The Shell of the Invasive Bivalve Species Dreissena polymorpha: Biochemical, Elemental and Textural Investigations

The zebra mussel Dreissena polymorpha is a well-established invasive model organism. Although extensively used in environmental sciences, virtually nothing is known of the molecular process of its shell calcification. By describing the microstructure, geochemistry and biochemistry/proteomics of the shell, the present study aims at promoting this species as a model organism in biomineralization studies, in order to establish a bridge with ecotoxicology, while sketching evolutionary conclusions. The shell of D. polymorpha exhibits the classical crossed-lamellar/complex crossed lamellar combination found in several heterodont bivalves, in addition to an external thin layer, the characteristics of which differ from what was described in earlier publication. We show that the shell selectively concentrates some heavy metals, in particular uranium, which predisposes D. polymorpha to local bioremediation of this pollutant. We establish the biochemical signature of the shell matrix, demonstrating that it interacts with the in vitro precipitation of calcium carbonate and inhibits calcium carbonate crystal formation, but these two properties are not strongly expressed. This matrix, although overall weakly glycosylated, contains a set of putatively calcium-binding proteins and a set of acidic sulphated proteins. 2D-gels reveal more than fifty proteins, twenty of which we identify by MS-MS analysis. We tentatively link the shell protein profile of D. polymorpha and the peculiar recent evolution of this invasive species of Ponto-Caspian origin, which has spread all across Europe in the last three centuries.


Chitin-binding lectins / N-acetylglucosamine / N-acetyllactosamine
Triticum vulgare wheat germ WGA GlcNacβ(1,4) GlcNacβ(1,4) GlcNac > GlcNacβ(1,4) GlcNac > GlcNac >> sialic acid (Neu5Ac) >> GalNAc. Chitin-binding lectins. N-acetylglucosamine, with preferential binding to dimers and trimers of this sugar. WGA can bind oligosaccharides containing terminal N-acetylglucosamine or chitobiose. Bacterial cell wall peptidoglycans, chitin, cartilage glycosaminoglycans, and glycolipids can also bind WGA. Native WGA has also been reported to interact with some glycoproteins via sialic acid residues (see succinylated WGA). WGA reacts strongly with the chitobiose core of asparagine linked oligosaccharides, specifically with the Man β(1,4)GlcNAc β(1,4)GlcNAc trisaccharide. It has been suggested that WGA also has an affinity for N-acetylneuraminic acid (Neu5Ac , sialic acid), since the binding of WGA to animal cells can be decreased or eliminated by treatment of the cells with neuraminidase. The precipitation of ovine submaxillary mucin OSM by the lectin is indicative of the sialic acid specificity since OSM is devoid of GlcNAc residues. Desialylated OSM contains terminal O-linked α-GalNAc residues. WGA also recognizes this carbohydrate, but to a lesser degree than either GlcNAc or sialic acid. Several analogs of Neu5Ac are also inhibitors of WGA but N-acetylglycolylneuraminic acid (Neu5Gc) is not inhibitory. Both GlcNAc and Neu5Ac are commonly found in cellular glycoproteins and in various tissue types [37].
Triticum vulgare wheat germ sWGA GlcNacβ(1,4) GlcNacβ(1,4) GlcNac > GlcNacβ(1,4) GlcNac > GlcNac >> GalNAc. Succinylated Wheat Germ agglutinin does not bind to sialic acid residues, unlike the native form, but retains its specificity toward N-acetylglucosamine. Using conjugates of the native lectin and the succinylated form can provide a system to distinguish between sialylated glycoconjugates and those containing only N-acetylglucosamine structures. Native WGA can be modified by succinylation to yield a lectin which no longer reacts with sialic acid but which still retains its other carbohydrate binding properties [36].
Datura stramonium jimson weed DSL Chitotriose > Chitobiose > N-acetyl-D-glucosamine. The carbohydrate binding site recognizes (β-1,4) linked N-acetylglucosamine oligomers, preferring chitobiose or chitotriose over a single Nacetylglucosamine residue. Chitin-binding lectins. DSL also binds well to N-acetyllactosamine and oligomers containing repeating N-acetyllactosamine sequences. It is distinct among poly-Nacetyllactosamine-binding lectins in that it apparently does not require the presence of a GlcNAcβ(1-6)-linkage for interaction. A branched pentasaccharide including two N-acetyllactosamine disaccharides linked to mannose (β-1,6) and (β-1,2) was reported to be the most potent inhibitor of agglutination. Repeating units of GlcNAc and muramic acid, as found in the cell walls of Micrococcus luteus , are inhibitors of the lectin. This lectin binds well in the acidic pH range but its affinity decreases above pH 8.0 [42].
Lycopersicon esculentum tomato LEL Chitin-binding lectins.N-acetyl-D-glucosamineb(1,4)N-acetyl-D-glucosamine oligomers up to 4 carbohydrate units. The N-acetyl-D-glucosamine residues do not need to appear consecutively, a finding that has been noted for DSL, but not for WGA or STL. LEL requires 3 consecutive LacNAc residues, making it specific for poly N-acetyllactosamine glycoproteins. Recognition of high mannosetype N glycans by LEL. Repeating units of GlcNAc and muramic acid, as found in the cell walls of Micrococcus luteus , are strong inhibitors of the lectin [45].

