Junctional Peptide Research Articles

Investigating the Protective Antibody‐Circumsporozoite Complex of Plasmodium falciparum and its Role in Vaccine Development

Malaria is a parasitic disease associated with high fevers, shaking chills, and flu‐like symptoms. It is typically spread by mosquitoes and caused by infection with Plasmodium falciparum (Pf). Due to its high transmission rate and metastatic properties, malaria is a major public health concern. Insecticide resistance has led to alternative methods of disease management, such as vaccine development. The circumsporozoite protein of Plasmodium falciparum malaria is the foundation for many malaria vaccines, which are designed to block the transmission of Pf. The most promising vaccine candidate is RTS,S, a recombinant protein designed around the central NANP repeat and C‐terminal region of circumsporozoite protein. The repeat region consists of 19 repeating NANP amino acid sequences, allowing multiple antibodies to bind simultaneously. Molecular dynamics simulations and crystallography structures show the importance of the NANP amino acid sequences for binding. The Summit Country Day School MSOE Center for Biomolecular Modeling MAPS Team used 3D modeling and printing technology to examine the structure‐function relationship of the human Fab688 circumsporozoite complex and its associated binding of NANP repeat peptides to protective antibodies. Antibodies that bind to the fully extended peptide are perpendicular to the heavy chain‐light chain interface and rely on the first NANP repeat that follows the only NPDP sequence in the junctional region of Pf circumsporozoite protein, as this arrangement favors free energy contributions and results in superior protection. Junctional peptide antibody protection may relate to the ability to cross‐react with the NANP repeat region. Amino acids Asn11 and Asn13 on the junctional peptide form a hydrogen bond network with Fab668. This web of hydrogen bonds, which connects the residues that form the binding pocket, stabilizes it and contributes to stronger binding.Fab688 binds to the NANP repeat region with equal or better affinity, shedding light on the affinity and specificity for the junctional region of circumsporozoite. This research lays the framework for future advancements in vaccines by revealing important targets and thereby limiting the spread of infectious diseases.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP.

During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles.

Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.

Trans-spliced heat shock protein 90 modulates encystation in Giardia lamblia.

BackgroundHsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear.Principle FindingIn this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation.SignificanceOur results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.

Insights into the role of the junctional region of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase

BackgroundPlasmodium falciparum dihydrofolate reductase-thymidylate synthase (pfDHFR-TS) is a well-defined target of anti-malarial drug, such as pyrimethamine and cycloguanil. Emergence of malaria parasites resistant to these drugs has been shown to be associated with point mutations of the gene coding for the target enzymes. Although the 3D-structure of P. falciparum bifunctional pfDHFR-TS has been reported previously, relatively little is known about the interactions between the pfDHFR and pfTS domains and the roles of the junctional region that links the two domains together. Therefore, a thorough understanding of the interaction of the two domains and the role of the junctional region of this target is important as the knowledge could assist the development of new effective anti-malarial drugs aimed at overcoming drug-resistant malaria.MethodsA system was developed to investigate the interaction between pfDHFR and pfTS domains and the role of the junctional region on the activity of the recombinant pfTS. Based on the ability of co-transformed plasmids coding for pfDHFR and pfTS with truncated junctional region to complement the growth of TS-deficient Escherichia coli strain χ2913recA(DE3) on minimum media without thymidine supplementation, active pfTS mutants with minimal length of junctional region were identified. Interactions between active pfDHFR and the pfTS domains were demonstrated by using a bacterial two-hybrid system.ResultsUsing TS-deficient E. coli strain χ2913recA(DE3), the authors have shown for the first time that in P. falciparum a junctional region of at least 44 amino acids or longer was necessary for the pfTS domain to be active for the synthesis of thymidylate for the cells. Truncation of the junctional region of the bifunctional pfDHFR-TS further confirmed the above results, and suggested that a critical length of the junctional peptide of pfDHFR-TS would be essential for the activity of TS to catalyze the synthesis of thymidylate.ConclusionThe present study demonstrated the interactions between the pfDHFR and pfTS domains of the bifunctional pfDHFR-TS, and revealed that the junctional region linking the two protein domains is essential for the expression of catalytically active pfTS domain. The findings could be useful since inhibition of the pfDHFR-TS domain-domain interaction could form a basis for the development of new anti-malarial drugs based on targeting the non-active site region of this important enzyme.

