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R E Humphreys

Publications and source records attributed to R E Humphreys.

At least 37 records · Page 2Linked to original sources
PubMed1993

Characterization of the invariant chain C-terminus (Glu183-Glu193) epitope which is obscured in processed Ii, MHC alpha,beta trimers.

The E1 serum was developed against invariant chain peptide Ii (183-193) in order to study the function of the Ii protein which associates with class II MHC alpha,beta chains from time of synthesis until cleavage and release, possibly regulating the binding of antigenic peptides. Subpopulations of Ii, Ii(VIC) and Ii(E1), respectively, were demonstrated by sequential immunodepletions and immunoprecipitations with: (1) VIC-Y1 monoclonal antibody to an N-terminal epitope of Ii, and (2) E1 rabbit antiserum to Ii(183-193). In 3 hr radiolabeled cells, VIC-Y1 recognized Ii, Ii and N- and O-linked glycosylation (IpN, IpO), p41 and co-precipitated class II alpha,beta chains, while E1 recognized Ii, IpN and immature Ii-alpha complex. In 15 min radiolabeled cells, each antibody recognized similar, immature Ii forms without alpha,beta. Urea denaturation of Ii(VIC) rendered the main Ii species but not IpO immunoprecipitable with E1. E1 recognized O-glycanase-treated Ii (VIC). We conclude that the Ii(183-193) epitope was obscured by interactions of Ii with class II alpha,beta chains and by the O-linked glycosylation of Thr187, which may in part regulate association of Ii to class II alpha and beta chains.

Antibody Specificity↗
PubMed1993

Comparison of actual and random-positioning-model distributions of peptide scavenging and T cell-presented sites in antigenic proteins.

In a peptide with a T cell-presented epitope (T site), a folded structure with a hydrophobic surface, 'the scavenger (S) site', may regulate transfer to major histocompatibility complex class II molecules. Three procedures which were proposed to identify T sites selected for amphipathic helical patterns but not T sites. In testing whether S sites lay in or near T sites, we found their linkage was not greater than that generated by a model in which segments of equal length and number to the S and T sites for each protein were distributed at random. This study establishes criteria for evaluation of schemes to predict functional motifs in antigenic proteins.

Amino Acid Sequence↗
PubMed1993

Adsorption and helical coiling of amphipathic peptides on lipid vesicles leads to negligible protection from cathepsin B or cathepsin D.

The processing of antigenic peptides for presentation by MHC molecules to T cells, may depend upon the function of a second, consensus sequence in or near the T cell-presented epitope. One such processing-regulating sequence appears to be composed of amino acids Leu, Ile, Val, Phe, and Met recurring in a fashion to form a longitudinal, hydrophobic strip when the excised peptide is coiled as an alpha-helix. Such a hydrophobic strip-of-helix may: (a) scavenge peptides from lumens onto lipid membranes of digestion vesicles, (b) stabilize peptides there as protease-resistant helices, (c) specify recognition by the antigenic peptide-binding sites of chaperonin proteins, transmembranal transporters, or MHC molecules. By circular dichroism and electron paramagnetic resonance, we demonstrated that peptides with recurrent hydrophobic residues potentially forming longitudinal strips adsorbed to, and partially coiled as helices on, di-O-hexadecyl, D-L-alpha-phosphatidylcholine (DHPC) vesicles. Cathepsin B or cathepsin D cleavages of three such peptides were identified. With either enzyme, it made no significant difference whether a peptide substrate was in solution or bound to vesicles in terms of efficiency and specificity of peptide bond cleavages. We conclude that protease resistance, per se, of membrane-adsorbed, helically coiled peptides is not a major factor in the selection for T cell presentation of epitopes in peptides which have a motif with a longitudinal hydrophobic strip.

Adsorption↗
PubMed1992

Critical functional role of the COOH-terminal ends of longitudinal hydrophobic strips in alpha-helices of T4 lysozyme.

