ORIENTATION OF SUGARS BOUND TO THE PRINCIPAL C-TYPE CARBOHYDRATE-RECOGNITION DOMAIN OF THE MACROPHAGE MANNOSE RECEPTOR

The extracellular region of the macrophage mannose receptor, a protein involved in the innate immune response, contains eight C-type carbohy drate-recognition domains (CRDs). The fourth of these domains, CRD-4, is central to ligand binding by the receptor, and binds mannose, fucos e and N-acetylglucosamine by direct ligation to Ca2+. Site-directed mu tagenesis combined with NMR and molecular modelling have been used to determine the orientation of monosaccharides bound to CRD-4. Two reson ances in the H-1 NMR spectrum of CRD-4 that are perturbed on sugar bin ding are identified as a methyl proton from a leucine side chain in th e core of the domain and the H-2 proton of a histidine close to the pr edicted sugar-binding site. The effects of mutagenesis of this histidi ne residue, a nearby isoleucine residue and a tyrosine residue previou sly shown to stack against sugars bound to CRD-4 show the absolute ori entation of sugars in the binding site. N-Acetylglucosamine binds to C RD-4 of the mannose receptor in the orientation seen in crystal struct ures of the CRD of rat liver mannose-binding protein. Mannose binds to CRD-4 in the orientation seen in the CRD of rat serum mannose-binding protein and is rotated by 180 degrees relative to GlcNAc bound to CRD -4. Interaction of the O-methyl group and C-l of alpha-methyl Fuc with the tyrosine residue accounts for the strong preference of CRD-4 for this anomer of fucose. Both anomers of fucose bind to CRD-4 in the ori entation seen in rat liver mannose-binding protein.