Evidence that Actin-Myosy Cycling in Muscle may not Pass through Rigor Configuration

In 1989, Sutoh, Tokunaga and Wakabayashi (J Mol Biol 206:357) prepared a monoclonal antibody (IgG) directed to junctional peptide between 50- and 20-kDa segments of myosin heavy chain, and showed that the antibody attaches around the tip of myosin heads facing actin filaments in muscle. using the same antibody, we successfuly position-mark the tip of myosin heads in synthetic myosin filaments kept in hydrated state in the gas environmental chamber, and succeeded in recording ATP-induced myosin head movement electron microscopically (Sugi et al. PNAS 105: 17396, 2008). We have examined the effect of this antibody on both ATP-dependent in vitro actin-myosin sliding and Ca-activated contraction of permealized rabbit psoas muscle fibers. Unexpectedly, the antibody (concentration, up to 2mg/ml) had no appreciable effect on the velocity profile of actin-myosin sliding and the contraction characteristics of muscle fibers, including magnitude of the maximum Ca-activated isometric force, the maximum unloaded shortening velocity, ans MgATPase activity during contraction. Since the junctional peptide is located between the two main actin- binding sites of myosin heads during formation of rigor linkages, attachment of massive antibodies to myosin heads completely covers their actin- binding sites. The possibility that the antibody does not diffuse into muscle fibers is excluded by the fact that other kinds of antibody diffuse into muscle fibers to inhibit Ca-activated force generation (e.g., Sugi et al. PNAS 89: 6134, 1992). Our work, therefore, suggests strongly that attachment-detachment cycle between actin and myosin in muscle may not pass through rigor configuration. It seems possible that so-called strong actin-myosin binding might be due to some electrostatic mechanism.

Abstract 2395: Prediction of HLA-DRB1-restricted CD4+ T cell epitopes in the TEL-AML1 fusion oncoprotein of childhood ALL: population coverage, and design of heteroclitic peptides

Abstract In-frame fusion of two transcription factor genes, TEL (ETV6) on chromosome 12p and AML1 (RUNX1) on chromosome 21q, is thought to be an initiating event in ∼25% of childhood acute lymphoblastic leukemias (ALL). TEL-AML1 fusion leads to the expression of a chimeric oncoprotein which interferes with the function of the normal AML1 protein, leading to arrested B cell development, and enhanced B cell progenitor self-renewal. In vitro studies showed that a TEL-AML1 junctional peptide induced HLA-DP-restricted CD4+ T cell responses. Although expression of TEL-AML1 by pro-leukaemic cells makes it an attractive immunotherapeutic target, information on the number, position, and population distribution of HLA-DR-restricted CD4+ T cell epitopes in TEL-AML1 has not been published. Here, we used the artificial neural network algorithm, NetMHCIIpan (Nielsen et al, PLoS Computational Biology, 2008, 4(7), e1000107) to predict DR-restricted T cell epitopes in TEL-AML1. The 797 residue fusion oncoprotein was screened using NetMHCIIpan to identify 9-mer DR-binding core peptides. 15-mer peptide sequences were submitted in FASTA format to the NetMHCIIpan server (http://www.cbs.dtu.dk/services/NetMHCIIpan/) to facilitate discovery of peptides with strong binding affinity (IC50:<50 nM). Consensus T cell epitopes predicted to provide maximum coverage across common DR alleles in 4 ethnic groups were identified. Overlapping 15-mer junctional sequences were screened to identify a 9-mer DR-binding core sequence, and P1,4,6, and 9 substituted with all 20 amino acids to identify junctional sequences with enhanced (heteroclitic) binding affinity. NetMHCIIpan predicted 45 TEL-AML1 epitopes binding to 22 common DR alleles in 4 ethnic groups. Fourteen epitopes are in TEL and 31 in AML1, with 5 epitopes binding to 5-8 DR alleles, and 1 binding to 10 alleles. Three TEL peptides achieve the greatest population coverage across all ethnic groups, one peptide (266IQLMPSPIM274) giving maximal coverage (25.5-50.1%). Weak DRB1*0101-restricted binding of a junctional peptide (332IGRIA/ECIL340) (415 nM) was strongly increased (52 nM) by substituting serine for glutamic acid at the P6 position. A junctional peptide (332FGRMA/SCIV343) with substitutions at P1, P4 and P9 had enhanced DRB1*0101 affinity (13 nM). These predictions suggest wide population coverage of a small number of strongly binding TEL-AML1 epitopes. The binding affinity of a junctional peptide is enhanced by substituting key anchor residues. Functional studies are now required to distinguish CD4+ T cell-activating TEL-AML1 epitopes inducing anti-leukemic, autoimmune, and regulatory T cell responses. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2395.

