Peptide transport by the multidrug resistance protein MRP1.

Small hydrophobic peptides were studied as possible substrates of the multidrug resistance protein (MRP)-1 (ABCC1) transmembrane transporter molecule. As observed earlier for P-glycoprotein- (Pgp; ABCB1) overexpressing cells, MRP1-overexpressing cells, including cells stably transfected with the MRP1 cDNA, showed distinct resistance to the cytotoxic peptide N-acetyl-Leu-Leu-norleucinal (ALLN). Resistance to this peptide and another toxic peptide derivative, which is based on a Thr-His-Thr-Nle-Glu-Gly backbone conjugated to butyl and benzyl groups (4A6), could be reversed by MRP1 inhibitors. The reduced toxicity of 4A6 in MRP1-overexpressing cells was found to be associated with lower accumulation of a fluorescein-labeled derivative of this peptide. Glutathione (GSH) depletion had a clear effect on resistance to ALLN but hardly affected 4A6 resistance. In a limited structure-activity study using peptides that are analogous to 4A6, MRP1-overexpressing cells were found to be resistant to these peptides as well. Remarkably, when selecting A2780 ovarian cancer cells for resistance to ALLN, even in the absence of Pgp blockers, resulting cell lines had up-regulated MRP1, rather than any of the other currently known multidrug resistance transporter molecules including Pgp, MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCCS), and the breast cancer resistance protein ABCG2. ALLN-resistant, MRP1-overexpressing cells were found to be cross-resistant to 4A6 and the classical multidrug resistance drugs doxorubicin, vincristine, and etoposide. This establishes MRP1 as a transporter for small hydrophobic peptides. More extensive structure-activity relationship studies should allow the identification of clinically useful peptide antagonists of MRP1.

The primary structure of phospholipase A2 from porcine pancreas. A reinvestigation.

The primary structure of porcine pancreatic phospholipase A2 (EC 3.1.1.4) has been reinvestigated. A number of modifications have been introduced including the addition of a 7th disulfide bridge. The structure which is presented here shows a high degree of homology with the amino acid sequence of snake venom and horse pancreas phospholipase A2.

The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas.

The primary structure of porcine pancreatic isophospholipase A2 (EC 3.1.1.4) has been investigated. The sequence of procine isophospholipase differs from the sequence of porcine phospholipasy by four substitutions; viz. Ala12 leads to Thr; His17 leads to Asp leads to; Met20 leads to Leu and Glu71 leads to Asn.

Manipulation of antipeptide immune response by varying the coupling of the peptide with the carrier protein.

Antibodies were raised against synthetic peptides of two regions of the surface protein VP1 of foot-and-mouth disease virus. The peptides were conjugated with keyhole limpet hemocyanin via C- or N-terminal amino acid residues by use of different coupling agents. The fine specificity of the resulting antibodies was determined by PEPSCAN methods. In general, amino acid residues specific for antibody recognition tended to be located opposite to those used for coupling with the carrier protein. Depending on the method of conjugation, the orientation of the peptide at the carrier protein changes and directs the immune response. Thus, the method of conjugation can be used to manipulate the immune response and to improve the antiviral activity of antipeptide antibodies. The PEPSCAN method is an effective monitor in this process.

Domains of rat interleukin 1 beta involved in type I receptor binding.

In the present study the inhibitory effects of a panel of 21 monoclonal antibodies (moabs) to rat interleukin 1 beta (rIL-1 beta) on the binding of 125I-labeled rIL-1 beta to murine type I IL-1 receptors on EL4 cells were investigated. Furthermore, the epitopes of these moabs were determined by the use of the pepscan technique, and these epitopes were visualized on a three-dimensional model of rIL-1 beta. Some moabs (SILK 3, 4, 5, 6, and 22) inhibited receptor binding of radioiodinated rIL-1 beta at concentrations that are similar to the dissociation constant values of antibody-rIL-1 beta binding. Another group of moabs (SILK 7, 11, 20, 21, and 23) also inhibited receptor binding but only at concentrations that are 10-150 times higher than their dissociation constants. A large group of moabs did not affect receptor binding in the concentration range tested, and two moabs enhanced the binding of rIL-1 beta to type I receptors. The result of pepscan analysis shows that the moabs bound to one or more of the amino acid sequences 35-49, 66-85, 78-97, 106-124, and 123-143 of mature rIL-1 beta. Modeling of rIL-1 beta shows that the binding domains of SILK 4, 5, 6, and 22 (sequence 123-143) is located at the closed end of the molecule, indicating that this part of rIL-1 beta harbors domains that are crucial for type I receptor binding. The binding domain of SILK 3 (sequence 66-85) is also located at this end of the molecule. In contrast, the binding domains of SILK 7, 11, 20, 21, and 23 (sequence 78-97) are located at the open end of the molecule, which is at the same face as the amino- and carboxy-terminals. The binding domain of SILK 16 (sequence 106-124) is positioned at the center of the molecule. It is concluded that the closed end of rIL-1 beta contains sequences that are crucial for its binding to type I receptors on murine EL4 cells. Because of the high concentrations of antibodies to residues 78-97 of rIL-1 beta that are needed to interfere with receptor binding, the importance of these domains in binding to type I receptors remains uncertain.

