Biomedical subjects

P Pucci

Publications and source records attributed to P Pucci.

At least 19 recordsLinked to original sources

PubMed1997

Structural characterization of four genetic variants of human serum albumin associated with alloalbuminemia in Italy.

A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations: albumins Tregasio, Val122-->Glu, Bergamo, Asp314-->Gly, and Maddaloni, Val533-->Met. The fourth, albumin Besana Brianza, has the same Asp494-->Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.

Amino Acid Sequence↗

PubMed1997

Identification of the active site nucleophile in the thermostable beta-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli.

Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli was fully inactivated at 65 degrees C, according to pseudo-first-order kinetics, by [3H]conduritol B epoxide (DL-1,2 anhydro-myo-inositol) synthesized as the active site directed inhibitor by a slight modification of Legler's procedure [Legler, G. (1977) Methods Enzymol. 46, 368-381]. The determination of kinetic constants for the inactivation showed that the process took place through the formation of a stabilized inhibitor-enzyme intermediate. Inactivation and reactivation studies suggested that the inhibitor-enzyme intermediate complex was formed more rapidly and hydrolyzed at a lower rate than it was for other glycosidases. Moreover, the stoichiometry of the binding, determined by electrospray mass spectrometric analysis, revealed that one molecule of the inhibitor was covalently bound to each enzyme subunit. The binding site for [3H]conduritol B epoxide was identified by the isolation and partial sequence analysis of the radioactive peptide obtained by cyanogen bromide and pepsin digests. Electrospray tandem mass analysis of the labeled peptide showed that the inhibitor was covalently bound to E387. This result, in agreement with data obtained from sequence alignments of S. solfataricus beta-glycosidase with other gluco- and galactosidases of the glycosyl hydrolase family 1 [Henrissat, B. (1991) Biochem. J. 280, 309-316], indicates that the conserved E387 is the nucleophilic amino acid residue in the active site of the enzyme.

Amino Acid Sequence↗

PubMed1997

Surface topology of Minibody by selective chemical modifications and mass spectrometry.

The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.

Acetylation↗

PubMed1997

Microheterogeneity of odorant-binding proteins in the porcupine revealed by N-terminal sequencing and mass spectrometry.

Several odorant-binding proteins (OBP) have been previously purified from the nasal mucosa of the old world porcupine Hystrix cristata. In this paper, we report their N-terminal amino-acid sequences and accurate molecular weights, as measured by electrospray mass spectrometry. The partial amino acid sequences reveal significant similarity with OBPs of other mammalian species and segregate the eight proteins purified into two subclasses. Mass spectrometry has revealed microheterogeneity among the proteins belonging to each of these two groups, suggesting a total number of OBPs of at least nine. The molecular weight differences between OBPs cannot be readily accounted for by common post-translation modifications and indicate different gene products. Such a large number of different OBPs may represent further support to an odour discriminating role for these proteins.

Amino Acid Sequence↗

PubMed1997

Identification of the prion protein allotypes which accumulate in the brain of sporadic and familial Creutzfeldt-Jakob disease patients.

A characteristic feature of Creutzfeldt-Jakob disease (CJD) is the accumulation in the brain of the amyloid protease-resistant protein PrPres. PrPres derives from a host-encoded, protease-sensitive isoform, PrPsen. Mutations of this protein are linked to familial variants of the disease, and the presence of a methionine or valine residue at the polymorphic position 129 may be critical in sporadic CJD cases. We found that in the brain of patients heterozygous for the mutation in which isoleucine is substituted for valine at codon 210 (Val21Olle), the PrPres is formed by both the wild-type and mutant PrPsen. We also found that in a sporadic CJD patient, who was heterozygous (Met/Val) at position 129, PrPres is also formed by both allotypes. These data associate transmissible spongiform encephalopathies with other amyloidosis, although the nature of the transmissible agent remains unsettled.

Amino Acid Sequence↗

PubMed1997

In vitro reactivity of the antineoplastic drug carmustin and acrolein with model peptides.

