion be-
tween the cells of the sponge Microciona prolifera, ascribed to
these molecules in previous functional investigations (4 – 8).
To examine whether homophilic glyconectin to glyconectin
binding was also involved in primordial cellular adhesion and
recognition self-assembly processes associated with the emer-
gence of multicellularity, we studied the molecular basis of
specific xenogeneic cell interactions in the phylum Porifera.
Early work from the beginning of the 20th century on dissoci-
ated marine sponge cells provided important phenomenological
evidence for cell sorting (4 – 8). However, these and subsequent
experiments from 1960 to 1980 used semi-purified and chemi-
cally ill-defined extracts, termed aggregation factors, and thus
lacked quantitative and biochemical data about the underlying
molecular mechanisms (9 –12). In the last two decades, the first
structure-to-function-related studies have been performed on
M. prolifera glyconectins (1, 13–18). Here, we have isolated
glyconectins (GNs) from three marine sponge species, M. pro-
lifera (GN1), Halichondria panicea (GN2), and Cliona celata
(GN3). Physicochemical analyses and mass spectrometric fin-
gerprinting of the three purified glyconectin macromolecules
revealed species-specific proteoglycan-like structures that de-
fine a new class of glycocojugates. Using a quantitative func-

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15580 Molecular Recognition between Glyconectins
tional approach with color-coded beads, cells, and blotting, we
have shown that highly specific cell surface glyconectin to
glyconectin interactions indeed mediate cell recognition and
adhesion in the three Porifera species. Specific inhibition of cell
adhesion by three monospecific anti-GN carbohydrate poly-
clonal antibodies, together with species-selective coaggregation
of GN carbohydrate-coated beads with cells showed that GN
glycans from the three sponge species are functional in cell
recognition and adhesion. Thus, this study establishes a new
paradigm for the pivotal role of primordial glyconectin car-
bohydrates in the self-assembly and non-self discrimination
pathway of cellular adhesion prior to the evolution of
multicellularity.
EXPERIMENTAL PROCEDURES
Isolation of Glyconectin Proteoglycans—Glyconectins were extracted
from fresh cuts of sponges with artificial Ca2⫹- and Mg2⫹-free seawater
(ACMFSW;1 462 mM NaCl, 10.7 mM KCl, 7 mM Na2SO4, and 2.1 mM
NaHCO3) at ⫹4 °C for 12 h. Glyconectins were purified by sequential
centrifugation, 20 mM CaCl2 precipitation, and ultracentrifugation
(14, 15).
Ultracentrifugation Analysis—Molecular weight and sedimentation
coefficient of sponge glyconectins were determined by sedimentation
equilibrium and sedimentation velocity analyses in seawater-Tris
(SWT; 0.5 M NaCl, 2 mM CaCl2, 20 mM Tris, pH 7.4) at ⫹20 °C using a
Beckman Model E analytical Ultracentrifuge according to procedures
described previously (14, 15).
Agarose Electrophoresis—Electrophoretic separation of sponge gly-
conectins was performed on a 0.75% agarose gel in 50 mM Tris acetate,
pH 7.3, for 2 h at 100 V and 4 °C (13). Gels were stained with 0.02%
toluidine blue in 3% acetic acid followed by 0.1% Amido Black 10B in 3%
acetic acid.
Peptide Mapping of Glyconectins—GN1, GN2, and GN3 were reduced
with dithiothreitol and alkylated with acrylamide in the presence of
SDS 1%, 0.2 M Tris, pH 8.4 (19), and then digested by trypsin in 50 mM
ammonium bicarbonate at 30 °C for 12 h. Salts were removed on ZipTip
C18 (Millipore) and peptides analyze by mass spectrometry. Matrix-
assisted laser desorption ionization time-of-flight mass spectrometry
(MALDI-TOF MS) was performed on a “Voyager DE STR” time-of-flight
instrument (Applied Biosystems) equipped with a 337-nm UV laser.
The mass spectra were acquired in reflectron mode with positive detec-
tion and with 20 kV accelerating voltage. Aliquots (0.5 ␮l) of the elution
solution were mixed with an equal volume of the matrix solution,
␣-cyano-4-hydroxy-trans-cinnamic acid (10 mg/ml dissolved in acetoni-
trile-water, 50/50 by volume). External calibration was performed using
a peptide mix: des-Arg-bradykinin (904.4681 kD), angiotensin I
(1296.6853 kD), Glu-fibrinopeptide B (1570.6774 kD), ACTH(clip 1–17)
(2,093.0867 kD), and ACTH(clip 18 –39) (2,465.1989 kD).
Purification of Glyconectin Glycans—Purified glyconectins were pre-
cipitated with 70% ethanol and centrifuged at 2000 ⫻ g for 10 min. The
glyconectin pellet was resuspended again in 70% ethanol, and centrif-
ugation was repeated. Finally glyconectins were precipitated with a
water/chloroform/methanol (1/1/3 by volume) mixture and dried under
a N2 stream. Pronase (type E from Merck) was dissolved in 0.1 M Tris,
pH 8, 1 mM CaCl2 at a concentration of 1 mg/ml and preincubated for 20
min at 60 °C. Subsequently 1 ml of preincubated pronase (1 mg/ml) was
added to 20 mg of dried glyconectin pellet and incubated for 12 h at
60 °C. This proteolysis digestion procedure was repeated twice by add-
ing fresh aliquots of preincibated pronase. Released glycans were sep-
arated from free amino acids, salts, and small peptides by gel filtration
and subsequent ion exchange chromatography.
Preparation of Monospecific Polyclonal Antibodies against Glyconec-
tin Carbohydrates—Purified glyconectin carbohydrates were used for
preparation of polyclonal antibodies according to the procedure de-
scribed previously (14). Monospecific polyclonal antibodies were ob-
tained by affinity purification on GN glycans cross-linked to a solid
1
The abbreviations used are: ACMFSW, artificial Ca2⫹- and Mg2⫹-
free seawater; SWT, seawater-Tris; CSW, Ca2⫹- and Mg2⫹-free sea-
water buffered with Tris; GC/MS, gas chromatography-coupled mass
spectrometry; GN, glyconectin; EI-MS, electronic impact mass spec-
