Specific photoaffinity labeling of Tyr-49 on the light chain in the steroid-combining site of a mouse monoclonal anti-estradiol antibody using two epimeric 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amidoestradiol photoreagents

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody was photoaffinity labeled with two cross-reactive 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amido[17alpha-3H]estradiol photoreagents (6alpha- and 6beta-ANBA-[17alpha-3H]estradiol). Covalently bound radioactivity was found exclusively on the light chain. The maximal level of specific incorporation was 0.18 mol of label per mole of antibody for both photoreagents. In both cases, tryptic digestion of the photolabeled light chain, immunopurification with the immobilized antibody, reverse-phase liquid chromatography, and Edman degradation showed the presence of radioactive peptide GLM-([3H]X)-HGNTLEDGIPSR derived from peptide 46-61 of the light chain sequence (determined from cDNA) in which the unidentified amino acid corresponding to X is a Tyr residue. Two other radioactive peptides were also isolated, one corresponding probably to the methionine sulfoxide derivative of the peptide 46-61 photolabeled with the 6beta-reagent and the other to the N-terminal tetrapeptide 46-49 of the peptide 46-61 photolabeled with the 6alpha-reagent. In all cases, the main peak of radioactivity was released at the fourth Edman cycle, thus suggesting that the same Tyr-49 residue on the light chain was photolabeled. This residue is contiguous to the N-terminal amino acid of the second hypervariable complementary determining region 50-56 of light chain. Covalent labeling was confirmed by mass spectrometry of photolabeled peptides which showed molecular ion values corresponding to the addition of the photoactive 6alpha- or 6beta-ANBA-estradiol nitrene derivatives to the peptide.     

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha (2-8) polysialic acid. 2. Amino acid sequence of the heavy (H-) chain Fd' region

The complete amino acid sequence of the Fd' region including the VH part, the CH1 domain, and the hinge segment of the biologically relevant monoclonal mouse anti-alpha (2-8) polysialic acid antibody mAb735 is presented. The reduced and carboxymethylated H-chain was digested with trypsin and cyanogen bromide. For subfragmentation selected peptides were cleaved with thermolysin and endoproteinase Asp-N. The generated peptides were isolated by RP-HPLC and characterized by sequence analysis, plasma desorption mass spectrometry (PDMS), and amino acid analysis. The N-terminal sequence was determined after enzymatic deprotection with pyroglutamate aminopeptidase. According to Kabat et al. the variable region of the H-chain belongs to the subgroup II. Sequence data from the constant region indicate that mAb735 represents the gamma 2a isotype.     

Complete and rapid peptide and glycopeptide mapping of mouse monoclonal antibody by LC/MS/MS using ion trap mass spectrometry

Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized.     

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems.     

Monoclonal antibody against bovine Lactoferricin and its epitopic site

Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".     

Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.     

Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain

Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.     

Heterogeneity of crystalline antithyroid phytoprecipitin

The crystalline preparation of antithyroid phytoprecipitin has been resolved by chromatography on Whatman CM-32 cellulose on a preparative scale into two components, designed respectively A and B. Each component was further resolved into consisting polypeptide chains alpha (mol. weight 7.000) and beta (mol. weight 17.000) by gel filtration on sephadex G-50 in 1 M acetic acid 6 M urea. Homologous chains were comparatively studied by electrophoresis in acrylamide gel, amino acid analysis and peptide mapping technique. Electrophoresis in acrylamide gel with 6 M urea according to Takayama [8] revealed the identical mobility of beta chains in both components A and B, while alpha chains differed. alphaA chain was more basic, than alphaB, i.e. it had greater positive sharge. The amino acid analysis and peptide mapping showed that alphaA chain had one residue of lysine more than alphaB chain. The comparison of beta chains by peptid mapping confirmed their complete identity.     

