Proteome Analysis of Rheumatoid Arthritis Gut Mucosa.

Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular and membrane proteins and included known targets of anticitrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS data have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.

Excessive folic acid intake and relation to adverse health outcome.

The recent increase in the intake of folic acid by the general public through fortified foods and supplements, has raised safety concern based on early reports of adverse health outcome in elderly with low B12 status who took high doses of folic acid. These safety concerns are contrary to the 2015 WHO statement that "high folic acid intake has not reliably been shown to be associated with negative healeffects". In the folic acid post-fortification era, we have shown that in elderly participants in NHANES 1999-2002, high plasma folate level is associated with exacerbation of both clinical (anemia and cognitive impairment) and biochemical (high MMA and high Hcy plasma levels) signs of vitamin B12 deficiency. Adverse clinical outcomes in association with high folate intake were also seen among elderly with low plasma B12 levels from the Framingham Original Cohort and in a study from Australia which combined three elderly cohorts. Relation between high folate and adverse biochemical outcomes were also seen in the Sacramento Area Latino Study on Aging (High Hcy, high MMA and lower TC2) and at an outpatient clinic at Yale University where high folate is associated with higher MMA in the elderly but not in the young. Potential detrimental effects of high folic acid intake may not be limited to the elderly nor to those with B12 deficiency. A study from India linked maternal high RBC folate to increased insulin resistance in offspring. Our study suggested that excessive folic acid intake is associated with lower natural killer cells activity in elderly women. In a recent study we found that the risk for unilateral retinoblastoma in offspring is 4 fold higher in women that are homozygotes for the 19 bp deletion in the DHFR gene and took folic acid supplement during pregnancy. In the elderly this polymorphism is associated with lower memory and executive scores, both being significantly worse in those with high plasma folate. These and other data strongly imply that excessive intake of folic acid is not always safe in certain populations of different age and ethnical/genetic background.

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli.

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope ß is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a ß epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

Chemically self-assembled antibody nanostructures as potential drug carriers.

Chemically self-assembled antibody nanorings (CSANs) displaying multiple copies of single-chain variable fragments can be prepared from dihydrofolate reductase (DHFR) fusion proteins and bis-methotrexate (bisMTX). We have designed and synthesized a bisMTX chemical dimerizer (bisMTX-NH(2)) that contains a third linker arm that can be conjugated to fluorophores, radiolabels, and drugs. Monovalent, divalent, and higher-order AntiCD3 CSANs were assembled with a fluorescein isothiocyanate (FITC)-labeled bis-methotrexate ligand (bisMTX-FITC) and found to undergo rapid internalization and trafficking by HPB-MLT, a CD3+ T-leukemia cell line, to the early and late endosome and lysosome. Because the fluorescence of bisMTX-FITC when incorporated into CSANs was found to be significantly greater than that of the free ligand, the stability of the endocytosed AntiCD3 CSANs could be monitored. The internalized CSANs were found to be stable for several hours, while treatment with the nontoxic DHFR inhibitor trimethoprim resulted in a rapid loss (>80%) of cellular fluorescence within minutes, consistent with efficient intracellular disassembly of the nanorings. Over longer time periods (24 h), cellular fluorescence decreased by 75-90%, regardless of whether cells had been treated with DMSO or trimethoprim. Although bisMTX is a potent inhibitor of DHFR, it was found to be nontoxic (GI(50) > 20 µM) to HPB-MLT cells. In contrast, AntiCD3 CSANs prepared with bisMTX were found to be at least 13-fold more cytotoxic (GI(50) = 0.5-1.5 µM) than bisMTX at 72 h. Consistent with our findings from CSAN stability studies, no increase in cytotoxicity was observed upon treatment with trimethoprim. Taken together, our results suggest that cell receptor targeting CSANs prepared with trifunctional bisMTX could be used as potential tissue selective drug carriers.

