Biomedical subjects
Publications and source records attributed to R E Humphreys.
The regular recurrence of hydrophobic amino acid residues along a peptide sequence determines the formation of a longitudinal hydrophobic strip when the peptide forms an alpha-helix. An understanding of the ways this may affect both folding of nascent proteins and antigen presentation should facilitate vaccine and therapeutics design.
The effect of human serum with or without Epstein-Barr virus (EBV) antibodies was characterized on virus production in P3HR-1 cells. Cell culturing with EBV-seropositive sera reduced both production of infectious virus and amounts of virion DNA in the supernatants. EBV DNA was also reduced in the cells. Such reductions in cell-associated EBV DNA depended upon the concentration of seropositive serum and incubation time. Decreased frequencies of productive EBV DNA-replicating cells were observed in cell populations which had reduced levels of cell-associated EBV DNA. The inhibitory effect of seropositive serum was reversed upon switching the cells to medium with seronegative serum. In serial sera of an acute infectious mononucleosis patient the EBV DNA-reducing activity arose in parallel to antibodies against EBV membrane antigen and nuclear antigen. Possible mechanisms were discussed for antibody-mediated inhibition of EBV production.
We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.
Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.
Two proteins, p70 and p80, were found in chemically crosslinked complexes with class II MHC molecules and Ii after 3-12 hr labelings with [35S]methionine. Two-dimensional, nonreduced/reduced SDS gel electrophoresis of immunoprecipitated complexes revealed 1) endogenous disulfide linkages between Ii-Ii and Ii-p70 and 2) chemically crosslinked, nearest neighbors of alpha-beta, alpha-Ii, Ii-p70, and alpha-p80. Although such nearest neighbors within multimeric complexes were identified as dimers in nonreduced/reduced 2D gels, stoichiometries could not be determined in the high molecular weight complex(es), which included alpha, beta, Ii, p70, and p80, and were not separated in the first dimension. p80 was not the chondroitin-sulfate form of Ii (Ii-CS) because it was not electrophoretically heterogeneous and was not sensitive to chondroitinase ABC. p70 was not hsp72/74 detected with C92 or N27 mAbs, and p80 was not BiP detected with its respective mAb. While only these two proteins associated prominently with class II MHC antigens and Ii late after synthesis, their functions are unknown.
A comparison of three methods to predict T cell-presented sequences within antigenic proteins led to the view that recurrent hydrophobic residues might nucleate excised peptides as alpha-helices against hydrophobic surfaces. Such helices could be protease-protected structures on their way to desetope binding. The compared methods were: the amphipathicity algorithm of DeLisi and Berzofsky [Proc. natn. Acad. Sci. U.S.A. 82, 7048-7052. (1985)] as modified by Margalit et al. [J. Immun. 138, 2213-2229. (1987)] the strip of-helix hydrophobicity algorithm (SOHHA) of Stille et al. [Molec. Immun. 24, 1021-1027. (1987)] and the motifs algorithm of Rothbard and Taylor [EMBO J. 7, 93-100. (1988)]. Correct prediction was defined at two levels of stringency: (1) the predicted sequence overlapped the experimentally reported sequence when the ratio of the intersection of both to the union of both greater than or equal to 0.5 or (2) the sequences touched when there was a non-empty intersection of both sequences. We determined the sensitivity (correct predictions/number of reported T cell-presented sequences) and efficiency (correct predictions/number of predictions) at each level of stringency. In terms of overlap, the SOHHA was more sensitive (0.43) than the amphipathicity (0.29) (not significant) and motifs (0.0, 0.0) (p less than 0.05) predictions and more efficient (0.35) than the amphipathicity (0.14) and motifs (0.0, 0.0) predictions. At the less stringent criterion touching, the amphipathicity method (0.71) was as sensitive as motif Rothbard-4 (0.79) and more sensitive than SOHHA (0.57) and motif Rothbard-5 (0.43). At that criterion, the SOHHA was more efficient (0.47) than the amphipathicity (0.36) and motifs (0.25, 0.40) methods. We hypothesize that the comparability of these approaches reflected the common, predominant influence of recurrent hydrophobicity in their predictions.