Vector prime protein boost vaccination in the setting of myeloablative-induced lymphopenia suppresses growth of leukemia and solid tumors

We have developed a vaccine, which is designed to induce tumor-associated antigen (TAA)-specific T cells and antibodies in the setting of profound lymphopenia induced by myeloablative therapy and T-cell-depleted bone marrow transplantation. Test mice were injected subcutaneously (sc) with the 32DP210Bcr-Abl cell line, which is positive for the p210Bcr-Abl protein (Group 1). In Group 2, 7 days after injection of the 32DP210Bcr-Abl positive cell line, the mice received 900 cGy total body irradiation (TBI) followed in 1 h by the intravenous infusion of 10 million T-cell-depleted syngeneic bone marrow cells (TCDBMT) (Group 2). The leukemia-bearing group received an intravenous injection of 10 million spleen cells (donor lymphocyte infusions) from unvaccinated (Group 3) and TAA/ecdCD40L-vaccinated (Group 4) syngeneic mice 3 days after completion of the TBI and TCDBMT. Groups 3 and 4 mice received three additional sc vaccinations at 7-day intervals with the TAA/ecdCD40L vaccine, in which the TAA was taken from the junctional peptide of the P210bcr-Abl protein. The survival of Groups 3 and 4 mice was significantly longer than that in Groups 1 and 2 mice. Vaccinated mice from Group 4, which developed complete responses, survived up to 350 days post-injection of the leukemia cells without any evidence of leukemia regrowth.

TAA/ecdCD40L VPP Vaccination Induces Robust Adaptive Immune Response Even in Individuals with Post Transplantation Lymphopenia

