Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites |
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Authors: | Meng-Ru Ho Yuan-Chao Lou Shu-Yi Wei Wen-Chang Lin Chinpan Chen |
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Institution: | 1 Institute of Biomedical Sciences, Academia Sinica, 128 Section 2, Academia Road, Nankang, Taipei 115, Taiwan, ROC2 Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan, ROC3 Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan, ROC |
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Abstract: | Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of 15N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract. |
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Keywords: | CPMG Carr-Purcell-Meiboom-Gill CRD carbohydrate recognition domain EDTA ethylenediaminetetraacetic acid HSQC heteronuclear single quantum coherence hxNOE heteronuclear nuclear Overhauser effect LLR long loop region PDB Protein Data Bank RU response unit SPR surface plasmon resonance |
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