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• W2010968968 abstract "Dissociated sponge cells quickly reaggregate in a species-specific manner, differentiate, and reconstruct tissue, providing a very handy system to investigate the molecular basis of more complex intercellular recognition processes. Species-specific cell adhesion in the marine sponge Microciona prolifera is mediated by a supramolecular complex with a Mr = 2 × 107, termed aggregation factor. Guanidinium hydrochloride/cesium chloride dissociative gradients and rhodamine B isothiocyanate staining indicated the presence of several proteins with different degrees of glycosylation. Hyaluronate has been found to be associated with the aggregation factor. Chemical deglycosylation revealed a main component accounting for nearly 90% of the total protein. The cDNA-deduced amino acid sequence predicts a 35-kDa protein (MAFp3), the first sponge aggregation factor core protein ever described. The open reading frame is uninterrupted upstream from the amino terminus of the mature protein, and the deduced amino acid sequence for this region has been found to contain a long stretch sharing homology with the Na+-Ca2+ exchanger protein. A putative hyaluronic acid binding domain and several putative N- and O-glycosylation signals are present in MAFp3, as well as eight cysteines, some of them involved in intermolecular disulfide bridges. Northern blot data suggest variable expression, and Southern blot analysis reveals the presence of other related gene sequences. According to the respective molecular masses, one aggregation factor molecule would contain about 300 MAFp3 units, suggesting that sponge cell adhesion might be based on the assembly of multiple small glycosylated protein subunits. Dissociated sponge cells quickly reaggregate in a species-specific manner, differentiate, and reconstruct tissue, providing a very handy system to investigate the molecular basis of more complex intercellular recognition processes. Species-specific cell adhesion in the marine sponge Microciona prolifera is mediated by a supramolecular complex with a Mr = 2 × 107, termed aggregation factor. Guanidinium hydrochloride/cesium chloride dissociative gradients and rhodamine B isothiocyanate staining indicated the presence of several proteins with different degrees of glycosylation. Hyaluronate has been found to be associated with the aggregation factor. Chemical deglycosylation revealed a main component accounting for nearly 90% of the total protein. The cDNA-deduced amino acid sequence predicts a 35-kDa protein (MAFp3), the first sponge aggregation factor core protein ever described. The open reading frame is uninterrupted upstream from the amino terminus of the mature protein, and the deduced amino acid sequence for this region has been found to contain a long stretch sharing homology with the Na+-Ca2+ exchanger protein. A putative hyaluronic acid binding domain and several putative N- and O-glycosylation signals are present in MAFp3, as well as eight cysteines, some of them involved in intermolecular disulfide bridges. Northern blot data suggest variable expression, and Southern blot analysis reveals the presence of other related gene sequences. According to the respective molecular masses, one aggregation factor molecule would contain about 300 MAFp3 units, suggesting that sponge cell adhesion might be based on the assembly of multiple small glycosylated protein subunits. INTRODUCTIONSponges have been traditionally used as models to study cell adhesion, since their rather loose and porous extracellular matrix allows a mild cell dissociation and the recovery of intercellular structures in virtually native state. Three basic components were shown to be necessary to aggregate sponge cells (Humphreys, 20Humphreys T. Dev. Biol. 1963; 8: 27-47Crossref PubMed Scopus (140) Google Scholar), the cell surface, calcium ions, and an extracellular complex termed aggregation factor. Sponge aggregation factors are proteoglycan-like molecules showing either a linear appearance (Halichondria, Haliclona, Terpios) or a closed, sunburst-like morphology (Microciona, Geodia). Their active participation in species-specific cell-cell and cell-matrix interactions (Gramzow et al., 14Gramzow M. Schröder H.C. Uhlenbruck G. Batel R. Müller W.E.G. J. Histochem. Cytochem. 