New l-Rhamnose-Binding Lectin from the Bivalve Glycymeris yessoensis: Purification, Partial Structural Characterization and Antibacterial Activity

Abstract

In this study, a new l-rhamnose-binding lectin (GYL-R) from the hemolymph of bivalve Glycymeris yessoensis was purified using affinity and ion-exchange chromatography and functionally characterized. Lectin antimicrobial activity was examined in different ways. The lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as l-rhamnose, d-galactose, lactose, l-arabinose and raffinose. Using the glycan microarray approach, natural carbohydrate ligands were established for GYL-R as l-Rha and glycans containing the α-Gal residue in the terminal position. The GYL-R molecular mass determined by MALDI-TOF mass spectrometry was 30,415 Da. The hemagglutination activity of the lectin was not affected by metal ions. The lectin was stable up to 75 °C and between pH 4.0 and 12.0. The amino acid sequence of the five GYL-R segments was obtained with nano-ESI MS/MS and contained both YGR and DPC-peptide motifs which are conserved in most of the l-rhamnose-binding lectin carbohydrate recognition domains. Circular dichroism confirmed that GYL is a α/β-protein with a predominance of the random coil. Furthermore, GYL-R was able to bind and suppress the growth of the Gram-negative bacteria E. coli by recognizing lipopolysaccharides. Together, these results suggest that GYL-R is a new member of the RBL family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity.

Keywords: bivalve lectin; hemolymph; innate immune; l-rhamnose-binding lectin; microorganism binding.

Figure 1

(a) Size exclusion chromatography of lectin on Superdex 75. (b) Anion-exchange chromatography of lectin on SOURCE 15Q.

Figure 2

(a) SDS-PAGE of GYL-R. Protein bands were stained with Coomassie Brilliant Blue R-250 reagent. Lanes: M—molecular weight markers (kDa); 1—GYL-R in non-reducing conditions (without DTT); 2—GYL-R in reducing conditions (with DTT). (b) Molecular mass determination of GYL-R via MALDI-TOF mass spectrometry.

Figure 3

Effects of temperature (a) and pH (b) on the hemagglutination activity of GYL-R.

Figure 4

Secondary structure of GYL-R was measured by Far-UV CD spectrum (260 nm–190 nm) with protein concentration of 20 µM at 25 °C.

Figure 5

Interaction of GYL-R with PAMP adsorbed on a 96-well plate, determined by the ELLA. Data are presented as mean ± SD (n = 3).

Figure 6

GYL-R binding to microorganisms, determined by ELLA. PSM (porcine stomach mucin)—positive binding control; BSA (bovine serum albumin)—negative binding control. Data are presented as mean ± SD (n = 3). Significant difference (p < 0.05) with the negative control is indicated with asterisk.

Figure 7

Growth of bacterial cells in absence and presence GYL-R. BSA is negative binding control. Data are presented as mean ± SD (n = 3). Significant difference (p < 0.05) from the control is indicated with asterisk.