The in vitro interaction of the antineoplastic drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and acrolein with model peptides has been investigated in order to provide a detailed description of their electrophilic reactivity towards biological macromolecules. Following incubation with these substances, the modified species were separated by HPLC and identified by fast atom bombardment mass spectrometry, whereas the reactive amino acids within the peptide structure were assigned by tandem mass spectrometry. Incubation with BCNU led essentially to the formation of an N-terminal carbamoyl derivative that slowly decomposed to form three isomeric structures and a very minor ethylated adduct. Alkylation with acrolein gave rise to a mixture of different adducts due to the reaction of both the double bond and the carbonyl group. Two species containing intramolecular cross-links were also observed. These results constitute the prerequisite for in vitro and in vivo studies on the modification of haemoglobin in patients following treatment with antineoplastic drugs.

Acrolein↗

PubMed1997

Compound heterozygosity for Hb Lepore-Boston and Hb Neapolis (Dhonburi) [beta 126(H4)Val-->Gly] in a patient from Naples, Italy.

The simultaneous presence of two hemoglobin variants has been detected in a 14-month-old patient affected by thalassemia intermedia. The two variants were characterized by a combination of allele-specific amplification methods and mass spectrometric procedures carried out on isolated globins. These were identified as Hb Lepore-Boston and Hb Neapolis (also known as Hb Dhonburi) or beta 126 (H4)Val-->Gly. Hb Lepore-Boston is the most common hybrid variant detected in Campania and several cases of Hb Neapolis which causes a mild hypochromic microcytic anemia have been identified in this region in the last few years. This is the first report of a double heterozygosity involving Hb Lepore-Boston and Hb Neapolis.

Follow-Up Studies↗

PubMed1996

Structural characterisation of human recombinant glycohormones follitropin, lutropin and choriogonadotropin expressed in Chinese hamster ovary cells.

The alpha and beta chains from human recombinant gonadotropins follitropin, lutropin and choriogonadotropin expressed in CHO cells have been structurally characterised both at the protein and at the carbohydrate level by using advanced mass spectrometric procedures. The three alpha chains share the same amino acid sequence while they display different glycosylation patterns. The oligosaccharide structures detected belong to the complex-type glycans with different degree of sialylation. Partial proteolytic processing occurred at the N-terminus of the follitropin beta chain and at the C-terminus of the lutropin beta chain. The N-linked glycans from the three beta chains were found to contain fucosylated and sialylated diantennary and triantennary complex-type structures. The follitropin beta chain showed the presence of N-acetyllactosamine repeats on the antennae. The overall structure of the recombinant glycohormones corresponds to their natural counterparts with the exception that sulphated terminal glycosylation is missing.

Amino Acid Sequence↗

PubMed1996

Amino acid sequence and molecular modelling of glycoprotein IIb-IIIa and fibronectin receptor iso-antagonists from Trimeresurus elegans venom.

Low-molecular-mass Arg-Gly-Asp (RGD)-containing polypeptides were isolated from the venom of Trimeresurus elegans by a simple two-step procedure consisting of membrane filtration and reverse-phase HPLC. A combination of electrospray MS, fast-atom bombardment MS and Edman degradation allowed us to ascertain the presence in the venom of different isoforms and to determine their primary structures. The amino acid sequences resembled the structure of elegantin, the only disintegrin previously reported from the T. elegans venom [Williams, Rucinski, Holt and Niewiarowski (1990) Biochim. Biophys, Acta 1039, 81-89]. MS analyses indicated the occurrence of differential proteolytic processing at both the N-terminus and the C-termins of the polypeptide chains. The amino acid sequence alignment of the elegantin isoforms with known components of the disintegrin family demonstrated the complete conservation of the 12 cysteine residues involved in disulphide bridges. Molecular modelling of elegantins predicted an overall folding of these molecules quite similar to that reported for the kistrin solution structure. The newly identified polypeptide isoforms strongly inhibited ADP-induced aggregation in both human and canine platelet-rich plasma but showed a different species-dependent specificity. These molecules were also able to inhibit B16-BL6 murine melanoma cell adhesion to immobilized fibronectin. The comparison of the structures and biological activities of elegantin isoforms and kistrin allowed us to highlight some structural features that, in addition to the RGD locus might be involved in the interaction of these snake-venom polypeptides with the integrin receptors on the platelet and cell surface.

