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1.
A two-stage strategy was used to synthesize the 45-residue, four-disulfide-bonded ragweed protein allergen Ra5: (i) the protected linear chain was prepared by solid-phase, stepwise synthesis according to the sequence of the fully reduced protein; (ii) after removal from the resin and deprotection by hydrogen fluoride, the reduced chain was purified by gel filtration on Sephadex G-50 and allowed to fold “spontaneously” in the presence of oxidized dithiothreitol to form the disulfide links. The product (synthetic Ra5) was purified to disc-electrophoretic homogeneity by cycling through Sephadex G-50 and CM-Sephadex (overall yield: 6% theoretical). Synthetic Ra5 was closely similar in structure to the natural protein by amino acid analysis. polyacrylamide gel electrophoresis, tryptic peptide mapping, and circular dichroism spectra, as well asin vitro andin vivo immunoassays of antigenic/allergenic activities. 相似文献
2.
Two major human allergenic sites on ragweed pollen allergen antigen E identified by using monoclonal antibodies 总被引:1,自引:0,他引:1
Murine monoclonal antibodies specific for antigen E (AgE), the major allergen isolated from short ragweed pollen, have been produced and characterized. These monoclonal antibodies, when coupled to Sepharose and used as immunoadsorbents, specifically bound AgE when a crude pollen extract was passed through the column. Three antigenic sites (A, B, and C) on AgE were identified by using five of these monoclonal antibodies in both inhibition and double-bind solid-phase ELISA. These three antigenic sites appear to be nonoverlapping and nonrepeated, that is, present only once on each AgE molecule. Site C on AgE could readily be bound by the monoclonal antibody specific for that site, but only when AgE was in solution or "presented" by an anti-site A or anti-site B antibody. Site C appears to be only marginally available for binding when AgE is directly adsorbed to polyvinyl chloride microtiter wells. The majority of monoclonal antibodies isolated after immunization of BALB/c mice were specific for site A on AgE. In addition, the binding to AgE of pooled BALB/c polyclonal, hyperimmune antisera against AgE was blocked approximately 80% by a monoclonal antibody directed against site A, but was only blocked approximately 20% by an anti-site B monoclonal antibody. This suggests that site A on AgE is the predominant antigenic site in the BALB/c immune response and that site B represents a less dominant site. The binding of IgE in pooled human serum from ragweed-allergic individuals is blocked approximately 50% by a monoclonal antibody directed to site A on AgE and also approximately 50% by a monoclonal antibody directed against site B. A series of individual human short ragweed allergic antisera also showed significant, although varied, inhibition of IgE binding to AgE by both anti-site A and anti-site B monoclonal antibodies. Simultaneous addition of anti-site A and anti-site B was somewhat additive and inhibited up to 80% of the binding of human IgE specific for AgE. The conclusion from these data is that site A and site B defined by two murine monoclonal antibodies represent two very major allergenic sites in the human response to this molecule. 相似文献
3.
The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach. 总被引:6,自引:3,他引:6
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The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations. 相似文献
5.
T-cell recognition of human haemoglobin. Localization of the full T-cell recognition profile of the beta-chain by a comprehensive synthetic strategy.
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This paper reports the localization of the regions on the beta-chain that are recognized by T cells from mice immunized with haemoglobin. The 14 overlapping peptides encompassing the entire beta-chain were examined in vitro for their ability to stimulate lymph-node cells from haemoglobin-primed B10.D2 (H-2d) and SJL (H-2s) mice. Several regions of the molecule (T sites) were found to stimulate haemoglobin-primed lymph-node cells. This strategy has enabled the localization of the full profile of T-cell recognition of the beta-chain by these mouse strains. Some of the regions that stimulated T cells appeared to coincide with those recognized by antibodies (i.e. B cells). It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has other sites that are recognized exclusively by T cells and to which no detectable antibody response is directed. 相似文献
6.
