Showing papers on "Peptide sequence published in 1993"
TL;DR: The results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured aligo‐ and polypeptides.
Abstract: The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo- and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo- and polypeptides. The application of this approach to protein studies is discussed.
1,779 citations
TL;DR: An early step in ethylene signal transduction in plants may involve transfer of phosphate as in prokaryotic two-component systems.
Abstract: Ethylene behaves as a hormone in plants, regulating such aspects of growth and development as fruit ripening, flower senescence, and abscission. Ethylene insensitivity is conferred by dominant mutations in the ETR1 gene early in the ethylene signal transduction pathway of Arabidopsis thaliana. The ETR1 gene was cloned by the method of chromosome walking. Each of the four known etr1 mutant alleles contains a missense mutation near the amino terminus of the predicted protein. Although the sequence of the amino-terminal half of the deduced ETR1 protein appears to be novel, the carboxyl-terminal half is similar in sequence to both components of the prokaryotic family of signal transducers known as the two-component systems. Thus, an early step in ethylene signal transduction in plants may involve transfer of phosphate as in prokaryotic two-component systems. The dominant etr1-1 mutant gene conferred ethylene insensitivity to wild-type Arabidopsis plants when introduced by transformation.
1,482 citations
TL;DR: Primary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid, and the availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidsosis in AD.
Abstract: A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.
1,423 citations
TL;DR: A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database and each protein was uniquely identified from over 91,000 protein sequences.
Abstract: A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.
1,290 citations
TL;DR: Results suggest an important link between tumor initiation and cell adhesion and two cellular proteins that associate with APC were identified as the E-cadherin-associated proteins alpha- and beta-catenin.
Abstract: Mutations of APC appear to initiate sporadic and inherited forms of human colorectal cancer. Although these mutations have been well characterized, little is known about the function of the APC gene product. Two cellular proteins that associate with APC were identified by nucleotide sequence analysis and peptide mapping as the E-cadherin-associated proteins alpha- and beta-catenin. A 27-residue fragment of APC containing a 15-amino acid repeat was sufficient for the interaction with the catenins. These results suggest an important link between tumor initiation and cell adhesion.
1,255 citations
TL;DR: It is concluded that di peptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7-36)amide in human serum and inactivates these peptide hormones.
Abstract: Peptides of the glucagon/vasoactive-intestinal-peptide (VIP) peptide family share a considerable sequence similarity at their N-terminus. They either start with Tyr-Ala, His-Ala or His-Ser which might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growth-hormone-releasing factor (1-29)amide and gastric inhibitory peptide/glucose-dependent insulinotropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1(7-36)amide/insulinotropin [GLP-1(7-36)amide] and peptide histidine methionine (PHM) with terminal His-Ala were hydrolysed to their des-Xaa-Ala derivatives by dipeptidyl-peptidase IV purified from human placenta. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP-1(7-36)amide and PHM were analyzed in detail. For these peptides Km values of 4-34 microM and Vmax values of 0.6-3.8 mumol.min-1.mg protein-1 were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP-1(7-36)amide the same fragments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa-Ala peptides and Tyr-Ala in the case of GIP or His-Ala in the case of GLP-1(7-36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidyl-peptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ile), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7-36)amide in human serum. Since an intact N-terminus is obligate for the biological activity of the members of the glucagon/VIP peptide family [e. g. GIP(3-42) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidyl-peptidase-IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.
1,198 citations
1,181 citations
TL;DR: Northern blotting and in situ hybridization analyses revealed that the expressions of individual mRNAs for the NMDAR2 subunits overlap in some brain regions but are also specialized in many other regions.
977 citations
TL;DR: The structural constraints for the receptors are used to allocate particular helices to the peaks in the recently published projection map of rhodopsin and to propose a tentative three‐dimensional arrangement of the helices in G protein‐coupled receptors.