Selection of phage-displayed peptides mimicking an extracellular epitope of human MDR1-P-glycoprotein

To study the structural conformation of the MM4.17 monoclonal antibody (mAb) epitope, twenty-six mAb MM4.17-specific phage clones were affinity-isolated and their inserts characterized for amino acid composition and homology with MDR1 gene product (MDR1-P-glycoprotein). The resulting sequence alignment shows that a unique consensus sequence, which corresponds to the previously mapped TRIDDPET linear peptide identified through synthetic peptide scanning, could not be identified. However, similarities between the inserts of positive phage clones and P-glycoprotein primary structure, consisting in two or three amino acid-long sequences, were observed. An analysis of the over-represented amino acid residues in the inserts of positive clones, and their comparison with the sequence of the antigen was also performed. The two different procedures led to the identification of four regions in which these similarities are clustered, indicating that four different antigen regions, one of which includes the TRIDDPET linear amino acid sequence, might participate in forming the structure of monoclonal antibody MM4.17 epitope.     

Isolation and characterization of a 20 kD prolamin from kodo millet (Paspalum scrobiculatum) (L.): homology with other millets and cereals

The homologus 20 kD prolamin from kodo millet and other minor millets viz. barnyard, little and foxtail millets, were purified using preparative gel electrophoresis and reversed phase high performance liquid chromatography (RP-HPLC). The amino acid composition of the purified 20 kD prolamin protein from different minor millets revealed higher content of glutamic acid, alanine, leucine and serine and lower quantity of lysine and methionine. They contain 55 to 58 percent of non-polar amino acids which make them more hydrophobic than other protein fractions. The total number of amino acid residues per polypeptide chain ranged from 152 to 155 based on theoretical calculation. Peptide mapping of the 20 kD prolamin hydrolyzed with trypsin gave fewer cleavage products than expected. The antigenic relationships among these minor millets and cereals viz. wheat, maize, rice, sorghum, finger millet and pearl millet were studied using the antibody raised against the 20 kD prolamin. Cross reactivity was seen in all the minor millets at the 20 kD region. But in barnyard and little millets lower molecular weight polypeptides also cross reacted with the antibody. Immunoblotting studies revealed that the prolamins from other cereals and millets are related to the 20 kD prolamin of kodo millet. Rice was the only common cereal that did not cross react immunologically with the antibody raised against 20 kD prolamin of kodo millet.     

A humanized antibody with specificity for hepatitis B surface antigen

A murine monoclonal antibody H67 was characterized for the binding specificity, which showed that H67 recognizes a disulfide-bond-dependent conformational epitope of common a antigenic determinant on the hepatitis B surface antigen. The result suggested that this antibody may have the potential of replacing hepatitis B immune globulin in the prevention of hepatitis B virus (HBV) infection. Therefore, we have constructed the humanized antibody HuS10 by grafting the complementarity determining regions and some framework amino acid residues of H67 onto the most homologous human antibody variable regions, 21/28 for heavy chain variable region and B1 and J kappa 2 for light chain variable region, followed by combining with human constant regions C gamma 1 and C kappa. The affinity of the HuS10 was the same as that of the H67, 8 x 10(8) x 10(8)M-1, and the HuS10 neutralized the in vitro infection of adult human hepatocyte primary culture by adr or ayw subtype of HBV. The neutralization assay showed that the HuS10 had approximately 2,000-times higher specific activity than commercially available polyclonal HBIG. These results suggest that the humanized antibody will be useful in the prevention or treatment of HBV infection.     

Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching

Peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching with the set of measured peptide masses is now a powerful method for the identification of pure proteins. Protein mixtures--such as frequently occur due to comigration in polyacrylamide gel bands--have hitherto required protein sequencing. Here we demonstrate that such protein bands can also be analyzed by peptide mass mapping alone. Database searching with the complete list of peptide masses determined by delayed-extraction MALDI mass spectrometry with a mass error of less than 30 ppm retrieves the most prominent protein in a mixture. In a second step, the protein identity is further confirmed by matching as many of the measured peptide masses as possible to the retrieved amino acid sequence. Peptide masses remaining after this "second pass search" are searched again to identify the next component in the protein mixture. This iterative process is repeated until all major ion signals are accounted for. Protein mixtures consisting of two or more individual components in a single gel band can be analyzed, further increasing the general applicability of MALDI peptide mapping for protein identification.     