Design of synthetic peptides for diagnostics.

Due to the advantageous properties of synthetic molecules compared to biological ones biological molecules in diagnostic tests are replaced increasingly by synthetic ones, usually synthetic peptides or related molecules. The replacement of biological antigens by synthetic peptides is most advanced at present, as well as the use of site-specific antibodies induced with synthetic peptides. Moreover recent results indicate that synthetic molecules may also replace antibodies. Ultimately this will lead to diagnostic assays built of synthetic molecules only.

Identification of epitopes within beta lactoglobulin recognised by polyclonal antibodies using phage display and PEPSCAN.

Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunised with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overlapping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA procedure was used to locate which of these peptides bound the antibodies under investigation. Comparisons of PEPSCANs for antisera from six different rabbits showed that each rabbit recognized a similar set of epitopes within BLG. PEPSCAN analysis also showed that polyclonal antibodies from the mouse recognize a set of epitopes similar to those recognized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fused to the coat proteins of filamentous phage as a source of epitopes for analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recognized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti-BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive phage ELISA was used to screen several hundred single phage clones for binding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screens of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found to constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG recognised by IgG antibodies from immunised rabbits and mice.

Effects of monoclonal antibodies to specific epitopes of rat interleukin-1 beta (IL-1 beta) on IL-1 beta-induced ACTH, corticosterone and IL-6 responses in rats.

Recently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL-1 beta and found that MoAbs binding to the aminoacid sequences 66-85 and 123-143 of mature rIL-1 beta inhibited the binding of rIL-1 beta to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL-1 interfere with the biological effects of rIL-1 beta in adult male rats in vivo. Administration of rIL-1 beta (1 or 5 micrograms/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL-6 in a time- (0.5-4 h) and dose-dependent manner. Because 2 h after 5 micrograms/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL-1 beta was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37 degrees C or at 4 degrees C. Plasma ACTH, CORT and IL-6 responses to these mixtures are compared to those obtained after preincubation of rIL-1 beta with a non-IL-1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66-85 domain of rIL-1 beta, reduced the ACTH and IL-6 responses by 48 and 45% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of epitopes on follicle-stimulating hormone and FSH-antiserum-induced suppression of bioactivity of follicle-stimulating hormone and luteinizing hormone.

There are currently two major approaches to hormonal male contraception. One relies on testosterone (analogs) either alone or in combination with gonadotropin releasing hormone (GnRH) (analogs or immunizations), the other on immunizations against follicle-stimulating hormone (FSH). Theoretically, the latter method will suppress spermatogenesis whilst not interfering with libido. An absolute requirement is, however, that an anti-FSH vaccine does not include anti-luteinizing hormone (LH) antibodies (LH being responsible for the induction of testosterone which is necessary to maintain libido). In this report we show that when whole FSH is used for vaccination, in most cases in addition to biological activity against FSH, anti-LH activity is also induced. By systematic analysis of the antisera raised with FSH using systematic epitope scanning (PEPSCAN) we found differences between the FSH-specific and FSH-nonspecific sera. Only the FSH-specific antiserum contained antibodies that recognized amino acid sequence 37-55 on the beta-subunit in a linear manner. Because antibodies against this epitope have not been found in the cross-reactive sera this epitope forms a prime candidate for an anti-FSH contraceptive vaccine.

In vitro inhibition of the bioactivity of follicle-stimulating hormone by antisera against a peptide representing part of the FSH-receptor.

