Structural analysis of neutral glycosphingolipids bearing human blood group-like sugar units from the mantle skin of the jumbo flying squid ( <i>Dosidicus gigas</i> )

Glycosphingolipids (GSLs) from invertebrates often exhibit unique sugar chain structures distinct from those in vertebrates, reflecting evolutionary diversity in glycan biosynthesis. In this study, neutral GSLs were extracted and purified from the mantle skin of the jumbo flying squid Dosidicus gigas (Cephalopoda, Mollusca) using silicic acid (Iatrobeads) column chromatography. Their structures were characterized by gas-liquid chromatography, gas chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and 1H-nuclear magnetic resonance spectroscopy. The purified GSLs contained oligosaccharide chains comprising one to eight sugar residues, including mollusk-specific mannose-containing cores (Mollu-series). Notably, the octasaccharide fractions included GSLs bearing human blood group A- and B-like epitopes, which reacted specifically with anti-A and anti-B sera in thin-layer chromatographic immunostaining. The principal structures were identified as GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-2Manα1-3(Xylβ1-2)Manβ1-4Glcβ1-Cer (A-type) and Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-2Manα1-3(Xylβ1-2)Manβ1-4Glcβ1-Cer (B-type). Ceramide analysis revealed fatty acids ranging from C14 to C24, with C16:0, C22:1, and C24:1 as dominant components. Odd-chain and hydroxy fatty acids were also detected. Sphingoids such as d16:1, d17:1, and d18:1 were present, with d16:1 being the most abundant. These findings highlight the structural complexity of molluscan GSLs and provide the first molecular evidence of both A- and B-type blood group-like glycosphingolipids in cephalopods, offering novel insights into the evolution and functional diversification of glycan biosynthesis across animal lineages.