Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species

Abstract

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is “hijacked” by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. A screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). A phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. A glycan array analysis, however, revealed that unlike CvGal1 that preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Further, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and β-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for P. chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of Perkinsus species by the oyster galectins is consistent with their relative prevalence in oyster and clam species, and supports their role in facilitating parasite entry and infectivity in a host-preferential manner.

Structural, functional, and evolutionary aspects of galectins in aquatic mollusks: From a sweet tooth to the Trojan horse

“…The first species to be rigorously investigated in this regard has been the eastern oyster Crassostrea virginica [55–60]. Although the presence of multiple galactosyl binding lectins had been already reported in this species [55, 56], their lectin family identity had remained unresolved.…”

“…The 4-CRD galectins are proteins of approximately 560 amino acid-long polypeptides, of approximate MW of 64 kDa in which four similar but not identical CRDs are separated by short linker peptides ranging from 8 to 17 amino acid residues [57, 60]. The 2-CRD galectins consist of 280 to 300 amino acid-long polypeptides, of approximate MW of 32kDa in which two similar but not identical CRDs are separated by a 10–15 amino acid-long linker peptide [62].…”

“…Early studies in C. virginica implementing serological and biochemical approaches by agglutination of human (A, B and O) and animal erythrocytes and agglutination-inhibition revealed the presence of multiple soluble and cell membrane-associated lectins in tissues, hemocytes, and biological fluids with binding preferences for galactose and N-acetylgalactosamine [55, 56]. The identification of galectins among the various members of the oyster’s lectin repertoire and the characterization of their specificity by solid phase binding and binding-inhibition assays, together with the detailed analysis of specificity by a glycan array technology (Fig 2) revealed their specificity for ABH blood group oligosaccharides [57, 58, 60]. Furthermore, in contrast with most galectins analyzed so far, the initial studies on CvGl1 indicated that it displayed stronger binding to galactosamine and N-acetylgalactosamine relative to D-galactose [57].…”

“…In addition, availability of glycan arrays for the elucidation of the detailed carbohydrate specificity of the proteins and the recent development and optimization of bioinformatic tools for ligand docking experiments has enabled the visualization of the the 3-D structure of the binding site and the multiple direct or water-mediated interactions of the amino acid residues in the CRD with the oligosaccharide(s) recognized [58, 60]. Furthermore, the modeling and docking structural studies, combined with the glycan array analyses indicated not only that the 4-CRD galectins from C. virginica exhibit a unique specificity for ABH blood group oligosaccharides, but also that the 4 CRDs exhibit differences in their binding properties (Fig 3), suggesting that these proteins are endowed of uniquely broad recognition capabilities [58].…”

“…Therefore, the spatial distribution of galectin transcripts and mature proteins in selected tissues and cell types of aquatic mollusks has recently become a matter of great interest. At present time, however, documented information about the potential tissue-specific expression and localization of the galectins is only available for a limited number of aquatic mollusk species, including Crassostrea virginica, Biomphalaria glabrata, Solen grandis, Argopecten irradians, Ruditapes philippinarum and Pinctada fucata [57, 58, 60, 62–68]. The experimental approaches implemented to examine the spatial distribution of galectin transcripts in tissues and selected cell types such as hemocytes, have consisted of RT-PCR with primers based on galectin sequences obtained by cloning in the species of interest, or from public databases.…”