Comparison of antifreeze glycopeptides from several Antarctic fishes

Summary Antifreeze glycopeptides have been isolated from eight species of antarctic fish representing the families Nototheniidae, Channichthyidae, and Bathydraconidae. Amino acid analysis indicates that the glycopeptides have amino acid compositions identical to previously studied antarctic nototheniids. The sizes of the glycopeptides show some heterogeneity although they all span the molecular weight range of approximately 34,000 to 2,500 daltons. The species studied here are representative of most of the ecological habitats of the Antarctic Ocean.     

The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures

The research on antifreeze glycopeptides (AFGPs) from Antarctic and Arctic fishes has focused primarily on their interaction with ice crystals. This study reports results of experiments in which pig oocytes, known to be sensitive to hypothermic temperatures, were exposed to 4 degrees C for various periods of time, in solutions of different molecular weight AFGPs from Antarctic nototheniid fishes. The membrane potential was measured across the oolemma following hypothermic exposure. The results show that a physiological combination of the different molecular weight AFGPs protects the structural integrity of the oolemma and inhibits ion leakage across the oolemma at hypothermic temperatures. The results also show that the hypothermic protection is nonlinearly dependent on concentration and that separately, the different molecular weight glycopeptides do not stop ion leakage even at very high concentration. The protection of membranes at hypothermic temperatures is a new property of AFGPs which was not known prior to our work.     

Blood glycoprotein from antarctic fish. Possible conformational origin of antifreeze activity

Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a tetramer of molecular weight 95 000. The other enzyme, adenosine phosphorylase, was specific for adenosine and deoxyadenosine. The molecular weight of the native enzyme was 153 000 +/- 10% and the molecular weight of the subunits was 25 500 +/- 5%. This indicates a hexameric structure. The adenosine phosphorylase was inactivated by 10(-3) M p-chloromercuribenzoate and protected against this inactivation by phosphate, adenosine and ribose 1-phosphate.     

Evidence for a gamma-turn motif in antifreeze glycopeptides.

Knowledge of the secondary structure of antifreeze peptides (AFPs) and glycopeptides (AFGPs) is crucial to understanding the mechanism by which these molecules inhibit ice crystal growth. A polyproline type II helix is perhaps the most widely accepted conformation for active AFGPs; however, random coil and alpha-helix conformations have also been proposed. In this report we present vibrational spectroscopic evidence that the conformation of AFGPs in solution is not random, not alpha-helical, and not polyproline type II. Comparison of AFGP amide vibrational frequencies with those observed and calculated for beta and gamma-turns in other peptides strongly suggests that AFGPs contain substantial turn structure. Computer-generated molecular models were utilized to compare gamma-turn, beta-turn, and polyproline II structures. The gamma-turn motif is consistent with observed amide frequencies and results in a molecule with planar symmetry with respect to the disaccharides. This intriguing conformation may provide new insight into the unusual properties of AFGPs.     

Blood glycoprotein from antarctic fish possible conformational origin of antifreeze activity

High resolution ¹H NMR and circular dichroism (CD) measurements have been performed on aqueous solutions of antarctic fish antifreeze glycoprotein. The carbohydrate contribution to the observed CD spectrum has been estimated from closely analogous model compounds. The residual peptide contribution cannot be interpreted of the known spectral behaviour of α-helix, β-sheet and random coil. Instead it resembles the CD spectrum of β-structure in position, magnitude and spectral form, but is of opposite sign, indicating a specific but unusual peptide conformation, which we suggest may be stabilized by non-bonded interactions between the peptide backbone and the carbohydrate sidechains. Previous evidence which supports this interpretation is reviewed. NMR and CD measurements between ?2 and +30°C are consistent with conformational stability throughout the biologically relevant temperature range. The mechanism of the antifreeze activity is discussed in terms of the spatial and orientational correlations of sugar hydroxy groups and water in the liquid and solid states. The implication of an ordered peptide structure is explained by the comparison of the antifreeze glycoprotein with synthetic water-soluble polymers which also exhibit limited antifreeze properties.     

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.     

Adsorption to ice of fish antifreeze glycopeptides 7 and 8.