Subunit interacting surfaces of human haemoglobin. Localization of the alpha-subunit-beta-subunit interacting surfaces on the beta-chain by a comprehensive synthetic strategy. 总被引:1,自引:1,他引:1
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A synthetic approach is introduced for localization of subunit interacting surfaces in oligomeric proteins. It consists of studying the binding activity of consecutive uniform overlapping peptides encompassing an entire subunit to the other, radiolabelled, subunit. This permits the establishment of the full profile of peptides that bind the other intact subunit. This approach has been demonstrated with haemoglobin, and its application here with the beta-chain peptides has enabled the localization on the beta-chain of the submolecular regions responsible for its binding to alpha-chain in solution. There was good agreement between the binding surfaces found here in solution and those expected from the crystal structure. There were also, however, some significant differences in the levels of binding found in solution and those expected from the crystal. Peptide 21-35 possessed much higher binding activity than would be expected from its contribution to subunit association in the crystal. Conversely, other regions expected to possess considerable binding capacity for alpha-chain either showed low (peptides 111-125 and 121-135) or almost no binding (peptides 91-105 and 101-115) capacity. On the other hand, two interacting surfaces (within peptides 11-25 and 71-85) that make a contribution in solution do not appear to play a role in the crystal. It is concluded that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should serve as an effective method for localization of subunit interacting surfaces of unknown proteins, even those that can be isolated only in traces. 相似文献
7.
Plant reproduction has broad implications for ecology and society (e.g., production of allergenic pollen), and a number of processes that affect flowering are affected by climate. In this study, we tested for variation in traits related to pollen production by the allergenic plant, common ragweed, across a climate gradient in Massachusetts. We tested whether traits that are easily-measured in the field could be used to predict differences in spike length, a known proxy for allergenic pollen production, and the timing and duration of flowering. We also tested whether flowering time and allometric estimates of spike length varied across the climate gradient, to better understand climate effects on future pollen production. We found that height predicts inflorescence length, but the slope of the relationship between the two traits is steeper in cooler climates, suggesting ragweed growing in cool climates produces more pollen per unit of vegetative height than ragweed in warm climates. Cool climates were also associated with larger and higher numbers of flowers and earlier and longer periods of flowering. Thus, we provide improved estimates of local pollen exposure by establishing variation in timing and output of flowering across a regional climate gradient, and at a spatial scale that may be useful for developing management strategies for allergenic plants. 相似文献
8.
Immune responsiveness to Ambrosia artemishfolia (short ragweed) pollen allergen Amb a VI (Ra6) is associated with HLA-DR5 in allergic humans 总被引:1,自引:0,他引:1
David G. Marsh Linda R. Freidhoff Eva Ehrlich-Kautzky Wilma B. Bias Marianne Roebber 《Immunogenetics》1987,26(4-5):230-236
The relationship between HLA type and specific immune responsiveness toward ultrapure Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a VI (Ra6) was explored in a genetic-epidemiologic study of groups of 116 and 81 Caucasoid subjects who were skin-test \ positive (ST–) toward common environmental allergens. Specific immune responsiveness to Amb a VI was assessed by measuring serum IgE and IgG antibodies (Abs) by double Ab radioimmunoassay in both ST– groups. Significant associations were found between IgE Ab responsiveness to Amb a VI and the possession of HLA-DR5; P values for the two groups were, respectively, 7 × 10–7 and 1 × 10–3 by nonparametric analyses, and 4 × 10–11 and 5 × 10–8 by parametric analyses. The levels of significance for the associations between HLA-DR5 and IgG Ab responsiveness were highly dependent on the extent of ragweed immunotherapy (Rx) within the patient group; by parametric statistics, the associations were 10–11 for the group that had received relatively little Rx and 2 × 10–3 for the group that had received more intensive Rx. These results provide further striking evidence for the existence of specific HLA-linked human Ir genes involved in responsiveness toward inhaled allergens and illustrate the usefulness of the allergy model in studies of the genetic basis of human immune responsiveness. Extension of these studies to investigation of structure-function relationships involved in antigen recognition by Ia molecules and the T-cell receptor will lead to a better understanding of human susceptibility toward immunologic diseases.Abbreviations used in this paper Ab
antibody
-
Amb a VI
Amb a V, new IUIS nomenclature for Ambrosia artemisiifolia pollen allergens nos. 6 and 5 (short ragweed Ra6 and Ra5) (Marsh et al. 1986b)
-
Lol p II, III
new IUIS nomenclature for Lolium perenne pollen allergens II and III (perennial rye grass, Rye II and Rye III) (Marsh et al. 1986b)
- BBS
borate-buffered physiologic saline
- BSA
bovine serum albumin
- DARIA
double-antibody radioimunoassay
- Ia
immune-associated
- PAGE
polyacrylamide gel electrophoresis
- RIST
radioimmunosorbent test
- Rx
immunotherapy
- SDS
sodium dodecyl sulfate
- ST
skin test 相似文献
9.
