Analogues of peptide 9-21 of glycoprotein D of herpes simplex virus and their binding to group VII monoclonal antibodies.

Several analogues of the amino acid sequence of peptide 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, EII-24 and Ev-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9-21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10-18 and peptide 9-17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9-21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.

Virus neutralizing activity induced by synthetic peptides of glycoprotein D of herpes simplex virus type 1, selected by their reactivity with hyperimmune sera from mice.

Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30. 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9-21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9-21, 10-24, 151-165, 216-232, 282-301 and with peptide 340-354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10-24, 151-165, 216-232, 244-267, 260-274, 270-284, 260-284, 282-301, 300-314, 340-354, 348-362 and 355-369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10-24, which covers the same epitope as peptide 9-21, neutralized HSV-1 in vitro.

The influence of different adjuvants on the immune response to a synthetic peptide comprising amino acid residues 9-21 of herpes simplex virus type 1 glycoprotein D.

The immuno-modulating properties of different adjuvant systems on the murine humoral and cellular immune response to a synthetic peptide comprising amino acid residues 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were investigated. For immunization, the peptide was conjugated to ovalbumin or bovine serum albumin by glutaraldehyde and the adjuvants used in this study were Freund's complete adjuvant (FCA), aluminium hydroxide, the Ribi adjuvant system (RAS) and two non-ionic block polymer surfactants, viz. L101 and 31R1, in oil in water emulsions. High anti-peptide antibody titers were obtained after immunization with FCA, aluminium hydroxide, RAS and L101. All adjuvants, except RAS, stimulated the induction of delayed type hypersensitivity obtained after immunization with peptide 9-21 coupled to ovalbumin and elicited by injection of purified HSV-1 virions in the footpad. Challenge with a lethal dose of HSV-1 showed that mice immunized with peptide 9-21 coupled to ovalbumin in combination with FCA, RAS and L101, respectively, were significantly protected. Although immunization with peptide 9-21 coupled to ovalbumin combined with aluminium hydroxide stimulated induction of delayed type hypersensitivity, no significant protective immunity against the challenge was generated.

Controlled peptide-protein conjugation by means of 3-nitro-2-pyridinesulfenyl protection-activation.

The disulfide bond in S-3-nitro-2-pyridinesulfenyl (S-Npys) compounds is stable towards the acid treatment used in solid-phase peptide synthesis, yet the liability of S-Npys-peptides towards nucleophiles enables the conjugation to proteins to proceed under mild conditions. Thus Boc-Cys(Npys)-OH was coupled as N-terminal residue to a resin-linked peptide chain. After deprotection and cleavage from the resin the Npys-cysteinylpeptide was attached to a properly functionalized protein by reaction with a mercapto group. The amount of peptide conjugated to the protein was determined by measuring the amount of 3-nitro-2-thiopyridone liberated. The cysteinylpeptide which was detached from the protein by reduction of the disulfide bond was shown to be identical with the product obtained by reduction of the Npys-cysteinylpeptide.

Chemical modification of tyrosine-38 in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5'-p-fluorosulfonylbenzoyladenosine: a probe for the elucidation of the NADPH binding site? Involvement in catalysis, assignment in sequence and fitting to the tertiary structure.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens was covalently modified by the nucleotide analog 5'-(p-fluorosulfonylbenzoyl)-adenosine in the presence of 20% dimethylsulfoxide. The inactivation reaction is pH-dependent and does not obey pseudo-first-order kinetics, due to spontaneous hydrolysis of the reagent. The kinetic data further indicate that a weak, reversible enzyme-inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activity. The inactivation is strongly inhibited by NADPH and 2',5'ADP. Steady-state kinetics and 2',5'ADP bioaffinity chromatography of the modified enzyme suggest that the essential residue is not directly involved in NADPH binding. Sequence studies show that Tyr-38 is the main residue protected from modification in the presence of NADPH. From crystallographic studies it is known that the hydroxyl group of Tyr-38 is 1.84 nm away from the active site. Model-building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended conformation with the sulfonylbenzoyl portion in an orientation different from the nicotin-amide ring of NADPH.

Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro.

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro.

The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme-linked immunosorbent assay.

Rabbits were immunized with synthetic peptides of herpes simplex virus type 1 glycoproteins, coupled to a carrier protein with glutaraldehyde. Antibodies directed against the peptides were determined in an enzyme-linked immunosorbent assay (ELISA). Either free peptides or peptides coupled with glutaraldehyde to another carrier protein than the one used for immunization were used as the coating antigen. When conjugated peptides were used as the coat, it was necessary in some instances to correct the antibody titers for a substantial amount of antibody activity against glutaraldehyde. When free peptides were used, optimal coating conditions with regard to pH and ionic strength had to be determined, since some peptides failed to coat under standard conditions, at pH 9.6. The results show that some peptides needed stringent pH conditions while others could be coated in a broad pH range. The addition of 0.6 M NaCl had a favorable effect on peptide coating.

Immunological properties of an N-terminal fragment of herpes simplex virus type 1 glycoprotein D expressed in Escherichia coli.

The N-terminal fragment, comprising residues -5 to 55 of herpes simplex virus type 1 glycoprotein D was expressed as a beta-galactosidase fusion protein in Escherichia coli. This gD-fusion protein reacts with monoclonal antibody LP 14 directed against glycoprotein D of HSV. Antisera obtained after immunization of rabbits with purified gD-fusion protein react with HSV-1 gD in a Western blot and with N-terminal synthetic peptides of gD. In addition, these antisera are able to neutralize viral infectivity in vitro.

Structural properties and reactivity of N-terminal synthetic peptides of herpes simplex virus type 1 glycoprotein D by using antipeptide antibodies and group VII monoclonal antibodies.

To investigate the contribution of individual amino acids to the antigenicity of the N-terminal region of herpes simplex virus type 1 glycoprotein D, a series of 14 overlapping synthetic peptides within residues 1 to 30 were examined for their reactivity with monoclonal antibody LP14 (a group VII monoclonal antibody; in herpes simplex virus mutants resistant to LP14, arginine 16 is substituted by histidine) and two antipeptide antisera (antipeptide 9-21 and antipeptide 1-23). Maximal binding was achieved with peptides 9-21, 10-30, 9-30, and 8-30 and the chymotryptic fragment 9-17 of peptide 9-21, suggesting that a major antigenic site is located within residues 10 through 17. Lysine 10 was shown to be essential for high reactivity, either by binding directly to the antibody molecule or by stabilizing an ordered structure of the peptide. The importance of ordered structure was demonstrated by a decrease in reactivity after sodium dodecyl sulfate treatment of peptides 9-21 and 8-30.

Chemical modification of tyrosine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: assignment in sequence and catalytic involvement.

p-Hydroxybenzoate hydroxylase was modified by diethyl pyrocarbonate at pH values greater than 7 and by p-diazobenzoate. Modification of the enzyme by diethyl pyrocarbonate abolishes the affinity of the enzyme for the substrate p-hydroxybenzoate. Modification by p-diazobenzoate has the same effect on the enzyme. The enzyme is protected against these modifications by the effector p-fluorobenzoate. The data indicate that the modification of one tyrosine residue in the active center of the enzyme is responsible for the loss of enzyme activity. This tyrosine residue has been identified by sequence studies using radioactively labeled p-diazobenzoate and was found to be most probably Tyr-222. Diethyl pyrocarbonate reacts with a tyrosine residue in the active center other than Tyr-222; the former could not be identified. Sequence studies further showed that Cys-211 is also partially modified by p-diazobenzoate. In addition, the sequence of residues 343-345 was found to be Ser-Trp-Trp instead of the tentative assignment Ser-Tyr-Trp made earlier. The results are briefly discussed on the basis of the existing three-dimensional model of the enzyme.

Prediction of sequential antigenic regions in proteins.

Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363-373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B-subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions.

Chemical modification of sulfhydryl groups in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Involvement in catalysis and assignment in the sequence.

The cysteine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens were modified with several cysteine reagents. One of the five sulfhydryl groups reacts rapidly and specifically with N-ethylmaleimide without inactivation of the enzyme. Cysteine-116 was found to be the reactive cysteine by isolation of a labeled tryptic peptide. The enzyme is easily inactivated by mercurial compounds. The original activity can be fully restored by treatment of the modified enzyme with sulfhydryl-containing compounds. The rate of incorporation of mercurial compounds is pH-independent and is pseudo-first-order up to 90-95% loss of activity. The reaction shows saturation kinetics. The substrate p-hydroxybenzoate protects the enzyme from fast inactivation. The mercurial compounds themselves inhibit the inactivation reaction at concentrations higher than 80 microM. A spin-labeled derivative of p-chloromercuribenzoate reacts fairly specifically with only Cys-152 on use of enzyme prelabeled with N-ethylmaleimide, in contrast to p-chloromercuribenzoate which reacts with additional cysteine residues, i.e. Cys-211 and Cys-158. From these results it is concluded that modification of Cys-152 decreases drastically the affinity of the enzyme for the substrate. The results strongly indicate that the substrate binding site and Cys-152 are interdependent. This observation is not obvious when the three-dimensional data only are considered. The modified enzyme exhibits a somewhat higher affinity for NADPH than the native enzyme. Modification of N-ethylmaleimide-prelabeled enzyme by p-chloromercuribenzoate leads to absorbance difference spectra showing maxima at 250 nm, 290 nm and 360 nm. The intensities of the absorbance difference maxima at 290 nm and 360 nm are strongly dependent on the pH value of the solution. The intensities are very low at low pH values and increase with increasing pH values, reaching a maximum at about pH = 9. The ionizing group shows a pK value of about 7.6. The maximal molar difference absorption coefficient at 290 nm is 3200 M-1cm-1 at pH 9, suggesting that tyrosine residues ionize under the conditions of modification of the enzyme. The results are discussed in the light of the known three-dimensional structure.

Polypeptide chains with similar amino acid sequences but a distinctly different conformation. Bovine and porcine phospholipase A2.

The primary structures of bovine and porcine pancreatic phospholipase A2 differ only by about 15%. Nevertheless, a 12 residue loop, with only one substitution (Val leads to Phe) has a quite different conformation, whereas the rest of the molecules have a very similar folding indeed. From this observation it is concluded that prediction of a 3-dimensional structure on the basis of sequence similarity of short segments alone might give erroneous results.

Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis.

Endoproteinase Lys-C from Lysobacter enzymogenes, which is commercially available, proved to be useful in the determination of primary structures of proteins. The enzyme preferentially cleaves at the carboxyl side of lysine residues.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 2. Fitting of the amino-acid sequence to the tertiary structure.

The complete primary and tertiary structure of p-hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron-density map at 0.25-nm resolution. The parts of the polypeptide chain contributing the residues to the FAD-binding site and the residues of the substrate-binding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p-hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD. Many side chains from the C-terminal part of the polypeptide chain are involved in subunit-subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 1. Completion of the elucidation of the primary structure.

As a final step in the elucidation of the primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111-276), that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346-347) in peptide CB2. The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map. The sequence of residues 343-346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map. An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys-C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.

Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The amino acid sequence of the p-hydroxybenzoate hydroxylase (4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) monomer from Pseudomonas fluorescens has been determined. The sequence was elucidated by a combination of the results from an X-ray crystallographic study at 0.25 nm resolution (Wierenga, R.K., de Jong, R.J., Kalk, K.H., Hol, W.G.J. and Drenth, J. (1979) J. Mol. Biol. 131, 55-73) and from protein sequence analysis. The polypeptide chain of the monomer contains 394 amino acids and has a molecular weight of 44 299.

Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.