Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis.
The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins.
Department of Biochemistry, Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
P R
PR
Crocker
M
M
Vinson
S
S
Kelm
K
K
Drickamer
D001665:Binding Sites; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D063267:Sialic Acid Binding Ig-like Lectin 1; D012794:Sialic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; C571166:SIGLEC1 protein, human; D063267:Sialic Acid Binding Ig-like Lectin 1; D012794:Sialic Acids; C000020:N-acetylneuraminoyllactose; C026321:4-O-methyl-N-acetylneuraminic acid; D007785:Lactose