Identification, Expression And Functional Characterization Of Properdin From Zebrafish

Properdin, also known as P factor, is an upregulator of the alternative complement pathway and acts by stabilizing the alternative pathway convertase (C3bBb). To elucidate the relationship between structure and function of properdin, and find the binding sites for C3b and factor B, we cloned and expressed the properdin gene in zebrafish to identify its functional properties.The open reading frame cDNA of properdin is 1314bp, coding a polypeptide of 437 amino acids with a calculated molecular weight of about 48.2 kDa, composed of six thrombospondin repeat typeⅠdomains(TSR-1–TSR-6) too. There were forty Cys in the mature Properdin, predicted fifteen possible disulfide bonds, and two potential N-linked glycosylation site located at the residual position 31 and 58. Phylogenetic tree analysis of different Properdin protein sequences showed that zebrafish Properdin clustered in bony fish clade. On aligning the zebrafish properdin and TSR with other species, zebrafish properdins protein were found to have consensus sequence motifs WXXWXXW and RXR, The amino acid identity between zebrafish and that of humans, mouse, xenopus and trout pfc1, pfc2 and pfc3 was 42%,41%,43%,48%,48% and 49%, respectively. Zebrafish Properdin gene expression pattern at different developmental stages and in different tissues was analyzed by qRT-PCR. Results show Properdin to have maternal expression with a tendency to decrease gradually as the development continues. No expression was recorded at 30%-epiboly stage. However, from the 20-somite stage, the zygotic expression of Properdin commenced again. In adult zebrafish, Properdin was found to be expressed in all the tissues. Relatively lower expression was found in the muscles, fins, intestine, ovaries and brain; higher expression was found in the heart, testis, liver, eyes and heart.In Summary, zebrafish properdin resemble its mammalian counterpart, which coincides with the phylogenetic status of zebrafish. One of the predicted glycosylation sites located between Cys45 and Cys63, which were predicted to form a disulfide bond, possibly serving to bind saccharide. Zebrafish Properdin has maternal expression, indicating its role in the early development. This is the first report of properdin in zebrafish. We have purified the recombinant Properdin protein thus making it possible to conduct immunoassays and other biological investigations in order to study the structure, function and mechanism of the action of Properdin. Further characterization of Properdin will help us clarify the pathological mechanism of the human complement defect and help us understand the functioning of the complement system.