1988; 36: 205-212Crossref PubMed Scopus (7) Google Scholar) is in contrast to the rather passive mechanical functions generally ascribed to proteoglycans, although growing evidence is accumulating in favor of their relevant role in differentiation and proliferation of cells (Ruoslahti, 48Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (548) Google Scholar). Variability in the glycosaminoglycan moiety of proteoglycans seems to correlate with changes in adhesiveness of the cells to other cells or to the substrate (Sanderson et al., 51Sanderson R.D. Turnbull J.E. Gallagher J.T. Lander A.D. J. Biol. Chem. 1994; 269: 13100-13106Abstract Full Text PDF PubMed Google Scholar; Roughley et al., 47Roughley P.J. White R.J. Magny M.-C. Liu J. Pearce R.H. Mort J.S. Biochem. J. 1993; 295: 421-426Crossref PubMed Scopus (119) Google Scholar), much in the same way as it has been described for the neural cell-adhesion molecule, where electrostatic interactions between charged glycosaminoglycan chains are thought to modulate the strength of cell-cell interaction (Rothbard et al., 46Rothbard J.B. Brackenbury R. Cunningham B.A. Edelman G.M. J. Biol. Chem. 1982; 257: 11064-11069Abstract Full Text PDF PubMed Google Scholar). Variability in the sizes of proteoglycan core proteins has been described as well (Marynen et al., 32Marynen P. Zhang J. Cassiman J.-J. Van den Berghe H. David G. J. Biol. Chem. 1989; 264: 7017-7024Abstract Full Text PDF PubMed Google Scholar; Doege et al., 8Doege K.J. Sasaki M. Kimura T. Yamada Y. J. Biol. Chem. 1991; 266: 894-902Abstract Full Text PDF PubMed Google Scholar), although the physiological relevance of such changes has not yet been explained.The Microciona prolifera aggregation factor (MAF) 1The abbreviations used are: MAFM. prolifera aggregation factorPAGEpolyacrylamide gel electrophoresisRBITCrhodamine B isothiocyanateTFMStrifluoromethanesulfonic acidHFhydrogen fluoridePCRpolymerase chain reactionkbkilobase pair(s)PNGasepeptide-N-glycosidase. is a Mr = 2 × 107 complex containing roughly equal amounts of protein and carbohydrate (Henkart et al., 18Henkart P. Humphreys S. Humphreys T. Biochemistry. 1973; 12: 3045-3050Crossref PubMed Scopus (139) Google Scholar; Cauldwell et al., 4Cauldwell C.B. Henkart P. Humphreys T. Biochemistry. 1973; 12: 3051-3055Crossref PubMed Scopus (63) Google Scholar). MAF-promoted, species-specific sponge cell adhesion involves 1) a Ca2+-dependent MAF self-interaction site, and 2) a Ca2+-independent MAF-cell binding (Jumblatt et al., 22Jumblatt J.E. Schlup V. Burger M.M. Biochemistry. 1980; 19: 1038-1042Crossref PubMed Scopus (56) Google Scholar). Highly polyvalent structures have been shown to be involved in both functional domains (Misevic and Burger, 36Misevic G.N. Burger M.M. J. Biol. Chem. 1986; 261: 2853-2859Abstract Full Text PDF PubMed Google Scholar; Misevic et al., 40Misevic G.N. Finne J. Burger M.M. J. Biol. Chem. 1987; 262: 5870-5877Abstract Full Text PDF PubMed Google Scholar), and carbohydrate-carbohydrate interactions play a central role in MAF self-association activity (Misevic et al., 40Misevic G.N. Finne J. Burger M.M. J. Biol. Chem. 1987; 262: 5870-5877Abstract Full Text PDF PubMed Google Scholar; Misevic and Burger, 38Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar). Carbohydrate groups are involved in specificity (Turner and Burger, 59Turner R.S. Burger M.M. Nature. 