Immunochemical localization and amino acid sequences of crossreactive epitopes within the group A streptococcal M6 protein.

mAbs 10A11, 10B6, and 10F5, raised against the native group A streptococcal M6 protein, were examined for their crossreactivity with non-laboratory passaged clinical isolates, representing 58 M serotypes, by bacterial dot blot immunoassay. mAb 10A11 crossreacted with 9, mAb 10B6 with 30, and mAb 10F5 with 30 different non-M6 serotypes. To identify the epitopes for these antibodies, the native M6 protein was cleaved with pepsin or staphylococcal V8 protease. Resultant peptides were purified by HPLC, examined for binding to crossreactive mAbs in ELISA, and reactive peptides were subjected to amino acid sequence analysis. Peptides were aligned with the amino acid sequence of the entire M6 protein predicted by the DNA sequence of the M6 gene. Competitive inhibition studies using peptides synthesized on the basis of peptide and DNA sequences, in concert with selective blocking of amino acid residues, allowed for the further identification and placement of these crossreactive epitopes within the M6 molecule. The 10A11 epitope was located within the six amino acid residues at position 134-139, which repeat at positions 159-164 and 184-189 within the variable amino terminal half of the native molecule. The conserved 10B6 and 10F5 epitopes were positioned within a 15-amino-acid span at position 275-289, with the possibility that either epitope could have been repeated at residues 239-247. Chemical modification of amino acids within this sequence aided in the differentiation of these two epitopes. Such studies should aid in the recognition of a sequence(s) common to a greater number of M serotypes, which may be useful for future vaccine development or group A streptococcal identification.

Anaphylatoxin-induced histamine release with human leukocytes: studies of C3a leukocyte binding and histamine release.

Purified human C3a and synthetic COOH-terminal peptides of C3a, i.e., a pentapeptide, Leu-Gly-Leu-Ala-Arg (5R), and an octapeptide, Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg (8R) induced histamine release from human basophil granulocytes. On a molar basis, 5R was one-tenth and 8R was one-fifth as active as C3a in causing histamine release. It was found that 125I-C3a binds to whole leukocytes, interacting with both mononuclear cells and neutrophils and the binding was inhibited by preincubation of cells with unlabeled C3a, but not by C5a. 5R and 8R also inhibited the binding of 125I-C3a to the cells. However, on a molar basis, 2,000 times more 8R or 6,000 times more 5R is required for 50% inhibition of 125I-C3a binding as compared with native C3a. Autoradiography of cells using 125I-C3a and 125I-C5a showed preferential binding of 125I-C3a to eosinophils and basophils, whereas 125I-C5a binds primarily to neutrophils and eosinophils and to a lesser extent to basophils. The preferential binding of C3a and C5a to different cell types may herald significance related to their physiological functions.

Structural basis of the oligomerization of hepatitis delta antigen.

BACKGROUND: The hepatitis D virus (HDV) is a small satellite virus of hepatitis B virus (HBV). Coinfection with HBV and HDV causes severe liver disease in humans. The small 195 amino-acid form of the hepatitis delta antigen (HDAg) functions as a trans activator of HDV replication. A larger form of the protein containing a 19 amino acid C-terminal extension inhibits viral replication. Both of these functions are mediated in part by a stretch of amino acids predicted to form a coiled coil (residues 13-48) that is common to both forms. It is believed that HDAg forms dimers and higher ordered structures through this coiled-coil region. RESULTS: The high-resolution crystal structure of a synthetic peptide corresponding to residues 12 to 60 of HDAg has been solved. The peptide forms an antiparallel coiled coil, with hydrophobic residues near the termini of each peptide forming an extensive hydrophobic core with residues C-terminal to the coiled-coil domain in the dimer protein. The structure shows how HDAg forms dimers, but also shows the dimers forming an octamer that forms a 50 A ring lined with basic sidechains. This is confirmed by cross-linking studies of full-length recombinant small HDAg. CONCLUSIONS: HDAg dimerizes through an antiparallel coiled coil. Dimers then associate further to form octamers through residues in the coiled-coil domain and residues C-terminal to this region. Our findings suggest that the structure of HDAg represents a previously unseen organization of a nucleocapsid protein and raise the possibility that the N terminus may play a role in binding the viral RNA.

