Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberry.

BACKGROUND: Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. METHODS: A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). RESULTS: Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3(+) subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. CONCLUSIONS: Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs.

Hagfish hemoglobins: structure, function, and oxygen-linked association.

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528).

L-Glutamate dehydrogenase from the antarctic fish Chaenocephalus aceratus. Primary structure, function and thermodynamic characterisation: relationship with cold adaptation.

In order to study the molecular mechanisms of enzyme cold adaptation, direct amino acid sequence, catalytic features, thermal stability and thermodynamics of the reaction and of heat inactivation of L-glutamate dehydrogenase (GDH) from the liver of the Antarctic fish Chaenocephalus aceratus (suborder Notothenioidei, family Channichthyidae) were investigated. The enzyme shows dual coenzyme specificity, is inhibited by GTP and the forward reaction is activated by ADP and ATP. The complete primary structure of C. aceratus GDH has been established; it is the first amino acid sequence of a fish GDH to be described. In comparison with homologous mesophilic enzymes, the amino acid substitutions suggest a less compact molecular structure with a reduced number of salt bridges. Functional characterisation indicates efficient compensation of Q(10), achieved by increased k(cat) and modulation of S(0.5), which produce a catalytic efficiency at low temperature very similar to that of bovine GDH at its physiological temperature. The structural and functional characteristics are indicative of a high extent of protein flexibility. This property seems to find correspondence in the heat inactivation of Antarctic and bovine enzymes, which are inactivated at very similar temperature, but with different thermodynamics.

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.

Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus.

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.

NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.

Biosynthesis and immunobiochemical characterization of gp17/GCDFP-15. A glycoprotein from seminal vesicles and from breast tumors, in HeLa cells and in Pichia pastoris yeast.

The gp17 factor is a secretory product of human seminal vesicle cells which binds to CD4 and acts as a potent inhibitor of T lymphocyte apoptosis induced by CD4 crosslinking and subsequent T-cell receptor (TCR) activation. The protein is identical to gross cystic disease fluid protein-15 (GCDFP-15), a breast tumor secretory marker PIP (prolactin inducible protein), a prolactin-controlled and androgen-controlled protein; secretory actin binding protein (SABP), a seminal plasma actin binding protein and extra-parotid glycoprotein (EP-GP), a secretory protein from the salivary gland. The structure of this protein has not yet been elucidated and no biological function has been clearly attributed to date. Expression of recombinant gp17/GCDFP-15 cDNA in bacteria and insect cells leads to the production of a misfolded insoluble protein. In this study, we describe the production of gp17/GCDFP-15 in two different eukaryotic systems, namely HeLa cells and the Pichia pastoris yeast. Using constructs in which gp17/GCDFP-15 was tagged with enhanced green fluorescent protein (EGFP) in various combinations, we observed expression only when the fusion protein was directed to the secretory compartment by the correct signal peptide. The resulting fluorescent protein was inefficiently secreted, thus suggesting that gp17/GCDFP-15 is not appropriately post-translationally processed and/or transported in HeLa cells. The use of the P. pastoris secretory pathway allowed instead the accumulation in the culture medium of a GCDFP-15/gp17 species which retained the ability to bind to CD4 and also most of the biochemical and immunological properties of the native protein. The production of an active recombinant molecule opens the way to correlate the structural properties of this peculiar factor to its ability to bind several proteins, including CD4, and to block CD4-mediated T cell programmed death.

The hemoglobins of the antarctic fishes Atedidraco orianae and Pogonophryne scotti. Amino acid sequence, lack of cooperativity, and ligand binding properties.

The oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae, Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the alpha and beta chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation.

Enzymes in antarctic fish: glucose-6-phosphate dehydrogenase and glutamate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G6PD) and L-glutamate dehydrogenase (GDH) from Antarctic fish were isolated and characterized. G6PD was purified from the erythrocytes of red-blooded Dissostichus mawsoni and from the colorless blood of the icefish Chionodraco hamatus. Structural and functional characterization showed that the two enzymes do not differ significantly from each other. GDH was purified from the liver of the icefish Chaenocephalus aceratus. As in other fish ODHs, it showed a marked preference for NAD-. The amino acid sequence of the active-site peptide is virtually identical to that of other fish and vertebrate counterparts. Although the basic structural features of the Antarctic enzymes are similar to those of mesophilic organisms, some catalytic and thermodynamic properties make the Antarctic enzymes more suited to cold-adapted organisms.