1973; 244: 509-510Crossref PubMed Scopus (79) Google Scholar; Misevic and Burger, 37Misevic G.N. Burger M.M. J. Biol. Chem. 1990; 265: 20577-20584Abstract Full Text PDF PubMed Google Scholar), at least in the cell binding site, although whether such specificity lies in the glycan structure itself, in the distribution pattern of glycans on the protein backbone, or both, remains unknown. A biological activity for the protein component of MAF, other than being a mere scaffold for the attachment of glycans, is also possible. Specific matrix interactions mediated by proteoglycan core proteins have been demonstrated (Schmidt et al., 53Schmidt G. Robenek H. Harrach B. Glössl J. Nolte V. Hörmann H. Richter H. Kresse H. J. Cell Biol. 1987; 104: 1683-1691Crossref PubMed Scopus (128) Google Scholar; Heremans et al., 19Heremans A. De Cock B. Cassiman J.-J. Van den Berghe H. David G. J. Biol. Chem. 1990; 265: 8716-8724Abstract Full Text PDF PubMed Google Scholar; Yamaguchi et al., 64Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar), and convincing arguments that biological activity resides in certain proteoglycan core proteins are also appearing (Kjellén and Lindahl, 23Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1671) Google Scholar; Templeton, 57Templeton D.M. Crit. Rev. Clin. Lab. Sci. 1992; 29: 141-184Crossref PubMed Scopus (65) Google Scholar). Although progress has been made in unraveling the structure of the glycan moiety of MAF (Misevic and Burger, 37Misevic G.N. Burger M.M. J. Biol. Chem. 1990; 265: 20577-20584Abstract Full Text PDF PubMed Google Scholar, 38Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar; Spillmann et al., 55Spillmann D. Hård K. Thomas-Oates J. Vliegenthart J.F.G. Misevic G. Burger M.M. Finne J. J. Biol. Chem. 1993; 268: 13378-13387Abstract Full Text PDF PubMed Google Scholar), very limited information is available concerning the protein components. The complexity of sponge cell aggregation is suggested by the finding of many proteins associated with the aggregation factor, among them sialyltransferases (Müller et al., 42Müller W.E.G. Arendes J. Kurelec B. Zahn R.K. Müller I. J. Biol. Chem. 1977; 252: 3836-3842Abstract Full Text PDF PubMed Google Scholar), protein kinase C (Gramzow et al., 15Gramzow M. Zimmermann H. Janetzko A. Dorn A. Kurelec B. Schröder H.C. Müller W.E.G. Exp. Cell Res. 1988; 179: 243-252Crossref PubMed Scopus (7) Google Scholar), calpactin (Robitzki et al., 45Robitzki A. Schröder H.C. Ugarković D. Gramzow M. Fritsche U. Batel R. Müller W.E.G. Biochem. J. 1990; 271: 415-420Crossref PubMed Scopus (19) Google Scholar), and other bridge proteins that seem to link the factor to the cell membrane receptors (Gramzow et al., 13Gramzow M. Bachmann M. Uhlenbruck G. Dorn A. Müller W.E.G. J. Cell Biol. 1986; 102: 1344-1349Crossref PubMed Scopus (24) Google Scholar; Varner, 60Varner J.A. J. Cell Sci. 1995; 108: 3119-3126PubMed Google Scholar). Nevertheless, the nature of the main protein component of the aggregation factor core structure has remained elusive.DISCUSSIONThe aggregation factor from the sponge Microciona prolifera is a supramolecular structure containing hyaluronate and several glycosylated proteins. Since major impurities are discarded in the MAF preparation (Henkart et al., 18Henkart P. Humphreys S. Humphreys T. Biochemistry. 1973; 12: 3045-3050Crossref PubMed Scopus (139) Google Scholar), those components must be held together in the native complex. The ∼210-kDa subunit observed in SDS-PAGE is likely to be the same entity described by Varner et al. (61Varner J.A. Burger M.M. Kaufman J.F. J. Biol. Chem. 1988; 263: 8498-8508Abstract Full Text PDF PubMed Google Scholar) as a MAF-binding protein. The MAF complex then could be composed of several interacting units, which might make it difficult to draw the limits between what belongs to the factor and what associates with it. The presence of noncovalent bonds has been confirmed by PAGE and dissociative gradient results. Two proteins have been found to be associated with glycosaminoglycan-containing bands, but chemical deglycosylation revealed the presence of a distinct major protein component.Electron microscopy (Humphreys et al., 21Humphreys S. Humphreys T. Sano J. J. Supramol. Struct. 1977; 7: 339-351Crossref PubMed Scopus (33) Google Scholar) and atomic force microscope studies (Dammer et al., 6Dammer U. Popescu O. Wagner P. Anselmetti D. Güntherodt H.-J. Misevic G.N. Science. 