Abundant Members Of Scavenger Receptors Family And Their Dynamic Expression In Juvenile Larimichthys Crocea

Scavenger receptors(SRs) are a surface transmembrane glycoprotein family which have different structure and versatile function. In this paper, based on the whole genome sequence of large yellow croaker(Larimichthys crocea), bioinformatic analyses including codon bias, phylogenetic evolution and crucial domain interpretation were demonstrated through the cloned full length ORF sequence of all scavenger receptor family members in large yellow croaker. Using peritoneal infection of pathogenic V. alginolyticus, different mRNA expression patterns of various family members was interpreted, and chose significantly up-regulated family members to further study including line location and 3D structure model. These key points were as follow.1. To investigate the codon usage pattern(CUP) and understand the differences of eight SRs genes, the codon usage relation was interpreted by CodonW program. Twenty different amino acids appeared in all the SRs members. Moreover, the eleven codon bias indexes including codon adaptation index(CAI) fully indicated the codon usage pattern of SRs family. Compared with the codon usage pattern of human, the result did not show a strong codon bias in the large yellow croaker SRs family. Based on relative synonymous codon usage(RSCU) value calculated by CodonW program, the heat map of 59 synonymous codon was constructed by HemI software and showed also no strong codon bias. Additionally, Correspondence analysis(COA) was analyzed by CodonW and further exhibited the relation between eight genes and main contributors based on the 59 synonymous codons. The concentrate distribution(all the codons ending by A\T\G\C) was represented by points marked in the main two-dimensional Axis1-Axis2, which implied that the A-, T-, G-, C-ended codons concentrate distribution was neither clear characterization nor obvious bias.2. To reveal the molecular phylogenetic position of all SRs members, an unrooted phylogenetic tree on the basis of amino acid sequences was constructed by MEGA v4.0using the Maximun Parsimony method. Every gene formed their own separate branches with strong bootstrap support. The mammals and no-mammals were subdivided into different branches. Forthermore, LycCD68 owned a single branch, LycSCARA5 and LycMARCO were in the same cluster, LycSREC1 and LycSREC2 fell into another one, and then two branches was grouped together with LycSCARB1. Hence, the above findings exactly demonstrated that the SRs members in the large yellow croaker were unambiguously assigned to various family. The line construction, gene structures andfeature domains were analyzed by SMART and GeneMaper, which were further illustrated by IBS 1.0 program. All eight members are located in eight different scaffold positions,means that these genes may be in different chromosomes. The transcript directions of all SRs members are not exactly the same, which may express the different intensity and efficiency of transcription based on the whole genome sequence of large yellow croaker.The introns and exons were analyzed by GeneMaper software version 2.5. Different length and numbers of exons and introns led to the diverse mRNA length and translating various feature domains. In order to exhibit the structure details of all SRs members, 3D-structure models of seven members(except for LycSCARB1) were showed by Swiss-Model online tool to analyze the spatial position of the key functional domains comparison. These findings will help to understand the genetic origin, genetic types, linear cluster structures and chromosome composition for SRs family in the large yellow croaker.3. To detect their potential functions in large yellow croaker, the expression pattern of all SRs members mRNA in eight different tissues were analyzed, including heart, gill, liver,intestine, muscle, spleen, brain and head kidney, the plot was also illustrated by HemI, and the gill was used as the relative standard. All the SRs members were almost few expressed in heart, gill and intestine, but considerable high in other five tissues, especially liver and spleen. More specifically, the highest expression of the class A subfamily was in spleen and the remaining members were almost in liver. The temporal expression profile of all SRs members mRNA were up-regulated after V. alginolyticus injection, and the expressive peak of different SRs members mRNA were diverse, but basically concentrated in the 24 to 48 h post-injection, LycSREC2 in class F reached the highest at 6 h post-injection. Besides, the sub-cellular localization was predicated by ProtComp 9.0 and the result showed plasma membrane, lysosome and extracellular were mainly sub-cellular region. Intuitively, the five members in class A and F family, LycSCARA3, LycSCARA5, LycMARCO, LycSREC1 and LycSREC2, located in the plasma membrane. Two members of class B located in different positions, LycSCARB1 was in lysosome and LycCD163 was extracellular. The class D member LycCD68 was in two sites, extracellular and plasma membrane. The results showed that the members in the class A and F family of SRs were major immune moleculars and needed to further