RESUMO
BACKGROUND: Type II antifreeze protein (AFP) from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved. RESULTS: Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes. CONCLUSIONS: These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between 'higher' eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.
Assuntos
Proteínas Anticongelantes Tipo II/genética , Proteínas de Peixes/genética , Transferência Genética Horizontal , Osmeriformes/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos , Evolução Molecular , Etiquetas de Sequências Expressas , Lectinas Tipo C/genética , Anotação de Sequência Molecular , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short beta-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and beta-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca(2+)-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis.
Assuntos
Proteínas Anticongelantes Tipo II/química , Proteínas Anticongelantes Tipo II/genética , Cálcio/metabolismo , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes Tipo II/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Gelo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Perciformes , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Propriedades de SuperfícieRESUMO
In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca(2+)-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+)-dependent) lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+) and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+)-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+) through the coordination with a water molecule of the ice lattice. This Ca(2+)-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.
Assuntos
Proteínas Anticongelantes Tipo II/química , Proteínas Anticongelantes Tipo II/metabolismo , Cálcio/metabolismo , Evolução Molecular , Peixes/genética , Animais , Proteínas Anticongelantes Tipo II/genética , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Peixes/metabolismo , Lectinas/metabolismo , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The type II antifreeze protein of Atlantic herring (Clupea harengus harengus) requires Ca(2+) as a cofactor to inhibit the growth of ice crystals. On the basis of homology modeling with Ca(2+)-dependent lectin domains, five residues of herring antifreeze protein (hAFP) are predicted to be involved in Ca(2+) binding: Q92, D94, E99, N113, and D114. The role of E99, however, is less certain. A previous study on a double mutant EPN of hAFP suggested that the Ca(2+)-binding site of hAFP was the ice-binding site. However, it is possible that Ca(2+) might function distantly to affect ice binding. Site-directed mutagenesis was performed on the Ca(2+)-coordinating residues of hAFP in order to define the location of the ice-binding site and to explore the role of these residues in antifreeze activity. Properties of the mutants were investigated in terms of their structural integrity and antifreeze activity. Equilibrium dialysis analysis demonstrated that E99 is a Ca(2+)-coordinating residue. Moreover, proteolysis protection assay revealed that removal of Ca(2+) affected the conformation of the Ca(2+)-binding loop rather than the core structure of hAFP. This finding rules out the possibility that Ca(2+) might act at a distance via a conformational change to affect the function of hAFP. Substitutions at positions 99 and 114 resulted in severely reduced thermal hysteresis activity. These data indicate that the ice-binding site of hAFP is located at the Ca(2+)-binding site and the loop region defined by residues 99 and 114 is important for antifreeze activity.
Assuntos
Aminoácidos/química , Proteínas Anticongelantes Tipo II/química , Cálcio/química , Gelo , Alanina/genética , Amidas , Aminoácidos/genética , Animais , Proteínas Anticongelantes Tipo II/genética , Cloreto de Cálcio/química , Radioisótopos de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Cristalização , Medição da Troca de Deutério , Hidrólise , Mutagênese Sítio-Dirigida , Conformação Proteica , Serina Endopeptidases/química , Espectrometria de FluorescênciaRESUMO
Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.
Assuntos
Proteínas Anticongelantes Tipo II/química , Proteínas Anticongelantes Tipo II/metabolismo , Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Congelamento , Animais , Proteínas Anticongelantes/genética , Proteínas Anticongelantes Tipo II/genética , Peixes , Mariposas , Mutagênese Sítio-Dirigida , Mioglobina/química , Perciformes , Ligação Proteica/genética , Soluções , Tenebrio , Fatores de Tempo , alfa-Fetoproteínas/químicaAssuntos
Proteínas Anticongelantes Tipo II/fisiologia , Proteínas Anticongelantes/fisiologia , Animais , Proteínas Anticongelantes/biossíntese , Proteínas Anticongelantes/genética , Proteínas Anticongelantes Tipo II/biossíntese , Proteínas Anticongelantes Tipo II/genética , Sítios de Ligação , Glicoproteínas/fisiologia , Humanos , Gelo , Ligação ProteicaRESUMO
A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.
Assuntos
Proteínas Anticongelantes Tipo II/genética , Osmeriformes/genética , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes Tipo II/química , Proteínas Anticongelantes Tipo II/isolamento & purificação , Proteínas Anticongelantes Tipo II/metabolismo , Sequência de Bases , Cálcio/metabolismo , DNA Complementar/química , DNA Complementar/genética , Água Doce , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Osmeriformes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fases de Leitura/genética , Rutênio Vermelho/química , Homologia de Sequência de AminoácidosRESUMO
Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.