1995; 267: 1173-1175Crossref PubMed Scopus (376) Google Scholar) have shown that MAF has a sunburst-like structure, with a central ring of about 200 nm across and radiating arms, each 180 nm long. MAF-MAF self-binding has been found to be the result of carbohydrate-carbohydrate interactions (Misevic and Burger, 38Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar). Therefore, since two MAF molecules seem to bind through their arms (Dammer et al., 6Dammer U. Popescu O. Wagner P. Anselmetti D. Güntherodt H.-J. Misevic G.N. Science. 1995; 267: 1173-1175Crossref PubMed Scopus (376) Google Scholar), these might be enriched in carbohydrate, leaving for the ring most of the protein. Urea, SDS, or mercaptoethanol have little effect on MAF as long as Ca2+ is present (Cauldwell et al., 4Cauldwell C.B. Henkart P. Humphreys T. Biochemistry. 1973; 12: 3051-3055Crossref PubMed Scopus (63) Google Scholar). Humphreys et al. (21Humphreys S. Humphreys T. Sano J. J. Supramol. Struct. 1977; 7: 339-351Crossref PubMed Scopus (33) Google Scholar) described that the aggregation factor was stable in 0.5% SDS, and only after 4 weeks of incubation in the presence of EDTA were all the arms dissociated, although the central ring, containing protein and polysaccharide, remained unaltered even after further treatment with SDS, dithiothreitol, and heating for 5 min at 100°C. Only a drastic 4-h treatment at 80°C in 40 m EDTA and 5 urea was able to fragment about 60-80% of MAF in fractions entering a separating gel, with still as much as 20-40% retained by the stacking gel (Misevic et al., 39Misevic G.N. Jumblatt J.E. Burger M.M. J. Biol. Chem. 1982; 257: 6931-6936Abstract Full Text PDF PubMed Google Scholar). We have found that chemical deglycosylation disrupted the aggregated material observed in SDS-PAGE, suggesting that glycosaminoglycan chains play a crucial role in maintaining MAF structure. This idea is strongly supported by the result of the brief HF deglycosylation shown in Fig. 2B, which demonstrates that MAFp3 units, otherwise so difficult to isolate, begin to be released immediately after glycan chopping off starts. Covalent link between proteins through glycosaminoglycan chains has already been described (Enghild et al., 11Enghild J.J. Salvesen G. Thøgersen I.B. Valnickova Z. Pizzo S.V. Hefta S.A. J. Biol. Chem. 1993; 268: 8711-8716Abstract Full Text PDF PubMed Google Scholar), but we have no proof of such a bond existing in sponges. On the other hand, the high negative charge of MAF (Henkart et al., 18Henkart P. Humphreys S. Humphreys T. Biochemistry. 1973; 12: 3045-3050Crossref PubMed Scopus (139) Google Scholar) might hamper the interaction with other negatively charged molecules like EDTA and SDS, thus limiting their disruptive effects. In such situations, Ca2+ ions buried within MAF could be protected enough to form relatively stable ionic bonds with the abundant acid residues from MAF glycosaminoglycans and/or proteins, helping to stabilize the overall structure. In agreement with this hypothesis, two distinct groups of Ca2+-binding sites have been found in MAF, which might be related to the requirement of low Ca2+ concentrations to maintain the subunit organization of the aggregation factor complex and of higher Ca2+ concentrations for the factor to keep cells together (Cauldwell et al., 4Cauldwell C.B. Henkart P. Humphreys T. Biochemistry. 1973; 12: 3051-3055Crossref PubMed Scopus (63) Google Scholar).The presence of putative N- and O-glycosylation sites, PNGase F digestion data, and alkaline β-elimination results suggest that MAFp3 contains both N- and O-linked glycans, although its localization in an intermediate fraction of dissociative guanidinium hydrochloride/cesium chloride density gradients indicates a moderate degree of glycosylation. The carboxyl-terminal GSGLGSGIG (positions 293-301) closely resembles sequences found in rat serglycin (Bourdon et al., 2Bourdon M.A. Shiga M. Ruoslahti E. J. Biol. Chem. 1986; 261: 12534-12537Abstract Full Text PDF PubMed Google Scholar), syndecan (Saunders et al., 52Saunders S. Jalkanen M. O'Farrell S. Bernfield M. J. Cell Biol. 1989; 108: 1547-1556Crossref PubMed Scopus (370) Google Scholar), human fibroglycan (Marynen et al., 32Marynen P. Zhang J. Cassiman J.