Abstract: G protein-coupled receptors form a large family of integral membrane proteins whose amino acid sequences have seven hydrophobic segments containing distinctive sequence patterns. Rhodopsin, a member of the family, is known to have transmembrane alpha-helices. The probable arrangement of the seven helices, in all receptors, was deduced from structural information extracted from a detailed analysis of the sequences. Constraints established include: (1) each helix must be positioned next to its neighbours in the sequence; (2) helices I, IV and V must be most exposed to the lipid surrounding the receptor and helix III least exposed. (1) is established from the lengths of the shortest loops. (2) is determined by considering: (i) sites of the most conserved residues; (ii) other sites where variability is restricted; (iii) sites that accommodate polar residues; (iv) sites of differences in sequence between pairs or within groups of closely related receptors. Most sites in the last category should be in unimportant positions and are most useful in determining the position and extent of lipid-facing surface in each helix. The structural constraints for the receptors are used to allocate particular helices to the peaks in the recently published projection map of rhodopsin and to propose a tentative three-dimensional arrangement of the helices in G protein-coupled receptors.
973 citations
TL;DR: It is reported here that neuronal death results from chronic exposure of primary rat hippocampal cultures to micromolar concentrations of a peptide corresponding to residues 106–126 of the amino-acid sequence deduced from human PrP complementary DNA.
Abstract: The cellular prion protein (PrPC) is a sialoglycoprotein of M(r) 33-35K that is expressed predominantly in neurons. In transmissible and genetic neurodegenerative disorders such as scrapie of sheep, spongiform encephalopathy of cattle and Creutzfeldt-Jakob or Gerstmann-Straussler-Scheinker diseases of humans, PrPC is converted into an altered form (termed PrPSc) which is distinguishable from its normal homologue by its relative resistance to protease digestion. PrPSc accumulates in the central nervous system of affected individuals, and its protease-resistant core aggregates extracellularly into amyloid fibrils. The process is accompanied by nerve cell loss, whose pathogenesis and molecular basis are not understood. We report here that neuronal death results from chronic exposure of primary rat hippocampal cultures to micromolar concentrations of a peptide corresponding to residues 106-126 of the amino-acid sequence deduced from human PrP complementary DNA. DNA fragmentation of degenerating neurons indicates that cell death occurred by apoptosis. The PrP peptide 106-126 has a high intrinsic ability to polymerize into amyloid-like fibrils in vitro. These findings indicate that cerebral accumulation of PrPSc and its degradation products may play a role in the nerve cell degeneration that occurs in prion-related encephalopathies.
946 citations
TL;DR: Most of the peptides derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens, were derived from major histocompatibility complex-related molecules.
Abstract: Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.
TL;DR: The advantages of being able to predict the position of a protein of known structure within a two‐dimensional gel are shown.
Abstract: The focusing positions in narrow range immobilized pH gradients of 29 polypeptides of known amino acid sequence were determined under denaturing conditions. The isoelectric points of the proteins calculated from their amino acid sequences matched with good accuracy the experimentally determined pI values. We show the advantages of being able to predict the position of a protein of known structure within a two-dimensional gel.
TL;DR: A sequence in HA that has a high propensity for forming a coiled coil is identified, which leads to a model for the fusogenic conformation of HA: the coiled-coil stem of the native state extends, relocating the hydrophobic fusion peptide, by 100 A, toward the target membrane.
TL;DR: The peptide specificity of class I molecules and experimental evidence indicate that T cells are tolerant to only a small fraction of the expressed genomic sequences and are not tolerant to the remainder.
Abstract: MHC class I molecules are peptide receptors of stringent specificity which however still allow millions of different ligands. This is achieved by the following specificity characteristics summarized as allele specific peptide motifs: Peptides are of defined length, depending on the class I allele (either 8 or 9 residues; exceptions have been observed). Typically, 2 of the 8 or 9 positions are anchors that can only be occupied by a single amino acid residue, or by residues with closely related side chains. Location and characteristics of anchors vary with class I alleles. The C terminus of the peptide ligands is frequently an aliphatic or charged residue. Such allele-specific class I peptide ligand motifs, known so far for H-2Kd, Kb, Kk, Kkm1, Db, HLA-A*0201, A*0205, and B*2705, are useful to predict natural T cell epitopes. The latter can be determined by extraction from cells recognized by the T cell of interest. It is not known how the class I ligands are produced in the cell, although speculative models exist. The peptide specificity of class I molecules and experimental evidence indicate that T cells are tolerant to only a small fraction of the expressed genomic sequences and are not tolerant to the remainder. The function of class I molecules is to present a collection of self-peptide samples at the cell surface for surveillance by T cells.
