Sequence variety in the CC' loop of Siglec-8/9/3 determines the recognitions to sulfated oligosaccharides

Siglecs are important lectins found in different types of immune cells and function as regulatory molecules by recognizing self-associated glycans and converting extracellular interactions into signals for inhibiting immune cell functions. Although many Siglecs have been found to show broad specificities and recognize different types of sulfated oligosaccharides, Siglec-8 and Siglec-9 displayed a high degree of specificity for sialyl N-acetyllactosamine (sLacNAc) with sulfations at O6-positions of the galactose (6'-sulfation) and N-acetylglucosamine (6-sulfation), respectively. Siglec-3 was recently discovered to bind sLacNAc both sulfations. In addition to a conserved arginine residue for binding to sialic acid residue, the sequence variety in the CC' loop may provide binding specificities to sulfated oligosaccharides in Siglecs. Thus, the present study employed molecular models to study the impact of different residues in the CC' loops of Siglec-8/9/3 to the recognitions of 6-sulfations in Gal and/or GlcNAc of sLacNAc. The negatively charged residues in the CC' loop of Siglec-9 formed unfavorable electrostatic repulsions with the 6-sulfate in Gal and resulted no recognitions, in contrast to the favorable interactions formed between the positively charged residues in the CC' loop of Siglec-8 and the 6-sulfate in Gal resulting strong specificity. A two-state binding model was proposed for Siglec-3 recognizing 6-sulfations in Gal and GlcNAc of sLacNAc, as the neutral residues in the CC' loop of Siglec-3 could not form strong favorable interactions to lock the 6-sulfate in Gal within a single binding pose or strong unfavorable interactions to repel the 6-sulfate in Gal. The oligosaccharide adopted two distinctive binding poses and oriented the sulfate groups to form interactions with residues in the CC' loop and G-strand. The present study provided a structural mechanism for the sequence variety in the CC' loop of Siglec-8/9/3 determining the recognitions to the sulfated oligosaccharides and offered insights into the binding specificities for Siglecs.