Identify Interacting Membrane Proteins Of LSECtin By Expression Cloning

Liver and lymph node sinusoidal endothelial cell lectin, LSECtin, is a new kind ofC-type lectin cloned from human liver cDNA library. LSECtin can not only negativelyregulate hepatic T-cell immune response and inhibit CTL-dependent virus clearance inmouse models of viral hepatitis, but also promote adhesion and migration of coloncarcinoma cells to liver. Membrane proteins including LSECtin can regulate signaltransduction between cells depending on membrane protein interactions. So screeningand identifying interacting proteins of LSECtin, especially membrane proteins isneeded to reveal the mechanism above.There are several methods to screen and identify interacting proteins includingCo-immunoprecipitation, Pull-down, Yeast two-hybrid, Far Western Blot and so on.Membrane proteins tend to be insoluble due to their hydrophobic nature and can beheterogeneous in size as a result of post-translational modifications, such asglycosylation. So it is a tough work to identify interacting membrane proteins bycommon methods. Expression cloning is one of the classical methods to identifyinteracting proteins especially membrane proteins. The principle is to find anappropriate screen method, and reduce the positive library by repeating the screen.After three rounds screen the clones in the positive library are identified by thesequencing.To find new interacting proteins of LSECtin by expression cloning, the cellstransfected with human spleen cDNA library were adhered to LSECtin recombinantprotein. The cells adhered by LSECtin were separated by FACS. After Hirt extraction,electroporation transform and sequencing, the potential interacting proteins ofLSECtin were identified. We have find nine potential proteins by three independentscreens, two of them CD48and HLA-A are confirmed to new interactive membraneproteins of LSECtin.