We here report the purification of a novel member of the galectin family, the β-galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed for the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acid-long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving an N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan-binding properties of hRTL revealed that it had high affinity towards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galβ1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cytotoxicity.

We here report the purification of a novel member of the galectin family, the β-Galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acids long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving a N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan binding properties of hRTL revealed that it had high affinity to-wards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galβ1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cell death by activating MAP kinases, thereby inhibiting cancer cell proliferation.

Functional divergence among lectins can arise even in the absence of variation in carbohydrate recognition domains (CRDs), yet the underlying mechanisms remain poorly understood. Here, we investigated the structure–function relationships of hRTLs, a family of six prototype galectins from the sponge Chondrilla australiensis. Recombinant hRTLs lacking the predicted 23-amino-acid non-canonical signal peptide were expressed in Escherichia coli and analyzed for glycan-binding specificity using glycan arrays. Four members (hRTL-1, 2, 5, and 6) exhibited glycan-binding activity, whereas hRTL-3 and 4 showed markedly weaker activity. hRTL-1 and 2 displayed similar binding profiles, including recognition of TF-related glycans, while hRTL-5 preferentially bound type-1 LacNAc and hRTL-6 recognized blood group-related glycans. Structural analyses revealed distinct oligomeric states, with dimeric and tetrameric species supported by native mass spectrometry and size-exclusion chromatography, while additional higher-molecular-weight (HMW) species were observed by non-heated SDS–PAGE. Far-UV CD spectroscopy further showed that representative active and low-activity hRTLs retained β-sheet-rich secondary structures. Notably, thermal stability assays showed that multimer-forming hRTLs retained hemagglutination activity after prolonged incubation at 60 °C, whereas non-multimerizing members exhibited reduced stability. These findings show that distinct oligomeric organizations are associated with functional diversification among structurally conserved galectin CRDs and suggest that modulation of multimerization may have contributed to the evolution of early metazoan lectins.

The structure of a lectin, OXYL, derived from the feather star, Anneissia japonica (phylum Echinodermata), was analyzed from biochemistry, informatics, and 3D structural prediction. OXYL’s primary structure resembled the globular domain of complement C1q in vertebrates. Structural simulations identified OXYL as part of the C1q/TNF superfamily, characterized by a jellyroll β-sandwich fold. The subunits can form monomer to nonamer (9-mer), with trimers being the most stable. However, gel permeation chromatography indicated tetramers as the significant form. In hemagglutination inhibition tests, OXYL bound type-2 N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc) and inhibited agglutination but did not bind lactose (Lac: Galβ1-4Glc). Glycan microarray analysis with 20 immobilized glycans confirmed OXYL’s specificity for type-2 LacNAc and its sialic acid α2-3 linkage. The structural simulation showed a suitable molecular docking between type-2 LacNAc and the lectin rather than lactose. OXYL bound to the LacNAc glycan on the microarray was also elucidated to capture human IgG. This finding suggests that the invertebrate C1q/TNF family lectin will mimic vertebrate C1qs, despite lacking evolutionally antibody systems, possess the potential to interact with antibodies.