Abstract
Site-directed mutagenesis has been used to identify residues that ligate Ca2+ and sugar to the fourth C-type carbohydrate-recognition domain (CRD) of the macrophage mannose receptor. CRD-4 is the only one of the eight CRDs of the mannose receptor to exhibit detectable monosaccharide binding when expressed in isolation, and it is central to ligand binding by the receptor. CRD-4 requires two Ca2+ for sugar binding, like the CRD of rat serum mannose-binding protein (MBP-A). Sequence comparisons 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 Ca2+ binding site in MBP-A indicates that only one, Asn728, is involved in ligation of Ca2+. Alanine-scanning mutagenesis was used to identify two other asparagine residues and one glutamic acid residue that are probably 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 protein and bound sugar in a manner analogous to MBP-A was obtained by mutation of one of the amino acid side chains at this site. Ring current shifts seen in the 1H NMR spectra of methyl glycosides of mannose, GlcNAc, and fucose in the presence of CRD-4 and site-directed mutagenesis indicate that a stacking interaction with Tyr729 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 interact with Tyr729, whereas C-2 of GlcNAc is closest to this residue, indicating that these two sugars bind to CRD-4 in opposite orientations. Sequence comparisons with other mannose/GlcNAc-specific C-type CRDs suggest that use of a stacking interaction in the binding of these sugars is probably unique to CRD-4 of the mannose receptor.
Highlights
Many proteins of both plants and animals are involved in recognition of complex carbohydrates attached to proteins or lipids (1, 2)
The results suggest that one Ca2ϩ is ligated to carbohydrate-recognition domain (CRD)-4 in the same way as in mannose-binding proteins (MBPs)-A but that CRD-4 has a unique binding site for the auxiliary Ca2ϩ
Each of the CRDs will probably have additional contacts with the bound sugar, such as that seen between the -carbon of His[189] of MBP-A and mannose (15), it is likely that in each case, the main interaction with sugar will be via direct ligation to Ca2ϩ at this conserved site
Summary
Many proteins of both plants and animals are involved in recognition of complex carbohydrates attached to proteins or lipids (1, 2) One such protein, the macrophage mannose receptor, binds terminal mannose, fucose, or N-acetylglucosamine residues of glycoconjugates in a Ca2ϩ-dependent manner. Apart from the mannose receptor, two other proteins, a phospholipase A2 receptor of muscle and an endocytic receptor called DEC-205, located on dendritic cells, have multiple CRDs in a single polypeptide (8 –10) These two proteins are very divergent from other groups of C-type lectins and probably do not bind carbohydrates. Ligand binding studies with expressed CRD-4 have shown that the domain requires two Ca2ϩ for sugar binding and that a ternary complex is formed between protein, sugar, and Ca2ϩ (13). CRD-4 and the CRD of MBP-A show very similar monosaccharide specificities
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