Spermine and Spermidine Priming against Botrytis cinerea Modulates ROS Dynamics and Metabolism in Arabidopsis.

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.

Using bispecific antibodies in forced degradation studies to analyze the structure-function relationships of symmetrically and asymmetrically modified antibodies.

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

Effects of N-Glycan Composition on Structure and Dynamics of IgG1 Fc and Their Implications for Antibody Engineering.

Immunoglobulin G1 (IgG1), a subclass of human serum antibodies, is the most widely used scaffold for developing monoclonal antibodies to treat human diseases. The composition of asparagine(N)297-linked glycans can modulate the binding affinity of IgG1 Fc to Fc γ receptors, but it is unclear how the structural modifications of N-glycan termini, which are distal from the binding interface, contribute to the affinity. Through atomistic molecular dynamics simulations of a series of sequentially truncated high-mannose IgG1 Fc glycoforms, we found that the C'E loop and the Cγ2-Cγ3 orientation are highly dynamic, and changes in N-glycan composition alter their conformational ensembles. High-mannose glycoform preferentially samples conformations that are more competent to FcγRIIIa binding, compared to the truncated glycoforms, suggesting a role of IgG1 Fc N-glycan in optimizing the interface with the Fc receptor for efficient binding. The trajectory analyses also reveal that the N-glycan has large amplitude motions and the carbohydrate moiety interconverts between Fc-bound and unbound forms, enabling enzymatic modification of the glycan termini.

Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry.

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6-9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.

Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae.

In autoimmune diseases, there have been proposals that exogenous "molecular triggers", i.e., specific 'non-self antigens' accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma.

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

Asn347 Glycosylation of Corticosteroid-binding Globulin Fine-tunes the Host Immune Response by Modulating Proteolysis by Pseudomonas aeruginosa and Neutrophil Elastase.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues upon elastase-based proteolysis of the exposed reactive center loop (RCL). However, the molecular mechanisms that regulate the RCL proteolysis by co-existing host and bacterial elastases in inflamed/infected tissues remain unknown. We document that RCL-localized Asn(347) glycosylation fine-tunes the RCL cleavage rate by human neutrophil elastase (NE) and Pseudomonas aeruginosa elastase (PAE) by different mechanisms. NE- and PAE-generated fragments of native and exoglycosidase-treated blood-derived CBG of healthy individuals were monitored by gel electrophoresis and LC-MS/MS to determine the cleavage site(s) and Asn(347) glycosylation as a function of digestion time. The site-specific (Val(344)-Thr(345)) and rapid (seconds to minutes) NE-based RCL proteolysis was significantly antagonized by several volume-enhancing Asn(347) glycan features (i.e. occupancy, triantennary GlcNAc branching, and α1,6-fucosylation) and augmented by Asn(347) NeuAc-type sialylation (all p < 0.05). In contrast, the inefficient (minutes to hours) PAE-based RCL cleavage, which occurred equally well at Thr(345)-Leu(346) and Asn(347)-Leu(348), was abolished by the presence of Asn(347) glycosylation but was enhanced by sialoglycans on neighboring CBG N-sites. Molecular dynamics simulations of various Asn(347) glycoforms of uncleaved CBG indicated that multiple Asn(347) glycan features are modulating the RCL digestion efficiencies by NE/PAE. Finally, high concentrations of cortisol showed weak bacteriostatic effects toward virulent P. aeruginosa, which may explain the low RCL potency of the abundantly secreted PAE during host infection. In conclusion, site-specific CBG N-glycosylation regulates the bioavailability of cortisol in inflamed environments by fine-tuning the RCL proteolysis by endogenous and exogenous elastases. This study offers new molecular insight into host- and pathogen-based manipulation of the human immune system.

Large-scale preparation of Asn-glycopeptide carrying structurally homologous antigenic N-glycan.