analyze.4. The class A scavenger receptors are important pattern recognition receptors of the innate immune system in living organisms. According to the whole-genome data of large yellow croaker, LycSCARA3, LycSCARA5 and LycMARCO were cloned from the spleen.The BLASTp analysis strongly suggested that the sequences shared high similarity withknown SCARA3, SCARA5 and MARCO. The phylogenetic relationship analysis illustrated that different subtype of SRs formed their own separate branches, LycSCARA3 and LycSCARA5 were placed in SCARA3 and SCARA5 branch of Osteichthyes fish respectively with strong bootstrap support. Curiously, the LycMARCO was clustered with Alligator sinensis. ClustalW analysis with amino acid sequences revealed that the proportion of identity with other species was 59~71% for LycSCARA3 and 55~72% for LycSCARA5, but the scale of LycMARCO was considerable lower than those of other two genes(only approximately 38%). There was an optional cysteine-rich(SRCR) domain containing 6 conserved cysteines residues(C-482, C-495, C-526, C-536, C-546 and C-556)in LycSCARA5. Likewise, the SRCR domains of LycMARCO also contained 6 conserved cysteines residues(C-333, C-346, C-374, C-384, C-394 and C-404). Particularly, there were the major TRAF2-binding consensus motif and two main motifs on internalization of receptor in LycSCARA3 and LycSCARA5. The gene structures of different species were analyzed with GeneMaper v2.5, and the number of introns and exons of LycSCARA3,LycSCARA5 and LycMARCO in DNA sequences were different, for example LycSCARA3 and LycMARCO were both 13 exons and 12 introns, whereas LycSCARA5 was less, only 12 exons and 11 introns. Furthermore, quantitative real-time PCR(qRT-PCR) analysis indicated the highest mRNA expression of LycSCARA3, LycSCARA5 and LycMARCO all appeared in spleen among eight detected tissues, also muscle, brain and liver witnessed a higher expression level. The expression of them were all up-regulated in spleen after V.alginolyticus injection. Due to the SRCR domain in the C-terminal, hence the response rate of defense and clearing pathogen in LycSCARA5 and LycMARCO were faster than that in LycSCARA3. The primary protein products of LycMARCO was obtained by prokaryotic expression, which can provide a reference for the study of protein function and signaling pathway for the future.5. The class F scavenger receptors(SCARFs) mainly play roles in the signal transmission and eliminating pathogens in host immune system. The complete ORF sequences of LycSREC1 and LycSREC2 in large yellow croaker were cloned and identified from the liver. The BLASTp analysis strongly suggested that the sequences shared high similarity with known SREC I and SREC II. Eukaryotic Linear Motif showed that there were total eight EGF or EGF-like domains in LycSREC1 and total seven EGF or EGF-like domains in LycSREC2. Multiple alignment analysis showed that several highly conserved amino acids were found compared with different species, especially EGF and EGF-like domains, TRAF2-binding consensus motifs and Tyrosine-based sorting. Two EGF domainsin LycSREC1 and five EGF domains in LycSREC2 were considerable conserved with homologous molecules of other species. However, there was no strong relevance of these EGF motifs between LycSREC1 and LycSREC2. The findings illuminated EGF domain in the long-term evolution was relatively conservative, and did not show a lot of variation sites, which may be related to the exercise of their functions. Given the priority to the length of DNA, LycSREC1 was 7603 bp and contained 12 exons and 11 introns, while LycSREC2 was only 4883 bp and merely 4 exons and 3 introns, which all shorter than Homo sapiens and Mus musculus. The location maps about eleven relative genes between H. sapiens and L. crocea were changed, while the five genes in ten species were also existed and changed. These interest phenomena showed that the evolution of genes might experience extremely complicated redistribution to contribute to the balance and optimization of individual survival and adjustment. Furthermore, qRT-PCR analysis indicated that the temporal expression profiles of LycSREC1 and LycSREC2 were both up-regulated in liver after V. alginolyticus injection. The expression of LycSREC2 expressed dramatically and jumped to the peak with approximately 40-fold at 6 h post-injection.However, LycSREC1 mRNA increased slowly, and the peak reached approximately 15-fold to the blank at 12 h post-injection.The aforementioned results fully demonstrated the SRs family in the large yellow croaker may participated in the fish innate immunity. All the findings would pave the way to understand not only the evolution of the SR-mediated immune response, but also the complexity of fish immunity. These bioinformatic analyses including codon bias and phylogenetic evolution explained the basic molecular mechanisms to defense from pathogen infection. The further study of the class A and F family will help to understand the immune role of scavenger receptor in family Sciaenidae and provide reference for the future to carry out prevention and vaccine research, however there are still some infection mechanism is currently no way to know pending further research.