Cloning And Functional Study Of Zebrafish Intelectin: ZITLN1, ZITLN2 And ZITLN3

The main function of immune system is to tell apart "self" from "non-self". Innate immune is the first defense line of immune system. Innate immune responses depend on germline-encoded receptors, the pattern-recognition receptors (PRRs). The PRRs recognize the highly conserved microbial structures such as cell wall saccharide and the dsRNA of virus, and induce series signal pathways to regulate the expressions of immune-related genes to clear the pathogens. Lectin which is a key member of PRRs, plays a significant role in host defense.Intelectin (ITLN) is a new type of glycan-binding lectin involved not only in many physiological processes, but also some human diseases such as asthma, heart disease, inflammatory bowel disease, chronic obstructive pulmonary disease and cancer. Up to now, intelectin orthologs have been identified in cephalochordates, urochordates, amphibians, fish and mammals. Although the sequences of ITLN in different species are conserved, their expression patterns, quaternary structures and functions diverse considerably among and in species. Zebrafish is an important model organism, the immune system of which is integrated, and its immune system is similar to mammals. So we choose zebrafish intelectin (zITLN) to research its mechanism in pattern-recognition.We first analyzed the position of zITLN in evolution using bioinformatics and choosed zITLN1, zITLN2 and zITLN3 from three different branches for our study. We cloned the three zITLNs, and detected the tissue distribution of adult zebrafish by real-time PCR. Our results revealed that the three zITLNs were expressed in the eye, liver, intestine, skin, cheek and tail (other tissues were not examined) but the expression mode and quantity were various. zITLN1 and zITLN2 were mainly expressed in the intestine while zITLN3 was mainly expressed in the liver and it was also high expressed in the intestine. We also tested the up-regulation of S. aureus to zITLNl, zITLN2, and zITLN3. S. aureus really could up-regulate the expression of the three zITLNs although in different levels. zITLN1 and zITLN3 were more sensitive to the S. aureus challenges. The expression of zITLN1 and zITLN3 were up-regulated in the early 4h and peaked at 8h, while the up-regulation of zITLN2 increased slowly in the first 8h and peaked suddenly at 10h. To further explore the physiological function, recombinant proteins were expressed in E. coli system. Recombinant zITLN1, zITLN2 and zITLN3 proteins can aggregate FITC-labeled bacteria at different degree in a calcium-dependent manner. zITLN1 and zITLN3 likely preferred to aggregate S. aureus to E.coli while zITLN2 didn't show any preference between the two bacterial. Western blotting analysis showed that recombinant proteins were left on bacteria surface after intensive washing. zITLN1 and zITLN3 had higher binding abilities to S. aureus while zITLN2 failed to discriminate between Gram-negative and Gram-positive bacteria. Furthermore, zITLN1 and zITLN3 proteins had a higher affinity to peptidoglycan (PGN) than lipopolysaccharide (LPS), while zITLN2 recognized LPS and PGN with no obvious difference, but zITLN2 really had a higher affinity to the glycan than zITLN1 and zITLN3. All the results suggested that zITLN1, zITLN2 and zITLN3 recognize PGN or LPS, the chief components of bacteria cell wall, and then cause direct bacterial binding and agglutinating. However the mechanism of their recognization and binding effection might be different from each other, to further investigate the CRD of the intelectin gene family, we designed deletions named zITLN1-D5, zITLN2-D5 and zITLN3-D5. Bacterial binding, bacterial agglutinating and polysaccharide binding assays indicated that Domain 5 (about 100 aa) at the C terminal was related to lectin-activity at the same levels as the full-length protein suggesting that the CRD of zITLN was D5 not FReD reported before. Meanwhile we confirmed three sites (G53-G54-G55) about calcium-binding through the bacterial binding, bacterial agglutinating and polysaccharide binding assays. Furthermore we built two chimeras zITLN1-2D5 and zITLN2-1D5 by changing the D5 domain between zITLN1 and zITLN2, and tested the lectin-activity of them. We found that the lectin-activities of zITLN1-2D5 and zITLN2-1D5 were consistent with the D5 domain they contained each other. These results showed that D5 was really an important CRD of zITLN, and the D5 domain decided the polysaccharide binding activity of the full-length.In addition, recombinant zITLN1, zITLN2, and zITLN3 can bind to bovine lactoferrin in vitro by enzyme-linked immunosorbent (ELISA) assay. But none of them display any apparent inhibition effect of bacterial growth.Above all the studies about the three zITLNs' recognizing PGN and LPS, binding to bacterials and agglutinating bacterials provided clues to host defense against bacterial infection. Our findings identified for the first time a new type of CRD in zITLN, which would provide new insight leading to a better understanding of pathogen-host interactions. The sequence of intelectin is highly similar with ficolin's, but their CRD is different, suggesting that the different 3D structure decided their different function. Our future study will concentrate on analyzing the zITLN's structure to obtain a more elaborate mechanism of its pattern recognization.