Beta2-microglobulin is involved in the immune response of large yellow croaker to Aeromonas hydrophila: A proteomic based study

Outbreaks of infectious diseases in cultured large yellow croaker have resulted in great economic losses. However, information regarding its immune defense is limited. In the present study, an approach by the combination of differential proteomics with EST resource was applied for investigation of a profile of serum immune response by large yellow croaker to Aeromonas hydrophila challenge after immunization and for characterizing of one of the targeted immune molecules. Of the twelve altered proteins involved in the response, eight were identified by MS, in which three were randomly selected for antiserum preparation and were further confirmed by Western blotting. Furthermore, three β₂m clones, one of the altered molecules, were obtained from a previously constructed Kidney Smart cDNA library of this fish, and were compared for their identity, which contributed to the identification of β₂m cDNA diversity. Meanwhile, the up-regulated β₂m in response to the bacterial immunization and challenge was further confirmed by Western blotting. Our results indicate that β₂m is involved in the immune response of large yellow croaker to the challenge by A. hydrophila after immunization, which suggests an efficient approach for characterizing of targeted molecules at both the gene and protein levels.     

Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea)

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5′-untranslated region of 99 nt and a 3′-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.     

Cloning, mRNA expression, and recombinant expression of peptidoglycan recognition protein II gene from large yellow croaker (Pseudosciaena crocea)

Peptidoglycan recognition protein II (pglyrp2) is a type of pattern recognition receptor (PRR) that has amidase activity and is structurally conserved through evolution. However, their contributions in immune defense are different between mammal pglyrp2 and its counterpart in insects. Hitherto, fish pglyrp2 was poorly known in its structure, expression pattern and its contribution in immune defense. In present study, the pglyrp2 gene of the large yellow croaker (Pseudosciaena crocea) was cloned by RACE approach; the full-length cDNA (1842 bp) of pglyrp2 of P. crocea contains a 1446 bp open reading frame that encodes a putative protein of 482 amino acids (aa) with one 21-residue signal peptide. The pglyrp2 fusion protein and mature peptide fusion protein of P. crocea expressed by pET28a vector in the insoluble inclusion bodies (IBs) of Escherichia coli BL21 were confirmed by SDS-PAGE and subsequently purified to homogeneity by Ni-NTA agarose affinity chromatography. In addition, quantitative Real-time PCR (QRT-PCR) assays indicated that large yellow croaker pglyrp2 could be strongly expressed in liver and weakly in gonad, intestine, and stomach. Also, maternally derived pglyrp2 mRNA displayed a high level in unfertilized eggs and low expression throughout embryogenesis and yolk-sac larvae stage. Moreover, as shown in an artificial infection model, the pglyrp2 of P. crocea was confirmed to be a constitutive and inducible acute-phase protein, inducibility of which correlated with activation of anti-oxidant defense response. Thus, pglyrp2 of P. crocea was believed to play an important role in defending the eggs, bacteria recognition and activation of downstream host immune defense.     

Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus)

Ferritin is a conserved iron binding protein existing ubiquitously in prokaryotes and eukaryotes. In this study, the gene encoding a ferritin M subunit homologue (SoFer1) was cloned from red drum (Sciaenops ocellatus) and analyzed at expression and functional levels. The open reading frame of SoFer1 is 531 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE) preserved in many ferritins. The deduced amino acid sequence of SoFer1 possesses both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Expression of SoFer1 was tissue specific and responded positively to experimental challenges with Gram-positive and Gram-negative fish pathogens. Treatment of red drum liver cells with iron, copper, and oxidant significantly upregulated the expression of SoFer1 in time-dependent manners. To further examine the potential role of SoFer1 in antioxidation, red drum liver cells transfected transiently with SoFer1 were prepared. Compared to control cells, SoFer1 transfectants exhibited reduced production of reactive oxygen species following H₂O₂ challenge. Finally, to examine the iron binding potential of SoFer1, SoFer1 was expressed in and purified from Escherichia coli as a recombinant protein. Iron-chelating analysis showed that purified recombinant SoFer1 was capable of iron binding. Taken together, these results suggest that SoFer1 is likely to be a functional ferritin involved in iron sequestration, host immune defence against bacterial infection, and antioxidation.     

Lack of genetic structure in endangered large yellow croaker Larimichthys crocea from China inferred from mitochondrial control region sequence data

The large yellow croaker Larimichthys crocea is an important commercial marine fish species in China. However, information about the population structure of this species is limited. In the present study, mitochondrial DNA (mtDNA) control region was sequenced from four populations of the yellow croaker in the southern Yellow Sea, East China Sea and South China Sea to investigate the genetic structure of this species. A total of 54 haplotypes were detected among 62 individuals of large yellow croaker. High levels of population genetic diversity were observed. Among the four populations, the haplotype diversity was between 0.9895 ± 0.0193 (Xiamen) and 1.0000 ± 0.0524 (Lvsi, Zhoushan). The nucleotide diversity ranged from 0.0208 ± 0.0108 (Xuwen) to 0.0246 ± 0.0138 (Lvsi). The results of AMOVA detected no significant differences among populations. The conventional F_ST statistics were negative and insignificant values. These indicated lack of population genetic structure throughout the Yellow Sea, East China Sea and South China Sea, and random mixing of individuals among the samples. Biological characters of large yellow croaker and lack of physical barrier in the studied area could be the reasons for lack of genetic structure in this species.     