Griffonia simplicifolia
formerly Bandeiraea simplicifolia GSL-II N-acetyl-D-glucosamine. GSL-II is specific for terminal, non-reducing α-or β-linked N-acetylDglucosamine. GSL-II is the only lectin isolated that is specific for only a terminal GlcNAc residue. The subterminal saccharide does play an important role in lectin binding. GlcNAc linked β(1,3) or α(1,6) to galactose is a poor inhibitor of the lectin while GlcNAc linked α(1,3) to galactose or glucose is a potent inhibitor.

N-acetylgalactosamine
Dolichos biflorus horse gram DBA Terminal α-N-acetyl-D-galactosamine. α-linked N-acetylgalactosamine Vicia villosa hairy vetch VVA N-acetyl-D-galactosamine. VVA recognizes preferentially α-or β-linked terminal N-acetylgalactosamine, especially a single α-N-acetylgalactosamine residue linked to serine or threonine in a polypeptide (the Tn antigen). Evidence suggests that this lectin also may require specific amino acid sequences at the receptor site of glycosylation. The disaccharide galactosyl (α-1,3) Nacetylgalactosamine is also a potent inhibitor of this lectin [38].

Galactose / N-acetylgalactosamine
Artocarpus integrifolia jackfruit Jacalin α-D-Galactose and oligosaccharides terminating with this sugar, lectin is also highly specific for the T-antigen, Gal-β(1,3)GalNAc. Jacalin is useful in the purification of O-linked glycoproteins. This lectin appears to bind only O-glycosidically linked oligosaccharides, preferring the structure galactosyl (β-1,3) N-acetylgalactosamine. This structure (the T-antigen) is the oligosaccharide to which peanut agglutinin (PNA) binds. However, unlike PNA, Jacalin will bind a mono-or disialylated form of this structure. Another difference with PNA, Jacalin apparently does not bind to galactosyl-Nacetylglucosamine [41].
Arachis hypogaea peanut PNA Lactose > β-D-Galactose. The T-antigen, Gal β(1,3)GalNac, is a more potent inhibitor of lectin activity than any of the monosaccharides tested. Lactose, Gal β(1,4)Glucose, is also a strong inhibitor of the lectin, indicating that the terminal non-reducing β-galactose is of primary importance. Glucose and GalNAc by themselves are not considered inhibitors of the lectin. Peanut agglutinin required this O-linked oligosaccharide to be devoid of sialic acid, whereas Jacalin will bind to the fully sialylated disaccharide [43].
Glycine max soybean SBA Terminal α-and β-N-acetyl-D-galactosamine. SBA also reacts with galactose. SBA has a slight preference for α-linked sugars and contains four complementary binding sites / protein. SBA reacts more strongly with neuraminidase treated glycoconjugates, indicating a preference for terminal carbohydrates. SBA preferentially binds to oligosaccharide structures with terminal α-or β-linked Nacetylgalactosamine, and to a lesser extent, galactose residues [37].

Griffonia simplicifolia
formerly Bandeiraea simplicifolia GSL-I α-D-Galactoside and α-linked galactose oligosaccharides. α-GalNAc-O-Ser/Thr. αGal, αGalNAc. GSL I is a family of glycoproteins with molecular weights of approximately 114 kDa. There are two types of subunits, termed "A" and "B", with slightly different molecular weights. These subunits combine to form tetrameric structures, resulting in five isolectins. The "A"-rich lectin preferentially agglutinates blood group A erythrocytes and thus appears to be specific for α-N-acetylgalactosamine residues, while the "B"-rich lectin preferentially agglutinates blood group B cells and is specific for α-galactose residues [44].
Ricinus communis castor bean RCA 120 RCA 120 exhibits a specificity for β-galactose residues, with a preference for terminal sugars. RCA 120 reacts more strongly with branched cluster glycosides than with a monosaccharide. β(1,4)linkage is important for binding since lactose is a potent inhibitor; Gal β(1,3)glucose is only about one-third as inhibitory as lactose. N-acetyl-D-galactosamine is a very poor inhibitor of the agglutinin [37].

N-acetyllactosamine / N-acetylgalactosamine
Erythrina cristagalli coral tree ECL N-acetyllactosamine > Lactose > N-acetyl-D-galactosamine > Galactose. Their primary reactivity is with terminal LacNAc structures, Galβ1,4GlcNAc. LacNAc structure is a major component of most N-linked glycoproteins . It is also reactive, to a lesser degree, with GalNAc and weaker still with galactose. Sialylation is not tolerated. Fucose prevent lectine binding [40].