The aim of the present work was to define an FSH receptor (FSHR) peptide that can induce antibodies that will inhibit the bioactivity of FSH. Therefore, the hFSHR sequence was aligned with that of all other known G-protein coupled receptors. An area with increased sequence homology was identified between the FSH-, LH-, TSH receptors, the C5a receptor and the IL8 receptor. The similarity consists of a richness in acidic (D and E) and hydrophobic (Y and F) residues. In hFSHR the sequence is EDNESSYSRGFDMTYTEFDYDLCNEVVD (amino acid 299-326). Research on both the C5a- and IL8-receptor has indicated that this part is responsible for hormone binding but not for signal transduction. Protamine. an antagonist for both the C5a- and IL8 receptor also inhibited the bioactivities of FSH and LH when tested in a bioassay. This suggests that in the hFSHR this region might also be involved in hormone binding. Specificity of this region towards the diverse ligands all binding to the C5a or to the IL8 receptor might be attributed to differences in the profile of alternating basic and hydrophobic residues. Therefore, the hypothesis was tested as to whether antisera raised against peptides of this FSHR-domain would inhibit FSH-bioactivity but not LH-bioactivity. Indeed antisera were found (anti-hFSHR 309-322) that inhibited the biological activity of FSH in a bioassay. These antisera proved to be specific since they did not inhibit the bioactivity of LH. These data suggest that the core sequence (hFSHR 309-322) of the aligned domain of the hFSHR, in analogy to the IL8- and C5a receptors, is involved in hormone binding and ligand specificity. This domain therefore forms a valuable tool in FSH- and FSHR research for scientific and medical purposes.

Mimotopes: realization of an unlikely concept.

Theoretically it seems highly unlikely that relatively small peptides could mimic functionally discontinuous epitopes of antigens. Nevertheless various recent reports show this to be the case. Peptide mimics of protein-, polysaccharide- and DNA-epitopes have been shown to be able to replace the native epitope. Moreover, some of them are able to induce, when used in a vaccine, antibodies with the same activity as that of the antibody used as a template. These mimics, called mimotopes, can be used in vaccines and diagnostics and can be developed more or less systematically using solely antibodies and random, semi-random and dedicated peptide arrays or libraries. Furthermore, the mimotope concept which seems to have proven itself for antibody and antigen interaction can be applied equally well to many receptor ligand interactions and thus may form a new generic approach to the development of drugs. Ltd.

Location of CD4 dimerization site explains critical role of CDR3-like region in HIV-1 infection and T-cell activation and implies a model for complex of coreceptor-MHC.

CD4 cross-linking by antibodies or its natural ligands triggers a tyrosine kinase activity that is one of the necessary steps in the mechanism of human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and full Th-cell activation. In this study we mapped a part of the dimerization site of human CD4 to amino acids 87-98 using a bivalent CD4 immunoadhesin and a series of overlapping 12-mer peptides of the D1 domain. The dimerization site we found is part of the complementary determining region (CDR) 3-like region of CD4. Using the three-dimensional structure of other immunoglobulin dimers as a basis, a molecular modeling study was performed to dimerize the D1 domains of CD4. Both the peptide binding studies and molecular modeling studies independently led to the conclusion that the CDR3-like region is part of the CD4 dimerization site. The suggested dimerization of CD4 through its CDR3-like region explains the important role that has been ascribed to this region in Th-cell activation and HIV-1-mediated fusion. Based on this model of the CD4 dimer and published results of different mutational analysis studies, a model was proposed for the complex of the CD4 dimer with two MHC-II molecules. The CD4 dimer allows tight binding to a large surface of MHC-II and the complex of CD4 and MHC-II reconciles mutational analysis studies that were previously incompatible. Moreover, the complex suggests how CD4 can dimerize through ligand binding.

Semisynthesis of phospholipase A2. The effect of substitution of amino-acid residues at positions 6 and 7 in bovine and porcine pancreatic phospholipases A2 on catalytic and substrate-binding properties.