trometry; MALDI-TOF MS, matrix-assisted laser desorption ionization
time-of-flight mass spectrometry; Py(4,6)Gal, 4,6-O-(1-carboxyethyli-
dene)-␣-D-Galp; ACTH, adrenocorticotropic hormone; HPLC, high pres-
sure liquid chromatography.
matrix using previously a reported methodology of immobilization (13).
Immunoglobulins bound to GNs were eluted with 1 M NaCl and subse-
quently dialyzed against CSW (Ca2⫹- and Mg2⫹-free seawater buffered
with 20 mM Tris, pH 7.4). The monospecificity of these isolated anti-GN
carbohydrate antibodies to their respective glycan antigens was con-
firmed in immunodot assay (14).
Polyacrylamide Electrophoresis—Electrophoretic separation of puri-
fied glyconectin glycans was performed on a polyacrylamide gradient
gel (7.5–15%) at 350 V in 0.09 M Tris borate, pH 8.3, 2.4 mM EDTA (15,
20). Gels were stained with 0.3% alcian blue in 3% acetic acid and 25%
isopropanol solution.
Matrix-assisted Laser Desorption Ionization Time-of-flight Mass
Spectrometry of Oligosaccharides—The molecular mass of the oligosac-
charides was measured by MALDI-TOF MS on a Vision 2000 time-of-
flight instrument (Finnigan Mat) equipped with a 337 nm UV laser. 1
␮l of sample at a concentration of 100 pmol/␮l was mixed with an equal
volume of matrix and allowed to crystallize. We used 2,5-dihydroxyben-
zoic acid and 3-aminoquinoline for the neutral and acidic oligosaccha-
rides, respectively.
Electrospray Mass Spectrometry—All measurements were carried
out in positive-ion mode on a triple quadrupole instrument (Micromass
Ltd., Altrincham, UK) fitted with an atmospheric pressure ionization
electrospray source. A mixture of polypropylene glycol was used to
calibrate the quadrupole mass spectrometer. Underivated oligosaccha-
rides were dissolved in methanol/water (50/50) and permethylated oli-
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gosaccharides in acetonitrile at a concentration of 10 pmol/␮l. Samples
were infused using the nanoflow probe at 50 nl/min. Quadrupole was
scanned from 200 to 2000 Da with a scan duration of 3 s and a scan
delay of 0.1 s. The samples were sprayed using 1.4-kV needle voltage,
and the declustering (cone) was typically set at 70 V. For collision-
induced dissociation (CID) experiments, the pressure of argon in the
cell was set at 4 ⫻ 10⫺3 millibar and the collision energy was set to
values ranging from 25 to 75 eV.
NMR Spectroscopy—The NMR experiments were performed on
Bruker® ASX400 and DMX600 spectrometers, both equipped with a
5-mm 1H/13C mixed probe-head operating in the pulse Fourier trans-
form mode and controlled by an Aspect 3000 computer. Each glyconec-
tin sample was dissolved in 400 ␮l of 2H2O after three exchanges with
2
H2O (99.97% atom 2H, Euriso-top, CEA group, Gif-sur-Yvette France)
and intermediate lyophilizations. The glyconectins were analyzed at
300 K. The chemical shifts (␦) were referenced to internal acetone
(␦1H ⫽ 2.225 and ␦13C ⫽ 31.55 ppm under the conditions used). Two-
dimensional homonuclear (COSY90, TOCSY) and heteronuclear
(HMQC) experiments were performed by using standard Bruker® pulse
programs. The main pulses and variable delays were optimized for each
pulse program and sample.
Gas Chromatography—Monosaccharides were analyzed by GC/MS
as per-trimethylsilyl (21) and per-heptafluorobutyryl derivatives (22).
Methylation of oligosaccharides was conducted according to Ciucanu
and Kerek (23) before analysis on GC/MS.
Other Analytical Methods—The size of the oligosaccharides was as-
sessed by gel filtration on a Bio-Gel P2 or P4 column (Bio-Rad) equili-
brated in 0.5% acetic acid and calibrated with hydrolyzed dextran in
combination with thin layer chromatography as already described.
Amino acid composition was assessed by Pico-Tag HPLC and SO2⫺ 4
content by Dionex high pH anion-exchange chromatography, both after
6 M HCl hydrolysis at 100 °C for 12 h.
Preparation of Cells and Glyconectin-coated Beads—Sponge cells
were dissociated and washed in ACMFSW at 0 °C. In some experiments
cells were fixed with 1% glutaraldehyde (14, 15).
A volume of 200 ␮l of 2% latex-amidine bead suspension, 0.6 ␮m in
diameter (Molecular Probes, Inc., Eugene, OR), was washed three times
with 1 ml of SWT by centrifugation at 3000 ⫻ g for 10 min. Beads were
resuspended in 200 ␮l of SWT and bath-sonicated for 5 min. To allow
adsorption to beads on the basis of charge, 100 ␮g of either glyconectins
or glycans isolated from these glyconectins was added to a 100 ␮l of
bead suspension that was then incubated at 22 °C for 15 min followed
by three washes as described above. The glyconectin-coated beads were
resuspended in 100 ␮l of SWT. Because the diameter of the beads is 600
nm and the size of glyconectins are in the same range, each bead bound
at least 1 molecule of glyconectin and about 20 –50 large glycan
molecules.
Recognition, Adhesion, and Overlay Assays with Cells and Beads—
Cell-cell adhesion assays were performed with 106 cells bearing surface
glyconectins in 100 ␮l of ACMFSW, without or with 10 mM Ca2⫹, in the
presence or absence of anti-GN-glycan polyclonal antibodies. For glut-
araldehyde-fixed cells assays were done in SWT with or without Ca2⫹.
For the cell-bead assay, 5–10 ␮l of 2% bead suspension in SWT was
 Molecular Recognition between Glyconectins 15581
TABLE I
Physicochemical properties of sponge glyconectins
Molecular weight (Mr) and sedimentation coefficient (s20,w) of sponge
GNs were determined by analytical centrifugation. The carbohydrate
and amino acid composition (mol %) of glyconectins were obtained from
gas chromatography and HPLC, respectively. Carbohydrate/protein ra-
tio, content of uronic acids, and SO2⫺
4 were calculated from amino acid,
monosaccharide, and ion composition. The standard deviation from
three analyses was maximally 1% of each value (see “Experimental
Procedures”).
GN1 GN2 GN3

