| C-reactive protein(CRP)is a typical acute phase plasma protein and within this phase its serum concentration could rise to 1000-fold.It is also relevant with many chronic diseases and often used as a nonspecific inflammation marker in clinical examination.CRP is a pentameric protein consisted of five identical,non-covalently joint subunits.Each subunit contains 206 amino acids with intra-subunit disulfide bond between 36-Cys and 97-Cys and presents a recognition face and an effector which bind with PCh,calcium and C1 q respectively.We transfected COS-7 cells with CRP coding sequences(with its own signal peptides attached)and successfully obtained the secreted native pentameric CRP.Then we constructed a slew of CRP mutants with site-directed mutagenesis in certain essential motifs including N terminal,C terminal,intra-subunit disulfide bond,α helix,ionic bond,calcium binding sites and also introduced inter-subunit disulfide bond to express in COS-7 cells.Through ELISA and Western blotting,we found that the subunit folding and pentamer assembly are two separated events.Guided by N terminal aa1-31,aa32-101 could form a stable hydrophobic core.The formation and locating of the α helix formed by aa168-176 conduced to the formation and stabilization of the intra-subunit disulfide bond.The disulfide bond integrity is essential for CRP subsequent folding and act as a lock for 1 and 6β sheets in the same domain.Calcium is necessary for the native structure formation of the CRP,and the 61 N,138E,139 Q,140D,150 Q are the indispensable amino acids of the calcium binding sites.Ionic bonds are also involved in the subunit assembly process with 118R-155 D as a major factor.According to the above,we can obtain the basic structure formation process of CRP in eukaryotes.The folding and assembly of CRP subunits are sequential steps.First,N terminal guides aa168-176 to form the hydrophobic core structure and with the formation and locating of the α helix,they both help the formation of the intra-subunit disulfide bond.Second,subunit further folding.At last,subunits form the calcium binding sites and assemble into native pentameric CRP. |
