A comparison of the leech Theromyzon tessulatum angiotensin I-like molecule with forms of vertebrate angiotensinogens: a hormonal system conserved in the course of evolution

After five steps of purification including gel permeation, anti-angiotensin I affinity column chromatography followed by reverse-phase HPLC, a peptide immunoreactive to two different antisera (anti-angiotensin I) was purified to homogeneity from extracts of the leech Theromyzon tessulatum. The first 14 amino acid residues of the purified peptide (DRVYIHPFLLXWG) established by automated Edman degradation, reveal the existence in leeches of an angiotensin I-like molecule close to human angiotensin I. The sequence of the purified peptide presents 78.5% of homology with the N-terminal part of human angiotensin. Moreover, in its sequence, this peptide presents the cleavage sites of vertebrate angiotensin metabolic enzymes, i.e. the renin and the angiotensin-converting enzyme. This finding constitutes the first biochemical characterization of an angiotensin I in Invertebrates. It also reflects the high conservation of angiotensins in the course of evolution, suggesting a fundamental role of this family in fluid homeostasis.     

Covalently linked peptides for enzyme-linked immunosorbent assay

A general method is described, by which synthetic peptides are covalently linked via their carboxyl group to microtiter plates (CovaLink) for enzyme-linked immunosorbent assay (ELISA). Plates were prepared by this method with an angiotensin II peptide and with an HIV-2 peptide and attachment detected by rabbit anti-angiotensin serum and with a positive serum from an HIV-2-infected patient, respectively, using the common ELISA procedure in the last steps. The method is simple to perform, it constitutes an alternative to the common ELISA method, and eliminates the risk of inadvertent loss of peptide during the procedure. The method is highly reproducible and has a high sensitivity. It may be used for either antigen or antibody detection.     

Interaction of the octapeptide angiotensin II with a high-affinity single-chain Fv and with peptides derived from the antibody paratope

The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of the filamentous M13 phage and was compared with the full-length antibody (Ab). The scFv showed the same specificity profile and affinity constant as the intact antibody (5.0x10(10) and 8.0x10(10) M(-1), respectively, by Scatchard analysis). Several peptides from the set of overlapping dodecapeptides covering the variable domains of 4D8 mAb were found to specifically bind biotinylated angiotensin II: peptides from the L1, L2, L3 and H1 regions had the strongest capacity to bind the antigen.     

Exogenous beta 2-microglobulin is required for antigenic peptide binding to isolated class I major histocompatibility complex molecules

Binding of antigenic peptides to purified class I major histocompatibility complex (MHC) molecules, as measured by antigen-specific cytolytic T lymphocyte (CTL) degranulation, was found to occur in the presence of serum but not in its absence. The role of soluble beta 2-microglobulin (beta 2m), a normal component of serum, in class I-peptide complex formation was therefore examined. Sera depleted of beta 2m did not support effective peptide binding to class I, but binding was restored in the presence of low concentrations of purified human beta 2m. Sequential incubation of immobilized class I with human beta 2m first, followed by peptide, resulted in antigenic complex formation, while reversing the order of pulsing could not. Similar results were obtained in experiments examining H-2Db, Kb and Kd with appropriate peptides and CTL. These results demonstrate that mature class I proteins are not able to directly bind peptide, but that interaction with exogenous beta 2m results in a structure that will subsequently bind peptide. Binding of exogenous beta 2m appears to result in "empty" class I molecules, possibly by exchange for endogenous beta 2m, with a concomitant loss of endogenous peptide.     