The T-lymphocyte as a diploid reference standard for flow cytometry.

T-lymphocytes prepared from the peripheral blood of normal individuals exhibit uniform postmitotic DNA contents that are constant from individual to individual when fixed in ethanol and stained with mithramycin, or when fixed in formalin and stained with acriflavine-Feulgen and analyzed by flow microfluorometry. T-lymphocytes fixed by either method are stable on storage for several months. These cells are suitable for use as an external diploid reference standard for flow cytometric detection of cells with abnormalities in postmitotic content exceeding 5% of diploid reference. Cells with abnormalities in postmitotic DNA content of less than 5% can be detected in mixtures containing both normal and malignant cells by multiparameter analysis.

Molecular basis for the temperature-dependent insolubility of cryoglobulins--XI. Sequence comparison of the heavy-chain variable regions of the human cryoimmunoglobulins McE and Hil by metric analysis.

The amino acid sequences of the VH-domains from two human cryoimmunoglobulins have been compared with one another and with the VH-domains of noncryoglobulins by the mathematical method of metric analysis. The VHII sequence of McE [Gerber-Jenson et al. (1981), J. Immun. 126, 1212-1216] and the VHIII sequence of Hil [Chiu et al (1979), Biochemistry, 18, 553-560] resemble much more closely the VH-sequences of noncryoglobulins from their own subgroups than they resemble one another. Neither cryoglobulin sequence contains an unusual insertion or deletion of residues. Based on the crystallographic structure of the VHII domain in the Fab fragment of the human noncryoglobulin Newm [Saul et al. (1978), J biol. Chem., 253, 585-597], McE and Hil each contain two unprecedented residues in the outer beta-sheet structure of the VH-domain. The inwardly directed sidechains of Gly-71 and Ile-84 in McE may perturb the internal hydrophobic interactions and normal folding of adjacent segments of the outer beta-sheet from the third framework region. In contrast, the outwardly directed sidechains of Ile-23 and Arg-77 in Hil may perturb the external hydrophilic properties and folding of adjacent segments of the outer beta-sheet from the first and third framework regions. Thus, the monoclonal immunoglobulins McE and Hil may display cold-induced insolubility by different perturbations of the outer surface of the VH-domain. In each case, the unprecedented framework residues that mnay be responsible could have arisen by two point mutations involving single base changes.

The active site of human C4a anaphylatoxin.

