Biomedical subjects

L Otvos

Publications and source records attributed to L Otvos.

At least 73 records · Page 4Linked to original sources

PubMed1994

Rabies ribonucleocapsid as an oral immunogen and immunological enhancer.

The administration of rabies ribonucleocapsid (RNP) by oral as well as parenteral routes was found to prime specific T cells and elicit N-protein-specific antibodies. per os and intramuscular immunization led to the production of antibodies of the IgA and IgG isotypes, respectively. Mice primed orally with RNP produced significantly enhanced amounts of virus-neutralizing antibody, compared with non-immune controls, upon subsequent parenteral booster immunization with inactivated rabies virus. Thus oral immunization with rabies RNP primed cells capable of mediating a secondary systemic response to rabies virus. The results of experiments in which peptide and protein antigens were administered either physically coupled to or mixed with RNP indicate that RNP has an inherent capacity to enhance immune responses.

Adjuvants, Immunologic↗

PubMed1994

Glycosylation of synthetic T helper cell epitopic peptides influences their antigenic potency and conformation in a sugar location-specific manner.

The immunodominant T helper cell epitopes 31D and VF13N of rabies virus nucleoprotein and glycoprotein, respectively, correspond to peptide sequences AVYTRIMMNGGRLKR and VVEDEGCTNLSGF, and are expressed between amino acids 404-418 and 29-41, of the appropriate proteins. We investigated how internal or external glycosylation affects the biological activity and conformation of the peptides 31D and VF13N. Mid-chain incorporation of maltobiose or N-acetylglucosamine moieties into the asparagine residues greatly diminished the T-cell stimulatory activity in vitro (due to the diminished ability of the glycopeptides to bind to major histocompatibility complex determinants) and reduced the characteristic alpha-helicity of the peptides in aqueous trifluoroethanol solutions. In contrast, addition of maltobiose- or N-acetylglucosamine-coupled asparagines to the N-termini of peptides 31D and VF13N resulted in unchanged T-cell activity. Furthermore, N-terminal glycosylation of peptide 31D, as indicated by the functional assay, decreased the sensitivity of the peptide to degradation in human serum and did not affect the alpha-helical conformation. These data indicate that glycosylation of T-cell epitopes is not a preferable method for the preparation of antagonists, but incorporation of the sugars to appropriate positions may be advantageous in the design of T-cell agonists and peptide-based vaccines.

Amino Acid Sequence↗

PubMed1994

Comparative and optimized dabsyl-amino acid analysis of synthetic phosphopeptides and glycopeptides.

The optimal conditions for amino acid analysis of phosphopeptides and N-acetylglucosamine- and N-acetylgalactosamine-containing glycopeptides were investigated by dabsyl-Cl derivatization and reversed-phase high-performance liquid chromatographic separation. By comparing the chromatographic behaviour of the dabsylated phosphoamino acids and dabsylated aminosugars on three different columns, it appears that the mechanism of binding to the column is different for the two modified dabsyl derivatives. The acid sensitivities of sugar and phosphate groups were also investigated. We found that while the optimal hydrolysis conditions for phosphopeptide analysis are peptide sequence-dependent, there is generally an applicable condition for the highest recovery of glycopeptides. A 1-h gas-phase hydrolysis time seems to be appropriate for the majority of glycopeptides and 1.5 h is suitable for the majority of phosphopeptides. The analysis was extended to the successful verification of the presence of the N-acetylglucosamine and the N-acetylgalactosamine moieties when these sugars were incorporated as parts of a disaccharide side chain of glycopeptides.

Acetylgalactosamine↗

PubMed1994

An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants.

Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete increased amounts of long A beta, thereby fostering amyloid deposition.

Alzheimer Disease↗

PubMed1994

Identification of overlapping class I and class II H-2d-restricted T cell determinants of influenza virus N1 neuraminidase that require infectious virus for presentation.

