Characterization of HOL-30: a novel tandem-repeat galectin from the marine sponge Halichondria okadai

We here report the novel primary structure of a new member in the galectin family, the β-galactoside-binding lectin HOL-30, from the marine sponge <i>Halichondria okadai</i>, whose full-length sequence was determined thanks to the combination between Edman degradation and transcriptome analysis. The HOL-30 polypeptide is a tandem-repeat dimeric galectin, consisting of 281 amino acids, which includes two carbohydrate recognition domains (CRDs). Unlike most other galectins described in Porifera, HOL-30 did not have a signal peptide sequence for secretion. In solution, HOL-30 exhibited a molecular weight of 60 kDa, indicating a dimeric organization consisting of two 30 kDa tandem-repeat subunits stabilized by non-covalent interactions. Although the two CRDs had a similar predicted 3D structure, they displayed low pairwise sequence identity, approximately 20 %. HOL-30 exhibited glycan-binding affinities for type-1 (Galβ1-3GlcNAc) and type-2 (Galβ1-4GlcNAc) LacNAc. Furthermore, it also recognized blood type B-oligosaccharides on type-1 and type-2 LacNAc (Galα1-3Gal[Fucα1-2]β1-3/4GlcNAc), and blood type H-oligosaccharide on type-3 (Gal[Fucα1-2]β1-3GalNAcα). The glycan-binding properties of HOL-30 were compared with those of the hRTL galectin, previously identified in <i>Chondrilla australiensis</i>, consisting of tetrameric 15 kDa prototype subunits. The two sponge galectins displayed similar, but not identical, carbohydrate-binding properties, as evidenced by the fact that despite effectively binding to vertebrate cultured cells, HOL-30 had minimal impact on cell growth. Antiserum analysis revealed a mosaic distribution of HOL-30 in the parenchymal cells of sponge tissues within dense cell clusters surrounding the spicules.