Mothes-Luksch N Stumvoll S Linhart B Focke M Krauth MT Hauswirth A Valent P Verdino P Pavkov T Keller W Grote M Valenta R 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(7):4864-4873
The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines. 相似文献
10.
Essentially complete assignment of the proton resonances in the allergenic protein Amb a V has been made by analysis of two-dimensional NMR experiments. Conformational constraints were obtained in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb a V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a small segment of antiparallel beta-sheet, and several loops. A hydrophobic core exists at the interface of the alpha-helix and beta-sheet. The derived structure accounts for the several anomalous proton chemical shifts that are observed. The structure determined here for Amb a V is topologically similar to the structure determined previously for the homologous allergenic protein Amb t V [Metzler, W. J., Valentine, K., Roebber, M., Friedrichs, M. S., Marsh, D., & Mueller, L. (1992) Biochemistry 31, 5117-5127]; however, significant differences exist in the packing of side chains in the hydrophobic core of the molecules. Comparison of the detailed structural features of these two proteins will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes. 相似文献
11.
Patty Zwollo Eva Ehrlich-Kautzky Stephen J. Scharf Aftab A. Ansari Henry A. Erlich David G. Marsh 《Immunogenetics》1991,33(2):141-151
We investigated the molecular basis for the striking association between HLA-DR2,Dw2 and human immune responsiveness to the Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a V by sequencing the second exons of the DRB and DQBI genes of 17 selected ragweed-allergic Caucasoid subjects. We also studied the DQA1 allelic polymorphic regions (APRs) in these patients by dot-blotting using sequence-specific oligonucleotides (SSOs). The deduced amino acid sequences of the respective class II and polypeptides were compared, with particular emphasis on residues in the APRs that are implicated in antigen binding. No evidence for new HLA-DRB or DQB sequences unique to Amb a V responders were found on sequencing seven Dw2+ subjects. This suggests that the presence of a particular Dw2-associated class II molecule usually provides a necessary, but not always sufficient condition for responsiveness to Amb a V. The HLA phenotypes of three subjects suggest that they possess novel recombinant haplotypes containing either DRB1
*
1501 and DRB5
*
0101 (Dr2.2-associated) or DQB1
*
0602 (DQ1.2-associated) sequences. In these subjects, responsiveness to Amb a V was associated with the DR2.2 but not the DQ1.2 sequences, suggesting that DRI or DRV class II molecules are involved in antigen presentation. We investigated whether there may be shared HLA-D-encoded responder sequences present in all responders, including some exceptional DR2–
Amb a V responders. The 13 subjects producing antibody (Ab) responses to Amb a V [either from natural exposure and/or after ragweed immunotherapy (Rx)] possessed DRB1
*
1501, 1601, 1602, 0103, 0402, 0404, 0801 or 1101 sequences, which share the majority of their aa residues in the APRs 2–4. Some of these shared residues might be important for the binding of a common Amb a V agretope prior to presentation of the class II Amb a V complex to the T-cell receptor (Tcr). An alternative postulate is that the recognition of two different Amb a V agretopes may be determined by the I polypeptides of molecules having the DR2 and DQw3 specificities. 相似文献
12.
W J Metzler K Valentine M Roebber M S Friedrichs D G Marsh L Mueller 《Biochemistry》1992,31(22):5117-5127
Analysis of two-dimensional NMR experiments has afforded essentially complete assignment of all proton resonances in the allergenic protein Amb t V. Conformational constraints were obtained from the NMR data in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb t V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a short stretch of triple-stranded antiparallel beta-sheet, and several loops. In addition, the cystine partners of the four disulfide linkages (for which there are no biochemical data) have been assigned. The refined structures of Amb t V will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes and will assist us in defining the Ia/T cell epitopes that interact with the MHC class II (or Ia) molecule and the T cell receptor leading to the induction of the immune response to Amb t V. 相似文献
13.
Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides 总被引:9,自引:0,他引:9
The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
T Rafnar I J Griffith M C Kuo J F Bond B L Rogers D G Klapper 《The Journal of biological chemistry》1991,266(2):1229-1236
To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins. 相似文献
15.
