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J. Biol. Chem., Vol. 279, Issue 15, 15579-15590, April 9, 2004
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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, ||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
Received for publication, August 12, 2003 , and in revised form, December 30, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To examine whether homophilic glyconectin to glyconectin binding was also involved in primordial cellular adhesion and recognition self-assembly processes associated with the emergence 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 dissociated 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 chemically 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. prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3). Physicochemical analyses and mass spectrometric fingerprinting of the three purified glyconectin macromolecules revealed species-specific proteoglycan-like structures that define a new class of glycocojugates. Using a quantitative functional 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 polyclonal 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 carbohydrates in the self-assembly and non-self discrimination pathway of cellular adhesion prior to the evolution of multicellularity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 glyconectins 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 detection and with 20 kV accelerating voltage. Aliquots (0.5 µl) 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 acetonitrile-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 precipitated with 70% ethanol and centrifuged at 2000 x g for 10 min. The glyconectin pellet was resuspended again in 70% ethanol, and centrifugation 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 adding fresh aliquots of preincibated pronase. Released glycans were separated from free amino acids, salts, and small peptides by gel filtration and subsequent ion exchange chromatography.
Preparation of Monospecific Polyclonal Antibodies against Glyconectin Carbohydrates—Purified glyconectin carbohydrates were used for preparation of polyclonal antibodies according to the procedure described previously (14). Monospecific polyclonal antibodies were obtained by affinity purification on GN glycans cross-linked to a solid matrix using previously a reported methodology of immobilization (13). Immunoglobulins bound to GNs were eluted with 1 M NaCl and subsequently 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 confirmed in immunodot assay (14).
Polyacrylamide Electrophoresis—Electrophoretic separation of purified 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 oligosaccharides was measured by MALDI-TOF MS on a Vision 2000 time-of-flight instrument (Finnigan Mat) equipped with a 337 nm UV laser. 1 µl of sample at a concentration of 100 pmol/µl was mixed with an equal volume of matrix and allowed to crystallize. We used 2,5-dihydroxybenzoic acid and 3-aminoquinoline for the neutral and acidic oligosaccharides, 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 oligosaccharides were dissolved in methanol/water (50/50) and permethylated oligosaccharides in acetonitrile at a concentration of 10 pmol/µl. 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 x 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 transform mode and controlled by an Aspect 3000 computer. Each glyconectin sample was dissolved in 400 µl of 2H2O after three exchanges with 2H2O (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 assessed by gel filtration on a Bio-Gel P2 or P4 column (Bio-Rad) equilibrated 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 
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 µl of 2% latex-amidine bead suspension, 0.6 µm in diameter (Molecular Probes, Inc., Eugene, OR), was washed three times with 1 ml of SWT by centrifugation at 3000 x g for 10 min. Beads were resuspended in 200 µl of SWT and bath-sonicated for 5 min. To allow adsorption to beads on the basis of charge, 100 µg of either glyconectins or glycans isolated from these glyconectins was added to a 100 µl 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 µl 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 µl of ACMFSW, without or with 10 mM Ca2+, in the presence or absence of anti-GN-glycan polyclonal antibodies. For glutaraldehyde-fixed cells assays were done in SWT with or without Ca2+. For the cell-bead assay, 5-10 µl of 2% bead suspension in SWT was added to 100 µl of cell suspension containing 106 cells in the presence or absence of 10 mM Ca2+. In bead overlay assays 50-100 µl of 2% bead suspension in SWT was added to nitrocellulose membrane or agarose gel equilibrated in 1 ml of CSW in the presence of 10 mM Ca2+.
The degree of cell-cell and bead-bead adhesion in the presence of variable calcium or magnesium ion concentrations or anti-GN-glycan polyclonal antibodies was quantified after a 20-min rotation either by counting single cells and aggregates of at least 40 µm in diameter or by spectrophotometrically measuring the absorbance of supernatants after sedimentation at 100 x g for 1 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and 
anomers. This structure was confirmed as a pyruvated hexose in methyl-glycoside heptafluoryl derivative because of a [M + NH4] ion at m/z 698 in chemical ionization mass spectrometry and a very intense [M - COOMe] ion at m/z 611 in EI-MS (Fig. 2) (27). The exact nature of this residue was established as 4,6-O-(1-carboxyethylidene)--D-Galp (Py(4,6)Gal) by trimethylsilyl derivatization of methylglycoside and comparison with standard EI-MS spectra from the literature (Fig. 2) (27). Indeed, spectra was dominated by an ion at m/z 243, characteristic of a 4,6-O-(1-carboxyethylidene)-D-galactose structure (27), and by an intense [M - COOMe] at m/z 363. The intense ion at m/z 204, resulting from the cleavage of two adjacent trimethylsilyl groups, also characterized the 4,6-linkage position of the pyruvate group. Subsequently, this attribution was confirmed by NMR experiments of glyconectin hydrolysis fragments from GN2 (see accompanying article (40)). This is in agreement with previous work that identified a Py(4,6)Gal-containing oligosaccharide obtained by hydrolysis of glyconectin from M. prolifera (28). The stability of the 4,6-O-(1-carboxyethylidene)-galactose pyranose ring to methanolysis (27) enabled us to estimate, among the other monosaccharides, the amount of this particular component in each sample (Table I).