Mr ⫻ 10 6
19 10 8
s20,w (S) 58 42 46
Carbohydrate/protein ratio (w/w) 63/37 21/79 36/64
Monosaccharides (mol %)
Ara 0 0 11
Fuc 28 7 31
Man 9 16 20
Gal 29 43 12
GalNAc 1 1 7
GlcNAc 17 14 13
Py(4,6)Gal 4 16 0
GlcA 11 3 6
Amino acids (mol %)
Asx 9.1 7.8 11.1 FIG. 1. Electrophoretic separation of sponge glyconectins. A,
0.75% agarose gel stained with 0.02% toluidine blue followed by 0.1%

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Glx 10.4 9.2 9.5
Ser 6.7 7.0 11.3 Amido Black 10B. a– c, GNs from M. prolifera GN1, H. panicea GN2,
Gly 9.0 9.9 10.9 and C. celata GN3, respectively (10 ␮g each). B, 0.75% agarose gel
Arg 5.6 7.6 6.0 stained with color-coded fluorescent beads coated with GN1 (pink) (a),
Thr 9.9 10.2 14.1 GN2 (green) (b), and GN3 (blue) (c) in the presence of SWT with 10 mM
Ala 7.1 7.0 7.7 CaCl2.
Pro 7.3 8.2 10.7
Tyr 2.8 4.0 0.7 at first visualized by gas chromatography as methyl-glycoside
Val 9.2 8.6 6.1 heptafluoryl derivative as two peaks tentatively attributed to
Met 2.4 2.5 2.4 its ␣ and ␤ anomers. This structure was confirmed as a pyru-
Cys 0.1 0.1 0.2
Ile 5.4 6.0 3.9 vated hexose in methyl-glycoside heptafluoryl derivative be-
Leu 7.8 5.5 5.1 cause of a [M ⫹ NH4] ion at m/z 698 in chemical ionization
Phe 5.1 4.8 3.6 mass spectrometry and a very intense [M ⫺ COOMe] ion at m/z
Lys 0.2 0.2 0.1 611 in EI-MS (Fig. 2) (27). The exact nature of this residue was
SO2⫺ (mol/mol) 820 620 700
4
established as 4,6-O-(1-carboxyethylidene)-␣-D-Galp (Py(4,6)
Gal) by trimethylsilyl derivatization of methylglycoside and
added to 100 ␮l of cell suspension containing 106 cells in the presence or comparison with standard EI-MS spectra from the literature
absence of 10 mM Ca2⫹. In bead overlay assays 50 –100 ␮l of 2% bead (Fig. 2) (27). Indeed, spectra was dominated by an ion at m/z
suspension in SWT was added to nitrocellulose membrane or agarose 243, characteristic of a 4,6-O-(1-carboxyethylidene)-D-galactose
gel equilibrated in 1 ml of CSW in the presence of 10 mM Ca2⫹. structure (27), and by an intense [M ⫺ COOMe] at m/z 363.
The degree of cell-cell and bead-bead adhesion in the presence of The intense ion at m/z 204, resulting from the cleavage of two
variable calcium or magnesium ion concentrations or anti-GN-glycan
adjacent trimethylsilyl groups, also characterized the 4,6-link-
polyclonal antibodies was quantified after a 20-min rotation either by
counting single cells and aggregates of at least 40 ␮m in diameter or by age position of the pyruvate group. Subsequently, this attribu-
spectrophotometrically measuring the absorbance of supernatants after tion was confirmed by NMR experiments of glyconectin hydrol-
sedimentation at 100 ⫻ g for 1 min. ysis fragments from GN2 (see accompanying article (40)). This
is in agreement with previous work that identified a
RESULTS Py(4,6)Gal-containing oligosaccharide obtained by hydrolysis
Purification and Physical Characterization of Glyconectins of of glyconectin from M. prolifera (28). The stability of the 4,6-
Three Sponge Species—GNs were purified by CaCl2 precipita- O-(1-carboxyethylidene)-galactose pyranose ring to methanol-
tion and sequential ultracentrifugation of isotonic Ca2⫹-free ysis (27) enabled us to estimate, among the other monosaccha-
seawater extracts from three different marine sponge species, rides, the amount of this particular component in each sample
M. prolifera (GN1), H. panicea (GN2), and C. celata (GN3). This (Table I).
procedure is similar to the one we originally used for M. pro- As shown in Table I, glyconectins from the three analyzed
lifera and is thus different from guanidinium hydrochloride or species showed differences regarding their sugar compositions.
urea extraction techniques applied for classical mammalian GN1 was characterized by a high quantity of fucose (about
proteoglycans (24 –26). Analytical ultracentrifugation and aga- one-third of the total monosaccharide content) and minute
rose electrophoresis of isolated glyconectins showed that each amounts of Py(4,6)Gal, whereas GN2 was characterized by a
species expressed a unique type of multi-million macromole- lower proportion of fucose and a molar ratio of Py(4,6)Gal four
cule with sedimentation greater then 40 S composed of one or times higher than GN1. On the other hand, GN3 was the only
few highly negatively charged glycosylated subunits (Table I one to contain arabinose (11 mol %) as well as high quantities
and Fig. 1). of fucose but no Py(4,6)Gal at all. This last species was also the
Carbohydrate and Amino Acid Compositional Analysis of only one to exhibit significant amounts (⬎5%) of GalNAc.
Native Glyconectins—The three purified native glyconectins Amino acid analysis of three sponge glyconectins showed
were analyzed for their sugar compositions by gas chromatog- species-specific differences with some common features such as
raphy (Table I). Common monosaccharides, Fuc, Ara, Man, the presence of over 7% serine, threonine, and asparagine/
Gal, GlcNAc, GalNAc, GlcA, and a single type of pyruvated aspartic acid (Table I).
hexose were found in various amounts. Pyruvated hexose was Peptide Mapping of Native Glyconectins—Native GN1, GN2,
15582 Molecular Recognition between Glyconectins
FIG. 2. EI-MS identification of 4,6-
O-(1-carboxyethylidene)-D-galactose.
A, trimethylsilyl derivative. B, permeth-
ylated derivative.