Individuals of advanced chronological age exhibit an impaired immune response to vaccines. This may be due to a reduction in the ratio of antigen naïve/memory CD4 and CD8 T cells and acquisition of functional defects in activated “helper” CD4 T cells (eg diminished CD40 ligand (CD40L) expression) during the aging process. The absence of the CD40L on activated CD4 helper T cells reduces the magnitude of expansion of antigen specific T and B cells induced by vaccination. In order to circumvent this defective response to vaccines among individuals in the fifth and sixth decades of life, our laboratory has developed an adenoviral vector (Ad-sig-TAA/ecdCD40L) vaccine which is designed to overcome the absence of CD40L expression in activated CD4 helper T cells in older individuals. The subcutaneous (sc) injection of this vector leads to the release of a fusion protein composed of a TAA linked to the extracellular domain (ecd) of the CD40L, which binds to the CD40 receptor on DCs, activates the DCs, and leads to the presentation of TAA fragments on Class I MHC. Two sc injections of the TAA/ecdCD40L protein as a booster following the sc administration of the Ad-sig-TAA/ecdCD40L vector (we call this the TAA/ecdCD40L VPP vaccine) expands the magnitude of the cellular and humoral immune response induced by the vector in 18 month old aged mice as well as in younger mice. In order to explore ways of further amplifying the immune response induced by this vaccine, we decided to test the feasibility of using this vaccine following treatments which reduce the number of T cells in the body of the test subject. We hypothesized that during states of chemotherapy or radiation induced lymphopenia, the number of negative regulatory CD4CD25FoxP3 T cells would be reduced, and all of the regulatory signals in the T cell compartment would be promoting expansion of T cells, thus creating an ideal state for vaccination. To test this hypothesis, we injected 100,000 cells from an established neoplastic cell line sc. Three days later, we administered myeloablative doses of total body irradiation (TBI) followed by a T cell depleted syngeneic bone marrow transplant (TCDBMT) to reconstitute neutrophil and platelet production. Three days following the TBI and TCDBMT, we intravenously infused donor lymphocytes (DLI) from a TAA/ ecdCD40L VPP vaccinated syngeneic donor. Four weeks later, we vaccinated the recipient mouse further with TAA/ecdCD40L sc injections. We tested this for a TAA composed of a junctional peptide from the p210Bcr-Abl protein of chronic myelogenous leukemia (CML) and for the E7 protein of the human papilloma virus (HPV). We found that in the case of the BcrAbl/ecdCD40L VPP vaccine, 50% of the mice treated with TBI, TCDBMT, ten million lymphocytes (DLI) from BcrAbl/ecdCD40L VPP vaccinated syngeneic donors followed in 4 weeks by 3 BcrAbl/ecdCD40L protein sc injections of the recipient test mouse, developed a complete response with the vaccination and that these mice remained disease free beyond 250 days after injection of the P210Bcr-Abl positive 32D leukemia cells, whereas C56BL/6J test mice treated with TBI and TCDBMT without DLI from vaccinated donors nor sc BcrAbl/ecdCD40L sc booster vaccination following injection with the p210Bcr-Abl positive 32D myeloid leukemia cell line all died by day 32. Mice treated with TBI, TCDBMT, DLI from unvaccinated donors followed by vaccination of the recipient with 3 sc BcrAbl/ecdCD40L protein injections exhibited a degree of leukemia suppression that was equal to mice receiving TBI, TCDBMT, DLI from a BcrAbl/ecdCD40L VPP vaccinated donor and BcrAbl/ecdCD40L vaccination. To test the effect of the TAA/ecdCD40L VPP vaccine against an antigen associated with an epithelial neoplasm, we injected 100,000 E7 positive TC-1 mouse cancer cells into syngeneic C57BL6J mice followed in 3–5 days by myeloablative doses of TBI and engrafting doses of TCDBMT. Three days later, the mice received 10 million spleen cells from syngeneic donor mice previously vaccinated with the E7/ecdCD40L VPP vaccine. Finally, 4 weeks later, the test mice received sc E7/ecdCD40L protein booster injections. The vaccinated mice achieved much greater degrees of tumor suppression than was seen following TBI and TCDBMT without DLI from vaccinated donors. These studies show that it is possible to induce a robust adaptive immune response by vaccination with the TAA/ecdCD40L VPP vaccine even in severely lymphopenic individuals.

Clinical BCR-ABL Peptide Vaccination in Chronic Myeloid Leukaemia: Results of the EPIC Study.

