Abstract
The major function of extracellular matrix (ECM) is aggregating cells into tissues, signaling cells to grow, proliferate, and differentiate, coordinating the diverse function of cells of different types, and providing a path for cell migration. Multiadhesive matrix proteins, such as fibronectin and laminin, which contain several binding domains including GAG-binding regions and cell-binding sequences, attach cells to the matrix. It has been proposed that two putative GAG-binding regions, E286-312 and E391-416, capable of binding to the GAGs on the surface of cells reside in the envelope protein of Japanese encephalitis virus. Virus can attach to cells by binding to the GAGs on the surface of cells, which was considered as an indispensable step in the early stage of virus infection. Hence, by measuring the adhesion of cells to the immobilized fragments of E protein will we be able to understand the interaction between cells and protein and to estimate the potential of a cell-adhesive material out of this protein. Briefly, the gene of JEV E protein was constructed into pET32a vector as JEV E292-402/32a and JEV E277-420/32a, in addition, a single amino acid mutation was introduced to create an RGD-motif-carrying mutant, JEV RGD/32a. After expressed in an E.coli. system, pure proteins obtained by on-column refolding process and typical IMAC procedure were subsequently ready for cell adhesion assay. JEV E292-402/32a, JEV E277-420/32a, and JEV RGD/32a coated microplates promoted BHK-21 cell attachment in a dose-dependent manner while heparin inhibited BHK-21 cell attachment to JEV E292-402/32a, JEV E277-420/32a, and JEV RGD/32a coated microplates. Under serum condition, however, the results were quite different. When heparin added to cells, on the contrary, BHK-21 cell attachment to JEV E277-420/32a and JEV RGD/32a coated microplates was profoundly increased, but cell attachment to JEV E292-402/32a coated microplates remained inhibited. If heparin was mixed with proteins, an enhanced BHK-21 cell attachment to JEV E277-420/32a and JEV RGD/32a coated microplates was detected, and no prominent effect was found on JEV E292-402/32a coated microplates. JEV E277-420/32a and JEV RGD/32a coated microplates enhanced BHK-21 cell attachment whether we combine heparin with cells or proteins, which possibly indicated the involvement of certain serum factor in cell adhesion.