and GN3 were reduced with dithiothreitol, alkylated with ac-
rylamide in the presence of SDS, and subsequently treated
with trypsin and analyzed by mass spectrometry as described
under “Experimental Procedures.” All three glyconectins had
different peptide maps, indicating distinct core structures and
species-specific sequences (Fig. 3). Detailed analyses of MS
spectra showed that GN1 has 67, GN2 84, and GN3 44 pep-
tides. A comparison of GN1, GN2, and GN3 tryptic fragment
masses revealed 10 peptides with the same mass. Whether
these peptides are identical would have to be verified by direct
sequencing. Furthermore, the cloning and sequencing of GN2
and GN3 genes for their respective core proteins will show the
exact differences to the already cloned gene of GN1 (16, 18).
Purification and Biochemical Analyses of Glyconectin Gly-
cans—Purified glyconectins GN1, -2, and -3 were extensively
treated with pronase, as described under “Experimental Pro-
cedures,” to digest the protein part. After removal of small
peptides and amino acids by gel filtration and ion-exchange
chromatography, glycans of GN1, -2, and -3 were analyzed for
their monosaccharide and amino acid contents (Table II). Mon-
osaccharide analysis of the total GN glycans showed a compo-
sition almost identical to the native GNs, with the exception of
GN3, where the ratio of fucose and GlcNAc were different.
Recovery of total carbohydrates was more than 90%. These
data indicated that there was neither selective nor quantita-
tively significant loss of carbohydrates during pronase treat-
ment. Amino acid analyses showed that glycans are essentially
free of protein excluding the linkage and their neighboring
amino acids (Table II). Sulfate analyzes of the three GN total
glycan fractions revealed over 90% recovery from the intact
GNs.
A study of glyconectin glycan heterogeneity after pronase
treatment was performed by polyacrylamide gel electrophore-
sis (Fig. 4). Staining of gels by alcian blue revealed that GN1
has two major species of acidic glycans with molar masses of
200 and 6 kDa as reported previously (14, 15, 18, 29). GN2 had
one major large acidic glycan type with a molar mass of 180
kDa, representing more then 60% of the total carbohydrate
content, and heterogeneous populations of acidic oligosaccha-
rides with sizes below 10 kDa very faintly stained by alcian
blue because of the larger area spread (Fig. 4). The amount of
this glycan was more closely estimated after gel filtration on a
Bio-Gel P6. GN3 also contained one major acidic glycan species
(50% total carbohydrates) with a molar mass of 110 kDa and a
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size-heterogeneous population of smaller glycans shown tailing
from the major band (Fig. 4). According to the size, composition,
and fingerprinting (presented in the accompanying article (40))
the two large glycans of GN2 and GN3 should be built by
repetitive units as in the case of GN1 (14 –16, 18, 29).
One- and two-dimensional homonuclear NMR (COSY90,
TOCSY) and heteronuclear (HMQC) spectroscopy of three GN
total glycans indicated new types of species-specific repetitive
acidic polysaccharide structures with a high degree of complex-
ity (Fig. 5). These sequences were established by mass spec-
trometry analyses (reported in the article (40)). The large size
and great complexity of the GN glycans do not allow identifi-
cation of the complete arrangement of these sequences within
the polymer. It was nevertheless possible to confirm that fucose
is represented more in GN1 and GN3 than in GN2 glycan, as
shown by the intensity of the methyl signal resonances on the
one-dimensional spectrum (Fig. 5A). The GN2 glycan spectra
showed an intense signal relative to the methyl group of the
pyruvate unit. This signal was significantly smaller in GN1
and was completely absent in GN3. The one-dimensional NMR
spectra of the GN glycans also indicated the presence of ␣ and
␤ anomeric protons. In the case of GN1 glycans, these were
assigned as ␣Fuc, ␣Gal, ␤Gal, and ␤GlcNAc. For GN2 glycans,
␤ anomers were predominant. The ␣ or ␤ anomericity was
confirmed by two-dimensional NMR, based on the characteris-
tic H-2 chemical shifts (Fig. 5B). Moreover, the H-3 and H-4
signals of a ␤Gal unit with a sulfate group at the C-3 position
were easily assigned in the case of GN1, whereas the spin
system of pyruvated ␤Gal clearly appeared in the COSY spec-
trum of GN2. The NMR spectra of GN3 indicated that the
distribution of anomeric signals, the absence of pyruvate, and
the presence of pentose could be attributable to novel type of
sequences. These NMR results were in agreement with the
mass spectrometry fingerprinting of glycan sequences pre-
sented in the following article. Isolation of large quantities of
each glycan fragment and comparative NMR analyses with the
intact polysaccharides would allow construction of the overall
sequence maps of glyconectin glycans.
Enzymatic treatment of either intact glyconectins or their
isolated glycans with different types of glycosaminoglycan-de-
grading enzymes such as hyaluronidases, chondroitinases, and
heparinases were not able to degrade any of GN1, -2, and -3
glycans under conditions that completely digest the respective
enzyme substrates. In conclusion, NMR and enzymological
 Molecular Recognition between Glyconectins 15583