New developments in malaria diagnostics: monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase, heme detoxification protein and glutamate rich protein.

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, KD) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have KD values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (KD of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1-13), making them promising reagents for further test development.

Knockout of the dhfr-ts gene in Trypanosoma cruzi generates attenuated parasites able to confer protection against a virulent challenge.

BACKGROUND: Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes. METHODS AND FINDINGS: By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts(+/-) mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8(+) T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection. CONCLUSION: This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections.

Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition.

Although it has been known for many years that antibodies display properties characteristic of allosteric effectors, the molecular mechanisms responsible for these effects remain poorly understood. Here, we describe a single-domain antibody fragment (nanobody) that modulates protein function by constraining conformational change in the enzyme dihydrofolate reductase (DHFR). Nanobody 216 (Nb216) behaves as a potent allosteric inhibitor of DHFR, giving rise to mixed hyperbolic inhibition kinetics. The crystal structure of Nb216 in complex with DHFR reveals that the nanobody binds adjacent to the active site. Half of the epitope consists of residues from the flexible Met20 loop. This loop, which ordinarily oscillates between occluded and closed conformations during catalysis, assumes the occluded conformation in the Nb216-bound state. Using stopped flow, we show that Nb216 inhibits DHFR by stabilising the occluded Met20 loop conformation. Surprisingly, kinetic data indicate that the Met20 loop retains sufficient conformational flexibility in the Nb216-bound state to allow slow substrate turnover to occur.

Potent control of tumor growth by CEA/CD3-bispecific single-chain antibody constructs that are not competitively inhibited by soluble CEA.

Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.

Complete regression of large solid tumors using engineered drug-resistant hematopoietic cells and anti-CD137 immunotherapy.

Combining chemotherapy and immunotherapy is problematic because chemotherapy can ablate the immune responses initiated by modulators of the immune system. We hypothesized that protection of immunocompetent cells from the toxic effects of chemotherapy, using drug resistance gene therapy strategies, would allow the combined use of chemotherapy and immunotherapy. In wild-type mice, the antitumor effectiveness of an immunotherapy regimen employing an agonistic anti-CD137 antibody is diminished with escalating doses of the antifolate trimetrexate (TMTX). Using retroviral gene transfer of a mutant form of dihydrofolate reductase (L22Y-DHFR), hematopoietic stem cells were genetically engineered to withstand the toxic effects of TMTX. Mice transplanted with L22Y-DHFR-modified bone marrow were then challenged with AG104 sarcoma cells and treated with TMTX only, anti-CD137 only, or a combination of chemotherapy and immunotherapy. Although tumor burden was transiently decreased during TMTX administration, no mice treated with TMTX alone survived the tumor challenge, whereas approximately 40% of transplanted mice treated with anti-CD137 alone survived. However, 100% of mice survived with complete tumor regression after transplantation with L22Y-DHFR-transduced bone marrow followed by combined treatment with TMTX and anti-CD137. In addition, adoptive transfer of splenocytes from cured mice extended the survival of tumor- bearing animals by approximately 3 weeks compared with controls. Therefore, protection of the hematopoietic system can allow for the combined administration of chemotherapy and immunotherapy, which results in complete tumor clearance.

Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum.

BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64-100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64-100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits. RESULTS AND CONCLUSIONS: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

Epitope mapping of ligand-receptor interactions by diffusion NMR.

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation. Therefore, this diffusion artifact can be used to characterize the ligand binding epitope. The concept was investigated using dihydrofolate reductase (DHFR) and its ligand trimethoprim (TMP), and the epitope map of TMP on DHFR generated with this method is in excellent agreement with the structural and dynamic studies by crystallography and NMR, as well as the medicinal chemistry results.

A dhfr-ts- Leishmania major knockout mutant cross-protects against Leishmania amazonensis.

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.

Induction and persistence of a cytotoxic T lymphocyte (CTL) response against a herpes simplex virus-specific CTL epitope expressed in a cellular protein.