In our previous report (M. Okano, et al., Clin. Exp. Allergy, 34, 770-778 (2004)), we found that free plant complex type N-glycans suppressed the production of IL4 from Th2 cells of Japanese cedar pollinosis patients, suggesting that plant complex type N-glycan can be used as a leading compound for developing immuno-pharmaceuticals. Although immunoreactive plant complex type N-glycans occur ubiquitously on glycoproteins expressed in plants, an appropriate procedure has not been established to prepare non-labeled immunoreactive glycans or glycopeptides bearing structurally homologous immunoreactive glycans in large amounts. In this study, therefore, we developed a new preparative procedure for the large-scale preparation of Asn-glycopeptide bearing plant complex type N-glycan using a combination of gel-filtration and the hydrophilic partitioning method. By this new method, about 103 mg of Asn-glycopeptide bearing the antigenic N-glycans was obtained from 1.9 kg of shelled Ginkgo biloba seeds.

N332-Directed broadly neutralizing antibodies use diverse modes of HIV-1 recognition: inferences from heavy-light chain complementation of function.

Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332) is characterized by an N-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type) was generally consistent (58 ± 10 µg/ml). In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.

Use of site-directed mutagenesis to model the effects of spontaneous deamidation on the immunogenicity of Bacillus anthracis protective antigen.

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)--the major component of new-generation anthrax vaccines--affects vaccine immunogenicity, we created a "genetically deamidated" form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

Immunization with aspartate-ß-hydroxylase-loaded dendritic cells produces antitumor effects in a rat model of intrahepatic cholangiocarcinoma.

UNLABELLED: Dendritic cells (DCs) capture and process proteins and present peptides on the cell surface in the context of major histocompatibility complex I and II molecules to induce antigen-specific T cell immune responses. The aims of this study were to (1) employ an expanded and purified DC population and load them with aspartate-ß-hydroxylase (ASPH), a highly expressed tumor-associated cell surface protein, and (2) to determine if immunization induced antitumor effects in an orthotopic rat model of intrahepatic cholangiocarcinoma. Splenocytes were incubated with ASPH-coated beads and passed through a magnetic field to yield an 80% pure DC OX62+ population. This DC subset was stimulated with granulocyte-macrophage colony-stimulating factor, interleukin-4, CD40L, and interferon-γ, resulting in a 40-fold increase in interleukin-12A messenger RNA expression to subsequently generate a T helper 1-type immune response. After incubation with the cytokine cocktail, DCs were found to have matured, as demonstrated by increased expression of CD40, CD80, and CD86 costimulatory molecules. Immunization with ASPH-loaded DCs induced antigen-specific immunity. A clone of the parental tumorigenic rat BDEneu cholangiocyte cell line, designated BDEneu-CL24, was found to have the highest number of cells expressing this surface protein (97%); it maintained the same phenotypic characteristics of the parental cell line and was used to produce intrahepatic tumors in immunocompetent syngeneic Fisher-344 rats. Immunization with ASPH-loaded DCs generated cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed intrahepatic tumor growth and metastasis, and was associated with increased CD3+ lymphocyte infiltration into the tumors. CONCLUSION: These findings suggest that immunization with ASPH-loaded DCs may constitute a novel therapeutic approach for intrahepatic cholangiocarcinoma, because this protein also appears to be highly conserved and expressed on human hepatobiliary tumors.

N-linked glycosylation selectively regulates autonomous precursor BCR function.

Developing B cells express distinct classes of B cell antigen receptors (BCRs) that differ in their heavy chain (HC). Although only muHC is expressed in early stages, deltaHC-containing BCRs dominate on the surface of mature B cells. The reason for the tightly regulated expression of these receptors is poorly understood. Here we show that muHC was specifically required for precursor BCR (pre-BCR) function and that deltaHC was unable to form a functional pre-BCR. A conserved asparagine (N)-linked glycosylation site at position 46 (N46) in the first conserved domain of muHC was absolutely required for pre-BCR function, and swapping that domain with deltaHC resulted in a functional deltaHC-containing pre-BCR. When tested in the context of the BCR, muHC with a mutant N46 showed normal function, which indicated that N46-glycosylation is specifically required for pre-BCR function. Our results suggest an unexpected mode of pre-BCR function, in which binding of the surrogate light chain to N46 mediates autonomous crosslinking and, concomitantly, receptor formation.

Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs.

We report the presence of oligosaccharide structures on a glutamine residue present in the V(L) domain sequence of a recombinant human IgG2 molecule. Residue Gln-106, present in the QGT sequence following the rule of an asparagine-linked consensus motif, was modified with biantennary fucosylated oligosaccharide structures. In addition to the glycosylated glutamine, analysis of a lectin-enriched antibody population showed that 4 asparagine residues: heavy chain Asn-162, Asn-360, and light chain Asn-164, both of which are present in the IgG1 and IgG2 constant domain sequences, and Asn-35, which was present in CDR(L)1, were also modified with oligosaccharide structures at low levels. The primary sequences around these modified residues do not adhere to the N-linked consensus sequon, NX(S/T). Modeling of these residues from known antibody crystal structures and sequence homology comparison indicates that non-consensus glycosylation occurs on Asn residues in the context of a reverse consensus motif (S/T)XN located on highly flexile turns within 3 residues of a conformational change. Taken together our results indicate that protein glycosylation is governed by more diversified requirements than previously appreciated.

The anti-asparagines antibodies correlate with L-asparagines activity and may affect clinical outcome of childhood acute lymphoblastic leukemia.

The primary aim of the study was to evaluate the importance of anti-asparaginase antibodies for l-asparaginase activity in children with standard and medium risk acute lymphoblastic leukemia (ALL). Forty-seven children with newly diagnosed ALL were included into the prospective study. Enzyme activity and the presence of anti-asparaginase antibodies (IgG and IgM class) were determined. Anti-asparaginase antibodies were identified in 13/47 (IgM class) and 10/47 (IgG class) patients in the induction and in 19/47 (IgM class) and 20/47 (IgG class) patients in the reinduction phase of treatment. The enzyme activity was lower in patients that were positive for anti-asparaginase antibodies, especially in reinduction phase (median 37 (20 - 180) vs 355 (141 - 499), p = 0.001). An association between anti-asparaginase antibodies and the allergic reaction to the drug was found. Besides, the children who developed anti-asparaginase antibodies in the induction phase of treatment showed lower event-free survival as well as overall survival in comparison with children without antibodies. Since our study was carried out in a small number of patients, this observation is only speculative and needs to be confirmed by a further study on a larger sample size, with multivariable analysis. However, our data suggest that L-asparaginase activity together with anti-asparaginase antibodies measurements may become useful for effective therapy of ALL.

Deamidation of asparagine in a major histocompatibility complex-bound peptide affects T cell recognition but does not explain type B reactivity.

We have analyzed a panel of T cell hybridomas specific for the chemically dominant epitope of hen egg-white lysozyme 48-61 which has asparagine 59 as an important T cell receptor contact residue. A number of T cells recognize 48-61 with asparagine at position 59, but not the aspartic acid or isoaspartic acid derivatives. Conversely, we find T cells that specifically recognize 48-61 bearing an isoaspartic acid at residue 59, but not asparagine. For other T cells, asparagine, aspartic acid, or isoaspartic acid at residue 59 is irrelevant. We present evidence that our previous distinction between type A and type B T cells is not explained by asparagine deamidation at residue 59.

Two clusters of acidic amino acids near the NH2 terminus of complement component C4 alpha'-chain are important for C2 binding.

Previous work has indicated a role for the NH2-terminal segment of the C3 alpha'-chain in the binding interactions of C3b with a number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the NH2 termini of their respective alpha'-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases, as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the NH2-terminal segment of the C4 alpha'-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic peptide (C4 residues 740-756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cell-associated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 alpha'-chain contributes importantly to the interaction of C4b with C2.

D-LysAsnProTyr tetrapeptide: a novel B-cell stimulant and stabilized bursin mimetic.

