Molecular Design Of Antisense Peptides For Human C5a Anaphylatoxin And Study On Its Functions

The C5a anaphylatoxin is generated via either classical or alternative pathway by the clearage of a -chain of the fifth component of complement C5. C5a has shown to be the important constitution of innate unspecific immune and has significant function in immune regulation and adhesion molecule regulation. At the same time, it acts as a potent pro-inflammatory mediator and associated with the pathophysiological progressive development of many inflammatory diseases. Therefore, to blockade of the combination of C5a and C5a receptor would play a great role in the prevention or treatment of ALI, ARDS and other diseases associated with C5a anaphylatoxin. Up To date, there are three approaches that show experimental efficacy. The current studies suggested that C5a might be an inviting target in clinical settings, either by employment of blocking antibody or by use of nonantibody-based antagonists of C5a, such as peptides or reactive oligonucleotide preparations. This study is based on the theory of selective interaction between sense peptides and anti-sense peptides. It is to be a new pathway to the molecule design of neutralizing C5a anaphylatoxin.To obtain the anti-sense peptides of C5a and inhibit its biological effects, the following experiments were performed in this paper.1). By means of hydrophilic flexibility and accessibility schemes, the functional domains of human C5a binding to C5a receptor were predicated. Results showed that the functional domains were probably at or adjacent to residues 1-8th, 28-39th and 65-70th.2). According to the results of prediction above and referencing to the sequences of anti-sense homology box (LR3, LR4) reported in Baranyi's study, the residues 28-40th and 61-74th were defined to be the main function domains, and named sense peptide LI, L2 respectively. On the basis of models of anti-sense peptides molecule design, four anti-sense peptides were designed corresponding to this two sense peptides. The specific sequences showed as follows:Sense peptide LI: V28 N N D E T C E Q R A A R40Antisense peptide Rl: QLLLLWTLDARRAAntisense peptide R2: PCSSQLTGFIIINsense peptide L2: L61R A N I S H K D M Q L G R74Antisense peptide R3: PSQLHVFMRDISTEAntisense peptide R4: EARLQRVFLYVNPS3). The biospecific interaction analysis within C5a and its anti-sense peptides was studied by use of Biosensor technology. The results demonstrated that only one piece of anti-sense peptide R4 has been screened from the four pieces of anti-sense peptides and which can bind to C5a and sense peptide L2 specifically. The dissociation equilibrium constant(KD) between R4 and human C5a is 6.62 × 10-6M, and the KD between R4 and L2 is 4.508 ×10-6M.4). In vitro, the effects of anti-sense peptide R4 on the adhesion were studied between PMN and VEC resulting from human C5a anaphylatoxin. The results showed that in response to C5a after interactions with several concentrations of anti-sense peptide R4, the expressions of P-Selectin on VEC decreased significantly (P<0.01) at the concentration of 1000ng/ml. The activity of MPO in PMN reached the minimum at the concentration of 5000ng/ml and reduced from 1.16× 103u/well to 0.71 × 103u/well, decreased nearly 40% by rate when compare with the group of C5a stimulated merely.5). The concentration of C5a in post-ALI patients' serum was determinedby sandwich ELISA. The changes of adhesion molecule expressions on VEC were measured by Flow cytometry and the activity of myeloperoxidase (MPO) in PMN was determined after adhesion. The serum concentration of C5a increased significantly after ALI, in response to ALI patients' serum, the expressions of P-Selectin increased strikingly (p<0.01) at 5 min and reached the peak at 20 min, then decreased after 60 min. The intercellular adhesion molecules-1 (ICAM-1) was up-expression in VEC gradually and reached the peak at 12h. In addition, the activity of MPO in PMN increased after 3h and reached the peak at 12h.Conclusion: (1) According to the results pr...