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FIG. 3. Peptide mapping of glyconectins by mass spectrometry. Tryptic peptide maps of GN1 (top), GN2 (middle), and GN3 (bottom) are
shown.
15584 Molecular Recognition between Glyconectins
TABLE II
Monosaccharide composition of GN glycans
Carbohydrate and amino acid composition (mol %) of glyconectin glycans were obtained from gas chromatography after methanolysis and HPLC
after HCl hydrolysis, respectively, as described under “Experimental Procedures.” Carbohydrate/protein ratios and content of uronic acids and
SO2⫺
4 were calculated from amino acid and monosaccharide composition. The standard deviation from three analyses was maximally 1% of each
value (see “Experimental Procedures”). The total amount of SO2⫺
4 recovered in GN glycans after pronase treatment of each GN was more than 90%.

GN1 glycans GN2 glycans GN3 glycans

mol % mol % mol %

Monosaccharides (mol %)
Ara 0 0 7
Fuc 21 5 18
Man 6 13 13
Gal 30 37 16
GalNAc 2 0 8
GlcNAc 23 15 32
Py(4,6)Gal 8 26 0
GlcA 10 4 6
Amino acids (mol %)
Asx 39 33 35
Glx 22 20 25
Ser 9 12 13
Gly 15 16 13
Arg 0 0 1
Thr 10 12 10
Ala 2 3 1

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Pro 0 0 0
Tyr 0 0 0
Val 0 0 1
Met 0 0 0
Cys 0 0 0
Ile 0 0 0
Leu 2 3 1
Phe 1 1 0
Lys 0 0 0
Monosaccharide/amino acid ratio (mol %) 97/3 94/6 95/5
SO2⫺
4 (moles recovered from intact GN) 750 600 650

data together with compositional and electrophoretic analyses
specify that three GN carbohydrates have a novel type of spe-
cies-specific and repetitive structure residing in the large acidic
polysaccharides. More detailed chemical sequence analyses of
GN glycans combined with NMR and mass spectrometry are
described in the accompanying article (40).
The linkage type of GN glycans to the protein core was
studied using a chemical and enzymological approach. We an-
alyzed the total glyconectin glycans using the method of Maes
et al. (30), which shows that classical methanolysis conditions
quantitatively cleave the N-glycosidic bond (96%), liberating
glucosamine (and not its O-methylglycosides). Because other
N-acetyl-D-glucosamine (GlcNAc) residues are quantitatively
liberated as the O-methylglycosides of glucosamine, the Glc-
NAc residue involved in the N-glycosidic bond is separated
from the others using gas chromatography of heptafluorobu-
tyrate derivatives. GC/MS indicated that all three GNs have
N-linked glycans. Because of the large size of the glycans, the
great amounts of internal GlcNAc, and the comparatively small
amount of possible linkage GlcNAc, gas chromatography and
mass spectroscopy data of GN-type molecules cannot be abso-
lutely quantitative in terms of the exact number of moles of
linkage GlcNAc but rather are of a qualitative nature. Classical
alkali treatment of intact glyconectins was able to only par-
tially release glycans (about 30 –50%), as in the classical exam-
ples of N-linked glycans. Peptide-N-glycanase was not able to
release any of the large glycans but was able to release the
small 6-kDa glycan in the case of GN1 as reported previously
(29). Most likely peptide-N-glycanase did not cleave because of
the well known steric inaccessibility of the linkage due to the
high charge and large size of the glycans. Hydrazinolysis of
intact GNs under standard conditions released glycans; how-
ever, these conditions are now known to cleave both N-linked
and O-linked glycans. Gas chromatography coupled to mass
spectroscopy analyses and alkali treatment of GNs indicate
that the large glycans of GN2 and -3 are N-linked to the protein
core as in the case of both GN1 glycans (16, 18, 29). To confirm
the nature of the glycan-protein linkage, a more quantitative
analysis of the isolated linkage region must be made. This will
require a new methodology to take into account the spacing of
glycan chains, their internal sequences and charge distribu-
tion, as well as the peptide sequences of the core protein (which
greatly influence enzymatic and chemical cleavage). Our struc-
tural data of GN polysaccharides presented in the accompany-
ing article (40) promote the development of sequencing tech-
niques applicable to novel classes of glycans. Such potential
methods, when used on the purified GN fragments close to the
core protein together with cloning of the GN genes, will defin-
itively confirm the exact nature of GN glycan core and linkage
to protein.
Taking in account the amount of carbohydrate present per
intact glyconectin, the quantity of each individual glycan spe-
cies recovered from the respective glyconectin, the size of the
glycans, and the size of the intact glyconectins, a rough esti-
mation was made that: 1) GN1 would have 20 chains of 200
kDa and 950 chains of 6 kDa glycans as discussed previously
(14, 15, 18); 2) GN2 would have 10 repeats of 180 kDa glycan
and 300 glycans with an average size of 6 kDa; and 3) GN3
would have 13 copies of 110 kDa glycans. The rest of the
size-heterogeneous population of GN3 polysaccharides showing
tailing from the 110 kDa glycan band on the gel is most likely
either the result of degradation or the unfinished synthesis
product of this major glycan species (see accompanying article
(40)). We cannot completely exclude the possibility that this
product is also mixed with some less abundant yet undefined
polysaccharide species.
Glyconectin Glycoconjugates are Cell Adhesion Molecules—
The cell adhesive function of two new sponge glyconectins
purified from H. panicea (GN2) and C. celata (GN3) was tested
and compared with that of M. prolifera (GN1) in a rotary
 Molecular Recognition between Glyconectins
FIG. 4. Polyacrylamide gel electrophoresis of purified gly-
conectin glycan fraction. Electrophoresis of glyconectin glycans was
performed on a polyacrylamide gradient gel (7.5–15%). Gels were
stained with 0.3% alcian blue in 3% acetic acid in aqueous 25% isopro-
panol. Lane a, 20 ␮g of GN1 glycans; lane b, 20 ␮g of GN2 glycans; lane
c, 20 ␮g of GN3 glycans; lane d, 50 ␮g of hyaluronic acid (Sigma, from
bovine vitreous humor), 200 kDa, partially degraded; lane e, 50 ␮g of
chondroitin sulfate (Sigma, from shark cartilage), 80 kDa.
reaggregation assay with live metabolically attenuated and/or
fixed cells depleted of endogenous GNs. All three glyconectins,
at concentrations mimicking in vivo conditions, mediated cell
adhesion in the presence of physiological seawater with 10 mM
CaCl2 and not below 1 mM CaCl2 (Fig. 6A). In the absence of
GNs, independently of CaCl2 concentration, no aggregation
could be observed (data not shown). Magnesium ions could not
replace Ca2⫹ (Fig. 6D). These data indicate the essential and
specific role of the three tested glyconectins and Ca2⫹ in sponge
cell adhesion.
Ca2⫹-dependent Glyconectin to Glyconectin Interactions Me-
diate Cell Adhesion—We have previously shown by atomic
15585
force microscopy and glyconectin-coated bead adhesion that
Ca2⫹-dependent self-interactions between glyconectin 1 from
M. prolifera sponge provides the major driving force for cell
adhesion (1, 14, 15). To quantitatively test whether glyconec-
tins 2 and 3 from sponges H. panicea and C. celata also utilize
the same adhesive molecular mechanism via Ca2⫹-dependent
glyconectin to glyconectin interactions, we used two assay sys-
tems, Ca2⫹-induced aggregation of glyconectin-coated beads
(Fig. 6B) and glyconectins self-interaction monitored by gela-
tion (Fig. 6C). All three glyconectins revealed 100% self-asso-
ciation at 10 mM CaCl2 and no interactions in the absence of
calcium ions in either assay system (Fig. 6, B and C; see also
bead recognition assay in Figs. 8 and 9). CaCl2 could not be
substituted with MgCl2 indicating a specific role of Ca ions
(Fig. 6, E and F). Titration experiments of Ca2⫹ concentration
dependence of sponge glyconectin self-interactions revealed a
transition at 5 mM and 100% interactions at physiological 10
mM CaCl2 (Fig. 6, B and C), identical to that of Ca2⫹-dependent
glyconectin-promoted cell adhesion (Fig. 6A). These experi-
ments indicated that Ca2⫹-dependent glyconectin to glyconec-
tin interactions play a pivotal role in the cell adhesion of the
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three selected marine sponge species.
Specific Glyconectin to Glyconectin Interactions Mediate Po-
rifera Cellular Recognition and Adhesion—To test whether
cell-adhesive glyconectin-glyconectin interactions can also me-
diate xenogeneic recognition in sponges, we performed ex vivo
color-coded cell-cell, bead-bead, and bead-surface recognition
experiments. This type of approach is essential to understand-
ing the molecular mechanism underlying previously reported
phenomenological and biochemical studies on the role of pro-
teoglycan-like glycoconjugates in binary assays of dissociated
sponge cells (4 –12).
First, we tested the specificity of adhesion of GNs bearing
cells in a ternary species combination. Living dissociated and
metabolically attenuated cells in ACMFSW (at 0 °C) from M.
prolifera (Fig. 7A), H. panicea (Fig. 7B), and C. celata (Fig.
7C) were mixed in the presence (Fig. 7E) and absence (Fig.
7D) of 10 mM Ca2⫹ (physiological concentration in seawater).
In a rotary assay, within 5–15 min species-specific recogni-
tion and adhesion occurred only with 10 mM Ca2⫹ (Fig. 7E).
Fig. 7D shows a mixture of dissociated cells before CaCl2
addition. Upon removal of GNs from cell surface by repetitive
washing, none of the three species displayed aggregation in
the presence of 10 mM Ca2⫹ (results similar to that of Fig.
7D). Adding back the purified GNs to the same live cells at
0 °C completely restored species-selective cohesion (results
similar to that of Fig. 7E). Similar results were obtained with
non-living fixed cells (not shown). These experiments indi-
cated that glyconectins and Ca2⫹ mediate the initial steps of
xenogeneic cell recognition and adhesion of the three selected
sponge species.
In the second type of recognition assay we reconstituted the
observed cell recognition by using artificial system of glyconec-
tin color-coated beads. Glyconectin 1 was attached by adsorp-
tion to fluorescent pink, glyconectin 2 to fluorescent green, and
glyconectin 3 to fluorescent blue latex-amidine beads. Unla-
beled glyconectins were immobilized on a nitrocellulose mem-
brane in such a manner that the three molecules were used to
draw the subsequent letters of the words “GLYCONECTIN
RECOGNITION.” The three bead types were mixed and added
to the coated membrane in the presence of 10 mM CaCl2 (Fig.
8B) or absence of calcium ions (Fig. 8A). Within 5 to 15 min of
constant rotation, species-specific bead-bead aggregation and
homophilic recognition between membrane-bound and bead-
bound glyconectins were observed through three separate color
aggregates and selective staining of each letter only with 10 mM
15586 Molecular Recognition between Glyconectins