Chronic myeloid leukaemia (CML) is characterised by the BCR-ABL oncoprotein. The amino-acid sequences spanning the junctional region are completely leukaemia-specific. In vitro pulsing of antigen presenting cells elicits immune response to CML cells. Vaccination of CML patients with peptides from this junctional region could therefore elicit/augment immune responses to CML cells. In our Evaluation of Peptide Immunisation in CML (EPIC) study, the patient's entry requirements were as follows:first chronic phase of CML,expression of e14a2 (b3a2) BCR-ABL transcript, andprior treatment with imatinib daily (at least 400mg) at a stable dose for at least 6 months.Each patient received intradermally a cocktail of 3 BCR-ABL peptides:a 9-mer spanning the e14a2 region,the same 9-mer linked to PADRE (a 15-mer non-natural peptide shown to activate CD4+ T cells), anda 13-mer consensus e14a2 junctional peptide linked to PADRE.Peptides were administered at either 100 (5 patients), 300 (5 patients), 600 (5 patients), or 1000μg (4 patients) with sargramostim on 6 occasions over 2 months. Immune responses to the vaccine were monitored by IFN-γ and IL-5 ELISPOT assays on peripheral blood mononuclear cells. Molecular responses were assessed by quantitative real-time PCR of BCR-ABL mRNA. At entry no patient showed a detectable immune response to PADRE, but all 19 patients had detectable CD4+ T cells responses within 3 months of commencing vaccination. This indicated that the vaccination protocol was capable of stimulating T cell responses in all 19 patients. Immune responses to the 9-mer BCR-ABL junctional peptide used in the vaccine were detected in 11/19 patients, and demonstrated to be CD8+ T cells by cytokine analysis in flow cytometry. BCR-ABL immune responses were also assessed against a longer 18-mer peptide spanning the whole e14a2 junctional region. CD4+ T cells specific for this 18-mer peptide were detected in 14/19 patients. Interestingly, immunophenotyping indicated that these BCR-ABL-specific T cells were of a memory phenotype (CD45RO+). Serial molecular responses were available for at least 12 months on all cases. Of the 6 patients not in major cytogenic response (MCR) at baseline, molecular improvement was only observed in one case. However 12/13 patients in at least MCR at baseline had at least a 1 log fall in BCR–ABL transcripts, though this occurred several months after completing vaccination. Moreover, vaccination improved the fall in BCR–ABL transcripts in patients who had received imatinib for more than 12 months. These data show that peptide immunisation in CML can elicit anti-BCR-ABL peptide responses in CD4+ and CD8+ T cells. It also demonstrates that BCR-ABL peptide vaccination may improve control of CML, especially in patients responding well to imatinib.

B3a2 BCR-ABL fusion peptides as targets for cytotoxic T cells in chronic myeloid leukaemia.

Peptide sequences spanning the BCR-ABL protein junction potentially constitute novel leukaemia-specific antigens. 9-mer b3a2 fusion peptides have been reported to bind with high affinity to HLA-A3, -A11 and -B8. We have studied the effect of b3a2 BCR-ABL junctional peptides on the cytotoxic T-cell (CTL) response against normal and chronic myeloid leukaemia (CML) cells. Antigen-presenting cells (APCs) were prepared from HLA-A3- or -B8-positive peripheral blood mononuclear cells (PBMCs) by incubation with phytohaemagglutinin (PHA) and interleukin (IL)-2 for 7 d. These APCs were pulsed with the respective b3a2 junctional peptide in the presence of beta2-microglobulin and were then used to challenge autologous PBMCs at 7-d intervals in the presence of IL-2, IL-6, IL-7 and IL-12. On subsequent exposure to target cells (either further pulsed normal APCs or unpulsed CML cells), specific HLA-restricted CTL responses were observed against all HLA-A3/-B8 matched normal target cells tested, but not to targets that were HLA mismatched. Cytotoxicity was also induced against HLA-A3/-B8 unpulsed CML cells, but not against unmatched CML cells. These data indicate (i) that endogenous BCR-ABL junctional peptides may be presented by CML cells and (ii) that exogenous peptides are potential stimulators of autologous antileukaemic CTLs.

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoan Paramecium tetraurelia.

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan, Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a approximately 500 kb macronuclear chromosome and is transcribed as an mRNA of approximately 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of approximately 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, the P. tetraurelia gene is most similar to that from Toxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.

Transforming growth factor β1 (TGF-β1) inhibits GAP junctional communication and peptide hormone production during human trophoblast differentiation

Transforming growth factor β1 (TGF-β1) inhibits GAP junctional communication and peptide hormone production during human trophoblast differentiation

Analysis of Amelogenin Proteins Using Monospecific Antibodies to Defined Sequences