Apelin/APJ系统在心血管系统中的作用

Apelin是孤儿受体APJ的天然配体，apelin/APJ系统对心血管系统的作用主要为：可以通过拮抗血管紧张素Ⅱ的作用而舒张血管降低血压；可以作用于心肌引起强烈的正性肌力作用；在心力衰竭早期表达增加而心力衰竭晚期表达降低，因此，推测apelin/APJ系统可能是防止心力衰竭发生的有效的代偿方式。Apelin/APJ系统还可能参与调节胰腺对胰岛素的分泌，并且该系统还可以促进视网膜毛细血管的增生。Apelin/APJ系统在心血管系统的这些作用提示该系统可能在高血压、糖尿病血管病变、心力衰竭中发挥了一定的作用。     

Dot immunobinding and immunoblotting of picogram and nanogram quantities of small peptides on activated nitrocellulose

Nitrocellulose was activated with divinyl sulfone, a spacer of ethylenediamine, and glutaraldehyde. The aldehyde groups on the activated nitrocellulose, Nit-CHO, were stable through one month at 4 degrees C. Peptides were attached to the membrane by reaction of the amino group with the free carbonyl, forming peptide bonds. The decapeptide angiotensin I (AI), the octapeptide angiotensin II (AII), angiotensin analogues, Met- and Leu-enkephalin (Met-E and Leu-E) were tested on the membranes with specific rabbit antibodies (sRaAb) against the peptides, and visualized by horseradish peroxidase conjugated anti-rabbit antibody (HRP-anti-RaAb). With this technique AII could be detected with a sensitivity of 20 pg/cm2 and AI by 500 pg/cm2. Substitution of Ala7 for Pro7 in AI and AII caused a marked reduced binding of anti-AI and antid-AII antisera, respectively, and it completely abolished crossreactivity of anti-AI with Ala7-AII as well as anti-AII with Ala7-AI. Peptides from the gp41 and gp36 antigens corresponding to the sequence aa596-618 of the human immunodeficiency viruses type 1 and 2, HIV-1 and HIV-2, were tested on Nit-CHO with two human sera from infected patients. The serological reactions were specific for both the HIV-1 and HIV-2 peptide, respectively. This indicated that the technique could be exploited for serological testing of humans. Separation of peptides by high performance thin layer chromatography (HPTLC) and identification by immunoblotting was demonstrated with angiotensin analogues. After separation by HPTLC on silica aluminium plates the peptides were electrotransfered by semidry electroblotting on Nit-CHO, followed by specific antibody overlays and developed as for the dot immunobinding technique. This combined method enabled us to differentiate between closely related peptide analogues and it improved the sensitivity of peptide detection 100-1000 fold as compared to visualization by quenched fluorescence on chromatography plates.     

抗Resistin合成多肽抗体的制备及鉴定

目的制备抵抗素合成肽及其抗体.方法根据人抵抗素cDNA编码的氨基酸序列合成抵抗素分子22～34位的13个氨基酸的多肽(CSMEEAINERIQE),并利用MBS双功能试剂将人工合成多肽成功地与KLH进行偶联,免疫家兔制备抗抵抗素合成多肽的抗体,并经亲和层析进行了纯化.结果对此抗体进行鉴定的结果表明,抗人抵抗素合成多肽抗体可与天然抵抗素分子发生特异性反应,并可用于免疫沉淀和Western blot.结论该抗体的制备为Resistin分子功能的研究及肥胖和2型糖尿病的机制研究提供了有用的工具.     

Characterization of an HLA-A3 restricted human kidney specific T cell clone

BACKGROUND: The tissue specificity of a cytolytic T lymphocyte is determined by the MHC class I bound peptide it recognizes. We have developed an allorestricted human CTL clone, DBS 1.5, that recognizes an epitope found on HLA-A3+ kidney epithelial cells but not on HLA identical B-lymphoblastoid cells. The peptide recognized by this clone has been isolated from HPLC separated, acid eluted peptides from purified HLA class I molecules from HLA-A3+ kidney tissue. This peptide shares no sequence homology with any known protein. METHODS: To confirm the tissue specificity of the HLA-A3 restricted clone and the peptide it recognizes we have transfected the gene for HLA-A3 into a number of tumor cell lines both human and murine not expressing this antigen. The resulting transfected lines, confirmed by immunofluorescent staining, were used as targets to determine if expression of HLA-A3 alone was sufficient to allow recognition and lysis by the HLA-A3 restricted T cell clone. RESULTS: The HLA-A3 restricted T cell clone recognized HLA-A3 when expressed on human kidney epithelial cells and to a lesser extent on human lung epithelium and human epidermal cells. Of the tumor lines transfected with HLA-A3 only the human kidney tumor cell line was lysed at a level equal to the original kidney epithelial cell used to develop the clone. CONCLUSION: These results confirm that this allorestricted human CTL clone is tissue specific recognizing a peptide found in human epithelial tissue that must be presented in the context of HLA-A3 for recognition.     