Experimental results show that fish antifreeze glycopeptides (AFGPs) 8 and 7 (with 4 and 5 repeats respectively of the Ala-Ala-Thr backbone sequence) bond onto ice prism planes aligned along a-axes, and inhibit crystal growth on prism planes and on surfaces close to that orientation. The 9.31-A repeat spacing of the AFGP in the polyproline II helix configuration, deduced from NMR studies, matches twice the repeat spacing of ice in the deduced alignment direction, 9.038 A, within 3%. A specific binding model is proposed for the AFGP and for the alpha-helical antifreeze peptide of winter flounder. For AFGP 7-8, two hydroxyl groups of each disaccharide (one disaccharide is attached to each threonine) reside within the ice surface, so that they are shared between the ice crystal and the disaccharide. This provides 24 hydrogen bonds between AFGP 8 and the ice and 30 for AFGP 7, explaining why the chemical adsorption is virtually irreversible and the crystal growth can be stopped virtually completely. The same scheme of sharing polar groups with the ice works well with the alpha-helical antifreeze of winter flounder, for which an amide as well as several hydroxyls are shared. The sharing of polar groups with the ice crystal, rather than hydrogen-bonding to the ice surface, may be a general requirement for adsoprtion-inhibition of freezing.     

Brain and egg tubulins from antarctic fishes are functionally and structurally distinct.

The multitubulin hypothesis proposes that chemically distinct tubulins may possess different polymerization properties or may form functionally different microtubules. To test this hypothesis, we have examined the functional properties and the structures of singlet-specific nonneural and neural tubulins from Antarctic fishes. Tubulins were purified from eggs of Notothenia coriiceps neglecta, and from brain tissues of N. coriiceps neglecta or N. gibberifrons, by DEAE ion-exchange chromatography and cycles of microtubule assembly/disassembly. At temperatures between 0 and 20 degrees C, each of these tubulins polymerized efficiently in vitro to yield microtubules of normal morphology. Critical concentrations for polymerization of egg tubulin ranged from 0.057 mg/ml at 3 degrees C to 0.002 mg/ml at 18 degrees C, whereas those for brain tubulin at like temperatures were 4-10-fold larger. Polymerization of both tubulins was entropically driven, but the apparent standard enthalpy and entropy changes for microtubule elongation by egg tubulin (delta Happ0 = +33.9 kcal/mol, delta Sapp0 = +151 entropy units) were significantly greater than values observed for brain tubulin (delta Happ0 = +26.5 kcal/mol, delta Sapp0 = +121 entropy units). Egg tubulin was composed of approximately six alpha and two beta chains and lacked the beta III isotype, whereas brain tubulin was more complex (greater than or equal to 10 of each chain type). Furthermore, egg alpha tubulins were more basic, and their carboxyl termini more resistant to cleavage by subtilisin, than were the alpha chains of brain. We conclude that brain and egg tubulins from the Antarctic fishes are functionally distinct in vitro, due either to qualitative or quantitative differences in isotypic composition, to differential posttranslational modification of shared isotypes, or to both.     

Evolution and adaptive radiation of antarctic fishes

There are few instances where a knowledge of the thermal physiology, habitats and lifestyles of a group of closely related species can be mapped onto a well-supported phylogeny and a detailed climatic history. The unique fish fauna of the Southern Ocean, dominated by a single group of fish whose phylogeny is known from traditional and molecular techniques, provides one such opportunity. Furthermore, these fish are living at an extreme temperature for marine organisms. Physiological and molecular studies are revealing details of the mechanisms of temperature compensation and, combined with knowledge of the thermal history, are throwing new light on the process of evolution in this unique group of fish.     

Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).     

Synthesis and characterization of natural and modified antifreeze glycopeptides: glycosylated foldamers

In Arctic and Antarctic marine regions, where the temperature declines below the colligative freezing point of physiological fluids, efficient biological antifreeze agents are crucial for the survival of polar fish. One group of such agents is classified as antifreeze glycoproteins (AFGP) that usually consist of a varying number (n = 4–55) of [AAT] _n -repeating units. The threonine side chain of each unit is glycosidically linked to β-d-galactosyl-(1 → 3)-α-N-acetyl-d-galactosamine. These biopolymers can be considered as biological antifreeze foldamers. A preparative route for stepwise synthesis of AFGP allows for efficient synthesis. The diglycosylated threonine building block was introduced into the peptide using microwave-enhanced solid phase synthesis. By this versatile solid phase approach, glycosylated peptides of varying sequences and lengths could be obtained. Conformational studies of the synthetic AFGP analogs were performed by circular dichroism experiments (CD). Furthermore, the foldamers were analysed microphysically according to their inhibiting effect on ice recrystallization and influence on the crystal habit.     