The sensitivity of bacteriophage T4 lysozyme function to amino acid substitutions at defined positions in and around the longitudinal, hydrophobic strips of 9 alpha-helices was assessed after systematic replacement of each residue in the protein with a series of 13 amino acids. The hydrophobic strips were defined by identifying the longitudinal sectors in the helices with the highest mean residue hydrophobicities. Sensitivity to mutation (the percentage of replacements leading to loss of function) was calculated for each residue in the following positions: whole protein, helices, hydrophobic strips, other positions within the helices, and various positions within the hydrophobic strips as well as their extensions beyond the helices. Substitutions at positions in the hydrophobic strips led more frequently to loss of function than substitutions in the protein as a whole. One subset, the COOH-terminal hydrophobic strip residues, is apparently critical; substitutions of these residues (but not of their NH2-terminal counterparts) led at least as frequently to loss of function as substitutions of solvent-inaccessible residues, and nearly as frequently as substitutions of the most highly conserved residues.

Algorithms↗
PubMed1992

Residues in the longitudinal, hydrophobic strip-of-helix relate to terminations and crossings of alpha-helices.

An alpha-helix terminates when the virtual extension of its most hydrophobic, longitudinal strip containing Leu, Ile, Val, Phe, and Met lacks those residues. In each of 247 helices a template was fitted to maximize the mean hydrophobicity of positions forming a longitudinal strip-of-helix. The template was then extended into sequences beyond the ends of the helices. Leu, Ile, Val, Phe, and Met occurred in positions in the longitudinal strip-of-helix at an increased frequency (p less than 0.001), but in the first and second positions beyond either end of each true helix, they occurred at the same frequency as for their empirical distribution over all the proteins. Excesses of Asp and Glu were found in the N-terminal loop, and of Arg, His, and Lys in specific positions about the C terminus of helices. The longitudinal hydrophobic strip, the smallest amino acid in that strip, and charged amino acids in that strip, related to rotational and longitudinal orientation of alpha-helices in 15 proteins. Adjacent helices generally crossed through their longitudinal hydrophobic strips. They usually crossed through the smallest residue in the strip. Charged residues, when they occurred in the strips, were excluded from the crossing regions.

Amino Acid Sequence↗
PubMed1992

Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies.

A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.

Amino Acid Sequence↗
PubMed1992

Phosphorylation of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA.

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product ZEBRA is a first step in the cascade of the virus-productive cycle. ZEBRA protein was detected by immunoblotting as a single band at 38 kDa in Akata cells after crosslinkage of membrane immunoglobulin G (IgG) with anti-IgG antibody. Immunoprecipitation of [32P]phosphate-labeled, anti-IgG-stimulated Akata cells with anti-ZEBRA antibody showed that ZEBRA was phosphorylated. Phosphoamino acid analysis demonstrated phosphorylation of serine, but not threonine or tyrosine, and tryptic-peptide mapping showed multiple phosphorylated peptides of ZEBRA. Treatment with 8-bromo cAMP and blockage of phosphodiesterase by theophylline in anti-IgG-stimulated cells increased the phosphorylation of three ZEBRA peptides. Incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced the phosphorylation of these three ZEBRA peptides, while treatment with staurosporine, a protein kinase C (PKC) inhibitor, enhanced their phosphorylations. These data suggest that activation of PKC with TPA induces the ZEBRA dephosphorylation and that activation of cAMP-dependent protein kinase A enhances the ZEBRA phosphorylation at the specific sites.

Cell Line↗
PubMed1992

Biophysical mechanism of the scavenger site near T cell-presented epitopes.

We seek to identify consensus sequences in digested fragments of antigenic proteins regulating selection and major histocompatibility complex (MHC)-restricted presentation to T cells of epitopes within those fragments. One such pattern, of recurrent, hydrophobic sidechains forming a longitudinal hydrophobic strip when a sequence is coiled as an alpha-helix, is found in or near most T cell-presented epitopes. Such recurrent hydrophobicity may lead to protease-protected coiling of the fragment against endosomal membranes and transfer to MHC molecules. This concept leads to better identification of T cell-presented sequences and possible to engineering of T cell-presented vaccines to affect their potency and MHC restriction.

Amino Acid Sequence↗
PubMed1992

Hypothesis for the control of clotting factor VIII inhibitory antibodies by decreasing potency of helper T-cell-recognized epitopes in factor VIII.