Expression of anticardiolipin cofactor, human beta 2-glycoprotein I, by a recombinant baculovirus/insect cell system.

A full-length cDNA coding a human beta 2-glycoprotein I (beta 2-GPI) was introduced into the baculovirus genome to construct a recombinant baculovirus. Spodoptera frugiperda (Sf9) cells were infected with the recombinant baculovirus. A protein (mol. wt 43,000) reactive with anti-beta 2-GPI antisera was produced in the insect cells and secreted into the culture medium. The recombinant beta 2-GPI was purified from the culture supernatant by sequential cardiolipin (CL)-affinity column chromatography and gel filtration. The N-terminal amino acid sequence of the protein was identical to that of the native beta 2-GPI purified from human sera, and a putative signal peptide was cleaved from the secreted form of the recombinant protein. The purified recombinant protein had a cofactor activity which enhances CL binding of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE) patients, as well as the native beta 2-GPI. Thus, the beta 2-GPI expressed in insect cells is an immunologically active cofactor.     

Protaminase activity of plasma. III. Role of conversion enzyme (kininase II) in protaminase activity of plasma

Kininase I (carboxypeptidase N) and kininase II (angiotensin converting enzyme) were isolated from human plasma by gel filtration on Sephadex G 200, then separated and partially purified by ion exchange chromatography. These two partially purified enzymic preparations allowed us to demonstrate that protamine underwent an extensive degradation only when both kininases acted simultaneously. The effects of CoCl2, an activator, and of several inhibitors, amongst which captopril, suggest that the same enzymatic system is responsible for the in vitro protaminasic activity of diluted unfractionated plasma.     

A common epitope on major allergens from non-biting midges (Chironomidae)

A synthetic peptide corresponding to sequence 91-101 of the Chironomus thummi thummi haemoglobins (Chi t I) components III and IV was used to investigate binding and cross-reactivity with polyclonal human IgE and rabbit IgG antibodies and murine IgG1 subclass monoclonal antibodies (MABs). The synthetic peptide reacted with antibodies from all three mammals. The specificity of the reaction, especially that with IgE antibodies was shown by dose dependent inhibition with native Chi t I component III. Epitope(s) reacting with these antibodies were also found in haemoglobins from 14 of the 15 chironomid species analyzed. The synthetic peptide III/IV 91-101 enabled the identification of an important antigenic/allergenic determinant of the broadly distributed insect family Chironomidae. The antigenic potency of this synthetic peptide as shown by testing with human IgE, rabbit IgG and mouse MABs, and the widespread occurrence of the epitope in an identical or homologous sequence and/or superficial location, qualifies the peptide for therapeutic applications in medicine.     

Autoantibodies from patients with systemic lupus erythematosus bind a shared sequence of SmD and Epstein-Barr virus-encoded nuclear antigen EBNA I

