Research On Motional & Conformational Changes Of CRD In LSECtin Studied By Site-directed Spin Labeling EPR

The C-type lectin family plays important roles in the innate and adaptive immune responses, either as pathogen recognition receptors or cell adhesion receptors. Classical C-type lectins contain so-called carbohydrate recognition domains (CRDs) that bind carbohydrate structures in a calcium (Ca2+)-dependent manner. Ca2+ ions are directly involved in both ligand binding as well as in maintaining the structural integrity of the CRD that is necessary for the lectin activity. LSECtin is a novel C-type lectin originally identified by Liu W. It is found predominantly on sinusoidal endothelial cells of the liver and lymph nodes (LSEC) and denominated it as liver sinusoidal endothelial cell C-type lectin (LSECtin). LSECtin is a type II integral membrane protein and it shares 32% and 31% amino acid sequence identities with DC-SIGNR and DC-SIGN respectively. It has been shown to bind in vitro with mannose, GlcNAc, and fucose, but not to glactose. Interestingly, LSECtin can be colocalized with DC-SIGNR on the LSECs. Recent studies showed that LSECtin could bind to Ebola virus surface glycoprotein, filovirus glycoproteins, and the spike protein of SARS coronavirus. In addition, LSECtin was also demonstrated to be expressed in human peripheral blood and thymic dendritic cells isolated ex vivo. LSECtin expression confers Ebola virus-binding capacity to leukemic cells, participates in antigen uptake and internalization, and therefore it functions as a pathogen receptor. However, despite a voluminous literature describing some of the family's properties in great detail, we feel that the study on the structure of CRD would be useful, as structure determines function.Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structure of proteins. Application of SDSL is experiencing a stage of rapid evolution. The site-directed spin labeling (SDSL) technique involves the covalent attachment of a spin label side chain, which contains a stable unpaired electron, to a cystein residue on a protein or peptide. The most commonly used spin label is the sulfhydryl-specific nitroxide,2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate (MTSL). It can provide specific information on the location and environment of an individual residue within large and complex protein structures. There are three main categories of information that can be gained from SDSL-EPR experiments:motional dynamics (due to the motion of the protein, the spin labeled side chain, and/or the protein backbone), the accessibility of the spin label to paramagnetic broadening reagents, and distances between two introduced spin labels.. These events can be monitored in the millisecond or nanosecond time-scale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins investigated, and sometimes only 50-100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems. The purpose of this research is to construct the method of studying protein conformations with application of site-directed spin labeling electron paramagnetic resonance spectroscopy and to get the information of the mobility and structure changes during function of CRD. Methods:SOE-PCR (sequence overlapped extension PCR) methods are used to introduce cysteines into the CRD domain of hLSECtin. Based on not-so-conserved Ca2+ binding site 1, we selected six amino acids to be substituted by cysteine. Construct pET-28a-hLSECtin CRD expression vector, then express and purify the wild and mutant CRD proteins by Ni-NTA agarose affinity chromatography. DEAE-Cellulose ion exchange chromatography is used for renaturation and ultrafiltration for concentration of the mutant CRD proteins. The biological function of the refolded protein is verified by protein-glycan interaction. Finally, this reseach ends with MTSL labling and EPR detecting and analysis. Results:Successfully constructed pET-28a-hLSECtin CRD expression vector and mutants. Well established prokaryotic bacterial expression system and Ni-NTA protein purification system as well as DEAE-Cellulose ion exchange chromatography renaturation system. After MTSL labeling and EPR detecting, we get the preliminary information of the mobility and structure changes during function of CRD. Conclusions:Extracted from the EPR spectrum, MTSL could effectively label the designed cystein of mutant CRD. The motion of MTSL bound to mutant CRD protein is restricted obviously. Once in addition to Ca2+ and monosaccharide, the MTSL labeld with mutant CRD protein moves faster and the rotational correlation timeτc value decreases, which reflects relative mobilities and changes in the spin label motion. The accessibility data show that the mutant site(A:V264C) is on the surface of CRD spatial structure.As initial study on CRD structure of LSECtin, this research lay the foundations of the further study on conformational changes when LSECtin binding saccharide chain or viral protein. Then we can move forward to explore structural and functional differences among LSECtin, DC-SIGN, DC-SIGNR and CD23, which belongs the same family. These deep investigations may provide a new thinking on evolution of the C-type lectin and roles in immune system and provide a target to design novel small molecule drugs for preventing and curing relative diseases.