TL;DR: The high‐affinity H‐2Db‐binding peptide and putative CTL epitope E7 49‐57 (RAHYNIVTF) was used in vaccination studies against HPV 16‐transformed tumor cells and rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumors in vivo, and induced a CTL response which lysed the tumor cells in vitro.
Abstract: Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2Kb and H-2Db MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2Kb and H-2Db and the predictive value of these motifs is shortly discussed. The high-affinity H-2Db-binding peptide and putative CTL epitope E7 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E7 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.
TL;DR: Examples are given of changes that occur in the carbohydrates of soluble and cell-surface glycoproteins during differentiation, growth and malignancy, which further highlight the important role of these substances in health and disease.
Abstract: During the last decade, there have been enormous advances in our knowledge of glycoproteins and the stage has been set for the biotechnological production of many of them for therapeutic use. These advances are reviewed, with special emphasis on the structure and function of the glycoproteins (excluding the proteoglycans). Current methods for structural analysis of glycoproteins are surveyed, as are novel carbohydrate-peptide linking groups, and mono- and oligo-saccharide constituents found in these macromolecules. The possible roles of the carbohydrate units in modulating the physicochemical and biological properties of the parent proteins are discussed, and evidence is presented on their roles as recognition determinants between molecules and cells, or cell and cells. Finally, examples are given of changes that occur in the carbohydrates of soluble and cell-surface glycoproteins during differentiation, growth and malignancy, which further highlight the important role of these substances in health and disease.
TL;DR: This chapter emphasizes an application for which surface expression vectors are particularly well suited: construction of epitope libraries, in which the displayed ligands are whole folded domains.
Abstract: Publisher Summary A fusion phage is a filamentous virion displaying on its surface a foreign peptide fused to a coat protein and harboring the gene for the fusion protein within its genome This chapter emphasizes an application for which these surface expression vectors are particularly well suited: construction of epitope libraries In such a library—the kind so far constructed—the phages display random foreign peptides encoded by degenerate synthetic oligonucleotides spliced into the coat protein gene, the library as a whole representing up to billions of peptide sequences Affinity purified phages are eluted without destroying their infectivity, and the peptide sequences responsible for binding are easily ascertained by infecting the eluted phages into bacteria, propagating the resulting phage clones, and sequencing the relevant part of their viral DNAs The number of peptides that can be accommodated with this technology exceeds by a factor of 100–1,000 the number that can be screened with conventional expression systems, in which the epitope is not displayed as part of the propagatable unit that encodes it It also discusses more complex libraries, in which the displayed ligands are whole folded domains
TL;DR: A method was developed to clone, without the use of specific functional assays, complementary DNAs (cDNAs) that carry specific amino-terminal signal sequences, such as those encoding intercellular signal-transducing molecules and receptors.
Abstract: A method was developed to clone, without the use of specific functional assays, complementary DNAs (cDNAs) that carry specific amino-terminal signal sequences, such as those encoding intercellular signal-transducing molecules and receptors. The vector used in this system directed the cell surface expression of interleukin-2 receptor fusion proteins when inserts with signal sequences were cloned in-frame with the correct orientation. An expression cDNA library was constructed from a bone marrow stromal cell line, which contained 5' portion-enriched cDNAs (the average size was 400 base pairs). Two cDNAs that encoded putative cytokine molecules, stromal cell-derived factor-1 alpha (SDF-1 alpha) and SDF-1 beta, which belong to the intercrine-macrophage inflammatory protein superfamily, were cloned.
TL;DR: The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal–response system controlling density‐dependent expression of luminescence remains to be identified.
Abstract: Summary
Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two–component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal–response system controlling density-dependent expression of luminescence remains to be identified.
TL;DR: Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences.
Abstract: The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins.
TL;DR: The abundance of structurally altered aspartyl residues may profoundly affect the conformation of the beta A protein within plaque cores and thus significantly impact normal catabolic processes designed to limit its deposition, and contribute to the production and stability of beta-amyloid deposits in Alzheimer brain tissue.
