Human RegIV protein adopts a typical C-type lectin fold but binds mannan with two calcium-independent sites.
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 (15)N 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.
Ho
Meng-Ru
MR
Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 115, Taiwan, ROC.
Lou
Yuan-Chao
YC
Wei
Shu-Yi
SY
Luo
Shih-Chi
SC
Lin
Wen-Chang
WC
Lyu
Ping-Chiang
PC
Chen
Chinpan
C
D001665:Binding Sites; D002118:Calcium; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037181:Lectins, C-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000378:metabolism*; D009154:Mutation; D000073718:Pancreatitis-Associated Proteins; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D037181:Lectins, C-Type; D008351:Mannans; D000073718:Pancreatitis-Associated Proteins; D011134:Polysaccharides; C428528:REG4 protein, human; D006493:Heparin; D002118:Calcium