-J. Van den Berghe H. David G. J. Biol. Chem. 1989; 264: 7017-7024Abstract Full Text PDF PubMed Google Scholar), and in glypican (David et al., 7David G. Lories V. Decock B. Marynen P. Cassiman J.-J. Van den Berghe H. J. Cell Biol. 1990; 111: 3165-3176Crossref PubMed Scopus (219) Google Scholar; Fig. 5A). Two glutamate residues in positions −5 and −6 complete a good consensus sequence for glycosaminoglycan attachment (Bourdon et al., 3Bourdon M.A. Krusius T. Campbell S. Schwartz N.B. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3194-3198Crossref PubMed Scopus (188) Google Scholar). MAFp3 does not contain sequences, like RGD, often found in adhesive proteins (Kreis and Vale, 26Kreis T. Vale R. Guidebook to the Extracellular Matrix and Adhesion Proteins. Oxford University Press, New York1993Google Scholar), thus leaving open the question whether the protein moiety of MAF participates actively in the binding of the factor or only carbohydrate structures are involved in the recognition process.We have found 30% identity between the region enclosed by residues −149 and −16 from the sequence shown in Fig. 3 and the cytoplasmic domain of the Na+-Ca2+ exchanger (Fig. 5B), a membrane protein responsible for the maintenance of low intracellular Ca2+ levels. Canine and rat isoforms of the Na+-Ca2+ exchanger exhibit for the same region a mere 51% identity (Nicoll et al., 43Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (627) Google Scholar; Li et al., 29Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar), which makes the similitude with the sponge sequence even more striking. We regard this homology as most suggestive since, besides the classical secretory vesicle pathway, an alternative route to export MAFp3 to the extracellular matrix could be its synthesis as a larger polypeptide together with a protein targeted to the membrane, to be cleaved afterward. The deglycosylation results shown in Fig. 2A have been regularly reproduced with aggregation factor isolated in all seasons from pools of several individuals, without ever detecting any other amino-terminal or deglycosylation products different from those presented under “Results,” thus ruling out the existence of a longer MAFp3 variety that could be incorporated in the factor under certain conditions. Although the structure-function relationship of MAFp3 for sponge cell adhesion remains to be investigated, regulation of its expression might be related to the yearly life cycle (sexual and asexual reproduction, budding, repair after injury), environmental changes (temperature, light, salinity), or both. During winter, the sponge body degenerates and in spring it will grow again from groups of dormant cells. Since most of the physiological uproar takes place in spring and summer, a correlation between phenomena such as gemmulation, release of larvae or sperm cells, and the adhesiveness of the sponge tissue is to be expected. Our results showing clear differences in MAFp3 expression during this physiologically active period point in that direction.Whether the several bands identified in Southern blots represent different forms of MAFp3 or other related proteins is currently under investigation in our laboratory. The variety of products observed after chemical deglycosylation of MAF is most likely the result of an incomplete removal of glycosaminoglycan chains, although the possibility of the existence of several MAFp3 forms sharing the same amino terminus should not be ruled out. Considering the protein content of MAF, between 250 and 300 MAFp3 units are expected to be found in a single molecule of aggregation factor. This is the number of pyruvate epitopes suggested to be present in each molecule (Spillmann et al., 55Spillmann D. Hård K. Thomas-Oates J. Vliegenthart J.F.G. Misevic G. Burger M.M. Finne J. J. Biol. Chem. 1993; 268: 13378-13387Abstract Full Text