Biomedical subjects

G Grandi

Publications and source records attributed to G Grandi.

At least 19 recordsLinked to original sources

PubMed2001

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

Amino Acid Sequence↗

PubMed2001

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

Amino Acid Sequence↗

PubMed2001

Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis.

The most popular approach for proteomics analysis is based on the combination of two-dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two-dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture.

Animals↗

PubMed2001

Antibacterial vaccine design using genomics and proteomics.

After 200 years of practice, vaccinology has proved to be very effective in preventing infectious diseases. However, several human and animal pathogens exist for which vaccines have not yet been discovered. As for other fields of medical sciences, it is expected that vaccinology will greatly benefit from the emerging genomics technologies such as bioinformatics, proteomics and DNA microarrays. In this article the potential of these technologies applied to bacterial pathogens is analyzed, taking into account the few existing examples of their application in vaccine discovery.

Animals↗

PubMed2001

Iris alteration using mechanical iris retractors.

A 74-year-old woman had a 3-port pars plana vitrectomy for a dropped nucleus after complicated phacoemulsification. Flexible nylon iris retractors were positioned to dilate the small pupil. The patient died of a pulmonary embolus 1 day after surgery. The eyeball was removed at autopsy, and pathology of the iris was investigated. Localized damage to the iris where the retractor hooks were positioned was noted, but no other alterations to the iris were observed. These findings may explain the recovery of pupil function that is common after surgery with mechanical iris retractors.

Aged↗

PubMed2001

Crystallization and preliminary crystallographic studies on the large extracellular domain of human CD81, a tetraspanin receptor for hepatitis C virus.

The large extracellular domain of CD81, a member of the tetraspanin family and a receptor protein for hepatitis C virus envelope E2 glycoprotein, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.6 A resolution were obtained at the ID14 beamline of the European Synchrotron Radiation Facility from a flash-frozen crystal at 100 K. The crystals belong to space group P2(1), with unit-cell parameters a = 31.5, b = 77.2, c = 38.5 A, beta = 107.4 degrees, and are likely to contain two extracellular domains (2 x 99 residues) per asymmetric unit.

Antigens, CD↗

PubMed2000

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Antigenic Variation↗

PubMed2000

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Amino Acid Sequence↗

PubMed2000

Structure-function analysis of hepatitis C virus envelope-CD81 binding.

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

Amino Acid Sequence↗

PubMed2000

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Animals↗

PubMed1999

Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity.

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.

Cloning, Molecular↗

PubMed1999

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

Amino Acid Sequence↗

PubMed1998

Characterization of the syringomycin synthetase gene cluster. A link between prokaryotic and eukaryotic peptide synthetases.

With this work we have completed the characterization of the syringomycin synthetase gene cluster. In particular, by sequencing additional 28.5 kilobase pairs we show that the nine modules involved in the binding of the nine amino acids of syringomycin are localized on SyrB and SyrE, with SyrE carrying eight modules. The recombinant SyrB and the first and second modules of SyrE (SyrE1 and SyrE2) have been expressed in Escherichia coli and purified. The biochemical data indicate that SyrB binds threonine, the putative precursor of the last amino acid of syringomycin, whereas SyrE1 and SyrE2 bind serine, the first and the second amino acids of syringomycin, respectively. On the basis of the sequence analysis and the biochemical data presented here, it appears that syringomycin synthetase is unique among peptide synthetases in that its genetic organization does not respect the "colinearity rule" according to which the order of the amino acid binding modules along the chromosome parallels the order of the amino acids on the peptide. This feature, together with the absence of a single transcription unit and the absence of epimerase-like domains make syringomycin synthetase more related to the eukaryotic peptide synthetases than to the bacterial counterparts.

Amino Acid Sequence↗

PubMed1998

Binding of hepatitis C virus to CD81.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Amino Acid Sequence↗

PubMed1998

Coordinate transcription and physical linkage of domains in surfactin synthetase are not essential for proper assembly and activity of the multienzyme complex.

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.

Bacillus subtilis↗

PubMed1998

Multi-plasmid DNA vaccination avoids antigenic competition and enhances immunogenicity of a poorly immunogenic plasmid.

DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single- and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.

Animals↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Engineering of peptide synthetases. Key role of the thioesterase-like domain for efficient production of recombinant peptides.

Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.

Amino Acid Sequence↗