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

Isolation and Gene Expression of Yellow Grouper Ferritin Heavy Chain Subunit After Lipopolysaccharide Treatment

Ferritin is a ubiquitous and conserved iron storage protein that plays a central role in iron metabolism. The ferritin heavy chain subunit (FerH) homolog was isolated from yellow grouper (Epinephelus awoara) spleen using suppression subtractive hybridization and RACE-PCR. The nucleotide sequence of FerH full-length cDNA was 1173 bp and contained an open reading frame of 534 bp, encoding a putative protein of 177 amino acids. The encoded protein shows 78-94% identity with homologs. Based on phylogenetic analysis, yellow grouper FerH is highly conserved throughout evolution and is closer to European seabass than to other species. RT-PCR analysis demonstrated that FerH was widely expressed in various healthy tissues and significantly up-regulated in liver, spleen, and anterior kidney by lipopolysaccharide. The results suggest that yellow grouper FerH may play a role in immune response.     

Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771 bp, including a 5′-terminal untranslated region (UTR) of 36 bp, 3′-terminal UTR of 279 bp and an open reading frame (ORF) of 456 bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24 h) as much as that in the control (P < 0.05) challenged by poly I:C and 5.0 times (at 24 h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.     

Characterization of ferritin and ferritin-binding proteins in canine serum

Ferritin and ferritin-binding proteins in canine serum were characterized. A certain percentage of ferritin in canine serum, but no tissue ferritin, was precipitated by centrifugation at 16,000×g for 30 min. The precipitated ferritin was found to contain two subunits corresponding to the H and L subunits of canine liver ferritin by immunoblotting, the H subunit being predominant. More ferritin was precipitated from canine sera which had been incubated with anti-rat liver ferritin antibody than from untreated sera, and the H chain also predominated. To evaluate the possibility that the autoantibody was responsible for the precipitation of canine serum ferritin, the ferritin-binding activities of canine antibodies were examined using liver ferritin-coated microtiter plates and alkaline phosphatase-labeled antibodies specific for canine IgM, IgA, and IgG heavy chains. The results showed that IgM and IgA, but not IgG, had considerable ferritin-binding activities. Given these results, we suggest that there is H-chain-rich isoferritin in canine serum, and that ferritin exists as an immune complex.     

EST analysis and identification of gonad-related genes from the normalized cDNA library of large yellow croaker,Larimichthys crocea

On grounds of the especially limited numbers of identified gonad-specific or gonad-related genes of large yellow croaker Larimichthys crocea which may represent a major obstacle for the study of gonad development and sex differentiation, we initiated a sequencing program of Expressed Sequence Tags (ESTs) in large yellow croaker. In this study, we firstly constructed a normalized gonad cDNA library using the combination of SMART technique and DSN treatment. The titer of amplified cDNA library was 4.8 × 10¹¹ and the percentage of unique cDNA sequences of the library was 82.49%. 2916 unique cDNAs were clustered from the 3535 high quality ESTs. Among the 1785 ESTs which had significant homology with known genes in the NCBI database, about 64 significant gonad-related genes were found, accounting for 3.59% of the total unique cDNAs. Specifically, the testis-specific LRR gene and testis-specific chromodomain Y-like protein gene were identified from fish for the first time. Six gonad-related microsatellite-containing ESTs were identified from the 129 ESTs containing 149 microsatellites. Expression patterns of 10 of these gonad-related gene homologues in ovaries and testes were examined by qRT-PCR. The results will be powerful resources for our further investigation to establish the molecular mechanisms of gonad development and sex differentiation in large yellow croaker.     

Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus)

Ferritin is an evolutionarily conserved protein that plays an important role in iron storage and detoxification. In this study, the gene encoding a ferritin H subunit homologue (SmFer1) was cloned from turbot (Scophthalmus maximus) and analyzed at the expression and functional levels. The open reading frame of SmFer1 is 534 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE). The deduced amino acid sequence of SmFer1 shares extensive sequence identities with the H ferritins of a number of fish species and contains the ferroxidase center that is preserved in ferritin H subunits. Examination of tissue specific expression indicated that SmFer1 expression was most abundant in muscle, liver, and blood. Experimental infection with bacterial pathogens induced significant induction of SmFer1; however, the magnitudes of induction effected by Gram-negative pathogens were much higher than that induced by Gram-positive pathogen. Consistently, lipopolysaccharide (LPS) challenge drastically augmented SmFer1 expression. In addition to bacterial pathogens and LPS, poly(I:C) also induced a strong but transient induction of SmFer1 which differs in profile from those induced by bacterial pathogens. In vitro iron-chelating analysis showed that recombinant SmFer1 purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. To examine whether SmFer1, with its iron-chelating capacity, could have any effect on the infection of bacterial pathogens, purified recombinant SmFer1 was subjected to bacteriostatic analysis and proved to be able to inhibit the growth of the fish pathogen Listonella anguillarum which enhanced SmFer1 expression upon infection. Taken together, these results suggest that SmFer1 is likely to play a role in both iron storage and immune defense against microbial infections.