The requirement for endogenous viral protein synthesis in helper and cytotoxic T cell (CTL) recognition of influenza virus was studied at the level of individual epitopes. The viral envelope glycoprotein neuraminidase (NA) contains class I and class II major histocompatibility complex (MHC)-restricted T cell determinants that are presented by virus-infected antigen-presenting cells (APC). We had previously shown that recognition of NA by class II I-Ed-restricted T cells required either active viral infection of APC or introduction of uv-inactivated virus to the cytosol, similar to the well-established requirements for class I-restricted responses. Detailed mapping of T cell epitopes was undertaken using vaccinia virus vectors encoding truncated segments of the influenza NA molecule and by synthetic peptides. Class I MHC-restricted CTL were found to recognize two regions of NA: residues 69-89 in the context of Dd and 191-201 presented by Kd. Analyses of T cell proliferation and T hybridoma clones revealed that class II-restricted responses recognized the same two regions as the CTL, presented by I-Ed and I-Ad, respectively. Interestingly, both class I and class II MHC-restricted T cells showed similar requirements for endogenously synthesized antigen, responding poorly or not at all to endocytosed uv-inactivated virus. This extends previous observations that specific epitopes can be preferentially presented by class II molecules from endogenously synthesized antigens and shows that the same antigenic determinants can have access to both class I and class II antigen presentation pathways.

Amino Acid Sequence↗

PubMed1994

Spectroscopic evidence that monoclonal antibodies recognize the dominant conformation of medium-sized synthetic peptides.

Spectroscopic methods have amply documented that small- and medium-sized peptides tend to assume unordered conformations in water. The conformational tendencies, however, manifest in halogenated alcohols, and the preferred secondary structures are apparent from the circular dichroism (CD) spectra. Here we report the results of immobilizing peptide and protein antigens from various mixtures of trifluoroethanol and water during enzyme-linked immunosorbent assays. The increased recognition by the appropriate monoclonal antibodies (mAbs) is correlated with the increase of the alpha helical, beta turn, or beta pleated sheet content of the peptides presented in the different solvent mixtures. Remarkably, the antibody binding can be detected at considerably lower antigen levels if the antigen is immobilized from trifluoroethanol. The antigens we used corresponded to fragments of normal human neurofilaments and tau protein found in the paired helical filaments of Alzheimer's disease, and the nucleoprotein of rabies virus. The conformation of myoglobin is as stable in water as in trifluoroethanol, and therefore acted as a negative control. Indeed, the recognition of myoglobin did not increase upon increasing the trifluoroethanol concentration in the solvent used to apply the antigen to the plate. The possibility of imperfect binding to the plastic carrier or nonspecific binding to irrelevant antibodies is excluded by using control experiments. We offer the first direct evidence that the mAbs recognize the secondary structure of epitopes, and that it is possible to correlate the binding conformation of the epitopes with CD measurements made in trifluoroethanol-water mixtures.

Animals↗

PubMed1994

Plaque-associated neuronal proteins: a recurrent motif in neuritic amyloid deposits throughout diverse cortical areas of the Alzheimer's disease brain.

Diffuse and neuritic plaques are sites of accumulation of beta-amyloid peptides (A beta) in the brains of Alzheimer's disease (AD) patients. Although amyloid fibrils are formed from A beta, the contribution of other plaque-associated proteins and peptides to the pathogenesis of AD amyloidosis is unknown. To pursue this issue, we sought to identify proteins and peptides that were consistently associated with neuritic plaques in six different cortical areas of AD and control brains. We accomplished this by using quantitative, single and double label immunohistochemistry and a panel of antibodies to proteins or peptides that are known to be associated with neuritic plaques in the AD hippocampus. Our data showed that the molecular composition of neuritic plaques in association, limbic, sensory, and motor cortex was similar regardless of the type of cortex in which they were found or the apolipoprotein E genotype of the patient. Further, proteins and peptides associated with neuritic plaques in the cortical areas of the AD brain studied here were similar to those found in neuritic plaques of the AD hippocampus. Specifically, in addition to A beta 1-40 and A beta 1-42, these plaques contained immunoreactivity for other domains in A beta precursor proteins, neurofilament and tau proteins, as well as phosphotyrosine residues. We conclude that the recurrent association of a distinct group of neuronal and other proteins and peptides with neuritic plaques suggests that these plaque-associated components play a mechanistic role in the pathogenesis of amyloidosis in AD.

Aged↗

PubMed1994

Glycosylation of a synthetic peptide representing a T-cell determinant of influenza virus hemagglutinin results in loss of recognition by CD4+ T-cell clones.