Adenosine Diphosphate↗

PubMed1996

Transglutaminase from rat coagulating gland secretion. Post-translational modifications and activation by phosphatidic acids.

Structural and biochemical characteristics of transglutaminase purified by a rapid chromatographic procedure from the rat coagulating gland (anterior prostate) secretion are reported. Fast atom bombardment mapping and automated Edman degradation experiments allowed us to verify that at least 85% of the entire transglutaminase amino acid sequence is identical to that derived from the cDNA of the major androgen-dependent rat prostate protein called DP1. The enzyme was found NH2 terminally blocked and largely post-translationally modified, since the presence of N-linked oligosaccharides, as well as of complex lipidic structures, was observed. Mass spectral analysis showed that Asn-408 and -488 are the glycosylated sites, the N-linked structures identified belonging to both high-mannose and complex type glycans. The presence of myo-inositol, of glycerol bound fatty acids, and the high content of mannose residues, are in agreement with previous observations suggesting that a lipid anchor is bound to coagulating gland secretion transglutaminase. Furthermore, two tightly bound calcium ions per molecule of enzyme were detected. Finally, a strong stimulation of the enzyme activity in vitro by both SDS and a variety of phosphatidic acids was observed. The reported structural and functional peculiarities should definitively lead to consider the prostate enzyme as a new member (type IV) of the transglutaminase family.

Amino Acid Sequence↗

PubMed1996

Glutathione-dependent pathways of refolding of RNase T1 by oxidation and disulfide isomerization: catalysis by protein disulfide isomerase.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T1 (rd-RNase T1) and mixed disulfide between glutathione and reduced RNase T1 (GS-RNase T1) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding experiments were performed in the presence of different absolute concentrations of glutathione species while keeping the redox potential fixed, in order to evaluate the effect of the glutathione concentration on the distribution of the refolding intermediates. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T1 which occurs via the reiteration of two steps: (i) formation of a species containing both mixed disulfides with glutathione and free protein thiols, and (ii) formation of an intramolecular disulfide via thiol-disulfide interchange reaction between them. Refolding of rd-RNase T1 and GS-RNase T1 was also performed in the presence of protein disulfide isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. Refolding reactions carried out at different absolute concentrations of glutathione proved that GSH and/or GSSG participate directly in the reaction catalyzed by PDI. On the basis of these experiments and previous results on the refolding of RNase A [Torella, C., Ruoppolo, M., Marino, G., & Pucci, P. (1994) FEBS Lett. 352, 301-306], a hypothesis of a general pathway for folding of S--S containing proteins is proposed.

Animals↗

PubMed1996

Assignment of protein disulphides by a computer method using mass spectrometric data.

We designed a computer program for the assignment of protein disulphides using mass spectrometric data. All the theoretical linear peptides containing from one to three cysteines are generated on the basis of the protein sequence. Their combination ways are determined in order to create all the possible disulphide-bridged fragments containing from two to six cysteines and to calculate their molecular weight. One, two and three S-S bridges per linked fragment were considered, taking into account the possibility that the fragments contain unabridged residues. The mass data obtained from the spectral analysis of peptide mixtures of the digested protein are then associated to the fitting structures of disulphide-bridged peptides, giving rise to the primary output. This output can then be screened by using information on the specificity of the proteolytic agent(s) used and/or any further mass data provided by Edman degradation and/or carboxypeptidase treatment of the peptide mixtures. The need for such a computer aid is discussed and examples of application are given.