We synthesized three peptides, MA1 - Thr19-Val28(+Tyr) -, MA2 - Ser807-Ala816-, and MA3-Ser718-Glu729(+Tyr) from the sequence of Epstein-Barr virus gp350/220 and immunized rabbits with these peptides. Rabbit antisera to the peptides had antipeptide radioimmunoassay titers of 1:400 for anti-MA1, 1:200 for anti-MA2, and 1:1600 for anti-MA3. The anti-MA1 serum recognized gp350/220 in Western blotting to SDS-electrophoresed proteins from 12-O-tetradecanoylphorbol-13-acetate- and n-butyrate-treated B95-8 cells, but anti-MA2 and MA3 sera did not. None of the sera reacted with gp350/220 by membrane or cytoplasmic immunofluorescence or by immunoprecipitation of Triton X-100 solubilized proteins.
To test whether anti-idiotypic immunoregulation of factor VIII(FVIII)-inhibiting antibodies could be feasible in hemophiliacs, we assayed the minimal number and range of immunogenic, functional FVIII epitopes to which a series of murine anti-FVIII monoclonal antibodies (MAb) were directed. Rabbit anti-idiotypic sera to 7 MAb were prepared and used in competitive immunoradiometric assays to test Id similarities among 36 murine MAb. At least 12 immunogenic and functional epitopes were found on the FVIII molecule. The large range of target loci for FVIII inhibitors will make their immunoregulation by anti-idiotypic methods difficult.
The strip-of-helix hydrophobicity algorithm was devised to identify protein sequences which, when coiled as alpha or 3(10) helices, had one axial, hydrophobic strip and otherwise variably hydrophilic residues. The strip-of-helix hydrophobicity algorithm also ranked such sequences according to an index, the mean hydrophobicity of amino acids in the axial strip. This algorithm well predicted T cell-presented fragments of antigenic proteins. A derivative of this algorithm (the structural helices algorithm (SHA] was tested for the prediction of helices in crystallographically defined proteins. For the SHA, eight amino acid sequences, 2 cycles plus one amino acid in an alpha helix, with strip-of-helix hydrophobicity indices greater than 2.5, were selected with overlapping segments joined. These selections were terminated according to simple "capping rules," which took into account the roles of N-terminal Asn or Pro and C-terminal Gly in the stability of helices. In analyses of 35 crystallographically defined proteins with known alpha and 3(10) helices, the predictions with the SHA overlapped (had overlap indices x greater than or equal to 0.5) with 34% of known helices, touched (had overlap indices 0.5 greater than x greater than 0) or overlapped with 66% of known helices, or were neighboring (came within 6 residues) or touched or overlapped with 82% of known helices. At each level of judging the quality of prediction, the SHA was usually less sensitive (correct predictions/total number of known helices) and more efficient (correct predictions/total number of predictions) than the Chou-Fasman and Garnier-Robson methods. It was simpler in design and calculation. The chemical mechanisms underlying these algorithms appear to apply both to protein folding and to selection of T cell-presented antigenic sequences.
To determine how changing forms of class II major histocompatibility complex proteins and associated Ii molecules in intracellular compartments of human B lymphocytes might regulate or catalyze antigen processing or presentation, we analyzed immunoprecipitates of such molecules from subcellular fractions of [35S]methionine pulse-chase-labeled, 3-day-activated B lymphocytes after homogenization and distribution in Percoll density gradients. Two-dimensional gel electrophoresis of immunoprecipitates of subcellular fractions demonstrated: 1) progressive sialic acid addition to class II major histocompatibility complex beta chains and Ii but not to gamma 2, gamma 2', gamma 3, gamma 3' (p35), or p41 and its satellites; 2) association of p35 and p41 with class II complexes at 30-60 min after pulse labeling; 3) cleavage of an immature form of Ii without sialic acid at 15-30 min after pulse labeling to a COOH-terminal, 25,000-dalton fragment, p25, with a 60-90-min half-life; 4) the presence of Ii-related p29 at only 30-min chase times; 5) an effect of chloroquine or monensin, at maximal nontoxic doses, to increase (a) the time for associations of p35 and p41 with class II complexes and (b) the half-life of p25, which was then formed from Ii at reduced levels. In addition, while the half-lives of class II alpha and beta chains and Ii were comparable within intracellular fractions of any one density, in intracellular fractions of intermediate densities the complexes appeared to be longer lived (much greater than 6 h) than in lighter fractions (2-3-h half-lives).