Synthetic glycopeptides were used to study possible mechanisms for the reduction observed in the response of influenza virus-specific CD4+ T-cells to strains of virus in which amino acid substitution in the hemagglutinin has led to attachment of a carbohydrate side chain. The peptide NCTLIDALLGDPH stimulates vigorous proliferation of hemagglutinin-specific T-cell clones F1-36 and F1-40 but addition of a heptasaccharide, which approaches the size of natural carbohydrate antennae, eliminated the stimulatory capacity of the peptide. This occurs even though the site of carbohydrate attachment at the N-terminal asparagine lies outside the T-cell determinants encompassed by this sequence. A glycopeptide with only two sugar units was stimulatory for F1-36 but not F1-40, suggesting that peptides with a carbohydrate side chain are able to bind to MHC molecules but that approach of the T-cell receptor of certain clones to the glycopeptide-MHC complex is hindered. Loss of T-cell recognition following attachment of a long carbohydrate side-chain to T-cell determinants is not a general finding because attachment of six carbohydrate units to the peptide, NKYVKQNTLKLA, had little or no effect on the stimulation of a T-cell clone specific for this sequence.

Amino Acid Sequence↗

PubMed1994

Neuritogenic Lewis rat T cells use Tcrb chains that include a new Tcrb-V8 family member.

The P2 protein obtained from Schwann cells induces a population of T cells which, upon adoptive transfer, causes the disease experimental allergic neuritis (EAN), an animal model for Guillain-Barre syndrome. In this report, a truncated peptide, FR22, derived from a previously reported neuritogenic T-cell determinant, was used to generate from Lewis rats T cells that were shown to cause EAN. Since our previous studies showed that Tcrb-V8 was used by a majority of T-cell hybridomas specific for the neuritogenic peptide P26, which contains the FR22 sequence, we sequenced the Tcrb-V8+ mRNA from FR22-specific T-cell lines, and compared the sequences obtained with those obtained from similarly generated myelin basic protein (MBP) 68-88-specific Lewis rat T-cell lines. We found that in the EAN lines, several members of the Tcrb-V8 family were used, including a new family member, Tcrb-V8E. This was more diverse than the MBP-68-88-specific response in which only a single Tcrb-V8 family member was used. Also, in the EAN lines, the beta chain sequences did not show the same conserved junctional regions seen in the MBP lines. Thus, T-cell receptor beta chain usage in the response to this dominant neuritogenic peptide appears to be less restricted than the response to the dominant encephalitogenic determinant of MBP both in V region usage and in CDR3 usage.

Amino Acid Sequence↗

PubMed1994

Tyrosine- versus serine-phosphorylation leads to conformational changes in a synthetic tau peptide.

One of the major immunodominant epitopes of the paired helical filaments (PHF) of Alzheimer's disease is the peptide sequence GAEIVYKSPVVSGD (T3), comprising amino acids 389-402 of the microtubule-associated protein, tau, when it is phosphorylated at the first serine residue. While the corresponding anti-PHF monoclonal antibody recognizes the peptide phosphorylated at either serine, it does not recognize the tyrosine-phosphorylated peptide. Here we describe the effect of serine- versus tyrosine-phosphorylation on the conformation of a synthetic tau peptide. While adding a phosphate to the serine residue has practically no impact on the structure of the non-phosphorylated peptide, phosphorylation of the tyrosine results in considerable conformational changes.

Alzheimer Disease↗

PubMed1993

Determination of amino sugars in synthetic glycopeptides during the conditions of amino acid analysis utilizing precolumn derivatization and high-performance liquid chromatographic analysis.

The growing number of synthetic glycopeptides required an in-house method for the analysis of the final products. A reversed-phase high-performance liquid chromatographic protocol was developed to verify the presence of N-acetylglucosamine and N-acetylgalactosamine in synthetic glycopeptides after hydrolysis and derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride. Different sugar and glycoamino acid standards were used to separate the two carbohydrate moieties, and the location of the derivatized 2-aminoglucose was established by the use of radiolabeled sugar. The utility of the approach is demonstrated by the amino acid analysis of N- and O-glycosylated synthetic peptides and the method could provide an alternative for sugar analysis of glycoproteins.

Amino Acid Sequence↗

PubMed1993

Comparative analysis of human and Dutch-type Alzheimer beta-amyloid peptides by infrared spectroscopy and circular dichroism.

