MECHANISM OF CA2-TYPE CARBOHYDRATE-RECOGNITION DOMAIN OF THE MACROPHAGE MANNOSE RECEPTOR( AND MONOSACCHARIDE BINDING TO A C)

Site-directed mutagenesis has been used to identify residues that liga te Ca2+ and sugar to the fourth C-type carbohydrate-recognition domain (CRD) of the macrophage mannose receptor. CRD-4 is the only one of th e eight CRDs of the mannose receptor to exhibit detectable monosacchar ide binding when expressed in isolation, and it is central to ligand b inding by the receptor. CRD-4 requires two Ca2+ for sugar binding, lik e the CRD of rat serum mannose-binding protein (MBP-A). Sequence compa risons between the two CRDs suggest that the binding site for one Ca2, which ligates directly to the bound sugar in MBP-A, is conserved in CRD-4 but that the auxiliary Ca2+ binding site is not. Mutation of the four residues at positions in CRD-4 equivalent to the auxiliary Ca2binding site in MBP-A indicates that only one, Asn(728), is involved i n ligation of Ca2+, Alanine-scanning mutagenesis was used to identify two other asparagine residues and one glutamic acid residue that are p robably involved in ligation of the auxiliary Ca2+ to CRD-4. Sequence comparisons with other C-type CRDs suggest that the proposed, binding site for the auxiliary Ca2+ in CRD-4 of the mannose receptor is unique . Evidence that the conserved Ca2+ in CRD-4 bridges between the protei n and bound sugar in a manner analogous to MBP-A was obtained by mutat ion of one of the amino acid side chains at this site. Ring current sh ifts seen in the H-1 NMR spectra of methyl glycosides of mannose, GlcN Ac, and fucose in the presence of CRD-4 and site-directed mutagenesis indicate that a stacking interaction with Tyr(729) is also involved in binding of sugars to CRD-4. This interaction contributes about 25% of the total free energy of binding to mannose. C-5 and C-6 of mannose i nteract with Tyr(729), whereas C-2 of GlcNAc is closest to this residu e, indicating that these two sugars bind to CRD-4 in opposite orientat ions. Sequence comparisons with other mannose/GlcNAc-specific C-type C RDs suggest that use of a stacking interaction in the binding of these sugars is probably unique to CRD-4 of the mannose receptor.