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.

The unique hemoglobin system of Pleuragramma antarcticum, an antarctic migratory teleost. Structure and function of the three components.

Pleuragramma antarcticum (suborder Notothenioidei, family Nototheniidae) is the most abundant fish in the antarctic shelf. This pelagic species has a circum-antarctic distribution and is characterized by spawning migration. This species displays the highest multiplicity of major hemoglobins (three); the other notothenioids have a single one (except one species, having two) with relatively low oxygen affinity regulated by pH and organophosphates. The hemoglobins of P. antarcticum display strong Bohr and Root effects; however, they reveal important functional differences in subunit cooperativity and organophosphate regulation and, above all, in the response of oxygenation to temperature. Despite the substitution ValbetaE11 --> Ile found in Hb 2, which decreases the affinity in human mutants, the hemoglobins have similar oxygen affinity, higher than that of the other notothenioids. Hb 1 has the alpha chain in common with Hb 2 and the beta in common with Hb 3. The amino acid sequence of all four chains has been established. Thus the hematological features of P. antarcticum differ remarkably from those of antarctic notothenioids. This unique and sophisticated oxygen transport system may adequately meet the requirements of the unusual mode of life of this fish.

Domain organization and DNA-induced conformational changes of an archaeal family B DNA polymerase.

Family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol) is a monomer of about 100 kDa with two associated catalytic functions: 3'-5' exonuclease and DNA polymerase activities. The structure of this enzyme in the free and DNA-bound states was probed by limited proteolysis and fluorescence spectroscopy measurements. The results of partial trypsin proteolysis experiments on the recombinant Sso DNA pol pinpointed three major sites of protease sensitivity: near the N-terminus, within the center, and near the C-terminal end of the polypeptide chain. When partial trypsin digestion was carried out in the presence of either activated calf thymus DNA or primed M13mp18 single-stranded DNA, changes in cleavage pattern and in susceptibility to protease were detected. This phenomenon was dependent on the nucleic acid concentration and suggested the occurrence of DNA-induced conformational changes. These were also probed by steady-state fluorescence spectroscopy measurements using acrylamide as a quencher. Fine mapping of the DNA-specific cleavage sites allowed us to precisely locate the protein subdomains which were affected by these structural changes. Importantly, a specific proteolytic fragment of about 8 kDa was recovered after partial digestion of Sso DNA pol only in the presence of nucleic acid ligands. It was found to start at residues 392-394 and to span the protease-hypersensitive central region of the polypeptide chain. Its involvement in critical polymerase functions, such as substrate binding and/or enzyme processivity, was discussed. In addition, we found that controlled trypsin digestion of Sso DNA pol did not inactivate either polymerase or 3'-5' exonuclease activity concomitantly with the disappearance of full-sized enzyme. Activity gel analysis revealed that proteolytic products corresponding to the amino- and carboxyl-terminal halves of the enzyme retained 3'-5' exonuclease and DNA polymerase activity, respectively. These results are in line with the model of modular organization proposed for Sso DNA pol in a previous report.

The cathodic hemoglobin of Anguilla anguilla. Amino acid sequence and oxygen equilibria of a reverse Bohr effect hemoglobin with high oxygen affinity and high phosphate sensitivity.

As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates this hemoglobin shows a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a side pH range by GTP (whose concentration in the red blood cells varies with ambient oxygen availability). GTP obliterates the reverse Bohr effects in the cathodic hemoglobin. The molecular basis for the reverse Bohr effect in fish hemoglobins has remained obscure due to the lack of structural data. We have determined the complete amino acid sequence of the alpha and beta chains of the cathodic hemoglobins of A. anguilla and relate it to the oxygen equilibrium characteristics. Several substitutions in crucial positions are observed compared with other hemoglobins, such as the replacement of the C-terminal His of the beta chain of Phe (that suppresses the alkaline Bohr effect) and of residues at the switch region between alpha and beta subunits (that may alter the allosteric equilibrium, thus causing the high intrinsic oxygen affinity and low cooperativity). The residues binding organic phosphate in the beta cleft of fish hemoglobins are conserved, which explains the strong effect of GTP on oxygen affinity and suggests that these residues contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. Moreover, His beta 143 that is considered to be responsible for the reverse Bohr effect in human and tadpole Hbs is replaced by Lys.

Purification of a 240 kDa protein from serum and follicular fluid of water buffalo and its identification as haptoglobin.