Antibodies to either Ii or class II major histocompatibility complex (MHC) antigens did not recognize cell surface forms of Ii in immunoprecipitates of cells that had been radioiodinated by the lactoperoxidase method, whereas they bound [35S]methionine metabolically labeled molecules. N-hydroxysuccinimidobiotin (NHS-B) and biotin hydrazide (B-H) were used to react more generally with cell surface proteins via amino groups and nitrene coupling, respectively. Each of these latter compounds labeled alpha and beta chains of class II MHC antigens as seen in Western-blotted, electrophoresed immunoprecipitates probed with 125I-labeled streptavidin but not Ii or its associated forms. Although tyrosine residues might have been inaccessible to radioiodination in carbohydrate-derivatized forms of Ii, the lack of Ii biotinylation in these controlled, sensitive studies was consistent with the view that Ii forms were not surface expressed, with the possible exception of the chondroitin sulfate-derivatized forms of Ii (Ii-CS).
The cleavage of a high-mannose form of Ii to p25 was demonstrated in an intracellular compartment of B cells. Subcellular fractions of 72 hr-activated B cells, separated by Percoll density gradient centrifugation, were immunoprecipitated with anti-class II or anti-Ii serum and characterized for 5'-nucleotidase, acid phosphatase, and radiolabeled transferrin. The cleavage of p25 from Ii as a C-terminal fragment occurred from 20 to 60 min after synthesis in an intracellular compartment which was intermediate in density between lysosomal and plasma membrane fractions and coincided with the lighter to two internalized transferrin compartments. Chloroquine or monensin treatments, at maximal nontoxic doses, which block Golgi and lysosomal functions, did not seem to alter the cleavage of Ii to p25. p25 molecules were reduced to about 10,500 daltons by treatment with endoglycosidases F or H. We conclude that p25 was generated from a high mannose form of Ii in the endoplasmic reticulum or cis-Golgi. This finding could either implicate that site for class II MHC desetope charging with foreign peptides or reflect a mechanism for degradation of "excess" Ii molecules.
Antibodies against coagulation factor VIII were detected in a 33-year-old who developed severe bleeding 2 months postpartum. After treatment with plasmapheresis and immunosuppressive agents, the inhibitor was no longer detectable, but anti-idiotypic antibodies were detected, as demonstrated by binding in immunoradiometric assays, and by in vitro neutralization of the initial factor VIII autoantibodies. The anti-idiotypic titer subsequently declined but was still detectable 1 year later. Three commercial immunoglobulin preparations and pooled multiparous IgG also bound to (but did not neutralize) the patient's factor VIII antibody. These studies suggest that anti-idiotypic antibodies, arising in the face of immunosuppressive therapy, might suppress autoimmunity to factor VIII and confirm the presence of anti-idiotypic antibodies in pooled normal immunoglobulins.
Intracellular cleavage of Ii was evaluated in immunoprecipitates of radiolabeled Raji cells treated with protease inhibitors (leupeptin, antipain, chymostatin, and pepstatin) or blockers of endosomal function (chloroquine and monensin). Immunoprecipitates with anti-class II and anti-Ii(12-28) sera and VIC-Y1 MoAb revealed Ii cleavage products of 21,000 and 10,000 daltons (p21 and p10) only in leupeptin- and antipain-treated cells. Both p21 and p10 were judged to be N-terminal products because they were recognized with anti-Ii(12-28) and not with anti-Ii(183-193) or anti-Ii(192-211) sera. p10 might be derived from p21 because its intensity was increased in inverse proportion to p21 as a function of leupeptin or antipain concentration. p21, but not p10, was recognized by anti-class II antibody and thus might originate from class II-associated Ii. In pulse-chase studies, p21 and p10 appeared at 2 hr and later after Ii synthesis. p25, an Ii C-terminal fragment, was about 60% reduced by leupeptin or antipain. Intracellular proteolytic cleavage of class II-associated Ii appeared to follow two pathways leading either to N-terminal p21 and p10 or to C-terminal p25. Such cleavages might regulate or catalyze foreign antigen binding to class II.