PDF PubMed Google Scholar), although this might be an underestimation due to the acid lability of the pyruvate group, which makes it sensitive to the hydrolysis conditions used for its preparation. If the three putative N-glycosylation sites in each MAFp3 molecule were substituted, that would give about 900 pyruvate epitopes per MAF molecule, a value very close to the 1100 sites suggested by Block 1 antibody binding assays (Misevic et al., 40Misevic G.N. Finne J. Burger M.M. J. Biol. Chem. 1987; 262: 5870-5877Abstract Full Text PDF PubMed Google Scholar). PNGase F digestion releases a repetitive glycan from MAF, termed G-6, which should be represented about 950 times, and contains the cell binding site of MAF (Misevic and Burger, 37Misevic G.N. Burger M.M. J. Biol. Chem. 1990; 265: 20577-20584Abstract Full Text PDF PubMed Google Scholar). Since Block 1 antibody binds to G-6 (Misevic, 35Misevic G.N. Methods Enzymol. 1989; 179: 95-104Crossref PubMed Scopus (17) Google Scholar; Misevic and Burger, 38Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar), it seems reasonable to suggest that MAFp3 carries G-6 and, therefore, the pyruvate epitope that would be involved in the cell binding.Highly polyvalent carbohydrate-carbohydrate interactions have been shown to mediate sponge cell aggregation. Our finding that the main protein of MAF is a relatively small 35-kDa molecule suggests that polyvalency at the protein level might also exist. Highly glycosylated proteoglycans consist of repeated sites in a large protein chain to which glycosaminoglycan substituents are attached. In the sponge aggregation factor, the whole protein itself seems to be the repeating unit, and its polymerization can bring along the carbohydrate polyvalency required for sponge cell aggregation. Yeast agglutinins are cell adhesion molecules sharing many characteristics with the sponge system; the high binding affinity of the sexual agglutination factor from Hansenula wingei was found to be the result of the additive effect of several of the individual binding sites, located on protein subunits interconnected through disulfide bonds (Taylor and Orton, 56Taylor N.W. Orton W.L. Biochemistry. 1971; 10: 2043-2049Crossref PubMed Scopus (17) Google Scholar; Yen and Ballou, 66Yen P.H. Ballou C.E. Biochemistry. 1974; 13: 2428-2437Crossref PubMed Scopus (56) Google Scholar). Saccharomyces cerevisiae a-agglutinin analogs are highly glycosylated disulfide-linked oligomers, containing a large core subunit mediating cell surface attachment and a small self-binding subunit, the total molecular weight being about 106 (Lipke and Kurjan, 30Lipke P.N. Kurjan J. Microbiol. Rev. 1992; 56: 180-194Crossref PubMed Google Scholar). In reducing conditions, we have not detected any measurable decrease of MAF-mediated aggregation efficiency of sponge cells, although monomerization of the protein might be overcome by the existence of several active carbohydrate binding units on each single MAFp3, thus suggesting again the existence of strong interactions between glycosaminoglycan chains. Moreover, the presence of hyaluronate in the aggregation factor preparation suggests that binding of MAFp3 to hyaluronic acid might also occur. MAFp3 contains a cluster of basic amino acids (RRYRNRVR, residues 107-114), which could determine the attachment to hyaluronate (Hardingham et al., 17Hardingham T.E. Ewins R.J.F. Muir H. Biochem. J. 1976; 157: 127-143Crossref PubMed Scopus (120) Google Scholar; Lyon, 31Lyon M. Biochim. Biophys. Acta. 1986; 881: 22-29Crossref PubMed Scopus (13) Google Scholar). This sequence resembles the hyaluronan binding motif B(X7)B, where B is either arginine or lysine and X is any nonacidic amino acid (Yang et al., 65Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (334) Google Scholar). We have observed that the addition of exogenous hyaluronate to aggregation factor preparations with low activity raised the aggregation efficiency of the factor to equal that found in the most active preparations. Our data indicating (i) that the main protein