Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus

Abstract

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.

Keywords: X-ray crystallographic analysis; lectin; rhamnose; sea urchin; toxin.

Figure 1

Mitogenic activity of rSUL‐I and native SUL‐I on murine splenocytes. Data show the mean ± SD of three experiments with determinations in triplicate.

Figure 2

Cytotoxicity of rSUL‐I for Vero cells. Cytotoxicity was measured with the inhibition of colony formation method. Each point represents an average of triplicate measurements.

Figure 3

Glycoconjugate microarray analysis of rSUL‐I. The binding specificities of Cy3‐labeled rSUL‐I for various glycoconjugates were measured. The vertical arrows indicate glycoconjugates containing terminal GalNAc residues (Supporting Information Figure S1). All of the glycoconjugate structures used in this analysis are listed in Ref. 26.

Figure 4

Frontal affinity chromatography analysis for the binding of rSUL‐I to PA‐labeled oligosaccharides. Association constants (K _a) were calculated for 130 PA‐oligosaccharides. The structures of the PA‐oligosaccharides27 are shown in Supporting Information Figure S2. The marks representing the structures in this figure are the same as in Figure 3.

Figure 5

Overall structures of the rSUL‐I/l‐rhamnose monomer (A) rSUL‐I/L‐rhamnose dimer (B), and CSL3/l‐rhamnose (C). A, the three domains of rSUL‐I are colored differently. B and C, the two polypeptides of rSUL‐I dimer and CSL3 (PDB ID: 2ZX2) are depicted in different colors. Bound l‐rhamnose molecules (marked by red circles) are depicted as stick models.

Figure 6

Superposition of the main‐chain structures of the three domains of rSUL‐I. Domains 1 (residues 1–93), 2 (residues 94–191), and 3 (residues 192–284) are shown in green, pink, and orange, respectively.

Figure 7

Structure of the three carbohydrate‐binding sites of rSUL‐I. Bound l‐rhamnose and the residues involved in its recognition in domains 1, 2, and 3 are shown in A, B, and C, respectively. The 2F _o‐F _c electron density maps of l‐rhamnose are contoured at 1.5σ. Panel D shows the putative galactose‐binding mode, depicted by substituting d‐galactose to l‐rhamnose in domain 1.

Figure 8

Amino acid sequence alignments of the domains of SUL‐I and CSL3. The residues involved in carbohydrate‐recognition are enclosed in boxes. For CSL3, the residues interacting with Gb₃ are marked by red boxes. The variable loop region, which varies in length among RBLs, is indicated by a red line. Asterisks, colons, and periods indicate the positions of identical, strongly similar, and weakly similar residues, respectively.

Figure 9

Interactions between the C‐6 atom of l‐rhamnose and the residues in the three binding sites of rSUL‐I (A, B, and C) and in the N‐terminal half domain of CSL3 (D) (PDB ID: 2ZX2). The van der Waals radii of l‐rhamnose and the involved residues are indicated by dots.

Figure 10

Comparison of the structures of the rSUL‐I/l‐rhamnose and CSL3/Gb₃ complexes (PDB ID: 2ZX4). The crystal structures of domain 1 of the rSUL‐I/l‐rhamnose complex (green) and the N‐terminal domain of the CSL3/Gb₃ complex (blue) are superposed. The variable loops are enclosed by a red line. Hydrogen bonds are indicated by yellow dashed lines. The second β‐galactoside moiety of Gb₃ was labeled as βGal.