Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8 Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.     

Intradermal DNA Immunization: Antisera Specific for the Membrane Lectin MR60/ERGIC-53

The trafficking of intracellular membrane proteins in Golgi apparatus, endoplasmic reticulum or intermediate compartment has not yet been fully elucidated. The human MR60/ERGIC-53 and the rat p58 proteins are one such protein; and to study them in cell-free and in situ systems, high quality monospecific antisera are required. Highly specific antisera have been obtained after immunization of mice with plasmids containing a gene encoding either the full length or a truncated protein. The best results were obtained after intradermal injections of a plasmid encoding a truncated protein comprising both the luminal carbohydrate recognition domain and the stem down to a cysteine residue close to the C-terminal end, but neither the transmembrane nor the cytosolic domains. Such antisera have a very high titer and are very efficient tools to visualize the MR60 protein in situ or to selectively precipitate the MR60 proteins from a whole cell lysate.     

三株益生菌粘附性质及机制的初步研究

目的研究3株益生菌株的粘附性质,初步探讨细菌与肠细胞的粘附机制。方法选取已被深入研究的LGG作为阳性对照,应用显微观察和平板计数的方法测定3株乳杆菌和肠上皮细胞Caco-2的粘附,选择疏水性、表面电荷和自聚合能力3项指标来描述细菌的表面性质,应用粘附抑制试验和酵母凝集试验来测定糖类专一性粘附。结果无论是用显微观察还是平板计数,ST-Ⅲ均是所测试的4株菌中粘附能力最强的,当加入细菌和细胞比约为60∶1时,ST-Ⅲ在Caco-2上的粘附数为(7.43±0.65)CFU/细胞,强于对照菌LGG[(3.99±0.57)CFU/细胞]。在所测试的4株菌中,粘附能力和疏水性具有很好的相关性,同时自聚合能力对粘附也表现出一定的促进作用。除LGG外,D-甘露糖和甲基-α-D-甘露糖苷均能抑制另外3株乳杆菌的粘附,同时这3株乳杆菌也能不同程度地凝集酵母。结论3株益生菌的粘附均涉及到非特异性的疏水结合和甘露糖特异性粘附两个过程;ST-Ⅲ是1株具有良好粘附能力的益生菌,有待进一步的研究。