Lateral Transfer of a Lectin-Like Antifreeze Protein Gene in Fishes

Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs) that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven) cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche

Freeze resistance in rainbow smelt (Osmerus mordax): Seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production

Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9\uc2\ub0C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5\uc2\ub06\uc2\ub0C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5\uc2\ub0C vs. 0.25\uc2\ub0C for glycerol and AFP, respectively) at a water temperature of -1.5\uc2\ub0C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids.Peer reviewed: YesNRC publication: Ye

Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition

A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria. Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host. In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting. A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl a--mannopyranoside to be additional ligands of this lectin. The SSL in serum detected by Western blotting was shown to form a complex oligomer. These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose. The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces. No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody. Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species. It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species.Peer reviewed: YesNRC publication: Ye

Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition

A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria. Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host. In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting. A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl a--mannopyranoside to be additional ligands of this lectin. The SSL in serum detected by Western blotting was shown to form a complex oligomer. These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose. The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces. No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody. Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species. It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species.Peer reviewed: YesNRC publication: Ye

Chemical chaperone action of glycerol on the antifreeze protein of rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol and moderate levels of trimethylamine oxide (TMAO) that lower the colligative freezing point of the serum and thereby contribute to seasonal freeze resistance. In the current study, the possibility that one or both of these compounds might also have a chaperoning role at low temperatures in smelt was investigated by studying their effects on the smelt antifreeze protein (AFP). Activity of the AFP in the presence of physiological levels of TMAO and glycerol was observed by means of ice crystal morphology and measured as thermal hysteresis. Ice crystals in AFP solutions were not visibly modi\ufb01ed by either compound and TMAO at 25 and 50 mM had no appreciable effect on hysteresis; however, glycerol at 250 and 500 mM increased hysteresis. An equiosmolar level of NaCl was not as effective as glycerol in enhancing hysteresis, suggesting that osmolarity had little or no role. Although cross-linking experiments showed dimerization of AFP to be unchanged in the presence of glycerol, circular dichroism and intrinsic \ufb02uorescence analyses revealed enhanced protein folding. As glycerol enhances the folding and consequent activity of smelt AFP, protein chaperoning appears to be an endogenous role of glycerol in this vertebrate species.Peer reviewed: YesNRC publication: Ye

Ligand and pathogen speci\ufb01city 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-af\ufb01nity and mannan-af\ufb01nity 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 speci\ufb01city of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand speci\ufb01city of SSL was examined using glycan array analysis and frontal af\ufb01nity chromatography. Results: SSL showed binding to bacteria and yeast including, Pseudomonas \ufb02uorescens, 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.Peer reviewed: YesNRC publication: Ye

Molecular analysis, tissue profiles, and seasonal patterns of cytosolic and mitochondrial GPDH in freeze-resistant rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) is an anadromous teleost that, beginning in late fall, accumulates plasma glycerol in excess of 200 mM, which subsequently decreases in the spring. The activity of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH) is higher (i) in liver of smelt than in that of Atlantic salmon and capelin (nonglycerol accumulators), (ii) in liver of smelt maintained at 1\ub0C than in that of smelt held at 8\ub0-10\ub0C, and (iii) in smelt liver than in smelt muscle, heart, brain, or kidney. In addition, transcript levels of cGPDH in liver peak in December during the onset of glycerol production and then decline over the remainder of the season. There are four cGPDH protein isoforms in smelt liver that are present regardless of glycerol production status. A minimum of four cGPDH gene copies identified by Southern blotting provide adequate genetic potential to yield multiple protein isoforms. A full-length cDNA for smelt mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) was cloned and characterized. The 2,790-bp cDNA contains a 109-bp 5'UTR, a 2,193-bp open reading frame, and a 488-bp 3'UTR; transcripts are ubiquitously expressed in both warm- and cold-acclimated smelt tissues. Smelt mGPDH encodes a 730-aa protein that clusters with that of zebrafish and frog and contains several common structural motifs. mGPDH transcript levels generally increase late in the seasonal glycerol cycle, and mGPDH enzyme activity increases significantly durduring the glycerol decrease phase. Taken together, these findings suggest that liver cGPDH and mGPDH play a key role in the glycerol accumulation and decrease phases, respectively. \ua9 2011 by The University of Chicago. All rights reserved.Peer reviewed: YesNRC publication: Ye

Cloning of GLUT3 cDNA from Atlantic cod (Gadus morhua) and expression of GLUT1 and GLUT3 in response to hypoxia

A putative facilitative glucose transporter, GLUT3, cDNA was cloned from Atlantic cod. It is ubiquitously expressed, with substantial levels in kidney. The 519 aa protein has the highest sequence identity (66.3%) to grass carp GLUT3. Atlantic cod were exposed to a hypoxic challenge (45% DO2) for 24 h and the effects on GLUT1 and GLUT3 expression assessed. GLUT1 expression in gill is upregulated; however, in spleen, there is a significant decrease in both GLUT1 and GLUT3 expression. The increase in GLUT1 mRNA is considered to be associated with an increased energy demand on gill, whereas, the decrease in gene expression in spleen potentially reflects a general decrease in rates of transcription.Peer reviewed: YesNRC publication: Ye

Accelerated hepatic glycerol synthesis in rainbow smelt (Osmerus mordax) is fuelled directly by glucose and alanine: a 1H and 13C nuclear magnetic resonance study

At seawater temperatures below 1\uc2\ub0C, rainbow smelt (Osmerus mordax) accumulate plasma levels of glycerol up to 400 mM. Aspects of the synthesis of glycerol in liver and its regulation were previously investigated, but the pathways leading to glycerol synthesis remained unconfirmed. Here, we report nuclear magnetic resonance (NMR) studies which elucidate, in more detail, the fuel sources for rapid glycerol synthesis in rainbow smelt. Initial NMR analysis of liver homogenates from fish held at cold (-1\uc2\ub0C) temperatures and from fish transferred from 8\uc2\ub0C to -1\uc2\ub0C showed elevated glycerol, whereas those from fish held at 8\uc2\ub0C had far lower glycerol levels. These results confirm a temperature-responsive glycerol synthesis and show that NMR is a suitable approach to investigate the phenomenon. Further studies with fish held at low temperature and injected with labelled L-[2,3-13C2] alanine or D-[U-13C6]glucose revealed conversion of both alanine and glucose to glycerol. 13C spectra showed satellites (1JCC=41.1 Hz) about the glycerol resonances indicating intact incorporation of a 13C-13C unit in liver glycerol of fish injected with L-[2,3-13C2]alanine and a 13C-13C-13C unit in liver glycerol of fish injected with D[U-13C6]glucose. Thus, glycerol can be efficiently produced directly from amino acid precursors by glyceroneogenesis, which is an abbreviated gluconeogenesis process leading to glycerol through dihydroxyacetone phosphate (DHAP). Glucose can also be metabolised to glycerol via an abbreviated form of glycolysis that similarly leads to glycerol through DHAP.Peer reviewed: YesNRC publication: Ye