Adaptive evolution of hepcidin genes in antarctic notothenioid fishes

Hepcidin is a small bioactive peptide with dual roles as an antimicrobial peptide and as the principal hormonal regulator of iron homeostasis in human and mouse. Hepcidin homologs of very similar structures are found in lower vertebrates, all comprise approximately 20-25 amino acids with 8 highly conserved cysteines forming 4 intramolecular disulfide bonds, giving hepcidin a hairpin structure. Hepcidins are particularly diverse in teleost fishes, which may be related to the diversity of aquatic environments with varying degree of pathogen challenge, oxygenation, and iron concentration, factors known to alter hepcidin expression in mammals. We characterized the diversity of hepcidin genes of the Antarctic notothenioid fishes that are endemic to the world's coldest and most oxygen-rich marine water. Notothenioid fishes have at least 4 hepcidin variants, in 2 distinctive structural types. Type I hepcidins comprise 3 distinct variants that are homologs of the widespread 8-cysteine hepcidins. Type II is a novel 4-cysteine variant and therefore only 2 possible disulfide bonds, highly expressed in hematopoietic tissues. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio test under site-specific models detected significant signal of positive Darwinian selection on the mature hepcidin-coding sequence, suggesting adaptive evolution of notothenioid hepcidins. Genomic polymerase chain reaction and Southern hybridization showed that the novel type II hepcidin occurs exclusively in lineages of the Antarctic notothenioid radiation but not in the basal non-Antarctic taxa, and lineage-specific positive selection was detected on the branch leading to the type II hepcidin clade under branch-site models, suggesting adaptive evolution of the reduced cysteine variant in response to the polar environment. We also isolated a structurally distinct 4-cysteine (4cys) hepcidin from an Antarctic eelpout that is unrelated to the notothenioids but inhabits the same freezing water. Neighbor-Joining (NJ) analyses of teleost hepcidins showed that the eelpout 4cys variant arose independently from the notothenioid version, which lends support to adaptive evolution of reduced cysteine hepcidin variants on cold selection. The NJ tree also showed taxonomic-specific expansions of hepcidin variants, indicating that duplication and diversification of hepcidin genes play important roles in evolutionary response to diverse ecological conditions.     

Molecular evolution of hemoglobins of antarctic fishes (Notothenioidei)

Amino acid sequences of α- and β-chains of human hemoglobin and of hemoglobins of coelacanth and 24 teleost fish species, including 11 antarctic and two temperate Notothenioidei, were analyzed using maximum parsimony. Trees were derived for the α- and β-chains separately and for tandemly arranged sequences, using the human and coelacanth sequences as outgroups in all analyses. The topologies of the trees of the α-and β-chains are highly congruent and indicate a specific pattern of gene duplications and gene expression of teleost hemoglobins which has not yet been investigated into more detail. The Notothenioid fish generally contain a single major hemoglobin and often a second minor component. The α- and β-chains of the major components form a monophyletic group in all investigated trees, with the nonantarctic Pseudaphritis as their sister taxon. The minor chains also are a monophyletic group and form an unresolved cluster with the major chains and the hemoglobins of tuna and red gurnard. The Notothenioid families Nototheniidae and Bathydraconidae appear to be paraphyletic. Received: 26 March 1997 / Accepted: 7 May 1997     

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.     

Structure and function of hemoglobin in antarctic fishes and evolutionary implications

Summary The hematological features of cold-adapted, red-blooded Antarctic teleosts has prompted this study on the relationship between hemoglobin molecular structure and oxygen-binding properties. The hemolysates from 21 species of 5 families contained one component (Hb 1), often accompanied by an additional, minor one (Hb 2, 5%–10% of total). On the other hand, 3 species of Zoarcidae, a non-endemic family, had 4–5 components. All purified hemoglobins from the former group, but only 1–2 of the 4–5 hemoglobins of Zoarcidae, showed a strong Root effect (pH regulation of oxygen binding). Globins from each hemoglobin have been purified and characterised with respect to molecular structure in several species. The similarity between the complete amino acid sequence of one -chain and those of non-Antarctic -chains is lower than that among the latter sequences, suggesting independent pathways of evolution.Presented at the 5th SCAR Symposium on Antarctic Biology, Hobart, Australia (August 29th-September 3rd, 1988)     

Synthesis of peptides and glycopeptides with polyproline II helical topology as potential antifreeze molecules

A library of peptides and glycopeptides containing (4R)-hydroxy-l-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-β-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability. The majority of the (glyco)peptides did not exhibit thermal hysteresis (TH) activity and were not capable of modifying the morphology of ice crystals. However, an unglycosylated Ala-Hyp-Ala repeat peptide did show significant TH and ice crystal re-shaping activity suggesting that it was capable of binding to the surface of ice. All (glyco)peptides synthesized displayed some ice recrystallization inhibition (IRI) activity with unglycosylated peptides containing the Ala-Hyp-Ala motif exhibiting the most potent inhibitory activity. Interestingly, although glycosylation is critical to the activity of native antifreeze glycoproteins (AFGPs) that possess an Ala-Thr-Ala tripeptide repeat, this same structural modification is detrimental to the antifreeze activity of the Ala-Hyp-Ala repeat peptides studied here.