The fluids from healthy growing follicles of water buffalo were previously found free of the polypeptides H (M(r) 36,000) and L (M(r) 21,000) which were instead detected in fluids from atretic follicles and blood. Here we report evidence that these two polypeptides, as selected from serum by specific anti-L antibodies, are the subunits of an oligomeric protein. The protein was purified from serum or follicular fluid, and its molecular weight (240 kDa), isoelectric point (6.5), and amino acid composition were determined. The NH2-terminal sequences of the subunits L and H were analyzed: 100% and 90% homology with alpha and beta chains of bovine haptoglobin, respectively, was found. Thus, haptoglobin can be used as a novel molecular marker to assess the physiological state of the blood-follicle barrier or discriminate between atretic and healthy follicles.

Glucose 6-phosphate dehydrogenase from human erythrocytes: identification of N-acetyl-alanine at the N-terminus of the mature protein.

Glucose 6-phosphate dehydrogenase from human erythrocytes has a blocked amino-terminus and no information could be obtained by direct sequencing of the intact protein. The peptide corresponding to the amino-terminal region was isolated from a tryptic digest of the whole protein and identified on the basis of its amino acid composition and of the failure to obtain Edman degradation. Determination of peptide mass by fast atom bombardment mass spectrometry allowed identification of the blocked amino-terminal residue as N-acetyl-alanine.

Primary structure of human erythrocyte nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein FX: identification with the mouse tum- transplantation antigen P35B.

Human erythrocytes contain a nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein, FX, whose levels are significantly increased in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals bearing the mediterranean variant of G6PD. Elucidation of the still unknown biologic functions of FX was approached by means of amino acid sequencing of its 25 tryptic peptides. Searching in the EMBL data bank allowed identification of extensive homology between these tryptic peptides and all sequence-aligned regions encompassing the complete structure of a putative protein encoded by the P35B gene in the mouse. This gene, which differs from the normal allele by a point mutation, has been previously cloned from a tum- variant of the murine tumor cell line P815, so defined because it is associated with low tumorigenicity compared with the progenitor P815. The reported P35B cDNA contains an open reading frame (ORF) of 813 bp and encodes a putative protein of 271 amino acids (30 kD), whereas FX protein is 320 amino acids in length (35.81 kD, in good agreement with previous studies). However, a single base shift at position 4,752 of the P35B gene suppresses the stop codon after Phe 271 and allows continuation of the ORF for up to 320 amino acids to reach the same length as FX. The remarkably high extent (92%) of homology indicates that erythrocyte FX protein is the human homolog of the P35B gene product.

Molecular characterization of the functionally distinct hemoglobins of the Antarctic fish Trematomus newnesi.

Antarctic fish of the family Nototheniidae usually have a single major hemoglobin (Hb 1), often a second, minor component (Hb 2, about 5% of the total), and traces of another component (Hb C, less than 1%). These are functionally similar Bohr and Root effect hemoglobins. All species of other highly endemic fish families so far investigated also have one single major hemoglobin. The hematological features of the nototheniid Trematomus newnesi are remarkably different. It is the only Antarctic species in which Hb 1 and Hb 2 display only a very weak Bohr effect and no Root effect. Perhaps consequentially, Hb C (the only component showing regulation of oxygen binding by protons and other effectors) is not present in traces but accounts for 20-25% of the total. The primary structure of the three hemoglobins of T. newnesi and of Root effect HbC present in trace amounts in another nototheniid (Pagothenia bernacchii) is discussed in relationship with oxygen binding and in terms of molecular and stereochemical models. The hemoglobin multiplicity, the oxygen binding features of Hb 1 and Hb 2, and the presence of functionally distinct components, thus reveal that the oxygen transport of T. newnesi has unique characteristics.

The amino acid sequence of the alpha chain of HB 2 completes the primary structure of the hemoglobins of the Antarctic fish Notothenia coriiceps neglecta.

1. The blood of Notothenia coriiceps neglecta (a cold-adapted notothenioid fish, widely distributed in Antarctic waters, and characterized by a relatively low content of erythrocytes and hemoglobin), contains two hemoglobin components, Hb 1 and Hb 2; the amino acid sequences of the beta chain of Hb 1 and Hb 2 are identical. 2. The amino acid sequence of the alpha chain of Hb 2 has been established, thus completing the elucidation of the primary structure of the two hemoglobins.