Maurizio Tamburrini

Haemoglobin of the antarctic fish Pagothenia bernacchii. Amino acid sequence, oxygen equilibria and crystal structure of its carbonmonoxy derivative.

The Antarctic fish Pagothenia bernacchii has one major haemoglobin, Hb1 (over 95% of the total blood content). Hb1 has a strong alkaline Bohr effect and at low pH exhibits the reduced ligand affinity and co-operativity that comprise the Root effect. We have determined the complete amino acid sequence of P. bernacchii Hb1 and also the structure of its carbonmonoxy derivative by X-ray crystallography, to a resolution of 2.5 A. The crystallographic R-factor of the refined structure is 18%. The three-dimensional structure of this fish haemoglobin is similar to that of human haemoglobin A, with a root-mean-square difference in main-chain atom positions of 1.4 A after superimposition of the two structures, despite only 48% homology of their amino acid sequences (including insertion of a single residue in the CD region of the fish alpha-chain). Large structural differences occur only at the N and C termini of both the alpha- and beta-chains. Neither these nor other smaller structural differences provide any obvious explanation of the Root effect of this or other fish haemoglobins.

Oxygen transport in extreme environments

Evolution has adopted different strategies to solve the problem of transporting oxygen to respiring tissues, according to needs dictated by the environment. A thermodynamic analysis of haemoglobins of organisms living in extreme polar environments (mammals and fish) provides elegant examples of such adaptations.

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.

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39‐residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N‐terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH‐dependent and voltage‐gated pore‐forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore‐forming peptides with potential effects on human health. Copyright

The myoglobin of Emperor penguin (Aptenodytes forsteri): amino acid sequence and functional adaptation to extreme conditions

In the framework of a study on molecular adaptations of the oxygen-transport and storage systems to extreme conditions in Antarctic marine organisms, we have investigated the structure/function relationship in Emperor penguin (Aptenodytes forsteri) myoglobin, in search of correlation with the bird life style. In contrast with previous reports, the revised amino acid sequence contains one additional residue and 15 differences. The oxygen-binding parameters seem well adapted to the diving behaviour of the penguin and to the environmental conditions of the Antarctic habitat. Addition of lactate has no major effect on myoglobin oxygenation over a large temperature range. Therefore, metabolic acidosis does not impair myoglobin function under conditions of prolonged physical effort, such as diving.

Molecular Dynamics Analysis of a Second Phosphate Site in the Hemoglobins of the Seabird, South Polar Skua. Is There a Site-Site Migratory Mechanism along the Central Cavity?

Hemoglobin function is modulated by several non-heme ligands; among these effectors, organic phosphates generally bind to heterotropic sites with a one-to-one stoichiometry. The phosphate binding site of human hemoglobin is located at the interface between the two beta chains. An additional binding site for polyanions has been studied at the molecular level (Tamburrini, M., A. Riccio, M. Romano, B. Giardina, and G. di Prisco. 2000. Eur. J. Biochem. 267:6089-6098) in the hemoglobins of the south polar skua (Catharacta maccormicki). It is formed by a cluster of six positive charges of both alpha chains (Val-1, Lys-99, Arg-141); the two Lys-99alpha have an essential role in the site structure. The present investigation, carried out on skua deoxyhemoglobins by using a molecular dynamics approach, confirms the structural feasibility of the additional site, possibly having the role of an entry-leaving site, and leads to the proposal of a novel migration pathway for phosphate along the central cavity of hemoglobin from one binding site to the other, occurring according to the hypothesis of a site-site migratory mechanism, which may assign a functional role to the central cavity. The role of Lys-99alpha was further confirmed by molecular dynamics experiments on the mutant Lys-99alpha-->Ala in which, at the end of the simulation, the phosphate was external to the additional site.

The hemoglobin system of Pleuragramma antarcticum: Correlation of hematological and biochemical adaptations with life style☆☆☆

The hematological properties and the oxygen-transport system of the antarctic fish Pleuragramma antarcticum were investigated. Most blood parameters are at the lower end of the range of values known for red-blooded antarctic fish, suggesting a link with the sluggish mode of life of this species. P. antarcticum is the only species of the family Nototheniidae and of the suborder Notothenioidei having three major hemoglobins, which were isolated and fully characterized. The complete amino acid sequence of the alpha- and beta-globin chains was determined. The three hemoglobins showed strong Bohr and Root effects, and their oxygen-binding properties were differently regulated by temperature. None of the three hemoglobins of P. antarcticum can be considered as evolutionary (or larval) remnants. Therefore, this oxygen-transport system is one of the most specialized ever found in fish. The data suggest a strong relationship between hematological/biochemical adaptation and life style.

Structure, stability, and IgE binding of the peach allergen Peamaclein (Pru p 7)

Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D‐structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N‐terminal region, and four out of six cysteine pairs have been assigned by 2D‐NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat‐denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D‐structure up to 90°C, but it becomes unfolded at temperatures of 100–120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE‐binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein.

Proteomic analysis in the identification of allergenic molecules

Conventional and innovative strategies can be exploited to identify and characterize new allergenic proteins. With the aim of obtaining suggestions for future improvements, this article describes our attempt to understand and describe some of the advantages and pitfalls of the methodologies and procedures often used in this field. The analysis includes the protein extract preparation, starting from the allergenic source, the separation of the proteins contained in a mixture and the detection, identification and characterization of IgE-binding molecules. Classic and emerging proteomic technologies, including mass spectrometry-based methodologies, Edman degradation procedure, microarray-based techniques and bioinformatics search strategies, have been explored. A comparative analysis of biochemistry-based proteomics and molecular biology strategies has also been given.

Evolutionary Studies on Teleost Hemoglobin Sequences

During cold adaptation, the blood of Antarctic fish has acquired some features which clearly differentiate these fish from fish of temperate and tropical climates. A much lower erythrocyte and hemoglobin content counteracts the temperature-induced viscosity increase in the blood and, consequently, cardiac work is greatly facilitated. At the extreme end of such evolution, the blood of the Notothenioid family Channichthyidae (a unique case among vertebrates) is characterized by the total lack of hemoglobin.