The 42 amino acid beta A4 peptide is the major constituent of the senile plaques, one of the hallmark neuropathological lesions of Alzheimer's disease. While C-terminally truncated variants were shown to be present in normal body fluids, a single Glu-->Gln change in the 39 amino acid form of beta A4 results in accelerated fibril formation in the brains of patients with Dutch-type hereditary cerebral hemorrhage with amyloidosis. In this study we used Fourier-transform infrared and circular dichroism spectroscopies on synthetic peptides to demonstrate that this mutation results in altered secondary structure in membrane mimicking solvents, characterized by a considerably higher beta-structure content for the mutant peptide. Moreover, extreme high and low pH were less effective in eliminating the beta-conformation for the Dutch-variant than for the normal human sequence.

Alzheimer Disease↗

PubMed1993

Recognition of the minimal epitope of monoclonal antibody Tau-1 depends upon the presence of a phosphate group but not its location.

The major constituent of the paired helical filaments (PHFs) of Alzheimer's disease is the abnormally phosphorylated form of the microtubule-associated protein, tau. Monoclonal antibody (mAb) Tau-1 is used extensively to stain normal human tau, and tau isolated from the brains of Alzheimer's disease patients after dephosphorylation. We used a panel of 6 synthetic peptides to localize the minimal epitope of Tau-1 between amino acids 192-204. All 4 serine residues within this fragment were later phosphorylated individually by chemical methods, and it was shown that none of the peptides carrying a single phosphate group were recognized by the antibody. The serines included those that are probably not transformed in AD and consequently, conclusions drawn about malfunctioning kinase activity, based on Tau-1 immunoreactivity, can be extremely misleading. The recognition was restored at a decreased level when one of the serines was replaced by an alanine residue. mAb AT8 was made by immunizing with the PHFs and was reported to recognize the same region of the protein in a phosphorylated form. AT8 did not, however, cross-react with any of the singly phosphorylated peptides, indicating that the recognition site of the two antibodies are not entirely complementary or the binding to AT8 needs multiple phosphorylation of the antigen. The abolished recognition of the phosphorylated peptides cannot be attributed to a conformational change due to phosphorylation, since all peptides exhibited reverse-turn secondary structures, as indicated by circular dichroism (CD) spectroscopy. Anti-tau mAbs may distinguish between phosphorylated and non-phosphorylated forms of epitopes regardless of the location of the phosphate group.

Amino Acid Sequence↗

PubMed1993

Human and rodent Alzheimer beta-amyloid peptides acquire distinct conformations in membrane-mimicking solvents.

The major constituent of senile plaques (one of the hallmark lesions of Alzheimer's disease) is a 42(43)-amino-acid polypeptide, termed the A4 or beta-amyloid peptide. The beta-amyloid peptide or A4 is derived from one or more larger beta-amyloid precursor proteins. The precursor protein from whence the A4 peptide is derived is highly conserved throughout evolution, and humans, monkeys, dogs, and bears develop brain deposits of A4 peptide in amyloid fibrils. However, similar accumulations of A4 amyloid are negligible in the brains of rats and mice for reasons that remain unexplored. Notably, the A4 sequence of rodents, deduced from the cDNA clones, differs only in three amino acids from the A4 isolated from the brain of humans. Hence, these differences could account for the inability of rodents to develop Alzheimer-like A4 amyloid plaques. To test this hypothesis directly, using physical and chemical model systems, we synthesized, purified, and characterized A4 peptides corresponding to the human and rodent sequences. Circular dichroic and Fourier-transform infrared spectroscopy were used with various membrane-mimicking solvents, different peptide concentrations, and variable pH to identify those environmental conditions that promoted beta-pleated sheet formation of the human versus rodent A4. At an intermediate alkaline pH (< or = 10), the rodent peptide has more beta-pleated sheet structure than the human sequence. The beta-pleated sheets for both peptides could be eliminated at very high pH (> or = 12). The amount of the beta-structure increased in an octyl glucoside solution, compared to that found in SDS, as well as in several of the other solutions tested here. This suggests that particles originated from prior membrane damage may play a role in the stabilization of beta-pleated sheets with subsequent formation of amyloid deposits. Finally, we found that higher beta-pleated sheet content was observed for the rodent sequences in acetonitrile/water mixtures. In contrast, more beta-pleated sheets were detected with the human A4 in trifluoroethanol/water mixtures at neutral pH. Remarkably, at relatively low peptide concentrations, only the human sequences assumed an extended secondary structure. These data suggest that subtle inter-species amino-acid differences may account for the inability of the rodent peptide to form amyloid fibrils in situ.