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FIG. 5. NMR analyses of glyconectin glycans. A, one-dimensional spectra of GN1 glycans (top), GN2 glycans (middle), and GN3 glycans
(bottom). B, two-dimensional COSY90 spectra of GN1 (top), GN2 (middle), and GN3 (bottom).

CaCl2 (Fig. 8B). Both processes occurred at apparently similar white beads, as expected, mixed-color aggregates were formed
rates for each of the three glyconectins. In control experiments upon the addition of 10 mM CaCl2 (not shown). In the absence
with glyconectin 1 separately attached to pink, yellow, and of 10 mM CaCl2, bead aggregation did not occur either in the
 Molecular Recognition between Glyconectins 15587

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FIG. 6. Glyconectin- and calcium ion-dependent cell aggregation, glyconectin-coated bead adhesion, and self-interaction of
glyconectins. A and D, cell aggregation promoted by GNs was performed with glutaraldehyde-fixed cells in CSW supplemented with Ca2⫹ ranging
from 0 to 20 mM or Mg2⫹ ranging from 0 to 50 mM as described (13, 14). B and E, GN-coated bead aggregation assay was done in CSW supplemented
with Ca2⫹ ranging from 0 to 20 mM or Mg2⫹ ranging from 0 to 50 mM as described under “Experimental Procedures.” C and F, self-interaction assay
was performed with 10 ␮g of GNs in 100 ␮l of CSW supplemented with Ca2⫹ ranging from 0 to 20 mM or Mg2⫹ ranging from 0 to 50 mM. After a
20-min incubation at room temperature samples were centrifuged at 5000 ⫻ g, and pellet and supernatant were analyzed in a colorimetric and
immunodot assay for the presence of GNs. A–C, CaCl2 range from 0 to 20 mM. D–F, MgCl2 range, 10 to 50 mM. All three assays used similar
conditions for self-interaction of glyconectins.

