Hb G-Waimanalo [alpha 64(E13)Asp-->Asn] observed in a Caucasian family.
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Adult↗
Biomedical subjects
P Pucci
Publications and source records attributed to P Pucci.
Human-immunodeficiency-virus transmembrane glycoprotein gp41 is an amino acceptor and donor substrate for transglutaminase in vitro.
Recombinant gp41, the transmembrane glycoprotein of the human-immunodeficiency-virus (HIV) envelope, is an amino acceptor and donor substrate for transglutaminase in vitro. Gln51, Gln52, Gln66 and Lys77 residues were suggested as reactive sites, recognized by the enzyme, for possible cross-linking reactions with gp120, CD4 or other receptor(s) occurring on the surface of HIV-target cells. Soluble CD4, even though unable to function as an amino-acceptor transglutaminase substrate, becomes active in the presence of gp41, negatively influencing the enzyme-catalyzed incorporation of the polyamine spermidine into the transmembrane protein. These results suggest a possible role for transglutaminase in virus entry into host cells, via receptor-mediated endocytosis, and/or in HIV-induced CD4+ T-cell depletion via apoptosis.
Amino Acid Sequence↗
Disulfide isoform intermediates in the reoxidation of recombinant human basic fibroblast growth factor.
The reoxidation of human recombinant basic fibroblast growth factor was investigated following treatment of the protein with a mixture of reduced and oxidized glutathione, both in the absence and in the presence of protein disulfide isomerase. The oxidative process took place throughout the formation of two transient intermediates and yielded a stable bFGF derivative, GS2-bFGF. All of these components were separated by HPLC and accurately characterized at the molecular level by advanced mass spectrometric procedures. When the reoxidation was carried out in the presence of PDI, a 4-fold increase in the reaction rate was estimated. A mixed disulfide with a single glutathione molecule was shown to occur in the two transient intermediates, each of which has different cysteine residues involved in the linkage. The final product GS2-bFGF was structurally different from other bFGF derivatives previously described [Thompson, S. A. (1992) J. Biol. Chem. 267, 2269-2273; Caccia et al. (1992) Eur. J. Biochem. 204, 649-655]. The four cysteine residues are all involved in disulfide bridges; Cys 34 and Cys 78 are linked to exogenous glutathione, whereas Cys 91 and Cys 101 form an intramolecular S-S bridge.
Chromatography, High Pressure Liquid↗
Hemoglobin Ozieri: a new alpha-chain variant (alpha 71(E20)Ala-->Val). Characterization using FAB- and electrospray-mass spectrometric techniques.
A new silent hemoglobin variant, Hb Ozieri (alpha 71(E20)Ala-->Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the alpha-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the alpha-globin gene. This observation brings the number of variants due to a mutation in the alpha-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.
Alanine↗
Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.
Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.
Amino Acid Sequence↗
Sheep haemoglobin I or beta B13(A10)Gly-->Ser: an example of a CpG mutation in vertebrates. Characterization using FAB-mass spectrometry and amino acid sequencing.
1. The amino acid substitution which characterizes the haemoglobin I variant from sheep has been ascertained using a combination of Fast Atom Bombardment mass spectrometry and protein sequencing. 2. A Ser for Gly substitution at position 13 (10 of the A helix) was found in a polypeptide with the overall sequence of the beta B globin. 3. On the basis of the nucleotide sequence of the beta B-globin gene, a C to T transition occurring on a CpG doublet is considered to be responsible for the amino acid substitution. 4. This represents the first observation of a variant sheep Hb due to a mutation which is rather common in the human genome. 5. Amongst ruminants, serine is normally present at position 13 of goat and sheep epsilon II and gamma chains and of bovine gamma chain which had an independent and more ancient evolutionary origin than the beta chains.
Amino Acid Sequence↗
Hb O-Arab [beta 121(GH4)Glu-->Lys]: association with DNA polymorphisms of African ancestry in two Mediterranean families.
Hb O-Arab [beta 121(GH4)Glu-->Lys] was detected in two Mediterranean families, one from Southern Italy and the other from Albania. The GAA-->AAA mutation at codon 121 was characterized by DNA sequencing. The mutant genes were associated with the same beta-globin gene framework variant and with the rare Hpa I/3' beta polymorphic restriction site generating a 7.0 kb fragment. However, at 5' the gene of the Italian family was associated with the restriction fragment length polymorphism subhaplotype [+ - - - +] and the Taq I/3'G gamma polymorphic site, while that of the Albanian family was associated with subhaplotype [- - - - +] but not with the Taq I/3'G gamma site. The particular features of these polymorphisms support the hypothesis of an African origin for the Hb O-Arab gene and a subsequent recombination event leading to the haplotype found in the Italian family.
Adolescent↗
Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites.
The primary structure of the Lewis lung carcinoma protein HMGY belonging to the nuclear group of proteins HMGI (high mobility group I) was determined using electrospray and fast atom bombardment mass spectrometry. It was demonstrated that the sequence of the tumor protein corresponds to the amino acid sequence derived from the cDNA from cultured cells and that the N-terminal serine residue is N-acetylated. Moreover, the two high performance liquid chromatography-purified forms Y1 and Y2 of the protein HMGY were shown to differ at the level of serine phosphorylation, since they contain three phosphate and two phosphate groups, respectively, in the C-terminal region. No other modification was detected in the remaining part of the molecule.
