Biochemical Characterization of Oyster and Clam Galectins: Selective Recognition of Carbohydrate Ligands on Host Hemocytes and Perkinsus Parasites

Abstract

Both vertebrates and invertebrates display active innate immune mechanisms for defense against microbial infection, including diversified repertoires of soluble and cell-associated lectins that can effect recognition and binding to potential pathogens, and trigger downstream effector pathways that clear them from the host internal milieu. Galectins are widely distributed and highly conserved lectins that have key regulatory effects on both innate and adaptive immune responses. In addition, galectins can bind to exogenous ("non-self") carbohydrates on the surface of bacteria, enveloped viruses, parasites, and fungi, and function as recognition receptors and effector factors in innate immunity. Like most invertebrates, eastern oysters (Crassostrea virginica) and softshell clams (Mya arenaria) can effectively respond to most immune challenges through soluble and hemocyte-associated lectins. The protozoan parasite Perkinsus marinus, however, can infect eastern oysters and cause "Dermo" disease, which is highly detrimental to both natural and farmed oyster populations. The sympatric Perkinsus chesapeaki, initially isolated from infected M. arenaria clams, can also be present in oysters, and there is little evidence of pathogenicity in either clams or oysters. In this review, we discuss selected observations from our studies on the mechanisms of Perkinsus recognition that are mediated by galectin-carbohydrate interactions. We identified in the oyster two galectins that we designated CvGal1 and CvGal2, which strongly recognize P. marinus trophozoites. In the clam we also identified galectin sequences, and focused on one (that we named MaGal1) that also recognizes Perkinsus species. Here we describe the biochemical characterization of CvGal1, CvGal2, and MaGal1 with focus on the detailed study of the carbohydrate specificity, and the glycosylated moieties on the surfaces of the oyster hemocytes and the two Perkinsus species (P. marinus and P. chesapeaki). Our goal is to gain further understanding of the biochemical basis for the interactions that lead to recognition and opsonization of the Perkinsus trophozoites by the bivalve hemocytes. These basic studies on the biology of host-parasite interactions may contribute to the development of novel intervention strategies for parasitic diseases of biomedical interest.

Keywords: biochemical characterization; bivalve hemocyte; carbohydrate recognition; galectin; perkinsus parasites.

Figure 1

Electron micrographs of Perkinsus sp. (A) Mature trophozoites of Perkinsus sp. isolated from the Baltic clam, Macoma balthica. Scale bar = 2 μm. A, cortical alveoli expansion; Mt, mitochondria; N, nucleus; Nu, nucleolus; P, vacuoplast precursors; Va, vacuole; W, wall; Mi, rectilinear micronemes; F, flagellum; C; conoid. Scale bar = 1 μm. (B) Longitudinal section of mature zoospores of Perkinsus sp. isolated from Macoma balthica. Insert shows detail of anterior end of another zoospore. (C) Phagocytosis of Perkinsus marinus by eastern oyster (Crassostrea virginica) hemocytes in a scanning electron micrograph [Adapted from Harvell et al. (1999) with permission from the American Association for the Advancement of Science].

Figure 2

Galectins in bivalves. (A) Alignment of bovine galectin−1, zebrafish Drgal1−L2, C. elegans LEC−6 (formerly N16), and CRD1 to −4 of CvGal1. (B) Homology modeling of CvGal1 CRDs. Bovine galectin−1, CRD−1,−2,−3, and−4 are shown in white, blue, yellow, red, and green, respectively. Numbering of amino acid residues is based on bovine galectin−1. Loop 4 of the CvGal1 CRDs (arrow b) is shorter than the loop 4 of BaGal1 (arrow a). (C) Coomassie stain of recombinant CvGal1, CvGal2, and MaGal1 containing either four or two CRDs. Arrows indicate the expected molecular weight of the proteins. (D) Schematic illustration of four CRDs of CvGal1 and CvGal2 and two CRDs of MaGal1 [Adapted from Tasumi and Vasta (2007) with permission from the American Association of Immunologists].

Figure 3

Selective binding of CvGals and MaGal to Perkinsus parasites. (A,B) Perkinsus marinus (Pm) or P. chesapeaki (Pc) were incubated with recombinant CvGal1 (A) and CvGal2 (B) with or without 0.1 M lactose. The galectin binding was detected with galectin-specific antibodies followed by Fitc-conjugated anti-rabbit secondary antibody incubated, and analyzed in C6 cytometer. (C) P. chesapeaki was incubated with purified MaGal1 (12 or 2 μg) with (+Lac) or without (–) 0.1 M lactose and the bound galectin was pelleted along with the parasites by centrifugation. The bound galectin was eluted and run in SDS-PAGE along with the unbound galectin remaining in supernatant, and subjected to Coomassie staining. The intensities of both fractions from each samples were quantified (NIH Image J) and % bound was calculated as bound/(bound + unbound) [Adapted from Feng et al. (2015) with permission from the American Chemical Society].