The human C4 activation peptide C4a has recently been shown to be biologically active and to share common tissue receptors with human C3a anaphylatoxin. Human C3a and C4a each induce contraction and cause cross-desensitization of isolated guinea-pig ileal strips. The essential active site of C3a is comprised in the model peptide containing the five COOH-terminal residues, Leu-Gly-Leu-Ala-Arg. The anaphylatoxic activities of the corresponding C4a pentapeptide, Ala-Gly-Leu-Gln-Arg, and several other synthetic peptides related to the COOH-terminal sequence of human C4a were examined. The C4a pentapeptide induced contraction of guinea-pig ileum at 1 X 10(-3) M and produced a wheal and flare reaction in human or guinea-pig skin when 2-5 mumols were injected intradermally. The corresponding C3a pentapeptide is 500-fold more active, since it induces contraction of guinea-pig ileum at 3-4 X 10(-6) M and only 4-10 nmole induce a visible skin reaction. Although the C4a pentapeptide is relatively inactive compared to the C3a pentapeptide, two analogs of these peptides, Leu-Gly-Leu-Gln-Arg and Ala-Gly-Leu-Ala-Arg, each exhibited significantly greater activity than Ala-Gly-Leu-Gln-Arg and each analog desensitized ileal smooth muscle towards contraction by either C3a or C4a. Thus it is a combination of two amino acid substitutions, the Ala for Leu-73 and Gln for Ala-76, in the COOH-terminal pentapeptide of C3a that accounts for the markedly reduced activity of C4a. The contribution of the COOH-terminal portion of C4a on its activity was further documented by examining the C4a octapeptide, Lys-Gly-Gln-Ala-Gly-Leu-Gln-Arg and a trialanyl analog, Ala-Ala-Ala-Ala-Gly-Leu-Gln-Arg. The C4a octapeptide, C4a (70-77), exhibited 5-fold greater biologic activity than the C4a pentapeptide, while the trialanyl analog was 40-fold more active. Anaphylatoxic activities of the C4a-(73-77) pentapeptide, C4a-(70-77) octapeptide, and the trialanyl octapeptide analog and their ability to specifically block the action of C3a and C4a on smooth muscle tissue support the conclusion that, as in C3a, the essential active site of C4a resides at its COOH terminus. Since C4a functions as an anaphylatoxin and significant quantities of this mediator may be generated in individuals with hereditary angioneurotic edema (HANE), the hypotheses that the kinin-like activity promoting edema in HANE patients is derived solely from component C2 and/or kininogens should be reappraised. The activities previously assigned to C4a and now confirmed by synthetic C4a analog peptides suggest that the kinin-like activity generated in HANE plasma may be derived in part from C4a.

Antigenic specificity of a rabbit antiserum raised against the 15-28 segment of thymosin alpha 1.

Thymosin alpha 1, an acidic 28-residue peptide, enhances immune function. We have described a radioimmunoassay for this thymic factor based on a rabbit antiserum raised against a thymosin alpha 1-(15-28) conjugate (Incefy et al., J. Immun. Meth. 1986, in press). The detailed antigenic specificity of this antiserum was determined by measuring the ability of synthetic segments and analogues of thymosin alpha 1 and related peptides to compete with radioiodinated Ac-Tyr-thymosin alpha 1-(15-28) in this radioimmunoassay. The antiserum bound segments Ac-(1-28), (15-28), (20-28) and (21-28) with nearly equal efficiency but failed to bind segments Ac-(1-10), (11-20), (19-24) and (22-28). Thus, the major immunoreactive site seen by the antiserum is the COOH-terminal segment (21-28) (Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH). Immunoreactivity of (21-28) was nearly abolished when the carboxylate groups of Glu-21, Glu-27 and Asn-28 were omitted separately. The antiserum bound to prothymosin alpha and thymosin alpha 11, which lack the alpha-carboxylate group of Asn-28, with 0.9 and 0.2%, respectively, of the efficiency of thymosin alpha 1. But it bound nonspecifically to parathymosin alpha, which contains the internal segment . . . -Glu-Val-Val-Glu-Glu-Glu-Glu-Asn- . . . . Residues Glu-21, Glu-27 and Asn-28 of thymosin alpha 1 may be important features of the antigenic site through their ability to induce helical structure, through the ability of their negatively charged carboxylate groups to bind to specific sites on the antibody or both.

Engineering of betabellin 14D: disulfide-induced folding of a beta-sheet protein.

The betabellin target structure consists of 2 32-residue beta sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14D, the disulfide-bridged double chain. The 32-residue nongenetic betabellin-14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = D-Ala, k = D-Lys) has a palindromic pattern of polar (p), nonpolar (n), end (e), and beta-turn (t,r) residues (epnpnpnttnpnpnprrpnpnpnttnpnpnpe). Each half contains the same 14-residue palindromic pattern (underlined). Pairs of D-amino acid residues are used to favor formation of inverse-common (type-I') beta turns. In water at pH 6.5, the single chain of betabellin 14S is not folded, but the disulfide-linked betabellin 14D is folded into a stable beta-sheet structure. Thus, folding of the covalent dimer beta-bellin 14D is induced by formation of the single interchain disulfide bond. The binary pattern of alternating polar and nonpolar residues of its beta-sheets is not sufficient to induce folding. Betabellin 14D is a very water-soluble (10 mg/mL), small (64 residues), nongenetic (12 D residues) beta-sheet protein with properties (well-dispersed proton NMR resonances; Tm = 58 degrees C and delta Hm = 106 kcal/mol at pH 5.5) like those of a native protein structure.