from the 2 × 104-kDa sponge aggregation factor is a relatively small 35-kDa molecule and (ii) the presence on MAFp3 of carbohydrate structures involved in the aggregation activity, suggest that the cooperative effect of multiple low affinity interactions required for MAF-mediated sponge cell aggregation might be based on the cross-linking of a small glycosylated protein into a large polymer, where the active carbohydrate sites would be correctly exposed. INTRODUCTIONSponges have been traditionally used as models to study cell adhesion, since their rather loose and porous extracellular matrix allows a mild cell dissociation and the recovery of intercellular structures in virtually native state. Three basic components were shown to be necessary to aggregate sponge cells (Humphreys, 20Humphreys T. Dev. Biol. 1963; 8: 27-47Crossref PubMed Scopus (140) Google Scholar), the cell surface, calcium ions, and an extracellular complex termed aggregation factor. Sponge aggregation factors are proteoglycan-like molecules showing either a linear appearance (Halichondria, Haliclona, Terpios) or a closed, sunburst-like morphology (Microciona, Geodia). Their active participation in species-specific cell-cell and cell-matrix interactions (Gramzow et al., 14Gramzow M. Schröder H.C. Uhlenbruck G. Batel R. Müller W.E.G. J. Histochem. Cytochem. 1988; 36: 205-212Crossref PubMed Scopus (7) Google Scholar) is in contrast to the rather passive mechanical functions generally ascribed to proteoglycans, although growing evidence is accumulating in favor of their relevant role in differentiation and proliferation of cells (Ruoslahti, 48Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (548) Google Scholar). Variability in the glycosaminoglycan moiety of proteoglycans seems to correlate with changes in adhesiveness of the cells to other cells or to the substrate (Sanderson et al., 51Sanderson R.D. Turnbull J.E. Gallagher J.T. Lander A.D. J. Biol. Chem. 1994; 269: 13100-13106Abstract Full Text PDF PubMed Google Scholar; Roughley et al., 47Roughley P.J. White R.J. Magny M.-C. Liu J. Pearce R.H. Mort J.S. Biochem. J. 1993; 295: 421-426Crossref PubMed Scopus (119) Google Scholar), much in the same way as it has been described for the neural cell-adhesion molecule, where electrostatic interactions between charged glycosaminoglycan chains are thought to modulate the strength of cell-cell interaction (Rothbard et al., 46Rothbard J.B. Brackenbury R. Cunningham B.A. Edelman G.M. J. Biol. Chem. 1982; 257: 11064-11069Abstract Full Text PDF PubMed Google Scholar). Variability in the sizes of proteoglycan core proteins has been described as well (Marynen et al., 32Marynen P. Zhang J. Cassiman J.-J. Van den Berghe H. David G. J. Biol. Chem. 1989; 264: 7017-7024Abstract Full Text PDF PubMed Google Scholar; Doege et al., 8Doege K.J. Sasaki M. Kimura T. Yamada Y. J. Biol. Chem. 1991; 266: 894-902Abstract Full Text PDF PubMed Google Scholar), although the physiological relevance of such changes has not yet been explained.The Microciona prolifera aggregation factor (MAF) 1The abbreviations used are: MAFM. prolifera aggregation factorPAGEpolyacrylamide gel electrophoresisRBITCrhodamine B isothiocyanateTFMStrifluoromethanesulfonic acidHFhydrogen fluoridePCRpolymerase chain reactionkbkilobase pair(s)PNGasepeptide-N-glycosidase. is a Mr = 2 × 107 complex containing roughly equal amounts of protein and carbohydrate (Henkart et al., 18Henkart P. Humphreys S. Humphreys T. Biochemistry. 1973; 12: 3045-3050Crossref PubMed Scopus (139) Google Scholar; Cauldwell et al., 4Cauldwell C.B. Henkart P. Humphreys T. Biochemistry. 1973; 12: 3051-3055Crossref PubMed Scopus (63) Google Scholar). MAF-promoted, species-specific sponge cell adhesion involves 1) a Ca2+-dependent MAF self-interaction site, and 2) a Ca2+-independent MAF-cell binding (Jumblatt et al., 22Jumblatt J.E. Schlup V. Burger M.M. Biochemistry. 