R Shapira W C Mobley S B Thiele M R Wilhelmi A Wallace R F Kibler 《Journal of neurochemistry》1978,30(4):735-744
Abstract— The enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase (CNP) has been assayed in fractions from a continuous sucrose density gradient zonal centrifugation of rabbit brain homogenates. Basic protein (BP) was also assayed by a radioimmunomethod. Fractions were examined by SDS-polyacrylamide gel electrophoresis and by electron microscopy. These studies show that the major membrane fractions in the gradient differ greatly in the content of CNP and BP, and of high molecular weight proteins (HMW). The lightest membrane fractions contained numerous multilamellae, the highest content of BP and the lowest content of CNP and HMW, while the heaviest membrane fractions contained single membrane fragments and vesicles of unknown origin, the lowest content of BP and the highest content of CNP and HMW. The fraction containing the largest amount of membrane measured by turbidity, protein content, and water-washed dry weight contained only half the CNP specific activity of a denser fraction in the gradient. CNP specific activity in the lightest fractions was insignificant compared to that of denser fractions. Thus, we conclude that this enzyme may be absent from the typical multilamellar myelin structures but present in the single-membrane structures associated with myelin, such as the glial membrane and the paranodal segments of myelin adjacent to the axon. BP appears to occupy the opposite positions, highest in the multilamellae and lowest in the single-membrane structures of myelin. These studies do not exclude the possibility that CNP may not be bound to myelin membranes, but rather to a membrane of different origin. Evidence that this enzyme is a myelin-marker enzyme is circumstantial. Our evidence indicates the enzyme could be present either in a unique portion of myelin membranes or in another membrane structure. 相似文献
16.
Unlocking the allergenic structure of the major house dust mite allergen der f 2 by elimination of key intramolecular interactions 总被引:1,自引:0,他引:1
We report on the structural background of the remarkable reduction of allergenicity in engineering of the major house dust mite allergen Der f 2. Disruption of intramolecular disulfide bonds in Der f 2 caused extensive conformational change that was monitored by circular dichroism and gel-filtration analysis. The degree of conformational change correlated well with the degree of reductions in the capacity to bind IgE and to induce histamine release from basophils in mite-allergic patients. Loosening the rigid tertiary structure by elimination of key intramolecular interactions is an effective strategy to reduce the number of high affinity IgE epitopes of allergen vaccine. 相似文献
17.
K H Roux J M Kilpatrick J E Volanakis J F Kearney 《Journal of immunology (Baltimore, Md. : 1950)》1983,131(5):2411-2415
C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed. 相似文献
18.
Chanprapaph S Saparpakorn P Sangma C Niyomrattanakit P Hannongbua S Angsuthanasombat C Katzenmeier G 《Biochemical and biophysical research communications》2005,330(4):1237-1246
The NS3 serine protease of dengue virus is required for the maturation of the viral polyprotein and consequently represents a promising target for the development of antiviral inhibitors. However, the substrate specificity of this enzyme has been characterized only to a limited extent. In this study, we have investigated product inhibition of the NS3 protease by synthetic peptides derived from the P6-P1 and the P1'-P5' regions of the natural polyprotein substrate. N-terminal cleavage site peptides corresponding to the P6-P1 region of the polyprotein were found to act as competitive inhibitors of the enzyme with K(i) values ranging from 67 to 12 microM. The lowest K(i) value was found for the peptide representing the NS2A/NS2B cleavage site, RTSKKR. Inhibition by this cleavage site sequence was analyzed by using shorter peptides, SKKR, KKR, KR, AGRR, and GKR. With the exception of the peptide AGRR which did not inhibit the protease at a concentration of 1mM, all other peptides displayed K(i) values in the range from 188 to 22 microM. Peptides corresponding to the P1'-P5' region of the polyprotein cleavage sites had no effect on enzymatic activity at a concentration of 1mM. Molecular docking data of peptide inhibitors to a homology-based model of the dengue virus type 2 NS2B(H)-NS3p co-complex indicate that binding of the non-prime site product inhibitors is similar to ground-state binding of the corresponding substrates. 相似文献
19.
Sandra Westphal Daniel Kolarich Kay Foetisch Iris Lauer Friedrich Altmann Amedeo Conti Jesus F Crespo Julia Rodríguez Ernesto Enrique Stefan Vieths Stephan Scheurer 《European journal of biochemistry》2003,270(6):1327-1337
Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens. 相似文献
20.
Reese G Viebranz J Leong-Kee SM Plante M Lauer I Randow S Moncin MS Ayuso R Lehrer SB Vieths S 《Journal of immunology (Baltimore, Md. : 1950)》2005,175(12):8354-8364
The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90-98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use. 相似文献