CD8+ cytotoxic T-lymphocytes recognize small epitope peptides in association with MHC class I molecules expressed on the cell surface. In this study, we have determined whether an 8 amino acid viral CTL epitope, when expressed in a cellular protein, can be appropriately processed, presented, and recognized by the corresponding epitope-specific CTL and whether it is capable of inducing a CTL response in vivo. An H-2Kb-restricted CTL epitope from herpes simplex virus type 1 (HSV-1) glycoprotein B (gB epitope, residues 498-505) was cloned into the mouse dihydrofolate reductase protein (DHFR) at amino acid position 87. The recombinant DHFRs were expressed in vaccinia virus recombinants. To distinguish the recombinant DHFR proteins from the endogenous DHFR, an antibody epitope, recognized by monoclonal antibody PAb 901 and derived from simian virus 40 (SV40) T antigen was tagged to the C-termini of recombinant DHFR proteins. In vivo expression of recombinant DHFR was demonstrated by immunoprecipitation with the monoclonal antibody PAb 901. The H-2b cells infected with recombinant vaccinia virus expressing the recombinant DHFR were specifically lysed by gB epitope-specific CTL. Furthermore, the recombinant DHFR was functional in inducing a long lasting HSV gB epitope-specific CTL response upon immunization of C57BL/6 (B6) mice. These results indicate that a viral epitope expressed in a cellular protein can be efficiently processed, presented, and recognized by epitope-specific CTL and show that cellular proteins expressing CTL epitopes can be used for induction of CD8+ T lymphocyte responses.

Immunological application of the dihydrofolate reductase handle carrying a small peptide, leucine enkephalin.

The "dihydrofolate reductase (DHFR) handle" [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45; Iwakura, M. & Tanaka, T. (1992) J. Biochem. 111, 638-642] fused with a pentapeptide, leucine enkephalin (LEK), has been applied in immunoassays for LEK and for the preparation of anti-LEK monoclonal antibody. DHFR fused with LEK (DHFR-LEK) was first utilized as an immobilized antigen in an enzyme-linked immunosorbent assay for LEK. By using a commercially available anti-LEK and peroxidase-linked anti-IgG, LEK could be quantified in the range between 0.1 ng/ml and 10 micrograms/ml. By using a commercially available anti-LEK and the DHFR-LEK as an enzyme-labeled antigen, LEK was quantified in the range between 0.1 ng/ml and 1 microgram/ml by monitoring the recovery of the DHFR activity from the immuno-precipitates. By using the DHFR-LEK as an immunogen, three mouse monoclonal antibodies against LEK, but not DHFR, were isolated. All three monoclonal antibodies were of IgG1 kappa type. The large-scale preparation of two of these monoclonal antibodies, designated as anti-LEK-36 and anti-LEK-74, was carried out and their recognition specificities were studied by competitive binding assays. The IC50 values of LEK for the anti-LEK-36 and anti-LEK-74 were 3.74 x 10(-6) and 4.66 x 10(-6) M, respectively. The competitive binding assays showed that recognition specificities of the two monoclonal antibodies were high and restricted to LEK and leucine-enkephalin (sulfated form). These results strongly suggest that the DHFR handle is useful in several immunological applications.

Evidence for kinetic and immunologic heterogeneity of dihydrofolate reductase in L1210 leukemia cells.

Two species of DHFR were identified in wild-type L1210 murine leukemia cells by analysis of the kinetics of the binding of MTX and dissociation of the MTX-enzyme complex at pH 5.0 and pH 7.2. The two forms of DHFR were also distinguished by immunoinhibition of the binding of MTX and the catalytic reduction of FH2 to FH4 using an antiserum raised to the purified high affinity form of DHFR. The Ka for the binding of MTX by the low affinity form of the enzyme is 4.5 x 10(7) M-1, substantially lower than the reported Ka for the binding of this drug by the high affinity enzyme. The low affinity form of the enzyme catalyzed the reduction of FH2 to FH4 at a rate slower than the high affinity form of DHFR.