Figure 4

Binding ligand analysis through glycan array and homology modeling. (A) Six best glycans ranked by their affinity for CvGal1 in glycan array analysis. The negative percentages are the evaluation of the % change in the fluorescent signal (F_j – F_i)/F_i × 100%). (B) Model of the four CRDs of CvGal1: Chain A in orange, chain B in cyan, chain C in magenta and chain D in yellow. (C) Overlay between the modeled CvGal1 CRDs and BaGal1: Loop 4 of the CvGal1 CRDs (chain colors as in A above) is shorter than the loop 4 of BaGal1 (in gray), allowing bulkier structure next to the N-acetylglucosamine residue. (D) A2 blood oligosaccharide docked at the binding pocket of the CvGal1 model of the first CRD, using the observed common N-acetyllactosamine disaccharide bound to the template. CvGal1-binding site is shown as semi-transparent solvent-accessible surface colored by its vacuum electrostatic potential (positive in blue to negative in red). The schematic model of the protein is visible across the surface showing the interacting residues in a stick representation. H-bonds recognizing hydroxyl groups of the A2 oligosaccharide are displayed as dashed lines with their distances (in Å) between heavy atoms indicated [Adapted from Feng et al. (2013) with permission from the American Society for Biochemistry and Molecular Biology].

Figure 5

Binding of recombinant CvGal1 and CvGal2 to blood group A antigen. (A,D) blood group A tetrasaccharide-BSA (A Tetra-BSA), blood group A trisaccharide-BSA (A Tri-BSA), or N-acetylgalactosamine-BSA (GalNAc-BSA) were added at the concentrations indicated (serial dilution starting from 10 μg/ml, 100 μl/well) into 96-well plates and the binding of 0.2 μg/ml of rCvGal1 (A) or rCvGal2 (D) was assessed by ELISA. Data show optical density at 450 nm (OD_450nm) in triplicates with standard error (SEM). (B,C,E,F) Blood group A tetrasaccharide-BSA (A Tetra-BSA) or blood group A trisaccharide-BSA (A Tri-BSA) were immobilized up to 1,000 response units on CM5 chips, and the binding of rCvGal1 (B,C) or rCvGal2 (E,F) was assessed by SPR with either lectin being injected as analyte. The SPR sensorgrams were recorded with 2-fold serial dilutions of the analyte starting from 100 μg/ml. Negligible responses were observed on sensorgrams for the GalNAc-BSA (data not shown) [Adapted from Feng et al. (2013) with permission from the American Society for Biochemistry and Molecular Biology].

Figure 6

Binding of CvGal1 and CvGal2 to oyster hemocytes. (A) Upon hemocyte attachment and spreading, CvGal1 is translocated to the periphery and secreted. Western blotting of unattached hemocytes, plasma, attached-spread hemocytes, and supernatant. (B) Immunofluorescence staining with anti-CvGal1 and DAPI staining of attached-spread hemocytes with (+) or without (–) Triton X treatment, showing the presence of CvGal1 in the cytoplasm, and on the external surface of the hemocyte plasma membrane, respectively. Scale bar, 10 μm. (C) Binding of rCvGal1 (100 μg/ml) to hemocytes in the presence of PSM (0–300 μg/ml), whereby the control (Ctrl) is sample without exogenous rCvGal1 and inhibitor. (D) Binding of rCvGal1 to unattached hemocytes with α-N-acetylgalactosaminidase treatment (GalNAc'ase) or no treatment (Untreated) were measured by flow cytometry, whereby a sample without rCvGal1 was the control (Ctrl). Data show mean fluorescence intensity (MFI) ± S.E. of each sample. (E) Fixed hemocytes were stained with dilutions of anti-blood group A antibody (red, 1:2000; blue, 1:500; yellow, 1:100) or buffer only (black) in flow cytometry analysis. (F) Fixed cells were preincubated with anti-blood group A antibody (1:100), and the binding of rCvGal1 (100 μg/ml) was measured by flow cytofluorometry; the control (Ctrl) was recorded in the absence of rCvGal1. *Indicates significant difference (p < 0.05) between samples from One-Way ANOVA analysis [Adapted from Tasumi and Vasta (2007) and Feng et al. (2013) with permission from the American Association of Immunologists and the American Society for Biochemistry and Molecular Biology].