1980; 19: 1038-1042Crossref PubMed Scopus (56) Google Scholar). Highly polyvalent structures have been shown to be involved in both functional domains (Misevic and Burger, 36Misevic G.N. Burger M.M. J. Biol. Chem. 1986; 261: 2853-2859Abstract Full Text PDF PubMed Google Scholar; Misevic et al., 40Misevic G.N. Finne J. Burger M.M. J. Biol. Chem. 1987; 262: 5870-5877Abstract Full Text PDF PubMed Google Scholar), and carbohydrate-carbohydrate interactions play a central role in MAF self-association activity (Misevic et al., 40Misevic G.N. Finne J. Burger M.M. J. Biol. Chem. 1987; 262: 5870-5877Abstract Full Text PDF PubMed Google Scholar; Misevic and Burger, 38Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar). Carbohydrate groups are involved in specificity (Turner and Burger, 59Turner R.S. Burger M.M. Nature. 1973; 244: 509-510Crossref PubMed Scopus (79) Google Scholar; Misevic and Burger, 37Misevic G.N. Burger M.M. J. Biol. Chem. 1990; 265: 20577-20584Abstract Full Text PDF PubMed Google Scholar), at least in the cell binding site, although whether such specificity lies in the glycan structure itself, in the distribution pattern of glycans on the protein backbone, or both, remains unknown. A biological activity for the protein component of MAF, other than being a mere scaffold for the attachment of glycans, is also possible. Specific matrix interactions mediated by proteoglycan core proteins have been demonstrated (Schmidt et al., 53Schmidt G. Robenek H. Harrach B. Glössl J. Nolte V. Hörmann H. Richter H. Kresse H. J. Cell Biol. 1987; 104: 1683-1691Crossref PubMed Scopus (128) Google Scholar; Heremans et al., 19Heremans A. De Cock B. Cassiman J.-J. Van den Berghe H. David G. J. Biol. Chem. 1990; 265: 8716-8724Abstract Full Text PDF PubMed Google Scholar; Yamaguchi et al., 64Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar), and convincing arguments that biological activity resides in certain proteoglycan core proteins are also appearing (Kjellén and Lindahl, 23Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1671) Google Scholar; Templeton, 57Templeton D.M. Crit. Rev. Clin. Lab. Sci. 1992; 29: 141-184Crossref PubMed Scopus (65) Google Scholar). Although progress has been made in unraveling the structure of the glycan moiety of MAF (Misevic and Burger, 37Misevic G.N. Burger M.M. J. Biol. Chem. 1990; 265: 20577-20584Abstract Full Text PDF PubMed Google Scholar, 38Misevic G.N. Burger M.M. J. Biol. Chem. 1993; 268: 4922-4929Abstract Full Text PDF PubMed Google Scholar; Spillmann et al., 55Spillmann D. Hård K. Thomas-Oates J. Vliegenthart J.F.G. Misevic G. Burger M.M. Finne J. J. Biol. Chem. 1993; 268: 13378-13387Abstract Full Text PDF PubMed Google Scholar), very limited information is available concerning the protein components. The complexity of sponge cell aggregation is suggested by the finding of many proteins associated with the aggregation factor, among them sialyltransferases (Müller et al., 42Müller W.E.G. Arendes J. Kurelec B. Zahn R.K. Müller I. J. Biol. Chem. 1977; 252: 3836-3842Abstract Full Text PDF PubMed Google Scholar), protein kinase C (Gramzow et al., 15Gramzow M. Zimmermann H. Janetzko A. Dorn A. Kurelec B. Schröder H.C. Müller W.E.G. Exp. Cell Res. 1988; 179: 243-252Crossref PubMed Scopus (7) Google Scholar), calpactin (Robitzki et al., 45Robitzki A. Schröder H.C. Ugarković D. Gramzow M. Fritsche U. Batel R. Müller W.E.G. Biochem. J. 1990; 271: 415-420Crossref PubMed Scopus (19) Google Scholar), and other bridge proteins that seem to link the factor to the cell membrane receptors (Gramzow et al., 13Gramzow M. Bachmann M. Uhlenbruck G. Dorn A. Müller W.E.G. J. Cell Biol. 1986; 102: 1344-1349Crossref PubMed Scopus (24) Google Scholar; Varner, 60Varner J.A. J. Cell Sci. 1995; 108: 3119-3126PubMed Google Scholar). Nevertheless, the nature of the main protein component of the aggregation factor core structure has remained elusive." @default.
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• W2010968968 title "A 35-kDa Protein Is the Basic Unit of the Core from the 2 × 104-kDa Aggregation Factor Responsible for Species-specific Cell Adhesion in the Marine Sponge" @default.
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