Rational design of a three-heptad coiled-coil protein and comparison by molecular dynamics simulation with the GCN4 coiled coil: presence of interior three-center hydrogen bonds.

alpha-Helical coiled coils have a 7-residue repeating pattern (abcdefg) where a and d are usually hydrophobic. We have designed a 2-stranded 44-residue coiled-coil protein (P44) consisting of 2 22-residue alpha-helices linked by 2 terminal disulfide groups to test whether the disulfide bridges could stabilize a 3-heptad coiled coil. P44 should be stabilized by intrahelical hydrogen bonds, interhelical disulfide and salt bridges, and interior hydrophobic interactions. A computer model of P44 was built and its stability was studied by molecular dynamics simulation with explicit water. This doubly crosslinked 3-heptad coiled coil did not unfold during a 300-ps simulation with explicit water. This doubly crosslinked 3-heptad coiled coil did not unfold during a 300-ps simulation. But reduced P44 with 4 thiol groups did unfold. For comparison, the 62-residue crystal structure of the 4-heptad coiled coil of transcription activator GCN4 did not unfold during a 300-ps simulation. Thus P44 may be a stable folded protein in aqueous solution. These simulations revealed the presence of 2 local hydrogen bond networks involving intra-helical 3-center hydrogen bonds in the hydrophobic interior of the coiled coils of GCN4 and P44. The NH hydrogen at d makes a 3-center hydrogen bond whose major component is to the i - 4 C = O oxygen at g and minor component is to the solvent-inaccessible i - 3 C = O oxygen at a. Likewise, the NH hydrogen at g makes a 3-center hydrogen bond with the i - 4 C = O oxygen at c and the buried i - 3 C = O oxygen at d.

Deuterium exchange of alpha-helices and beta-sheets as monitored by electrospray ionization mass spectrometry.

Deuterium exchange was monitored by electrospray ionization mass spectrometry (ESI-MS) to study the slowly exchanging (hydrogen bonded) peptide hydrogens of several alpha-helical peptides and beta-sheet proteins. Polypeptides were synthetically engineered to have mainly disordered, alpha-helical, or beta-sheet structure. For 3 isomeric 31-residue alpha-helical peptides, the number of slowly exchanging hydrogens as measured by ESI-MS in 50% CF3CD2OD (pD 9.5) provided estimates of their alpha-helicities (26%, 40%, 94%) that agreed well with the values (17%, 34%, 98%) measured by circular dichroic spectroscopy in the same nondeuterated solvent. For 3 betabellins containing a pair of beta-sheets and a related disordered peptide, their order of structural stability (12D > 12S > 14D > 14S) shown by their deuterium exchange rates in 10% CD3OD/0.5% CD3CO2D (pD 3.8) as measured by ESI-MS was the same as their order of structural stability to unfolding with increasing temperature or guanidinium chloride concentration as measured by circular dichroic spectroscopy in water. Compared to monitoring deuterium exchange by proton NMR spectrometry, monitoring deuterium exchange by ESI-MS requires much less sample (1-50 micrograms), much shorter analysis time (10-90 min), and no chemical quenching of the exchange reaction.

Engineering of betabellin-15D: a 64 residue beta sheet protein that forms long narrow multimeric fibrils.

The betabellin target structure is a beta-sandwich protein consisting of two 32 residue beta-sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin-15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p=DPro, k=DLys, and h=DHis) did not fold in water at pH 6.5. Air oxidation of betabellin-15S provided betabellin-15D, the 64 residue disulfide bridged two-chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of beta structure observed for betabellin-15D increased with the pH and ionic strength of the solution and the betabellin-15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin-15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin-15D fibrils are consistent with a single row of beta-sandwich molecules joined by multiple intersheet H-bonds.

