Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for beta-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a beta sandwich folding and the representative motifs for binding beta-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and iota-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Automated summary

Galectins are a family of lectins that have ancient evolutionary origins and can recognize and bind to beta-galactosides. In this study, a galectin called HrGal was identified and characterized from the mollusk Haliotis rufescens. HrGal has four tandem-repeat CRDs and showed specificity for galactosides. It belongs to a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. The sulfated sugars heparin and iota-carrageenan were the only ones that inhibited its hemagglutinating activity.