The N-terminal alpha-helical region of phospholipase A2 is an important part of the enzyme for catalytic activity and lipid binding. Porcine pancreatic phospholipase A2 has Arg-Ser at positions 6 and 7, whereas the bovine enzyme has Asn-Gly. To pursue further the effects of these variable residues on differences in enzymatic properties, we prepared and studied the following semisynthetic analogs of epsilon-amidinated phospholipase A2 (AMPA): porcine [Ala7]AMPA, [Gly7]AMPA, [Asn6]AMPA, [Asn6-Gly7]AMPA and bovine [Ser7]AMPA and [Arg6-Ser7]AMPA. As we had previously found for the Asn6 leads to Arg bovine substitution, an Asn6-Gly7 leads to Arg5-Ser7 bovine substitution similarly improves the catalytic activity, the affinity for neutral lipid-water interfaces and the capacity to penetrate lecithin monolayers, while just changing Gly7 leads to Ser produces almost no effect on these properties. Ser7 leads to Ala and Ser7 leads to Gly substitutions in porcine AMPA did not affect penetration or lipid binding, although they did diminish catalytic activity (which is true of all substitutions made in the porcine enzyme). Arg6 leads to Asn substitution in porcine AMPA decreases penetration of lecithin monolayers, but not as much as it was improved by the Asn6 leads to Arg substitution in bovine AMPA. In contrast to the dramatic increase in affinity for lipid-water interfaces of Asn6 leads to Arg substitution in bovine AMPA, no decrease in affinity was found for Arg6 leads to Asn substitution in porcine AMPA. This difference is most likely due to the fact that the porcine enzyme has positively charged Lys and His in place of the Lys10, Glu17 pair that lie very close to residue 6 in the bovine structure. It can thus be conclude that (with the exception of Gly7 leads to Ser in bovine AMPA) all the substitutions tried at positions 6 and 7 in bovine and porcine AMPAs have definite effects on the catalytic activity.

The primary structure of the phosphatidylcholine-exchange protein from bovine liver. Isolation and characterization of the staphylococcal protease peptides and the amino-acid sequence of the N-terminal half (residues 1--122).

The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S-carboxymethylated derivative of the exchange protein at one specific site (Arg14-Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N-acetyl-methionine was the N-terminal residue and gave the sequence of the first 41 residues. The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complex sequence of the N-terminal half up to position 122.

Modification of arginine residues in porcine pancreatic phospholipase A2.

Although phenylglyoxal monohydrate reacts with Arg-6 in porcine pancreatic phospholipase A2, concomittantly the alpha-amino group of the N-terminal Ala-1 residue is quantitatively transaminated. Due to this latter reaction the enzymatic activity toward micellar substrate is lost irrespective of the Arg-6 modification. Upon reaction of [7-(14)C]phenylglyoxal monohydrate with alpha-amino-blocked phospholipase A2 analogs, two molecules of the reagent were incorporated per protein molecule, which were found to be present on Arg-6. Removal of alpha-amino-blocking groups after the modification reaction furnished the corresponding Arg-6-modified phospholipases possessing 30-38% of their original specific enzymatic activities in the egg-yolk assay. After reaction of 1,2-[1-(14)C]cyclohexanedione with porcine phospholipase A2 the crude reaction mixture was purified by chromatography on quaternary diethyl-(2-hydroxypropyl)aminoethyl-Sephadex in the presence of borate. A fraction was obtained containing a pure protein in which one molecule of 14C-labeled reagent per protein molecule was incorporated which was found to be localized almost exclusively on Arg-6. Cyclohexanedione modification of Arg-6 in phospholipase A2 does not significantly influence its catalytic activity when assayed toward monomeric and micellar substrates. The results of direct binding experiments using substrate analogs and of monolayer studies of the phospholipase modified at Arg-6 by cyclohexanedione are in agreement with previous findings that Arg-6 is involved in the interaction of the enzyme with lipid-water interfaces.

Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis.

Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.

Structural aspects of antibody-antigen interaction revealed through small random peptide libraries.

Two small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 10(7) hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.

Screening of a small set of random peptides: a new strategy to identify synthetic peptides that mimic epitopes.

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10(6)-10(7) random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC50s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest.

Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide.

Active immunization to immunomodulate regulatory processes suffers from the disadvantage that the antigen is usually 'self' and therefore poorly immunogenic. This has been well illustrated by the long-standing experience with immunocastration vaccines targeting GnRH, a ten amino acid peptide. Not all animals vaccinated with these vaccines are equally affected, even after multiple vaccinations. This is a severe handicap when immunocastration vaccines are applied to male piglets to circumvent surgical castration. Surgical castration is universally practised to prevent boar taint, produced in the testicles of mature boars. Alternative immunocastration is only acceptable if all animals are equally affected using a minimum of vaccinations. Vaccines based on the GnRH peptide itself cannot meet these goals. We showed that using a GnRH-like peptide, a 20 amino acid tandem repeat of the amino acid sequence of the GnRH peptide, these goals can be attained. Using the tandem GnRH peptide to vaccinate male piglets completely abolished the development and endocrinological functioning of the testicles, in contrast to monomer GnRH. These results show that superior antigens can be made for effective immunomodulation by appropriate alteration of the antigen.