The tetrapeptide I (D-lysine-L-asparaginyl-L-prolyl-L-tyrosine or D-LysAsnProTyr), and analogue sequences, were synthesized and evaluated for the ability to stimulate immune cell subsets. These sequences were selected based on their perceived ability to readily adopt a beta-turn structure. In vitro immunological assays revealed a robust stimulation of mitogen activated B-cell proliferation and a modest to significant stimulation of cytotoxic T lymphocytes (CTLs). Further, this in vitro stimulation of B-cells was accompanied by an in vivo expansion of B-cells in C57BL/6 mice, as demonstrated by immunophenotyping experiments. Interestingly, a conformational analysis of the low energy conformers of I and the endogenous B-cell stimulant bursin (LysHisGlyNH2) shows that these molecules can be superimposed. However, I displayed significantly enhanced physiological stability. For a number of reasons, I may be a particularly useful vaccine adjuvant.

Cross-reactions in the latex-fruit syndrome: A relevant role of chitinases but not of complex asparagine-linked glycans.

BACKGROUND: Cross-reactions between latex and plant foods (mainly fruits) have been widely reported. Although the cross-reactive components have not been well identified, class I chitinases seem to be the most credible candidates in chestnut, avocado, and banana. OBJECTIVE: We sought to evaluate the potential role of chitinases and complex glycans as cross-reactive determinants linked to latex-food allergy. METHODS: Extracts from 20 different plant foods and from latex were obtained. These preparations were immunodetected with anticomplex glycans and antichitinase sera raised in rabbits, as well as with sera from patients with latex-fruit allergy and sera from patients allergic to latex without food allergy. Immunoblot inhibition assays were carried out by using a purified class I chitinase from avocado or latex extract as inhibitors. RESULTS: Reactive proteins of approximately 30 to 45 kd (putative class I chitinases) were recognized by both specific polyclonal antibodies to chitinases and sera from patients with latex-fruit allergy in chestnut, cherimoya, passion fruit, kiwi, papaya, mango, tomato, and flour wheat extracts. Prs a 1, the major allergen and class I chitinase from avocado, and the latex extract strongly or fully inhibited IgE binding by these components when tested in immunoblot inhibition assays. Additional bands of 16 to 20 kd, 23 to 28 kd, and 50 to 70 kd were detected by the antichitinase serum but not with the patients' pooled sera. The putative 30- to 45-kd chitinases present in different food extracts did not react with a pool of sera from subjects allergic to latex but not to fruit. Very different immunodetection patterns were produced with the anticomplex glycan serum and the sera from allergic patients. CONCLUSIONS: Putative class I chitinases seem to be relevant cross-reactive components in foods associated with the latex-fruit syndrome, but do not play a specific role in allergy to latex but not to fruit. Cross-reactive carbohydrate determinants are not important structures in the context of latex-fruit cross-sensitization.

Recognition of phage-expressed peptides containing Asx-Pro sequences by monoclonal antibodies produced against Plasmodium falciparum circumsporozoite protein.

The immunodominant region of the Plasmodium falciparum circumsporozoite protein is comprised mainly of a series of tetrapeptide repeats that can, depending on the starting cadence chosen, be described as (NANP)n, (ANPN)n, (NPNA)n or (PNAN)n in one-letter amino acid code. Data from several studies suggest that the NPNA cadence alone is structurally correct, in that each NPNA tetrapeptide effectively forms a structural unit initiated by an Asx-Pro turn. To explore this idea further and to assess the immunological relevance of peptide conformation as it relates to the cadence of these tetrapeptide repeats, we used ELISA to compare the abilities of monoclonal antibodies (MAbs) produced against P. falciparum sporozoites to recognize repeat-related heptapeptides expressed on the surface of filamentous bacteriophage. Having included representatives of both NANP and NPNA cadences and other peptides in which the number and location of Asx-Pro sequences varied, we provide evidence that Asx-Pro sequences play an important role in peptide conformation and antibody recognition, that peptide conformation is influenced by the cadence of the tetrapeptide repeats and that peptide conformation is important to the abilities of these MAbs to recognize their epitopes.