Amino Acid Sequence↗

PubMed1996

Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae.

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.

Amino Acid Sequence↗

PubMed1996

The gene, protein and glycan structures of laccase from Pleurotus ostreatus.

A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification. The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G.(1993) Appl. Microbiol. Biotechnol. 39, 632-636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast-atom-bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product. The protein was N-glycosylated Asn444. Glycan analysis showed the presence of only a high-mannose structure containing varying numbers of mannose residues. The presence of O-linked oligosaccharides as well as other post-translational modification could be ruled out by the mass analysis.

Amino Acid Sequence↗

PubMed1996

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.

Amino Acid Sequence↗

PubMed1996

The Role of Chirocephalus diaphanus in the Early Recognition of Environmental Pollution by Trace Elements

A study was undertaken to evaluate whether the fairy shrimp Chirocephalus diaphanus (Crustacea: Anostraca) could be considered as a species capable of biomonitoring environmental contamination by accumulating trace elements. This preliminary investigation aimed at assessing the levels of As, Cd, Cu, Hg, Pb, Se, and Zn in natural waters as well as in adults and cysts of fairy shrimps sampled from five different sites in central Italy. Trace elements were determined by inductively coupled plasma (ICP)-based spectrometry. In particular, ICP-mass spectrometry (ICP-MS) was resorted to in most cases due to its high detection power. Pretreatment of samples (adults and cysts) consisted basically in the HNO3-assisted microwave digestion of the material. The highest levels of trace elements were found in the anostracan cysts, with concentration ranges (μg/g) of 0.464-3.11 for As, up to 0.820 for Cd, 2.62-13.0 for Cu, 0.011-0.213 for Hg, 0.966-8.46 for Pb, 0.295-2.45 for Se, and 16.4-50.4 for Zn. On the other hand, the lowest concentrations were found in natural waters, with values close to the ICP-MS detection power for some elements. These data are probative of a bioconcentration process, the extent of which is proportional to the environmental level of each element. This may turn out to be useful in planning biological monitoring and assessing ecotoxicological consequences which might ensue from the diffusion of the exploitation of such organisms in aquaculture.

Journal Article↗

PubMed1996

Identification of disulphide bonds in the refolding of bovine pancreatic RNase A.

BACKGROUND: Comprehension of the rules that govern the folding process is still far from satisfactory, though it is nevertheless clear that all the information required to define the folding is encoded in the amino acid sequence. In proteins that contain disulphide bonds, folding is associated with disulphide bond formation. Protein species with different numbers of disulphides tend to accumulate during the process; these species can be trapped in a stable form, by quenching any remaining free SH groups, and then characterized in order to identify the disulphide bonds formed. RESULTS: The refolding pathway of reduced and denatured RNase A has been studied using mass spectrometric strategies which allow identification of the formation and rearrangement of disulphide bonds during the process. When reoxidation was carried out in the presence of B M urea, producing the classic "scrambled' RNase, three native and 11 non-native disulphide bonds were identified. When the reoxidation was performed under nondenaturing conditions, the formation of several well defined non-native as well as native S-S bonds was observed at early stages of the refolding process. Under appropriate conditions, all four native disulphide bonds were identified at later stages of refolding and non-native disulphides were greatly diminished or non-existent. This stage corresponded with the almost complete recovery of biological activity of the protein. CONCLUSIONS: The results presented here show that both native and non-native disulphide bonds are formed during the refolding of reduced and denatured RNase A in vitro under different experimental conditions. Essentially 14 disulphide bonds were observed of the 2B theoretically possible cysteine couplings. Although this number constitutes a significant fraction of the theoretical total, the occurrence of only a subset of disulphides clearly indicates that the formation of the S-S bridges does not occur at random, even when reoxidation takes place under denaturing conditions.

Animals↗

PubMed1995

Structural analysis of saposin C and B. Complete localization of disulfide bridges.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.

Amino Acid Sequence↗