Amino Acid Sequence↗

PubMed1993

Peptide stability in drug development. II. Effect of single amino acid substitution and glycosylation on peptide reactivity in human serum.

The determination of peptide stability in human serum (HS) or plasma constitutes a powerful screening assay for eliminating unstable peptides from further development. Herein we report on the stability in HS of several major histocompatibility complex (MHC)-binding peptides. Some of these peptides are in development for the novel treatment of selected autoimmune disorders such as rheumatoid arthritis and insulin-dependent diabetes. For most of the l-amino acid peptides studied, the predominant degradation mechanism is exopeptidase-catalyzed cleavage. Peptides that were protected by d-amino acids at both termini were found to be more stable than predicted, based on additivity of single substitutions. In addition, N-acetylglucosamine glycopeptides were significantly stabilized, even when the glycosylation site was several amino acids from the predominant site(s) of cleavage. This indicates that long-range stabilization is possible, and likely due to altered peptide conformation. Finally, the effect of single amino acid substitutions on peptide stability in HS was determined using a model set of poly-Ala peptides which were protected from exopeptidase cleavage, allowing the study of endopeptidase cleavage pathways.

Acetylglucosamine↗

PubMed1992

Fmoc-protected, glycosylated asparagines potentially useful as reagents in the solid-phase synthesis of N-glycopeptides.

1-Amino 1-deoxy derivatives of unprotected O-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyrano se, 2-acetamido-2-deoxy-D-galactose, D-galactose, lactose, D-fucose, D-mannose, and 2-deoxy-D-arabino-hexose were prepared and acylated with N-fluorenylmethoxycarbonylaspartic acid alpha-tert-butyl ester. The anomeric configuration of the N-glycosyl bond (including that of the mannose derivative) in each of the purified compounds was shown to be beta. The probable stability of the N-glycosyl and glycosidic bonds during the conditions of solid-phase peptide synthesis was investigated by treatment of the glycosylated asparagine derivatives with different concentrations of trifluoroacetic acid. Based on their stability, we found that Fmoc-Asn(sugar)-OH derivatives are excellent candidates for automated synthesis of biologically active glycopeptides.

Asparagine↗

PubMed1992

A serine-->proline change in the Alzheimer's disease-associated epitope Tau 2 results in altered secondary structure, but phosphorylation overcomes the conformational gap.

Monoclonal antibody Tau 2 was raised against bovine tau protein, was reported to recognize a conformational epitope, and stained tau was found in neurofibrillary tangles of Alzheimer's disease, but not normal human tau. We synthesized tetradeka peptides corresponding to the original bovine sequence, its serine-->proline substituted analog, the genuine human sequence of this region, and the bovine epitope phosphorylated on the crucial serine. The secondary structure of the peptides was determined by circular dichroism. It was found that only the original bovine epitope showed a tendency to form the beta-pleated sheets characteristic of the neurofibrillary tangles. The spectra of the human peptide, its analog, and the phosphorylated bovine sequence were very similar, featuring a weak, helical beta-turn character. Eventual phosphorylation of epitopes of this otherwise heavily phosphorylated protein may overcome inter-species conformational gaps.

Amino Acid Sequence↗

PubMed1992

Immunological and conformation characterization of a phosphorylated immunodominant epitope on the paired helical filaments found in Alzheimer's disease.

The immunological recognition pattern of one of the most commonly used monoclonal antibodies, PHF-1, which detects the paired helical filaments of Alzheimer's disease, exhibits a high degree of similarity with the recognition of a polyclonal antibody, anti-T3P, raised against a synthetic phosphopeptide, GAEIVYKS(Phospho)PVVSGD, corresponding to amino acids 389-402 of the microtubule-associated protein tau. A panel of 16 synthetic non-phosphorylated and phosphorylated peptides, excised from different regions of tau and peptide analogs thereof, were used to show that PHF-1 is indeed directed against the T3 fragment. Circular dichroism spectroscopy shows that the phosphorylated peptide exhibits a limited propensity to form intramolecular beta-pleated sheets, and alteration is found in the reverse-turn structure that dominates the middle section of the molecule. The shift in the turn-forming amino acids may also allow a stacking procedure, may interfere with microtubule assembly, and, consequently, may be accountable for deposit formation.

Alzheimer Disease↗