Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.     

Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout, Macrozoarces americanus, in Escherichia coli: improved expression by the modification of the secondary structure of the mRNA.

The cDNA clone coding for the ocean pout antifreeze polypeptide (AFP) was modified to improve translation of its mRNA in Escherichia coli. A recombinant AFP (rAFP), MetLys-AFP-Lys, was expressed successfully using the lambda PL promoter, and constituted 1-2% of total bacterial proteins. The rAFP was purified to homogeneity from the soluble fractions of bacterial extracts. Its identity was confirmed by amino acid analysis, automated Edman degradation, immuno-blot and activity measurements. Although the rAFP is indistinguishable from the authentic AFP in its secondary structure, thermal hysteretic activity and the alteration of ice crystal structure, it is, however, thermally more stable (approximately 4.5 degrees C increase in Tm) and is more effective in inhibiting ice growth along the a-axis. These investigations indicate that the extra amino acids in rAFP significantly improve the thermal stability and ice-binding activity of the polypeptide.     

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.     

The Refined Crystal Structure of an Eel Pout Type III Antifreeze Protein RD1 at 0.62-Å Resolution Reveals Structural Microheterogeneity of Protein and Solvation

RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62 Å. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the “pretzel fold” includes four short β-strands and a 3₁₀ helix. There is a novel internal cavity of 45 ų surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized.     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic Salmon

Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an “all fish” gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is regulated by the 5′ promoter and 3′ termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1α) integrated as a single functional copy into a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1α integrant was organized as follows: base pairs 1580–2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete ocean pout antifreeze 3′ region, and the first 1678 bp of the ocean pout antifreeze 5′ region. Sequence analysis of the EO-1α integrant and genomic flanking regions in F₂ and F₄ generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct. These results indicate that the EO-1α transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.     

Structures of antifreeze peptides from the antarctic eel pout, Austrolycicthys brachycephalus

The antarctic eel pout, Austrolycicthys brachycephalus, synthesizes two predominant antifreeze peptides (AFPs) which, based on purification yields, make up about 94 and 6%, respectively, of the antifreezes in its serum. The amino acid sequences of these two AFPs, AB1 and AB2, were determined using automated sequencing, and compositional analyses of peptide fragments from enzymatic digests, and verified by molecular masses obtained with Fast Atom Bombardment Mass spectrometry. Substantial homologies in amino acid sequence exist between the AFPs of Austrolycicthys and those of other Southern and Northern eel pouts. 72% of the residues of AB1, and 84% of AB2, are identical to those of an AFP from another antarctic eel pout, Rhigophila dearborni. Between AB1 and AB2, 83% of the residues are identical. Secondary structure data based on circular dichroism studies indicated AB1 to be a random chain, but a sharp thermal transition of CD spectra around 30 degrees C suggested the presence of definite secondary or tertiary structure.     

Development of an all-fish gene cassette for gene transfer in aquaculture.

To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.     

Interfacial adsorption of antifreeze proteins: a neutron reflection study

Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO₂ substrate. The layer thickness of 32 Å was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 Å. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity.     

Structure of an antifreeze polypeptide precursor from the sea raven, Hemitripterus americanus

The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action.     

Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (<Emphasis Type="Italic">Macrozoarces americanus</Emphasis>) antifreeze protein OP5a gene promoter in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5′ region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1α) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1α GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.     

一个抗真菌蛋白在绿色木霉中的分泌表达

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。     

Expression of human alpha-fetoprotein in yeast.

Human alpha-fetoprotein (AFP) was expressed in Saccharomyces cerevisiae, with a plasmid containing the cDNA sequence for human AFP fused with the rat AFP signal peptide. The recombinant AFP was purified from the yeast lysate by DEAE-cellulose and immunoaffinity chromatography. The amino acid composition and the molecular weight of the recombinant AFP were similar to those of hepatoma AFP. N-terminal amino acids sequence analysis indicated that the signal peptide had been processed. The recombinant and hepatoma AFP reacted identically in Ouchterlony immunodiffusion and radioimmunoassay tests. These observations indicated that the yeast recombinant protein had the properties of native AFP.     

Hypothermic protection--a fundamental property of "antifreeze" proteins

For the last two decades fish antifreeze proteins have been considered to function exclusively in conferring freeze-resistance to fish by binding to ice crystals and thereby depressing blood plasma freezing points non-colligatively. We report here the discovery of a second fundamental property of antifreeze proteins, the ability to protect cells and their membranes from hypothermic damage. Experiments were carried out exposing immature bovine oocytes to 4 degrees C for 24 h in the presence of type I alanine rich alpha helical antifreeze polypeptides (AFP) from winter flounder, type II cysteine-rich AFP from sea raven or type III AFP from ocean pout. The presence of AFP in the incubation medium resulted in an approximate four fold increase in the number of oocytes retaining an intact oolemma and a three fold increase in the number of oocytes able to undergo in vitro maturation. None of the control oocytes could be fertilized, whereas, of those incubated in AFP, the percentage which developed normally following fertilization was comparable to that observed for fresh oocytes. These results indicate that cold-sensitive mammalian cells can be rendered cold-tolerant through the addition of "antifreeze" proteins.     

Possible existence of high molecular precursor proteins for substance P biosynthesis in rabbit spinal ganglia

The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [³⁵S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, ³⁵S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of ³⁵S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP.     

Food of 0-group ocean pout [Macrozoarces americanus (Schneider)] in eastern Newfoundland: the importance of harpacticoid copepods

Stomachs of 0-group ocean pout from Newfoundland all contained harpacticoid copepods, which comprised 82% of the diet. 0-group ocean pout were found hiding among coarsely branched algae.     

Biosynthesis of antifreeze polypeptides in the winter flounder. Characterization and seasonal occurrence of precursor polypeptides

The precursor proteins for winter flounder antifreeze polypeptide (AFP) were isolated from liver using gel filtration chromatography and reverse-phase high-performance liquid chromatography. Two major pro-antifreezes (Mr 5000), corresponding to the precursors for AFP-6 and AFP-8, were characterized by amino acid analyses and automated Edman degradation. These precursors showed significant antifreeze activity. The pro-antifreezes were synthesized in the liver seasonally as demonstrated by immunoblotting and in vitro liver incorporation studies. No mature AFP were detected in liver, thus indicating that the processing of pro-antifreezes, including amidation of the C-termini, occurred mainly in the serum. The function(s) of the prosequences, if any, remain unclear.     

NMR analysis of type III antifreeze protein intramolecular dimer. Structural basis for enhanced activity

The structure of a new antifreeze protein (AFP) variant, RD3, from antarctic eel pout (Rhigophila dearborni) with enhanced activity has been determined for the first time by nuclear magnetic resonance spectroscopy. RD3 comprises a unique translational topology of two homologous type III AFP globular domains, each containing one flat, ice binding plane. The ice binding plane of the N domain is located approximately 3.5 A "behind" that of the C domain. The two ice binding planes are located laterally with an angle of 32 +/- 12 degrees between the planes. These results suggest that the C domain plane of RD3 binds first to the ice [1010] prism plane in the <0001> direction, which induces successive ice binding of the N domain in the <0101> direction. This manner of ice binding caused by the unique structural topology of RD3 is thought to be crucial for the significant enhancement of antifreeze activity, especially at low AFP concentrations.