The importance of loop region residues 40-46 in human dihydrofolate reductase as revealed by site-directed mutagenesis.

Site-directed mutagenesis has been used to delete 2 residues (Gly45-Lys46) from a flexible "loop" region between residues 40 and 46 of human dihydrolate reductase. Steady-state kinetic studies show that the Km values for the deletion mutant enzyme for both dihydrofolate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) as well as the pH rate profile are virtually identical to that of the wild type. In contrast, the Vmax value of the mutant enzyme is decreased 2.5-fold. The results suggest that the loop region may play a role in the catalytic efficiency but not necessarily in the binding of substrates. Agents such as KCl, urea, and organomercurials at concentrations which show activating effects on the wild-type human dihydrofolate reductase have little or no effect on the deletion mutant. Competitive enzyme-linked immunosorbent assay experiments using peptide-specific antibodies against cyanogen bromide fragments generated from human dihydrofolate reductase show that the binding of folate, NADPH, and methotrexate, either in binary or in ternary complexes with the wild-type enzyme, causes a striking reduction in the binding of the antibodies. Compared with wild type, the binding of these ligands with the deletion mutant enzyme causes much less inhibition (2-16-fold less) in the binding of all three antibodies. The altered properties of the mutant enzyme can be explained on the basis of a need for the flexible loop 40-46 for reversible protein unfolding during activation and also for conformational changes induced by ligand binding, thus "communicating" the effects of ligand binding.

Mapping the antigenic epitopes of human dihydrofolate reductase by systematic synthesis of peptides on solid supports.

All of the 181 possible overlapping hexapeptides as well as 179 octapeptides covering the amino acid sequence of human dihydrofolate reductase (hDHFR) were synthesized on polyethylene supports. The synthetic procedure of Geysen et al. (Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274) was modified to obtain up to 100 nmol of peptide on each pin. Peptides constituting antigenic epitopes on hDHFR were identified by examining the binding of antibodies raised against both native and denatured hDHFR to these peptides by enzyme-linked immunosorbent assay. The peptides bound in a similar pattern to polyclonal antibodies against both native and denatured dihydrofolate reductase (DHFR). Six major epitopes were located corresponding to residues 27-33, 45-51, 67-74, 133-139, 153-158, and 176-181 using both hexapeptides and octapeptides. An additional epitope, constituting residues 14-21, was found by the use of octapeptides. Most of the epitopes are hydrophilic and reside largely in "loop" regions at the boundaries of secondary structural elements of hDHFR. This observation is consistent with our previous results which suggested that ligand binding at the active site of the enzyme can cause a dramatic reduction in antibody binding to DHFR due to conformational constraints in flexible loop regions in various parts of the molecule. The similarity of the immunogenic profiles of native versus denatured hDHFR indicates that the two forms of the antigen share the same amino acid sequence-specific epitopes. Competitive enzyme-linked immunosorbent assay showed that the binding of anti-hDHFR antiserum to both native and denatured hDHFR was inhibited by approximately 30% by the seven antigenic peptides, indicating that a significant proportion of the antibodies elicited by this enzyme is specific for short peptides. Besides revealing the antigenic structure of DHFR our results provide a rational basis for the design of mutant DHFRs to study the importance of loop residues in the conformational dynamics of the enzyme.

New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein.

Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP). To search for new epitopes in the non-repetitive part of the CS protein, we expressed the non-repetitive regions of the protein in E. coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues. These fusion proteins were obtained at greater than 70% purity by a single Ni-chelate affinity chromatography step. Of the new epitopes defined in the C-terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C-terminal of the protein's central repetitive domain. Pooled sera from inhabitants of a malaria-endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites. Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein. The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates. One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites. Antibodies which react with non-repetitive regions of the CS protein could contribute to maintaining its genetic variability.