Figure 7

Binding ligands of CvGals on oyster hemocyte surface. (A) Isolation and identification of CvGal1 ligands from oyster hemocytes. Oyster hemocyte extracts were eluted from a rCvGal1-column (rCvGal1 cross-linked to Affi-Gel 15, BioRad) using 50 mM lactose, prior to analysis by SDS-PAGE. The Coomassie blue-stained protein bands (H1–H5) were excised from the gel and subjected to proteomic analysis as described (Feng et al., 2013). Peptide sequences were identified using Mascot software to search the NCBInr 167 database, processed in Scaffold and the proteins identified based on the identification of at least two peptides at 95% or greater confidence. (B) MS/MS analysis of oyster glycans carrying the histo-blood group A modification. a–c, N-glycans from selected NP-HPLC fractions (6.0, 5.7, and 7.7 g.u., respectively) were subject to MS/MS of their protonated forms in positive mode with the focus on the fragments in the range m/z 500–800, which are indicative of modification by the blood group A and methyl groups; for the glycan carrying both non-methylated and methylated forms of the blood group A, the differences of 14 Da between the sets of fragments are shown with dashed lines. The blue boxes emphasize the identified blood group A tetrasaccharide structure (for further details, see Feng et al., and Kurz et al., 2013).

Figure 8

CvGals mediate P. marinus trophozoites adhesion onto oyster hemocytes. (A) Carbohydrate -specific binding of rCvGal1 to P. marinus trophozoites in the presence and absence of thiodigalactose (inhibits rCvGal1 binding) or glucose (does not inhibit rCvGal1 binding) was analyzed by immunofluorescence staining. Scale bar, 10 μm. (B) Adhesion of P. marinus trophozoites to hemocytes was enhanced by addition of recombinant CvGal1 or CvGal2 (left) or inhibited by addition of anti-CvGal1 or anti-CvGal2 antibody (right). *Indicates significant difference (p < 0.05) from control sample [Adapted from Tasumi and Vasta (2007), and Feng et al. (2013) with permission from the American Association of Immunologists and the American Society for Biochemistry and Molecular Biology].

Figure 9

Binding of rCvGal1 to P. marinus trophozoites. (A) Binding of rCvGal1 (100 μg/ml) to P. marinus trophozoites was measured by flow cytometry analysis. Data show mean fluorescence intensity (MFI) ± S.E. of each sample. The binding of rCvGal1 (100 μg/ml) to P. marinus trophozoites in the presence of PSM (0–100 μg/ml) is shown on the right hand panel of A. *Indicates significant difference (p < 0.05) from sample without PSM inhibition (0). Sample without exogenous rCvGal1 and inhibitor was shown (Ctrl). (B) anti-A or anti-B binding to P. marinus revealed the absence of exposed A and B blood group moieties. (C) P. marinus trophozoites were stained with fluorochrome-labeled lectins (red lines) or buffer alone (black lines) in flow cytometry analysis. Black circles indicate significant staining of SBA (soybean agglutinin) and PNA (peanut agglutinin) over background, and red circle indicates no significant staining of UEA (Ulex europaeus agglutinin) [Adapted from Feng et al. (2013) and Kurz et al. (2013) with permission from the American Society for Biochemistry and Molecular Biology].

Figure 10

Schematic model of CvGals-mediated Perkinsus sp. infection. Yellow triangles represent the binding ligands on hemocytes (mainly ABH oligosaccharides on dominin) and blue triangles represent the chemically different binding ligands on Perkinsus sp. parasites.

Figure 11

Recognition of Perkinsus marinus trophozoites by the oyster (Crassostrea virginica) galectin CvGals facilitates infection: CvGals displays four canonical galectin carbohydrate-recognition domains (CRDs), a domain organization that is unlike any of the known galectin types. CvGal is translocated to the periphery and secreted by attached hemocytes, and binds to the cell surface. P. marinus trophozoites (a) ingested by filter-feeding are recognized by CvGal on the surface of hemocytes that coat the gut tubules, phagocytosed (b) and transported through the gut epithelium (c,d) into the internal milieu. The parasite inhibits intracellular killing by the host hemocytes and proliferates (e), thereby causing systemic infection and eventually death of the host [Adapted from Vasta (2009) with permission from the Springer Nature].