Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Gene duplication of the seventh component of complement in rainbow trout

The seventh component of complement is a single-chain plasma glycoprotein that is involved in the cytolytic phase of complement activation through a sequence of polymerization reactions with other terminal components. We have previously isolated and characterized a C7 gene in rainbow trout (Oncorhynchus mykiss). Here, we report the cloning of a second trout C7 gene (C7-2). The deduced amino acid sequence of the C7-2 gene exhibits 43 and 50% identity with human C7 and trout C7-1, respectively. The structural motifs of trout C7-2 resemble those of mammalian C7 more than trout C7-1, and the cysteine backbone shows a high degree of conservation. C7-2 presents a different tissue expression profile from trout C7-1, which correlates with that of mammalian counterparts. Although duplication of complement genes is a common observation in teleost fish, this is the first report of two gene isotypes of a terminal membrane attack complex/perforin complement component in any organism.     

The beta 2-microglobulin locus of rainbow trout (Oncorhynchus mykiss) contains three polymorphic genes

Beta2-microglobulin (beta2m) associates with MHC and related class I H chains to form cell surface glycoproteins that mediate a variety of functions in defense. In humans, monomorphism of a single beta2m gene contrasts with the diversity and polymorphism of the class I H chain genes, and a similar picture was seen in almost all other species examined. In this regard, rainbow trout (Oncorhynchus mykiss) appeared unusual: trout beta2m genes gave a complicated and polymorphic pattern in Southern blots, and a minimum of 10 different mRNA encoding two distinct types of beta2m were expressed by a single fish. Characterization of genomic clones from the same fish now shows that the rainbow trout beta2m locus consists of two expressed genes and one partial gene that are closely linked. Four copies of the locus were identified and allelic variants of each gene defined, largely through comparison of the noncoding regions. A dramatic variation in the lengths of introns is caused by variable repetitive elements and accounts for the complex pattern seen in Southern blots. By comparison to noncoding sequences, the coding regions are conserved but the three loci differ within a cluster of codons that encode residues of beta2m that do not interact with class I H chains. Additional diversity in the trout beta2m genes appears to be due to somatic mutation that might be facilitated by the abundance of repetitive DNA elements within the 12 beta2m genes of an individual rainbow trout.     

Bacterial-binding activity and plasma concentration of ladderlectin in rainbow trout (Oncorhynchus mykiss)

Soluble, defense lectins bind conserved microbial patterns leading to pathogen opsonization, enhanced phagocytosis and activation of complement. These immune functions, however, vary widely among individuals due to genetic and acquired differences affecting binding capacity or plasma concentration. Most evidence for the defensive function of soluble lectins is based on mammals, but several functionally homologous, but less well-characterized, lectins have been identified in fish. In this study, we compared binding of rainbow trout plasma ladderlectin to relevant, intact bacterial targets. A polyclonal antiserum raised against a synthetic peptide identical to the 20 N-terminal amino acids of the reduced 16 kDa rainbow trout ladderlectin subunit was used to detect plasma ladderlectin in immunoblots and indirect enzyme-linked immunosorbent assay (ELISA). Ladderlectin binding to Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, Yersinia ruckeri and Pseudomonas sp. was detected by PAGE and immunoblots of saccharide elutions from intact bacteria incubated in the presence of normal trout plasma. Although plasma concentrations of immunoreactive ladderlectin were low in the majority of trout, significant (P < 0.0001) variation between individual fish was observed in two separate populations. In addition, one population demonstrated a subset of individuals whose ladderlectin levels were approximately seven-fold higher than the population median. These findings indicate that rainbow trout have variable amounts of plasma ladderlectin capable of binding to the surfaces of several relevant bacterial targets.     

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.     

Seminal plasma of brown trout,Salmo trutta fario (L.) contains a factor able to retain iron at acid pH,typical feature of lactoferrin

Blood and seminal plasma of brown trout Salmo trutta fario were analyzed for their iron binding potential adopting two different methods. Seminal plasma showed an iron binding capacity that was retained even if samples were exposed at acid pH, similarly to mammalian lactoferrin that binds ferric iron also at acid pH. This suggests that the iron binding capacity is determined by a factor having a lactoferrin-like activity. Moreover, trout seminal plasma proteins were also analyzed in their pattern by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. When seminal plasma was subjected to immunoblotting using goat anti-bovine lactoferrin antibodies as a probe, only a single band having an apparent molecular weight of around 80 kDa was specifically detected, showing that this protein has homology with bovine lactoferrin.     

Urea production and transport in teleost fishes

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.     

Acute involution in the tammar wallaby: identification of genes and putative novel milk proteins implicated in mammary gland function

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution.     

The complement system in teleosts

Complement, an important component of the innate immune system, is comprised of about 35 individual proteins. In mammals, activation of complement results in the generation of activated protein fragments that play a role in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Fish appear to possess activation pathways similar to those in mammals, and the fish complement proteins identified thus far show many homologies to their mammalian counterparts. Because information about complement proteins, regulatory proteins, and complement receptors in fish is far from complete, it is unclear whether all the complement functions that have been identified in mammals also occur in fish. However, it has been clearly demonstrated that fish complement can lyse foreign cells and opsonise foreign organisms for destruction by phagocytes. There are also indications that complement fragments participate in inflammatory reactions. Fish possess multiple isoforms of several complement proteins, such as C3 and factor B. It has been hypothesised that the function of this diversity in complement proteins serves to expand their innate immune recognition capacity and response. Understanding the functions of complement in fish and the roles the individual proteins, including the various isoforms, play in host defence, is important not only for understanding the evolution of this system but also for the development of new strategies in fish health management.     

Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia

Discocotyle sagittata oncomiracidia were rapidly killed when incubated in na?ve plasma and immune sera from both rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), the killing proceeding at a faster rate with blood material from the latter fish species. The lethal activity of na?ve plasma and immune sera was comparable. This was abolished after incubation at 45 degrees C for 30 min and by the addition of EDTA but not EGTA supplemented with Mg(2+), indicating that complement acting via the alternative pathway is responsible for the parasiticidal effect observed. Scanning electron micrographs showed varying degrees of surface disruption in larvae exposed to fish plasma, suggesting that complement acts by breaching the oncomiracidial tegument. Control (untreated) oncomiracidia showed no damage. Ultrastructural damage was more extensive in oncomiracidia exposed to brown trout plasma than to rainbow trout plasma for equal periods, suggesting that the complement cascade may be involved in mediating host susceptibility.