Inhibition of thrombin by synthetic hirudin peptides.

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15-51 residues were synthesized. The C-terminal segment 40-65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. Central peptide 15-42 was insoluble at pH 7, but peptide 15-65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N-terminal loop peptide 1-15 had no inhibitory activity and did not affect the potency of peptide 15-65. These data suggest that the central region inhibits catalysis.

A radioimmunoassay for thymosin alpha-1.

A new radioimmunoassay (RIA) is described for the quantitation of thymosin alpha-1 (alpha-1). The assay employs an antiserum specific for the COOH-terminal half segment 15-28 of alpha-1, synthetic alpha-1-(15-28) as the hormone standard, and a radioiodinated N alpha-acetyltryrosyl-alpha-1-(15-28) as the tracer. Since alpha-1-(1-28) lacks a phenolic ring for direct radioiodination, the N alpha-acetyltyrosyl-alpha-1-(15-28) was synthesized by the solid-phase method. The peptide bears a Tyr in place of Lys in position 14 of the natural peptide. It showed full alpha-1-(15-28) immunoreactivity and its radioiodinated derivative served as tracer in the RIA. An anti-alpha-1-(15-28) antiserum was raised in a rabbit and was shown to recognize alpha-1-(15-28) or its tyrosyl analogue, and the peptide, alpha-1-(1-28). But it did not recognize other thymic hormones or the biologically active segment 32-36 of thymopoietin, or structurally unrelated peptides. It could also detect natural alpha-1 cross-reacting material in the cytoplasm of cultured human thymic epithelial cells as measured by indirect immunofluorescence. In the RIA, as little as 9 pg of alpha-1-(15-28) equivalents in a 50 microliter sample could be detected. In addition, alpha-1-(1-28)-like immunoreactivity was quantitated in 6 human thymus homogenates and ranged from 0.5 to 4.5 ng/mg of protein.

Radioimmunoassays for the thymic hormone serum thymic factor (FTS).

Four radioimmunoassays (RIA) are described for the quantitation of serum thymic factor (facteur thymique serique, FTS), a thymic peptide hormone. Each assay employs an antibody specific for FTS, synthetic FTS (Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn) as the hormone standard, and a radioiodinated FTS analogue as the tracer. Since FTS lacks a tyrosine residue, 2 FTS analogues were synthesized by the solid-phase method with tyrosyl-alanyl or 3-(2,6-dichlorobenzyl)tyrosyl-alanyl in place of the amino-terminal pyroglutamyl residue (Glp). They showed full FTS immunoreactivity and their radioiodinated derivatives served as FTS tracers. Two assays used the antiserum from a rabbit immunized with an FTS-protein conjugate. Two other assays used a monoclonal antibody against FTS produced by a hybridoma derived from mouse myeloma cells and splenocytes from a BALB/c mouse immunized with an FTS-mouse IgG conjugate (Ohga et al., 1982). All 4 RIAs were specific for FTS. The more sensitive rabbit antiserum can detect as little as 1 pg of FTS in a 50 microliters sample, which may allow quantitation of the FTS circulating in human peripheral blood.

Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein.

Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a beta structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry. The relative abundances of the ionic complexes suggested an order of affinities of Cu2+ > Zn2+ > Co2+ > Mn2+, consistent with solution-phase affinities for nitrogen- or sulfur-containing ligands. Limited proteolysis of betabellin 15D by immobilized pepsin, as measured by nanoelectrospray-ionization mass spectrometry, showed that the Phe12-Ser13 peptide bond of betabellin 15D was cleaved more slowly in the presence of Cu2+ than in its absence. Because Cu2+ has little or no effect on the catalytic rate of pepsin, the slower cleavage of the Phe12-Ser13 peptide bond may be due to its decreased accessibility caused by Cu(2+)-induced folding of betabellin 15D.