Isolation of an HLA-A2.1 extracted human minor histocompatibility peptide

Purified HLA-A2.1 molecules obtained by affinity chromatography of 6 x 10(10) Epstein Barr virus (EBV)-transformed B lymphocytes were used in an attempt to isolate the human HLA-A2.1-restricted minor histocompatibility (H) peptides H-Y and HA-2. Fraction 18 of the high-performance liquid chromatography (HPLC)-separated HLA-A2.1 peptide pool was found to contain the natural HA-2 peptide. An HA-2-specific, HLA-A2.1-restricted cytotoxic T lymphocyte clone lysed HLA-A2.1+ HA-2- EBV-transformed B lymphocyte cell lines reproducibly and in a concentration-dependent fashion in the presence of fraction 18, but not in the presence of other HPLC fractions. By contrast, H-Y sensitizing activity was not found in any fraction. Amino acid sequencing of peptide fraction 18 revealed a mixture of peptides with maximal length of nine amino acids, in which the presence of Leu at positions 2 and 9 was dominant. Surprisingly, the HA-2 peptide could not be mimicked by any of the peptide mixtures synthesized according to the amino acid sequences found in fraction 18. Our failure to obtain the actual amino acid sequence of the human minor H peptide HA-2 from a peptide pool with the established pattern for binding to HLA-A2.1 may indicate that this CTL defined minor H peptide does not represent an abundant HLA-A2.1 binding peptide.     

Definition of essential amino acid residues in the recognition of a peptide by a mouse monoclonal antibody

A mouse monoclonal antibody reacting in ELISA with a synthetic peptide representing a linear amino acid stretch of the protein antigen was tested on all overlapping 5-mer to 9-mer fragments of the peptide, as prepared by multi-pin synthesis. Analysis of the binding data suggests that several residues in the peptide might be relatively unrelevant for recognition, while few others seem to play a critical role as key residues. On the basis of such observations, we attempted to reconstruct an alternative essential epitope by introducing multiple amino acid substitutions in the 9-mer peptide exhibiting the best binding activity, and then tested its ability to be recognized by the monoclonal antibody.     

Mode of binding of anti-P-glycoprotein antibody MRK-16 to its antigen. A crystallographic and molecular modeling study

Monoclonal antibody MRK-16 recognizes a discontinuous extracellular epitope on the multidrug resistance-associated ATP-binding cassette transporter, P-glycoprotein. The atomic basis for specificity of this antibody is of interest because of its potential as a modulator of P-glycoprotein activity. The crystal structure of Fab MRK-16 is reported to a resolution of 2.8 A. A structure for a portion of the epitope was derived by comparison to regions of solved structures with similar primary sequence. This has permitted a proposal for the mode of binding of the peptide epitope to the antibody, in which the peptide makes specific contacts with complementarity-determining regions H1, H2, and H3 from the heavy chain and L3 from the light chain. These interactions are consistent with epitope mapping studies and with the observation that MRK-16 is specific for human class I P-glycoprotein. This result identifies side chains in MRK-16 that would be amenable to alteration in antibody engineering experiments to derive improved multidrug resistance inhibitors for clinical use during chemotherapy. In particular, Arg-H97 contacts both Glu-746 and Asp-744 of the peptide, Arg-L96 contacts Asp-743, and Thr-H33 interacts with Thr-747. All of these epitope residues were implicated in mediating specificity by epitope mapping studies.     

