Robert C. Richards

Sequence and expression of C-type lectin receptors in Atlantic salmon ( Salmo salar )

The diverse receptors of the C-type lectin superfamily play key roles in innate immunity. In mammals, cell surface receptors with C-type lectin domains are involved in pathogen recognition and in immune response, and in some cases are exploited by pathogens to gain entry into cells. This study reports on sequence and expression analysis of three paralogous group II C-type lectins from the teleost fish Atlantic salmon (Salmo salar). Each of the receptors showed similarity to immune-relevant mammalian receptors in terms of amino acid sequence and overall organization within the C-type lectin-like domain (CTLD). Two of the three have cytoplasmic motifs consistent with the immunoreceptor tyrosine-based activation motifs (ITAM), which are known to modulate downstream functions in leukocytes. All three C-type lectin receptors were expressed in multiple tissues of healthy fish, including peripheral blood leukocytes and salmon head kidney cells (SHK-1). Each receptor was up-regulated in salmon liver in response to infection by Aeromonas salmonicida and one receptor was substantially up-regulated in cultured SHK-1 cells in response to lipopolysaccharide (LPS). Putative binding sites for the CAAT-enhancer-binding protein (C/EBP) family of transcription factors in the regulatory regions of these C-type lectin genes may mediate their response to bacteria and LPS in salmon leukocytes. The identification of these types of receptors in distinct populations of cells within the immune system will provide important markers for identifying and categorizing the state of differentiation or activation of these cells and lead to further understanding of the interaction between the salmon host and multiple pathogens.

Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada

Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killifish, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these fish. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond fish compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killifish. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond fish.

Cloning and characterization of the Atlantic salmon serum lectin, a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.

Seasonal Freeze Resistance of Rainbow Smelt (Osmerus mordax) Is Generated by Differential Expression of Glycerol‐3‐Phosphate Dehydrogenase, Phosphoenolpyruvate Carboxykinase, and Antifreeze Protein Genes

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol‐3‐phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall‐winter‐spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.

Cloning of glycerol-3-phosphate dehydrogenase cDNAs from two fish species and effect of temperature on enzyme expression in rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) can accumulate extreme levels of glycerol in their blood during winter. Low temperatures are required for glycerol accumulation in smelt blood and the enzyme glycerol-3-phosphate dehydrogenase (GPDH) has been suggested to play a role in glycerol production/concentration in this species. In the present study, cDNA sequences encoding glycerol-3-phosphate dehydrogenase (GPDH) from rainbow smelt and Atlantic salmon (Salmo salar) were cloned. The encoded GPDH protein sequences were very similar to one another (88% identity). Using RT-PCR, GPDH mRNA was detected in skin, gill, heart, head kidney, brain and liver from both salmon and smelt obtained in December. However, GPDH was not detected in salmon intestine and spleen or in smelt intestine. Examination of GPDH expression in smelt liver during February by Northern blotting revealed temperature regulation. Elevation of the temperature resulted in a significant decrease in liver GPDH transcript level. Serum glycerol levels decreased concomitantly. These findings suggest a role for GPDH in the accumulation of glycerol in smelt at low temperatures.

Seasonal Changes in Hepatic Gene Expression Reveal Modulation of Multiple Processes in Rainbow Smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) are freeze-resistant fish that accumulate glycerol and produce an antifreeze protein during winter. Quantitative reverse transcription PCR (qPCR) and subtractive hybridization studies have previously revealed five genes in rainbow smelt liver to be differentially regulated in winter in comparison with the fall when water temperatures are warmer. In order to further define the suite of processes that are regulated seasonally, we undertook a large-scale analysis of gene expression by hybridization of smelt cDNA to the salmonid 16K cGRASP microarray. In total, 69 genes were identified as up-regulated and 14 genes as down-regulated under winter conditions. A subset of these genes was examined for differential regulation by qPCR in the individual cDNA samples that were pooled for microarray analysis. Ten of the 15 genes tested showed significant change in the same direction as microarray results, whereas one showed significant change in the opposite direction. Fructose-bisphosphate aldolase B and the cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase were among the most highly up-regulated genes, a result supporting a metabolic focus on glycerol synthesis during winter. Modulation of other processes, including endoplasmic reticulum stress, lipid metabolism and transport, and protein synthesis, was also suggested by the qPCR analysis of array-identified genes. The 15 genes were subsequently examined by qPCR for seasonal variation in expression over five sampling times between October and March, and ten showed significant variation in expression over the sampling period. Taken together, these results provide new understanding of the biochemical adaptations of vertebrates to an extremely low seasonal temperature.