Amelogenins are the predominant proteins found in the developing enamel matrix and are believed to play a crucial role in normal mineralization. Although the amelogenin gene is found as a single copy in all species in which it has been examined, multiple amelogenin polypeptides ranging in size from 5 to 25 kDa are obtained upon extraction of developing enamel matrix, making identification and characterization of individual components difficult. This heterogeneity may be ascribed to transcription of divergent genes located on the X and Y chromosomes, alternative splicing of the primary transcripts, physiologic degradative processing, and artefactual degradation. In order to characterize individual components, antibodies were produced to the following peptides: (1) QPLQPMQPMQPLQPLQPL (corresponding to the repeat sequence encoded only in the bovine X chromosome gene), (2) IRHPPLPP (corresponding to a unique sequence generated by alternative splicing found in leucine-rich amelogenin peptide (LRAP), (3) LPDLPLEAWPATDKTKREEVD corresponding to the amelogenin carboxy-terminus. Amelogenin proteins obtained from fetal bovine molars were subjected to SDS PAGE and Western electrotransfer, and immuno-ultrastructural analysis. These analyses demonstrated that: (1) the distribution of amelogenin polypeptides isolated from male fetuses differed appreciably from that of females, (2) the LRAP junctional peptide sequence can be specifically identified, and (3) the LRAP peptide can be immunolocalized in the enamel matrix of both males and females.

T cell receptor antagonism mediated by interaction between T cell receptor junctional residues and peptide antigen analogues.

A major objective of new rational immunosuppressive therapies is to be able to inhibit deleterious T cell responses in an Ag-specific manner. Recently, a novel approach to inducing Ag-specific nonresponsiveness in T cells, termed receptor or TCR antagonism, has been described. Several analogues of the influenza hemagglutinin (H3) peptide HA307-319 were shown to block recognition of the native HA307-319 peptide by a DR1-restricted T cell clone at concentrations 1,000- to 10,000-fold less than required to competitively inhibit HA307-319 binding to DR1. These Ag analogues that inhibited T cell recognition differed from the stimulatory antigenic peptide only at single positions. How such limited changes affect the ternary complex interactions of TCR, peptide, and DR remains unclear. In the present study, we have utilized two different DR5-restricted T cell clones that exhibit mutually exclusive specificities for HA peptides with or without substitutions at position 313 to investigate the molecular requirements for receptor antagonism. In this reciprocal model system, we show that a single peptide/DR complex can antagonize one T cell clone while stimulating another. A comparison of nucleotide sequences derived from the TCR of the T cell clones that were either antagonized or stimulated by the peptide analogue/DR5 complex indicated that the different responses of these T cells result from minor differences in the amino acid residues in junctional regions that most likely interact directly with position 313 of the peptide analogues. Our results suggest that TCR antagonism is an Ag-specific phenomenon in which T cells are inhibited by interactions involving TCR residues required for the recognition of conventional Ag and the altered residues in peptide analogues.

Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.

Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C.

Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (approximately 3 pS). Prior to complete electrical uncoupling, various discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC, isolated from rat brain, also caused electrical uncoupling. The presence of 0.1 mM dibutyryl cyclic AMP and 5 mM ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (less than or equal to 1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed.

Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins.

In protozoa, thymidylate synthase (TS; EC 2.1.1.45) and dihydrofolate reductase (DHFR; EC 1.5.1.3) exist as a bifunctional protein. Full-length cDNA and genomic DNA clones encoding the bifunctional TS-DHFR from Leishmania tropica have been isolated, characterized, and sequenced. There are no intervening sequences within the coding region for TS-DHFR, and the G+C content is high, with a strong bias for codons possessing guanine or cytosine in the third base. From the predicted protein sequence for TS-DHFR, we conclude that the enzymes exist as separate structural domains with DHFR located at the amino terminus and TS at the carboxyl terminus; in between, there is a junctional peptide predicted to consist of a random coil flanked by two alpha-helices that corresponds to the site of a putative gene fusion. Comparison of the predicted amino acid sequence of L. tropica TS-DHFR with sequences of the monofunctional enzymes from other sources shows that the TS domain is much more conserved than is the DHFR domain and that both enzymes are more related to their monofunctional counterparts in vertebrates than in prokaryotes. Most of the recognized elements of secondary structure of other DHFRs, as ascertained by x-ray crystallography, are found in the predicted structure of the DHFR domain of the bifunctional protein.

Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein.

Pig kidney microvillar proteins were extracted with octyl beta-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, 'right-side-out', orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. Electron microscopy of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5 to 9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.