Characterization of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresis/electrospray (ionspray) mass spectrometry and 252Cf plasma desorption mass spectrometry

A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass spectrometry and by 252Cf plasma desorption-time of flight mass spectrometry. The measured average molecular mass of the synthetic peptide was 4627.16 Da, which was only 0.08 Da higher than the calculated value (4627.08 Da). The plasma desorption mass spectrum of the synthetic peptide showed a protonated molecule at m/z 4624.1, which was 4 Da lower than the calculated one (4628.09 Da). The amino acid sequence of the peptide was confirmed in part by electrospray (ionspray) mass spectrometry using a high nozzle skimmer voltage difference. Five impurities were separated and identified by capillary electrophoresis/mass spectrometry and two of them also appeared in the plasma desorption mass spectrum.     

q analogs of the radial Schr?dinger equation in N space dimensions

Native parvovirus B19 was used as antigen to produce a mouse monoclonal antibody, R92F6, which reacted with B19 VP1 and VP2, neutralised the virus in bone marrow culture, and labelled infected cells in paraffin-embedded tissues from cases of B19-related fetal hydrops. The B19 epitope recognised by R92F6 (amino acids 328-344 from the amino terminal region of B19 VP2) appears to be highly conserved, since these tissue specimens were obtained over a 13 year period from widely spaced locations in the UK. This epitope was synthesised as a peptide (S7b) which was used as antigen to produce a mouse monoclonal antibody, 3H8, which specifically reacted with the B19 capsid proteins VP1 and VP2 in immunofluorescence and immunoblot assays. 3H8 was also capable of labelling formalin-fixed, paraffin-embedded, B19-infected fetal tissue and was shown to be of the same isotype as R92F6 (IgG1). Highly conserved epitopes derived from conserved amino acid sequences are valuable in the diagnosis of infectious disease. If these can be recognised and accurately synthesised, the production of specific mouse monoclonal antibodies may be possible for many human pathogens. Considering the vast amount of sequence data available in the literature, this approach seems to be both feasible and of wide potential.     

Engineering vaccines with heterologous B and T cell epitopes using immunoglobulin genes

Antibodies engineered in their variable domain to express epitopes of heterologous antigens-antigenized antibodies-function as immunogens. Only the third complementarity-determining region (CDR3) of the H chain has been used as the site of epitope expression, as this loop has the highest natural variability in length and amino acid composition. We demonstrate that the CDR2 can be engineered to express a 12-amino acid peptide, which is a T-cell determinant that enhances the response to a B-cell epitope peptide of Plasmodium falciparum expressed in the CDR3 of the same variable domain. Mice with this gene inoculated into the spleen mounted an antibody response against the B-cell epitope higher than mice receiving the gene coding for the B-cell epitope only. In vitro studies established that the two epitopes were independently immunogenic in vivo.     

Congenital central hypoventilation syndrome: an update

Beta-2 microglobulin (beta2m)has been shown to have an effect on the structural and functional constraints that facilitate proper class I antigen presentation. To date, no evidence has pinpointed the beta2m-specific amino acids that play an integral role in affecting structure in and around the peptide binding region of class I. To delineate beta2m amino acid positions that affect the alpha-1 helical region, we generated a series of mutant beta2m proteins bearing precise amino acid substitutions. The amino acid positions chosen were based upon previous results which demonstrated that human beta2m association with H2-Ld altered the structure of the alpha-1/alpha-2 super-domain. beta2m mutant proteins were used in beta2m exchange assays with cells expressing H2-Ld. Following exchange, cells were assayed to determine whether mutant beta2m association resulted in structural alteration of class I extracellular domains. The alteration in H2-Ld structure was evidenced by an increase in the binding of an antibody (34-1-2), specific for the alpha-1 helical region of H2-Ld. Results demonstrated that amino acid substitutions in beta2m positions 33 and 53 led to a dramatic increase in the reactivity of the alpha-1 domain-specific antibody 34-1-2. Identifying beta2m amino acid positions that influence the structure of the peptide binding region may allow for a better understanding of cellular immune responses that center upon class I/beta2m expression.