Amino Acid Sequence↗
Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.
The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.
Amino Acid Sequence↗
Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.
The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.
Animals↗
Factors affecting the fast atom bombardment mass spectrometric analysis of proteolytic digests of proteins.
The fast atom bombardment (FAB) mass spectrometric analysis of proteolytic digests of proteins is currently used in protein structural characterization. The major current limitation of this procedure is that not all the peptides generated by enzyme digestion can be observed in the spectra. Previous studies showed that in a mixture the more hydrophilic peptides are suppressed. Several enzymatic digests of 18 different proteins ranging from 10 kDa to 67 kDa in molecular weight were examined using FAB mass spectrometry. It was observed that, even though the hydrophobicity of peptides is a factor in determining their presence or absence in the spectra, the predictions of whether or not a peptide would be detected based on this criterion varies in a wide range of values. Moreover, present results seem to indicate that the presence of particular amino acid side chains within a peptide sequence capable of forming hydrogen bonds with the matrix heavily affects the behaviour of that peptide in the mixture, despite the overall hydrophobicity of the peptide itself.
Amino Acids↗
Characterization of hemoglobin lepore variants by advanced mass-spectrometric procedures.
We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.
Chromatography, High Pressure Liquid↗
Chemistry of the inactivation of 4-aminobutyrate aminotransferase by the antiepileptic drug vigabatrin.
The chemical modification of pig liver 4-aminobutyrate aminotransferase by the antiepileptic drug 4-aminohex-5-enoate (Vigabatrin) has been studied. After inactivation by 14C-labeled Vigabatrin, the enzyme was digested with trypsin, and automated Edman degradation of the purified labeled peptide gave the sequence FWAHEHWGLDDPADVMTFSKK. Chymotryptic digestion of the tryptic peptide and sequencing of a resulting tripeptide identified the penultimate lysine residue of this peptide as the site of covalent modification. This lysine normally binds the coenzyme. Absorption spectroscopy demonstrated the absence of coenzyme from the tryptic peptide, and mass spectrometry showed its mass/charge ratio to be increased by 128. All of the bound coenzyme released after denaturation of the inactivated enzyme was as pyridoxamine phosphate. The structural nature of the modification is deduced, and mechanisms for its occurrence identified. Initially, 1 mol of radiolabeled inhibitor was bound per mol of monomer of the enzyme, although approximately half was released during denaturation and digestion, while the remainder was irreversibly bound. Coenzyme not released as pyridoxamine phosphate retained the absorbance characteristics of the aldimine, although the enzyme was completely inactive. Mass spectrometry of the sample of purified radiolabeled tryptic peptide revealed the presence of an approximately equal amount of a second fragment that contained no modification and from which the second lysine was absent, indicating that at the time of proteolysis the active site lysine was unaltered in 50% of the enzyme molecules.
4-Aminobutyrate Transaminase↗
Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae.
The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudomonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin-producing strains afforded 3-hydroxydecanoyl-Dhb-Pro-Val-Val-Ala-Ala-Val-Val-Dhb-Ala-Val-Ala- Ala-Dhb-aThr-Ser-Ala-Dhb-Ala-Dab-Dab-Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin-producing strain and a syringotoxin-producing strain synthesize 3-hydroxydecanoyl-Dhb-Pro-Val-Ala-Ala-Val-Leu-Ala-Ala-Dhb-Val-Dhb- Ala-Val-Ala-Ala-Dhb-aThr-Ser-Ala-Val-Ala-Dab-Dab-Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue.
Amino Acid Sequence↗
Assignment of the five disulfide bridges in an alpha-amylase inhibitor from wheat kernel by fast-atom-bombardment mass spectrometry and Edman degradation.
The assignment of the five disulfide bridges in an alpha-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.
Amino Acid Sequence↗
Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector.
Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.
Amino Acid Sequence↗
Human alpha-fetoprotein primary structure: a mass spectrometric study.
The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.
Amino Acid Sequence↗
Mass spectrometric identification of the amino donor and acceptor sites in a transglutaminase protein substrate secreted from rat seminal vesicles.
Four different transglutaminase-modified forms of a protein secreted by the rat seminal vesicles (SV-IV) were synthesized in vitro and characterized. FAB maps of both the native protein and its derivatives, produced by the purified guinea pig liver enzyme in the presence or absence of the polyamine spermidine, were obtained by mass spectrometric analysis after proteolytic digestions. Two differently derivatized SV-IV molecular forms, both possessing only one glutamine residue out of two (Gln-86) cross-linked to endogenous lysine residues, were produced when spermidine was omitted from the reaction mixture: (i) an insoluble homopolymer in which Lys-2, -4, -59, -78, -79, and -80 were involved in the linkage; (ii) a soluble form of the protein with an intramolecular epsilon-(gamma-glutamyl)lysine isopeptide bond between Gln-86 and Lys-59. Two species of SV-IV-spermidine adducts were obtained when the protein was treated with transglutaminase in the presence of high concentrations of the polyamine. The first one was characterized by one spermidine molecule covalently bound to Gln-86 and the second one by two spermidine molecules respectively bound to Gln-9 and Gln-86.
Amino Acid Sequence↗