SmD is one of the small nuclear ribonucleoproteins frequently targeted by autoantibodies in systemic lupus erythematosus. We isolated and characterized the antibodies present in lupus sera that are specific for the C-terminal region of SmD (sequence 95–119). This region is highly homologous to sequence 35-58 of the EBNA I antigen, one of the nuclear antigens induced by infection with Epstein-Barr virus. Antibodies affinity purified over a peptide 95–119 column were able to recognize this sequence in the context of the whole SmD molecule, as they reacted with blotted recombinant SmD. Anti-SmD 95-119 antibodies bound also the EBNA I 35–58 peptide and detected the EBNA I molecule in a total cell extract from Epstein-Barr virus-infected lines. A population of anti-SmD antibodies is, therefore, able to bind an epitope shared by the autoantigen and the viral antigen EBNA I. To investigate the involvement of this shared epitope in the generation of anti-SmD antibodies, we immunized mice with the EBNA I 35-58 peptide. Sera from immunized animals displayed the same pattern of reactivity of spontaneously produced anti-SmD antibodies. They reacted in fact with the EBNA peptide as well as with SmD 95-119 and recombinant SmD. These data suggest that molecular mimicry may play a role in the induction of anti-SmD autoantibodies.     

Isolation of decay accelerating factor (DAF) by a two-step procedure and determination of its N-terminal sequence

Decay-accelerating factor (DAF) from human red cell membranes was purified by a two-step procedure involving anion exchange and immunoaffinity chromatography. The DAF preparations were purified to homogeneity as judged by silver staining. In several experiments, the final product yields were approximately 23% of the total DAF present in the initial membrane extracts. The purified DAF retained its ability to inhibit the classical pathway C3-convertase and to reincorporate into cell membranes. An amino-terminal sequence was obtained by gas-phase sequencing. Rabbit antibodies to a synthetic peptide representing part of this sequence reacted with purified reduced membrane DAF by Western blotting and by a solid-phase immunoradiometric assay.     

Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif

A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells.     

Identification of a cross-reactive continuous B-cell epitope in enterotoxigenic Escherichia coli colonization factor antigen I.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine by means of several antigenically distinct colonization factors (CFs). Several of these CFs have very significant amino acid sequence similarity or identity, particularly in the N-terminal end. We have previously shown that a monoclonal antibody (MAb) raised against the subunits of colonization factor antigen I (CFA/I) fimbriae, which reacts with a peptide corresponding to the 25 N-terminal amino acids of such subunits, can inhibit attachment to intestinal cells of ETEC expressing heterologous as well as homologous CFs, with related amino acid sequences. In this study we have, by means of Pepscan analysis, determined the sequence of the MAb-specific linear epitope to be 15IDLLQ19. Parenteral immunization of rabbits with an N-terminal 25-mer synthetic peptide of CFA/I fimbrial subunit, either covalently coupled to bovine serum albumin or uncoupled, induced high titers of specific antibodies against this peptide as well as against CFA/I fimbriae. Increased titers against several heterologous CF fimbriae with a related N-terminal sequence were also induced, whereas no increase was seen against fimbriae with an unrelated sequence. Neither antisera against the coupled peptide nor antisera against the uncoupled peptide inhibited binding of CF-expressing bacteria to the human intestinal cell line Caco-2 in spite of high titers. The difference in the inhibitory capabilities of the antipeptide sera and the MAb might be due to slightly different epitope specificities. Thus, whereas the antipeptide sera bound to several continuous epitopes in the N-terminal end, none of them reacted specifically with the epitope 15IDLLQ19.     

Generation of a monoclonal antibody to P-glycoprotein peptides using tuberculin-PPD as a carrier

 A novel immunization protocol together with stringent selection criteria have been employed to generate a new murine monoclonal antibody (’’D8’’, isotype IgG₁, kappa) which specifically recognizes the human p170 drug resistance glycoprotein. This antibody is directed towards a defined peptide sequence located in the −COOH terminal region of the first external loop of the molecule. It is reactive with its epitope within the intact native glycoprotein in formalin-fixed and conventionally processed histological tissues, in flow-cytometric preparations and by Western blotting. The antibody precipitates its target peptide sequence from solution, and thus may be a useful reagent with which to establish an ELISA, RIMA or other similar assay. The peptide epitope recognized by this monoclonal antibody is restricted to the human MDR1 gene product and is not contained within the rodent homologue of the P-170 molecule. Immunohistochemistry has consistently failed to detect this epitope in rodent tissues, thus confirming that it does not exhibit the cross-reactivity of other currently available anti-P-glycoprotein monoclonal antibodies. The experience of this study emphasizes the value of the tuberculin-PPD (purified protein derivative) immunization protocol as a powerful strategy when generating monoclonal antibodies to small synthetic peptides. The resulting monoclonal antibody (D8) will be an invaluable reagent with which to analyse P-170 glycoprotein expression when assessing the role of multidrug resistance in human cancers. Received: 11 August 1997 / Accepted: 15 September 1997     

Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification.

Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and intact pili. We are currently studying the importance of these defined epitope-containing regions as vaccine candidates.     

Reconstitution of class I MHC molecules expressed in E. coli and complexed with single antigenic peptides.

The HLA-Cw3 heavy chain has been expressed at high level as insoluble protein aggregates in E. coli. The protein aggregates dissolved in strong denaturant solution were efficiently reconstituted by removal of denaturant in the presence of an HLA-Cw3 binding peptide (FAM) and beta 2m. The reconstituted HLA-Cw3/FAM protein binds specifically to a p58 natural killer cell inhibitory receptor, a natural ligand. The HLA-A2 molecule has also been reconstituted in complex with either of a peptide from myelin associated glycoprotein (MAG) or a peptide from the GAG protein of human immunodeficiency virus. The HLA-A2/MAG protein crystallized under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. The reconstitution method has yielded an abundant supply of HLA molecules complexed with single antigenic peptides, and may be of general utility in reconstituting any class I MHC molecules. However, the HLA molecules could not be reconstituted either without a peptide or with an irrelevant peptide. Using this property, the reconstitution method could be used to determine whether a peptide is restricted/bound to certain class I MHC molecule.     

A novel radioimmunoassay for human erythropoietin using a synthetic NH2-terminal polypeptide and anti-peptide antibodies

Antibodies prepared by immunizing rabbits with a synthetic polypeptide representing the first 26 amino acids of the proposed NH2-terminal sequence of human erythropoietin cross-react with peptide, highly purified 125I-labeled erythropoietin and biologically active erythropoietin. We have now developed a rapid and convenient radioimmunoassay for the hormone using 125I-labeled peptide and anti-peptide antibodies. This peptide/anti-peptide approach completely circumvents the relative lack of availability of highly purified 125I-labeled erythropoietin and of erythropoietin for immunization purposes and makes a radioimmunoassay for the hormone universally available to investigators. This concept may be applied as a general method for designing radioimmunoassays of molecules available in severely restricted quantities.     

Correlation between chlamydial infection and autoimmune response: molecular mimicry between RNA polymerase major σ subunit from Chlamydia trachomatis and human L7

L7 is one of the ribosomal proteins frequently targeted by autoantibodies in rheumatic autoimmune diseases. A computer search revealed a region within the immunodominant epitope of L7 (peptide II) that is highly homologous to amino acid sequence 264 – 286 of the RNA polymerase major σ factor of the eubacterium Chlamydia trachomatis. Anti-L7 autoantibodies affinity purified from the immunodominant epitope were able to recognize this sequence as they reacted with purified recombinant σ factor. Immunofluorescence labeling experiments on C. trachomatis lysates revealed a punctate staining pattern of numerous spots when incubated with the affinity-purified anti-peptide II autoantibodies. Binding of autoantibodies to peptide II was inhibited by the homologous σ peptide. This is the first demonstration of epitope mimicry between a human and a chlamydial protein on the level of B cells. Antibody screening revealed a significant correlation between the presence of anti-L7 autoantibodies and C. trachomatis infection in patients with systemic lupus erythematosus and mixed connective tissue disease. Our results suggest that molecular mimicry is involved in the initiation of anti-L7 autoantibody response and may represent a first glance into the immunopathology of Chlamydia with respect to systemic rheumatic diseases.