Expression And Functional Analysis Of Complement Factor Properdin In Zebrafish Danio Rerio

Complement is a central component of innate immunity, and plays multiple rolessuch as immune cell activation, chemotaxis, opsonization, and lysis of antigens,protecting host from infection. Complement system can be activated by one of thethree pathways, i.e. the classical pathway (CP), the alternative pathway (AP), and thelectin pathway (LP). Complement factor properdin, also known as factor P (fP), wasinitially described by Pillemer et al., which is the only known naturally occurringpositive regulator of the complement system. Properdin binds and stabilizes theconvertase C3bBb that is formed from activated C3b and one of the major cleavageproducts of factor B (Bb). Properdin is composed of6typical TSR domains and atruncated TSR domain named TSR0. Our current knowledge regarding properdin ismainly from the study of mammalian species, and research of proterdin is ratherlimited in the lower vertebrates such as fish. Additionally, information regarding thestructure-activity relationship of properdin remains rather fragmentary. Zebrafish is awidely used model organism. However, little is known about properdin to date in thisimportant model species, therefore, the aims of this study were therefore to analyzethe expression pattern of zebrafish properdin gene and to examine its function as wellas structure-activity relationship.First, bioinformatics analysis of zebrafish properdin showed that the ORF ofzerafish properdin obtained was1314bp long and coded for a deduced protein of437amino acids including a signal peptide, six TSP domains numbered TSR1-6and atruncated TSR0. Like human properdin gene, zebrafish properdin was organized intoeleven exons interspaced by10introns. TSR0-5of zebrafish properdin were encodedby discrete exons and the sequence coding for the TSR6was split into two exons.Multiple alignment revealed that many amino acid residues were highly conserved.The CSVTCG motif conserved in mammalian TSR4and the WXXWXXW and RXRmotifs conserved in mammalian TSR1-6were also highly conserved in zebrafish properdin. The constructed phylogenetic tree using full-length properdin amino acidsequences demonstrated that fish properdins including zebrafish properdin wereclustered into a single group, which was positioned at the base of jawed vertebrates,well reflecting the established phylogeny of the chosen organisms.Second, tissue-and stage-specific expression of zebrafish properdin in thedifferent stages of embryos/larvae and the different tissues of adult zebrafish wastested by qRT-PCR and WISH. qRT-PCR analysis showed that zebrafish properdintranscript was detected in all the tissues examined. The zebrafish properdin transcriptwas the most abundant in the liver. qRT-PCR also showed that zebrafish properdinwas highly expressed in1hpf embryos and its expression was sharply decreased until10hpf and then gradually increased from10hpf to10dpf. The result of WISHshowed that strong maternal expression of zebrafish properdin was detected incleavage and blastula stages. It was ubiquitously expressed in the gastrula andsegmentation stage embryos, then expressed in the brain, neural plate, developing lensand neutrophils.Third, we constructed expression vectors and purified recombinant TSRs ofzebrafish properdin by prokaryotic expression system, then detected their function invitro. Recombinant TSRs proteins were demonstrated to be able to bind to C3b, LPS,LTA and both gram-negative and positive bacteria. In addition, assays for effects ofTSRs on phagocytosis were processed. Our results showed that only the binding ofTSR5to the microbes was able to enhance the phagocytosis of the microbes by themacrophages, indicating that TSR5plays a central role in the capacity of properdin topromote phagocytosis.In summary, these results together support the notion that properdin is a patternrecognition molecule capable of identifying non-self antigens/structures, and indicatethat TSR5plays a central role in the capacity of properdin to promote phagocytosis. Itis also suggested that properdin is associated with the pattern formation and immunedefense of early developing embryos/larvae.