We have defined the intracellular expression and localization of gp350/220, one of the Epstein-Barr virus (EBV) induced membrane antigens, on 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and n-butyrate-treated P3HR-1 cells. 1B6 monoclonal antibody (mAb) immunoprecipitated gp350/220 from [35S]-methionine-labeled cells, as confirmed with other mAbs (2L10, 72A1, and C1), to the same membrane antigen. The appearance of gp350/220 was observed about 14 h after TPA and n-butyrate activation and reached a maximal level at about 48 h. 1B6 mAb membrane immunofluorescence-positive and cytoplasmic fluorescence-positive cells appeared progressively in cell populations at the same frequencies. Cytoplasmic immunofluorescent staining with 1B6 mAb demonstrated a paranuclear complex which was identical to a rhodamine-labeled wheat germ agglutinin-stained pattern which has been ascribed to the Golgi apparatus. We investigated the effect of monensin on gp350/220 expression and processing. Monensin at 10(-7) M significantly inhibited membrane antigen expression in the Golgi apparatus and on the cell surface, but had a negligible effect on synthesis of viral capsid antigen, early antigen, and viral DNA. The inhibition of gp350/220 with monensin was further characterized by the immunoprecipitation of gp350/220 with anti-MA-positive human sera and mAbs. Monensin treatment resulted in the accumulation of a 165-kD molecule which was judged to be a precursor of gp350/220. These results were consistent with the view that the Golgi apparatus plays an important role as a place of synthesis, processing, and maturation of gp350/220.
The 25,000-Da protein that is seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii was shown to be a C-terminal fragment of Ii. [35S]Methionine pulse-chase labeling of polyclonally activated B lymphocytes or lymphoblastoid cell lines demonstrated maximal appearance of p25 in Percoll-separated endosomal fractions at 20- to 40-min chase times (studies in progress). This finding was consistent with the view that proteolysis of Ii to p25 and its release might catalyze the binding of digested foreign peptides to class II molecules and/or govern release of such charged complexes to traffic to the cell surface. We examined the structural relationship of p25 to Ii and the basis for cleavage of a relatively restricted site just external to the transmembranal segment. [35S]Methionine-labeled Ii and associated molecules were immunoprecipitated with a mAb to native Ii and then denatured, resolubilized, and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-terminal peptides of Ii (183 to 193 and 192 to 211), but not antibodies to an N-terminal peptide (12 to 28), did immunoprecipitate p25. The three antibodies to C-terminal and N-terminal peptides all immunoprecipitated denatured Ii proteins. The mAb to Ii immunoprecipitated [35S]methionine-labeled p25 but not [35S]cysteine-labeled p25. This finding was consistent with loss of a portion of Ii containing the only cysteine in Ii, Cys28. Digestion of class II MHC Ag-Ii complexes with various proteases yielded proteins migrating at and near p25 in two-dimensional electrophoretic gels. Upon increasing the duration of protease digestion, the 25,000-Da fragments were relatively resistant to further digestion. This observation was consistent with the presence of secondary structures (domains) leaving a relatively protease-sensitive (Ig hinge-like) region in Ii near its insertion into the membrane.
Hairy leukemic cells had altered expressions of accessory proteins (Ii, Ip, p70, p25) which associated intracellularly with class II MHC molecules. Immunoprecipitates from [35S]methionine, pulse-chase labeled leukemic cells, B-lymphoblastoid cells, and Burkitt's lymphoma cells were examined after 2-dimensional, polyacrylamide gel electrophoresis. The class II MHC-associated Ii chain was present in all three cell types, but the Ii-derived p25 protein was not found in HCL cells. Ip, the sialic acid-derivatized form of Ii, was well expressed in BLCL and Raji cells, but weakly in HCL cells. P70 was strongly expressed in BLCL and Raji cells, but negligibly in HCL cells. These alterations might reflect deficient mechanisms for antigen processing and presentation by these leukemic cells.
A strip-of-helix hydrophobicity algorithm to predict class II MHC-restricted peptides, on the basis of their structural similarity to an amphipathic, alpha-helix in Ii, also predicted peptides which were presented to cytotoxic T-cells by class I MHC molecules. This algorithm ranked peptides according to mean Kyte-Doolittle hydrophobicity values of amino acids at positions n, n + 4, n + 7, n + 11, n + 14 and n + 18 in a sequence which when coiled as a putative alpha-helix, had the indicated residues in an axial strip along one side of the helix. Sequences selected for highly scoring, hydrophobic strips were required to have at least 1 of the 4 adjacent strips scoring more negatively than -1 in the strip-of-helix hydrophobicity index and the entire sequence could contain no prolines. This algorithm predicted the class I MHC-restricted, T-cell-presented peptides in sequences of 4 proteins from which some class I MHC-restricted, T-cell-presented sequences had been experimentally determined. Since both class I and class II MHC-restricted peptides could be identified with this algorithm, one can propose that: (1) foreign peptide-binding sites (desetopes) of the class I and class II MHC molecules are structurally similar; and (2) any one T-cell-presented peptide can be presented by some specific allele of both a class I and a class II MHC antigen.