Seasonal expressed sequence tags of rainbow smelt (Osmerus mordax) revealed by subtractive hybridization and the identification of two genes up-regulated during winter

The rainbow smelt (Osmerus mordax) is freeze-resistant and maintains swimming and feeding activity during winter. In order to identify genes differentially expressed in smelt liver response to winter water temperatures, a large-scale analysis of gene expression using suppression subtractive hybridization was carried out using samples obtained in fall and winter. Forward and reverse subtractions were performed, subtraction-enriched products were cloned, and clones were sequenced from both of the resulting libraries. When 27 of these genes were screened by semi-quantitative RT-PCR to identify candidates for differential expression based generally on 2-fold changes in expression, one encoding FK506-binding protein 5 was classified as up-regulated in response to seasonal change, another encoding the mitochondrial solute carrier 25 member 25 (ATP-Mg/Pi carrier) was similarly classified with seasonal change and low temperature shift, and the one encoding the 78 kDa glucose-regulated protein was provisionally classified as down-regulated with low temperature shift. Analysis of fall (warm) and winter (cold) seasonal samples by quantitative PCR (qPCR) revealed significant up-regulation of genes encoding FK506-binding protein 51 and the mitochondrial solute carrier, whereas the gene encoding the glucose-regulated protein showed no significant change in expression. The mitochondrial solute carrier and FK506-binding protein results may relate to changes in cortisol action, as both are regulated by cortisol in other species.

Ligand and pathogen specificity of the Atlantic salmon serum C-type lectin

BACKGROUND An Atlantic salmon (Salmo salar) C-type lectin (SSL) binds to mannose and related sugars as well as to the surface of Aeromonas salmonicida. To characterize this lectin as a pathogen recognition receptor in salmon, aspects of its interaction with molecules and with intact pathogens were investigated. METHODS SSL was isolated using whole-yeast-affinity and mannan-affinity chromatography. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays. Microbial binding specificity of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand specificity of SSL was examined using glycan array analysis and frontal affinity chromatography. RESULTS SSL showed binding to bacteria and yeast including, Pseudomonas fluorescens, A. salmonicida, A. hydrophila, Pichia pastoris, and Saccharomyces cerevisiae, but there was no detectable binding to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against Escherichia coli, Bacillus subtilis, S. cerevisiae, or A. salmonicida, but it was found to agglutinate E. coli. The major surface molecule of A. salmonicida recognized by SSL was shown to be LPS and not the A-layer protein. LPS binding was mannose-inhibitable. Glycans containing N-acetylglucosamine were shown to be predominant ligands. CONCLUSION SSL has a distinct ligand preference while allowing recognition of a wide variety of related carbohydrate structures. GENERAL SIGNIFICANCE SSL is likely to function as a wide-spectrum pattern recognition protein.

Cystine-mediated oligomerization of the Atlantic salmon serum C-type lectin.

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a C-type lectin that binds to bacteria including salmon pathogens. SSL has been shown to be oligomeric in salmon serum and it displays a stoichiometric band-laddering pattern when analyzed by SDS-PAGE under non-reducing conditions. In this study, a model was generated for SSL isoform 2 in silico in order to identify cysteines that are available to form intermolecular disulfide bonds facilitating oligomerization. Then, recombinant SSL was expressed in E. coli and mutants were produced at positions Cys72 and Cys149. The SSL preparations were purified by metal-affinity chromatography and shown to be functional by carbohydrate-affinity chromatography. The recombinant SSL formed oligomers, which were evident by non-reducing covalent cross-linking and non-reducing SDS-PAGE; however, the band patterns were different for the mutants, with the maximal and predominant multimer sizes distinct from the wild-type recombinant lectin. Further examination of oligomerization by size exclusion chromatography revealed a subunit number from 35 to at least 110 for the wild-type recombinant SSL and subunit numbers below 9 for each mutant SSL oligomer. Thus, both cysteines were found to contribute to oligomerization of SSL.

A simple procedure for sulfation and 35S radiolabelling of paralytic shellfish poisoning (PSP) gonyautoxins

A method is described to sulfate PSP toxins at various positions in the molecule and to prepare 35S labelled compounds using H2(35)SO4 in the presence of dicyclohexylcarbodiimide (DCC). The 11-sulfates of saxitoxin and neosaxitoxin, known as gonyautoxins, are often the most abundant of the PSP toxins in algae and contaminated shellfish. Receptor site binding and antibody assays based on these analogues should, therefore, better reflect toxicity than those in which saxitoxin is used. Although the specific activity of 35S-gonyautoxins is lower than that of commercially available 3H-saxitoxin, the label is strongly bound and is not lost through proton exchange with water as occurs with tritiated saxitoxin. The labelling procedure is rapid, inexpensive and can be done on a small scale. Sulfate can be removed from the 11-position of GTXs in methanolic-HCl and from the 21-position by mild acid hydrolysis and H2(35)SO4 added in 5-10-fold excess. Addition or exchange occurs rapidly on mixing DCC in dimethylformamide with dry toxin and sulfate. Reaction conditions were optimized and reaction products identified by capillary electrophoresis, autoradiography and ionspray mass spectrometry. Together with methods for selective removal of sulfate, the sulfation reaction provides an additional way to prepare some of the naturally occurring derivatives of saxitoxin, many of which are sulfates.