In silico characterization of single and tandem-repeat galectin from terrestrial slug, Incilaria fruhstorferi

Abstract

Aim: Galectins are beta-galactoside binding proteins currently represented by 15 members (Galectin-1 through Galectin-15). The members are differentiated on the basis of the number of carbohydrate recognition domains (CRDs), the linker region connecting dual CRDs, and the conserved amino acid (aa) residues within CRDs. In this study, galactosyl-binding lectin family proteins were screened and characterized from transcriptome of terrestrial slug Incilaria fruhstorferi using bioinformatics analysis. Methodology: I. fruhstorferi unigene sequences showing identity to galectins in the orthologous species were used for prediction of ORFs using FGENESH software. Subsequently, the ORFs were validated for conserved domain analysis using BLASTp and SMART programs. The multiple sequence alignments and percent identity/distance was elucidated using Clustal X2 and represented in GeneDoc sequence visualization program. The phylogenetic tree was inferred using MEGA7. Results: A single-repeat and a tandem-repeat galectin (If_Gal6 and If_Gal4, respectively) were retrieved from the transcriptome profile of terrestrial slug, I. fruhstorferi. The predicted If_Gal4 and If_Gal6 comprised of 693 nucleotides translated to 230 aa residues and 435 nucleotides translated to 144 aa residues, respectively. If_Gal4 possessed two CRDs, each showing the highly conserved aa responsible for galactosyl-binding activity. Surprisingly, If_Gal6 is a single-CRD protein in contrast to the common tandem-repeat galectins illustrated for Gal6 in most other invertebrate and vertebrate species. At the level of aa sequence, If_Gal4 showed maximum of 54% identity and minimum distance of 0.459 with the sea snail, Haliotis discus Galectin-4 (Hd_Gal4). Cluster analysis showed higher similarities of If_Gal4 with selected molluscan Galectin-4 aa sequences. If_Gal6 showed 41% identity with the air-breathing freshwater snail, Biomphalaria glabrata Galectin-6 (Bg_Gal6) supposed to exist as dual-CRD molluscan galectin. Interpretation: Discovery and structural characterization of a single-CRD galectin from I. fruhstorferi is second in molluscs after the sequence was reported from the Pacific oyster, Crassostrea gigas.