The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype-anti-idiotype networks regulating FVIII inhibitors, we developed rabbit anti-idiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide, Ser1687-Thr1695, characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor-recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti-idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip-of-helix which is in or near many T-cell-presented epitopes.

Animals↗
PubMed1991

Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells.

Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells.

Antibodies, Monoclonal↗
PubMed1991

Effect of genistein, a tyrosine kinase inhibitor, on latent EBV activation induced by cross-linkage of membrane IgG in Akata B cells.

The activation of phosphoprotein tyrosine kinases was studied in the regulation of EBV activation in Akata cells after cross-linking membrane IgG with anti-IgG. Protein tyrosine phosphorylation was induced in Akata cells after stimulation with anti-IgG, as determined by immunoblotting with the PY20 anti-phosphotyrosine mAb. The frequency of phosphotyrosine-activated cells was also measured by immunofluorescence with the PY20 antibody. Genistein, an inhibitor of tyrosine kinases, at non-cytotoxic doses blocked EBV activation, as measured in the induction of EBV Ag, EBV immediate early BZLF1 mRNA, and its protein product ZEBRA. Such inhibitions were reversed upon removing genistein from the cultures. Genistein inhibition of early Ag induction depended upon the time of addition of genistein after stimulation with anti-IgG. These findings indicate that activation of tyrosine kinase is required for EBV activation after cross-linking membrane IgG in Akata cells.

Antibodies, Anti-Idiotypic↗
PubMed1991

Number and placement of hydrophobic residues in a longitudinal strip governs helix formation of peptides in the presence of lipid vesicles.

alpha-Helix formation of a peptidyl sequence is stabilized by hydrophobic residues recurring at positions which create a longitudinal hydrophobic strip upon folding of the sequence as a helix against a hydrophobic surface. To test that hypothesis, we measured by circular dichroism the helical coiling on lipid vesicles of nine analogs of a prototypic helix peptide PH-1.0, Leu-Tyr-Gln-Glu-Leu-Gln-Lys-Leu-Thr-Gln-Thr-Leu-Lys. In these analogs, Thr was substituted for 1 or 2 Leu residues in the longitudinal hydrophobic strip Leu1...Leu5..Leu8...Leu12 which forms in the alpha-helical configuration. We found that coiling of analogs of Leu-Tyr-Gln-Glu-Leu-Gln-Lys-Leu-Tyr-Gln-Thr-Leu-Lys on lipid vesicles depends upon the strength and structure of its longitudinal hydrophobic strip.

Amino Acid Sequence↗
PubMed1991

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains.

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1991

Highly restricted distributions of hydrophobic and charged amino acids in longitudinal quadrants of alpha-helices.

Helix formation in folding proteins is stabilized by binding of recurrent hydrophobic side chains in one longitudinal quadrant against the locally most hydrophobic region of the protein. To test this hypothesis, we fitted sequences of 247 alpha-helices of 55 proteins to the circular (infinite) template (symbol; see text) to maximize the strip-of-helix hydrophobicity index (the mean hydrophobicity of residues in (symbol; see text) positions). These template-predicted configurations closely matched crystallographic structures in 87% of four- or five-turn helices compared. We determined the longitudinal quadrant distributions of amino acids in the template-fitted, sheet projections of alpha-helices with respect to the best longitudinal, hydrophobic strip on each helix and to the N and C termini, interiors, and entire helices. Amino acids Leu, Ile, Val, and Phe were concentrated in one longitudinal quadrant (p less than 0.001). Lys, Arg, Asp, and Glu were not in the quadrant of Leu, Ile, Val, and Phe (p less than 0.001). Significant quadrant distributions for other amino acids and for termini of the helices were also found.

Amino Acids↗
PubMed1991

Relating homology between the Epstein-Barr virus BOLF1 molecule and HLA-DQw8 beta chain to recent onset type 1 (insulin-dependent) diabetes mellitus.