mixture of the three glyconectins or in one glyconectin coated to
three colored beads (same as Fig. 8A).
A similar type of experiment was performed by overlaying
agarose gel containing three electrophoretically separated gly-
conectins with color-coated glyconectin beads. After overnight
incubation at room temperature in the presence of 10 mM CaCl2
under gentle agitation, species-specific staining of gel glyconec-
tin bands identical to the ones stained with toluidine blue and
Amido Black showed that glyconectin to glyconectin interac-
tions are highly species-specific (Fig. 1).
The combinations of the above described experiments dem-
onstrate species-specific molecular self-recognition of glyconec-
tins in an elementary reconstituted bead adhesion system,
which fully resembles glyconectin-mediated cell-cell recogni-
tion and adhesion. Thus, glyconectins mediate self- and non-
self-discrimination in the initial step of sponge cell adhesion
and xenogeneic recognition. The degree of GN selectivity is
analogous to that of antibody-antigen interactions in similar
assay systems.
Self-assembly of a Biological and Artificial Cell-Bead System
via Species-specific Glyconectins Recognition—To evaluate the
exclusive role of glyconectins in self-recognition we tested spec-
ificity of homophilic glyconectin-glyconectin interactions in a
color-coded cell-bead adhesion assay. Each of the three gly-
conectins was attached to color-coded beads, as in the above
described experiments, and beads were mixed in nine combi-
nations with glyconectin-bearing cells of the three sponge spe-
cies in the presence of 10 mM Ca2⫹. To simultaneously follow
cells and beads, visible light microscopy was used. In visible
light, fluorescent pink beads with glyconectin 1 appear as pink,
fluorescent green beads with glyconectin 2 are yellow, and
fluorescent blue beads with glyconectin 3 are white. Bead-cell
coaggregation was observed for homotypic combinations, as
observed microscopically by the color and shape of uniformly
mixed beads and cells (Fig. 9, A, E, and I). The heterotypic
combinations always resulted in bead and cell segregation (Fig.
9, B, C, D, F, G, and H). Cell-bead recognition and adhesion
occurred at apparently equal rates and ended in similar aggre-
gate sizes for all three species. Such recognition took place only
when cells had glyconectin on their surfaces and in the pres-
ence of 10 mM CaCl2. This process did not require live cells
because the same results were obtained with glutaraldehyde-
fixed and/or metabolically attenuated cells at 0 °C (data not
shown). Self-assembly experiments with live cells, metaboli-
cally attenuated cells, fixed cells, and non-natural latex beads
showed that, independently of the carrier surface, glyconectins
are able to recognize self and distinguish self from non-self via
cohesive glyconectin to glyconectin interactions.
Glyconectin Carbohydrates Mediate Cell Recognition and Ad-
hesion—The final question was to examine whether glyconec-
tin carbohydrates are involved in cell adhesion and recognition
processes during reaggregation of Porifera cells. The facts that
GNs are carbohydrate-rich molecules and that GN1 glycans
specifically mediate cell adhesion in M. prolifera led us to test
for the cell adhesive function of GN2 and GN3 carbohydrates
using two different approaches. In the first immunological
method, we prepared affinity-purified monospecific polyclonal
antibodies against isolated GN1, -2, and -3 total glycans, es-
sentially free of proteins (see Table II), as described under
“Experimental Procedures.” Then we examined whether such
anti-GN-glycan antibodies can specifically inhibit cell aggre-
gation of the three respective sponge species. The results
presented in Fig. 10A showed that each of the three anti-GN-
glycan polyclonal antibodies, anti-GN1-glycan, anti-GN2-gly-
can, and anti-GN3-glycan, species-selectively inhibited cellular
adhesion in the presence of 10 mM CaCl2 of the respective
GN-bearing cells. In the second direct approach, we coated
preferentially large GN glycans, which were shown to be es-
sentially free of proteins (see Table II), to color-coded latex-
amidine beads using the same adsorption procedure as de-
scribed for the intact GNs. The adsorption is based on charge
interactions, which in high salt concentrations are much
15588 Molecular Recognition between Glyconectins
FIG. 7. Glyconectin glycoconjugates are cell adhesion and rec-
ognition molecules. Ca2⫹-dependent glyconectin to glyconectin inter-
actions mediate species-specific cell-cell recognition and adhesion. A–C,
M. prolifera (A), H. panicea (B), and C. celata (C) living sponges. Shown
are self- and non-self-discrimination and adhesion in the suspension of
mixed M. prolifera (orange), H. panicea (white), and C. celata (brown)
live cells bearing glyconectins. D and E, ACMFSW without 10 mM Ca2⫹
(D) and ACMFSW with 10 mM Ca2⫹ at 0 °C after 20 min of rotation (E).
The microscopically observed color of the cells is somewhat different
from that of the whole sponge. Early cell sorting experiments were
usually done with binary sponge combinations at room temperature
without rotation. The sorting is thus dependent on the presence of
recognition molecules at the cell surface, cell motility, and speed of new
synthesis and/or secretion of additional recognition molecules. Our ro-
tary assays using either metabolically attenuated or fixed cells reduce
the number of variable parameters.
greater in the case of the larger glycans. We verified, by gel
electrophoresis of the material coated to beads and the material
remaining in supernatants, that beads were coated principally
with the large glycans and not with the small ones. Of course,
in the case of a long glycan chain, many of its sites are not
bound to the surface of the bead and are therefore free to
interact with other molecules. The size of a single bead surface
(0.6 ␮m diameter) can accommodate either one GN molecule or
a roughly similar number of large glycan molecules as found in
a single intact GN molecule. Because polyvalency of adhesion
epitopes in GN1 was shown to be an imperative for its species-
specific cell adhesive function (14, 30, 33, 35), valency recon-
stitution experiments, performed by coating multiple copies of
large glycans on 0.6-␮m diameter latex beads, were necessary.
These reconstituted polyvalent GN-glycan-beads were tested in
coaggregation assays with cells using the following combina-
tions: M. prolifera cells bearing glyconectin 1 with GN1-glycan-
beads, GN2-glycan-beads, or GN3-glycan-beads; H. panicea
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FIG. 8. Simultaneous species-specific glyconectin to glyconec-
tin recognition in suspension and blotting assay. Letters were
drawn using 4 ␮l of 1.5 mg/ml glyconectins on a Hybond-C extra
nitrocellulose membrane (Amersham Biosciences) and probed in SWT
with pink, green, and blue fluorescent beads coated with glyconectin 1,
2, and 3, respectively. A, SWT without 10 mM Ca2⫹. B, SWT with 10 mM
Ca2⫹. All photographs were taken after 30 min of mixing.
cells bearing glyconectin 2 with GN1-glycan-beads, GN2-gly-
can-beads, or GN3-glycan-beads; and C. celata cells bearing
glyconectin 3 with GN1-glycan-beads, GN2-glycan-beads, or
GN3-glycan-beads (Fig. 10B). The results presented in Fig. 10B
demonstrated that only homotypic coadhesion of GN-glycan-
beads with cells occurred (images of homotypic coadhesion are
similar to those shown in Fig. 9, A, E, and I). The results of both
experimental approaches indicated that species-specific carbo-
hydrates of GNs are directly involved in Porifera cell recogni-
tion and adhesion, which may be implicated in xenogeneic
self-assembly and the non-self-discrimination pathway of cel-
lular interactions leading to multicellularity.
DISCUSSION
In 1987 it was demonstrated that the isolated cell surface
macromolecule from the M. prolifera sponge, then termed the
aggregation factor, mediates cell adhesion via a novel type of
carbohydrate-carbohydrate self-interactions (14). The distinct
functional and physicochemical properties of this compound
suggested that although it shares the general features of pro-
teoglycans such as large size and long, sulfated, negatively
charged glycans, it could define a new class of primordial cell
recognition and adhesion proteoglycan-like glycoconjugates,
named glyconectins. Recently reported partial sequence anal-
ysis of the carbohydrate moiety of the M. prolifera glyconectin
1 demonstrated new structural features distinct from glyco-
saminoglycans (17, 24, 28). The use of Block 1 and 2 monoclonal
antibodies established the role of 1 sulfated and 1 pyruvated
saccharide unit in the carbohydrate-carbohydrate self-interac-
tion of GN1 (14, 15, 17, 28). Plasmon resonance kinetic binding
 Molecular Recognition between Glyconectins 15589