TL;DR: The expression cloning of a functional ligand for P-selectin from an HL-60 cDNA library is reported, and the predicted amino acid sequence reveals a novel mucin-like transmembrane protein.
TL;DR: The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction, and comparison with the structure with the high affinity complex reveals only localized and relatively small changes.
Patent•
05 Feb 1993TL;DR: In this paper, a single-chain Fv polypeptide defining a binding site which exhibits the immunological binding properties of an immunoglobulin molecule which binds c-erbB-2 or a c-b-bb-2-related tumor antigen is described.
Abstract: Disclosed is a single-chain Fv (sFv) polypeptide defining a binding site which exhibits the immunological binding properties of an immunoglobulin molecule which binds c-erbB-2 or a c-erbB-2-related tumor antigen, the sFv includes at least two polypeptide domains connected by a polypeptide linker spanning the distance between the C-terminus of one domain and the N-terminus of the other, the amino acid sequence of each of the polypeptide domains includes a set of complementarity determining regions (CDRs) interposed between a set of framework regions (FRs), the CDRs conferring immunological binding to the c-erbB-2 or c-erbB-2-related tumor antigen.
TL;DR: X-ray crystallography results offer a structural framework for understanding the role of nonanchor peptide side chains in both peptide-MHC binding affinity and TCR recognition.
TL;DR: A simple binary code of polar and nonpolar residues arranged in the appropriate order can drive polypeptide chains to collapse into globular alpha-helical folds.
Abstract: A general strategy is described for the de novo design of proteins. In this strategy the sequence locations of hydrophobic and hydrophilic residues were specified explicitly, but the precise identities of the side chains were not constrained and varied extensively. This strategy was tested by constructing a large collection of synthetic genes whose protein products were designed to fold into four-helix bundle proteins. Each gene encoded a different amino acid sequence, but all sequences shared the same pattern of polar and nonpolar residues. Characterization of the expressed proteins indicated that most of the designed sequences folded into compact alpha-helical structures. Thus, a simple binary code of polar and nonpolar residues arranged in the appropriate order can drive polypeptide chains to collapse into globular alpha-helical folds.
TL;DR: Interestingly, vascular A beta contains considerably less isomerized and racemized aspartyl residues than does neuritic plaque A beta, suggesting that the vascular amyloid is "younger."
Abstract: Reinvestigation of the chemical structure of beta-amyloid peptide (A beta) deposits in the vascular tissue of Alzheimer disease brains revealed that the 42-residue form A beta-(1-42), rather than the more soluble A beta-(1-40) form, is the predominant peptide. Following removal of the surrounding tissue with SDS and collagenase, A beta was solubilized in formic acid and purified by Superose 12 chromatography. Peptides generated by enzymatic and chemical digestion of the A beta were purified by HPLC and characterized by amino acid analysis, sequence analysis, and mass spectrometry. In the leptomeningeal vessels, the average ratio of A beta-(1-42)/A beta-(1-40) was 58:42, whereas in the parenchymal vessels this ratio was 75:25. Interestingly, vascular A beta contains considerably less isomerized and racemized aspartyl residues than does neuritic plaque A beta, suggesting that the vascular amyloid is "younger." The discrete nature of the bands and spherical deposits of A beta associated with arterioles and capillaries, respectively, suggests that this amyloid arises from the vascular tissue itself. Increasing A beta deposition appears to lead to the distortion and occlusion of capillaries, which may contribute significantly to the pathology of Alzheimer disease.
TL;DR: The occurrence of two distinct classes of bacterial cytoplasmic repressor proteins which are homologous to two different clusters of periplasmic binding proteins suggests that the gene-splicing events which allowed functional conversion of these proteins with retention of domain structure have occurred repeatedly during evolutionary history.
TL;DR: Findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.
Abstract: A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pylori 84-183 by using lambda ZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pylori-infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pylori protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120- to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pylori isolates producing the 120- to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a lambda gt11 library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the lambda ZapII genomic library. Nucleotide sequencing of this insert (pYB 2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence of cagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.
TL;DR: Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity, and sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV proteinase substrate specificity.
Abstract: Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.