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Amino acid analysis of three sponge glyconectins showed species-specific differences with some common features such as the presence of over 7% serine, threonine, and asparagine/aspartic acid (Table I).
Peptide Mapping of Native Glyconectins—Native GN1, GN2, and GN3 were reduced with dithiothreitol, alkylated with acrylamide 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 peptides. 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).
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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 characteristic 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 spectrum 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 presented 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.
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The linkage type of GN glycans to the protein core was studied using a chemical and enzymological approach. We analyzed 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 GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate 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 absolutely 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 partially release glycans (about 30-50%), as in the classical examples 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; however, 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 distribution, as well as the peptide sequences of the core protein (which greatly influence enzymatic and chemical cleavage). Our structural data of GN polysaccharides presented in the accompanying article (40) promote the development of sequencing techniques 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 definitively 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 species recovered from the respective glyconectin, the size of the glycans, and the size of the intact glyconectins, a rough estimation 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 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.
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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 recognition 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 indicated that glyconectins and Ca2+ mediate the initial steps of xenogeneic cell recognition and adhesion of the three selected sponge species.
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A similar type of experiment was performed by overlaying agarose gel containing three electrophoretically separated glyconectins 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 glyconectin bands identical to the ones stained with toluidine blue and Amido Black showed that glyconectin to glyconectin interactions are highly species-specific (Fig. 1).
The combinations of the above described experiments demonstrate species-specific molecular self-recognition of glyconectins in an elementary reconstituted bead adhesion system, which fully resembles glyconectin-mediated cell-cell recognition 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 specificity of homophilic glyconectin-glyconectin interactions in a color-coded cell-bead adhesion assay. Each of the three glyconectins was attached to color-coded beads, as in the above described experiments, and beads were mixed in nine combinations with glyconectin-bearing cells of the three sponge species 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 aggregate sizes for all three species. Such recognition took place only when cells had glyconectin on their surfaces and in the presence 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, metabolically 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 Adhesion—The final question was to examine whether glyconectin 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, essentially free of proteins (see Table II), as described under "Experimental Procedures." Then we examined whether such anti-GN-glycan antibodies can specifically inhibit cell aggregation 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-glycan, 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 essentially free of proteins (see Table II), to color-coded latex-amidine beads using the same adsorption procedure as described for the intact GNs. The adsorption is based on charge interactions, which in high salt concentrations are much 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 µm 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 reconstitution experiments, performed by coating multiple copies of large glycans on 0.6-µm diameter latex beads, were necessary. These reconstituted polyvalent GN-glycan-beads were tested in coaggregation assays with cells 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 (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 carbohydrates of GNs are directly involved in Porifera cell recognition and adhesion, which may be implicated in xenogeneic self-assembly and the non-self-discrimination pathway of cellular interactions leading to multicellularity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The degree of selectivity observed in the evolutionarily advanced, heterophilic, self and non-self molecular recognition within the immunoglobulin superfamily is closely approached by the degree of specificity of homophilic carbohydrate-mediated glyconectin to glyconectin interactions in Porifera. However, the structural differences between these two systems imply conceptually distinct molecular mechanisms. First, glyconectins 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 antigens 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-carbohydrate 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 glyconectins may have been essential for the appearance of multicellularity. Clearly the evolution from primordial Metazoans to complex organisms required the development of additional cell recognition and adhesion mechanisms mediated via immunoglobulin, lectin, integrin, and cadherin families of molecules. Such an increase in molecular diversity enabled a corresponding increase in functions based on a more demanding self- and non-self-discrimination such as embryonal morphogenesis, renewal of adult tissues, and defense from foreign pathogens. Despite this evolving complexity of the components participating in cellular interactions, glyconectin-like structures have been preserved in mammalian systems, suggesting their versatility 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.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 332-35-14-69-08; Fax: 332-35-14-70-20; E-mail: gradimir{at}gradimir.com.
1 The abbreviations used are: ACMFSW, artificial Ca2+- and Mg2+-free seawater; SWT, seawater-Tris; CSW, Ca2+- and Mg2+-free seawater buffered with Tris; GC/MS, gas chromatography-coupled mass spectrometry; GN, glyconectin; EI-MS, electronic impact mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Py(4,6)Gal, 4,6-O-(1-carboxyethylidene)--D-Galp; ACTH, adrenocorticotropic hormone; HPLC, high pressure liquid chromatography.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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