FIG. 9. Species-specific glyconectin

to glyconectin interactions mediate
bead-cell recognition and adhesion.
Xenogeneic glyconectin self-recognition in
a mixture of glutaraldehyde-fixed cells
and glyconectin-coated beads in SWT in
the presence of 10 mM Ca2⫹. M. prolifera
cells bearing glyconectin 1 were incubated
with: glyconectin 1 (pink beads) (A), gly-
conectin 2 (yellow beads) (D), and gly-
conectin 3 (white beads) (G). H. panicea
cells bearing glyconectin 2 were incubated
with: glyconectin 1 (B), glyconectin 2 (E),
and glyconectin 3 (H) color-coded beads.
C. celata cells bearing glyconectin 3 were
incubated with: glyconectin 1 (C), gly-
conectin 2 (F), and glyconectin 3 (I) color-
coded beads (glutaraldehyde fixation
changes cell colors, i.e. M. prolifera, or-
ange to yellowish white; H. panicea, white
to yellowish brown; and C. celata, brown
to brownish orange. We did not observe
differences in adhesion properties be-
tween fixed and live metabolically atten-
uated cells in a rotary assay.

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FIG. 10. Glyconectin carbohydrates are involved in cell recognition and adhesion. A, cell aggregation was performed as described for
Fig. 6, A and D, in the presence of 50 ␮g of anti-GN monospecific polyclonal antibodies. Adhesion of GN-bearing cells from each of the three sponge
species was assayed in the presence of the three anti-GN-glycan polyclonal antibodies. B, cell and GN-glycan-bead coaggregation was performed
under the conditions described in the legend for Fig. 9 for the intact glyconectin-coated beads using the following combinations: M. prolifera cells
bearing glyconectin 1 with GN1-glycan-beads, GN2-glycan-beads, or GN3-glycan-beads; H. panicea cells bearing glyconectin 2 with GN1-glycan-
beads, GN2-glycan-beads, or GN3-glycan-beads; and C. celata cells bearing glyconectin 3 with GN1-glycan-beads, GN2-glycan-beads, or
GN3-glycan-beads.

studies on the synthetically prepared sulfated epitope of GN1
directly showed that this oligosaccharide is capable of self-
association (31). In the initial study using three sponge species
(M. prolifera, H. panicea, and C. celata), it was shown that the
two new glyconectins also are involved in species-specific ag-
gregation (13). Here we have isolated and performed detailed
physicochemical and functional characterizations of glyconec-
tin 2 from H. panicea and glyconectin 3 from C. celata and
extended the analyses of glyconectin 1 from M. prolifera. In this
comparative study of three glyconectins we have found that
they have species-specific and complex repetitive glycan struc-
tures with some common properties for isolated glycans such as
a large size, a high charge content (sulfate and/or pyruvate),
and N-acetylated hexose, pentose, or deoxyhexose, which clas-
sifies them as a new type of polysaccharide molecule distinct
from classical glycosaminoglycans. A more detailed description
of this new type of glycan sequences and their possible struc-
tures is reported and discussed in the accompanying article
(40).
The degree of selectivity observed in the evolutionarily ad-
vanced, heterophilic, self and non-self molecular recognition
within the immunoglobulin superfamily is closely approached
by the degree of specificity of homophilic carbohydrate-medi-
ated glyconectin to glyconectin interactions in Porifera. How-
ever, the structural differences between these two systems
imply conceptually distinct molecular mechanisms. First, gly-
conectins are 100 times larger and extend 10 times farther
from the cell surface than immunoglobulin molecules. Second,
the glyconectin 1 specificity and tight binding of ⬎109 M⫺1
reside in polyvalent carbohydrate-carbohydrate interactions of
⬎1000 sites, with a low affinity for the single site (⬍103 M⫺1)
(14, 15, 29, 32–34), whereas immunoglobulins recognize anti-
gens via higher affinity ranging from 104 to 109 M⫺1, with low
valency binding (34 –38). The low affinity of the monovalent
glyconectin oligosaccharide units needs reconstitution of the
naturally occurring valency in GNs to recover the adhesion
function. Therefore, the use of classical inhibition experiments
with monovalent glycan units, commonly utilized as evidence of
glycan function in the conceptually different lectin-carbohy-
drate type of interactions, is not applicable for GN glycans.
Here reported carbohydrate-mediated glyconectin-glyconectin
interactions may provide a new paradigm for initial molecular
self-recognition.
The close evolutionary relationship between the Porifera of
today and the ancestors of multicellular organisms (33–35)
indicates that xenogeneic discrimination of self-cohesive gly-
15590 Molecular Recognition between Glyconectins
conectins may have been essential for the appearance of mul-
ticellularity. Clearly the evolution from primordial Metazoans
to complex organisms required the development of additional
cell recognition and adhesion mechanisms mediated via immu-
noglobulin, lectin, integrin, and cadherin families of molecules.
Such an increase in molecular diversity enabled a correspond-
ing increase in functions based on a more demanding self- and
non-self-discrimination such as embryonal morphogenesis, re-
newal of adult tissues, and defense from foreign pathogens.
Despite this evolving complexity of the components participat-
ing in cellular interactions, glyconectin-like structures have
been preserved in mammalian systems, suggesting their ver-
satility in recognition and adhesion (39). A model system for
primordial xenogeneic self-recognition and adhesion based on
the simplest Metazoans of the phylum Porifera gives results
consistent with the hypothesis that the emergence of the first
multicellular organisms, as well as the divergence of species
and the appearance of more complex multicellular forms of life,
may have been achieved by the evolution of glyconectin-like
proteoglycan molecules with the capacity for self-recognition
and adhesion via carbohydrates.
Acknowledgments—We thank V. Norris for helpful discussions, D.
Florea for help in preparing samples, J. P. Zanetta for gas chromatog-
raphy and mass spectrometry analysis of N-type linkage, and C. Richet
for amino acid analysis.
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