A role for the Epstein-Barr virus in initiating Type 1 (insulin-dependent) diabetes mellitus has been proposed since Epstein-Barr virus BOLF1 (497-513) AVTPL RIFIVPPAAEY has an 11 amino acid identity with HLA-DQw8 beta (49-60) AVTPL GPPAAEY. Rabbit antisera to the BOLF1 (496-515) peptide crossreacted with the homologous DQw8 beta (44-63) peptide but not with the related DQw7 beta (44-63) peptide, which differed from the DQw8 peptide only in an ALA to ASP substitution in position 57. Antisera to DQw8 beta (49-60) reacted with the DQw8 beta (44-63) peptide and BOLF1 (496-515), but not with DQw7 beta (44-63). The antiserum to the BOLF1 peptide bound to denatured class II major histocompatibility complex beta chains from Epstein-Barr virus-transformed DQw8-positive lymphocytes in an immunoblotting analysis. Epstein-Barr virus antibodies were detected at equal frequencies and similar titres in sera of 30 patients with Type 1 diabetes (16 of 30; 63%) and in sera of 20 non-diabetic control subjects (13 of 20; 65%). Sera from diabetic patients did not bind to DQw8 beta (44-63) or BOLF1 (496-515) peptides. From these data we conclude that there is no simple relationship between serological evidence of Epstein-Barr virus infection and crossreactions between homologous Epstein-Barr virus and class II major histocompatibility complex peptides.

Adolescent↗
PubMed1991

Early events in Epstein-Barr virus genome expression after activation: regulation by second messengers of B cell activation.

RNA transcription from the BamHI Z and BamHI R and HindIII G regions of the Epstein-Barr virus (EBV) genome was studied after treatment of Akata cells with anti-immunoglobulin G (IgG), with second messenger agonists or antagonists to determine how latent EBV activation is regulated by B cell second messengers. Northern gel analysis demonstrated that BZLF1, BZLF1 + BRLF1, and BMLF1 + BSLF2 transcripts were induced at 2 hr and increased in concentration at 4 hr after induction with anti-IgG; transcripts from BRRF1, BaRF1, BMLF1, and BMRF1 were initiated at 4 hr; a transcript from BRRF2 appeared at 6 hr. The patterns of transcription from these genes after repeated stimulations with calcium ionophore A23187 + dioctanoylglycerol paralleled those with anti-IgG except that times of initiation were delayed by about 2 hr. Nuclear run-off assay of BZLF1 gene showed rapid increases in their transcriptions from 30 to 60 min after anti-IgG treatment. The protein kinase C antagonist, staurosporine, completely blocked the appearance of these transcripts, while 8-bromo cAMP + theophylline suppressed the transcription by about 40%. The regulation of EBV activation in Akata cells with anti-IgG or with second messenger agonists or antagonists can be explained by regulation at the level of transcription of immediate-early genes of EBV.

Alkaloids↗
PubMed1991

Prediction of alpha helices and T cell-presented sequences in proteins with algorithms based on strip-of-helix hydrophobicity index.

Recurrent aliphatic hydrophobic amino acids which occur in the sequence of a protein or a peptide at positions which form an axial, hydrophobic strip when the sequence is coiled as an alpha helix might stabilize coiling against hydrophobic surfaces. That effect can lead to helix formation against hydrophobic cores of nascent proteins or excised T cell-presented peptides and to protease protection and scavenging for presentation by MHC molecules. Such consensus sequences of recurrent hydrophobicity creating a scavenger "S" site might overlap to varying degrees the T cell-presented "T" epitope which actually sits in the antigen-binding site of a MHC molecules, as long as a cleavage "C" site does not fall between them when they are relatively separated. Cooperatively among the residues in an axial, hydrophobic strip to stabilize helix formation is reflected in the SOHHI, which is the mean hydrophobicity of residues in such potential strips. Algorithms based on the SOHHI, with additional considerations related to length and caps, lead to sensitive and efficient predictions of structural helices and of T cell-presented epitopes. In experimental tests of these ideas, the SOHHI was found to correlate to helical coiling of amphiphilic peptides in the presence of lipid vesicles. These principles lead to hypotheses to alter the potency and range of MHC restriction of peptide vaccines or to decrease the immunogenicity of therapeutic proteins.

Algorithms↗