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715 Modification of allergenic properties of a major natural rubber latex allergen Hev b 6.02 by site directed mutagenesis
S242
715
Abstracts
J ALLERGY CLIN IMMUNOL JANUARY 2000
Modification of Allergenic Properties of a Major Natural ber Latex Allergen Hev b 6.02 by Site Directed Mutagenesis Karisola*, Jari Mikkolat, niusf. Nisse Kalkkinent,
Kari Airenne*, Olli Laitinen*. Harri Jari He/in-#, limo Reunalall. Krisfiina
latex profilin was assayed by in vivo and in vitro techniques in two sample populations of spina bifida children (SB) and adults allergic to latex (AL). A method was developed to obtain natural profilin fron natural latex combining affinity chromatography (poly-L-proline Sepharose column) and ammonium sulphate fractionation. The removal of Hev b 1 traces co-eluted from PLP-Sepharose column using precipitation between 70-908 of ammonium sulphate saturation was ascertained by using monoclonal and monospecific polyclonal antibodies. Recombinant latex protilin isoform (rHev b 8) was cloned by PCR amplification. The entire coding-region of Hev b 8 was subcloned into the expression vector pKNl72 and a non fusion form of Hev b 8 was expressed in E. coli BL21 (DE3). Purified recombinant protein was obtained after a single passage through PLP-Sepharose column. Natural and recombinant purified Hev b 8 were cutaneously tested by intradermoreaction in I7 SB and I4 AL patients. They were both positive in IS and 14 with average wheal areas of 290 f 41 mm2 and 215 5 32 mm*. No wheals were produced when tested in non atopic control patients. Only 50% of sera from ID test positive patients revealed specific IgE titers class I or higher by enzymoimmunoassay and only 25% of them exhibited IgE binding by SDSPAGE immunoblotting with natural and recombinant Hev b 8. In conclusion, it seems that profilin is a relevant allergen for both group of patients from a frequency point of view but with scarce presence in natural latex extracts and low IgE binding properties.
RubPiia AleTur-
Time Palosuo$, Markku S. Kulomaa* *Department of Biological and Environmental Science, University of Jyvlskyl. Finland tFinnish Institute of Occupational Health, Helsinki, Finland SInstitute of Biotechnology. University of Helsinki, Finland [IDepartment of Dermatology, University Hospital of Tampere, Finland mational Public Health Institute, Helsinki, Finland Natural rubber latex (NRL) allergy is a growing worldwide problem particularly among health care employees as well as in patients. We have produced recombinant major latex allergen, hevein (Hev b 6.02). and its several mutants in baculovitus infected insect cells as a fusion protein with avidin (AvHev). Nine point mutations were designed by rotamer modeling with Insight97 program to search for the conformational IgE-epitopes. AvHev could be purified by affinity chromatography on a 2-iminobiotin column. The avidin tag formed the natural tetrameric structure, since AvHev bound tightly to a biotin surface. Its functional properties could thus be studied by an optical biosensor (IASyS, Affinity Sensor). Hevein was cleaved from AvHev by enterokinase and purified with reverse phase chromatography. Four extra amino acids (DDDK) were to our surprise detected at the N-terminus. Using another protease thrombin only two extra N-terminal amino acids were detected. MALDI-TOF mass spectrometric analysis indicated that like native hevein, both these recombinant heveins had four disulfide bonds. Site-directed mutagenesis (Megaprimer) was used to produce single amino acid or deletion mutants of hevein. An IgE-ELISA inhibition assay with sera from 23 allergic patients showed a significant decrease in the inhibition capacity of two mutants (HevD2 and HevD7). Amino acid substitution was directed to same amino acid in both of these mutants. Average inhibition percentages for HevD2, HevD7, recombinant hevein and native hevein (I mg inhibitor/ml) were 67%, 69%. 90% and 97%, respectively, when native hevein was the solid phase antigen. In skin prick tests, HevD2 was capable to induce clear-cut reaction only in two of five NRL-allergic patients tested, while HevD7, recombinant hevein and native hevein induced strong reactions in all five patients. More detailed analyses of the mutant heveins are in progress. Other mutated heveins showed no significant difference as compared to recombinant or native hevein in immunoreactivity with the patient sera. In conclusion, we have been able to detect one conformational IgE-epitope on the surface of hevein. Specific modification of this epitope eliminates the ability of modified hevein to cause reaction for a considerable proportion of NRL-allergic patients in skin prick tests and reduces significantly its inhibition capacity in IgE-ELISA inhibition assay. This epitope has not been recognized when studying overlapping synthetic peptides to detect linear IgG or IgE epitopes.
janmaall,
716 Assessment Patients Asturiasf,
of Protllin
as an Allergen
for
Latex
Sensitized
A Nieto*, A Mazdn*, M Boquetet. F Carballadaf. JA F Pinedat, J Martonezg, A Martonezf *Hospital Infan-
til La Fe. Valencia, Spain tComplexo Hospitalario tBial-Aristegui. Bilbao, Spain $Fac Farmacia, Univ Pais Vasco, Vitoria, Spain The presence of the actin-binding protein, profilin, has been demonstrated in natural latex extracts; but the significance of this molecule as an allergen for latex allergic patients still remains undocumented. In the present study, the allergenic reactivity of
717
Hev b 3, an Allergen Associated with Latex Bifida Patients, Harbours Multiple T Cell Bohle*, mannf,
Birgit .&o/t
Allergy Epitopes
in Spina-
Barbara Wagner*. Ute Vollmann*, Dietke Buckf, Bodo NiggeSzPpfalusi*. Otto Scheiner*. Dietrich Kraft, Heirno
Christof Ebner * *Department of General and Experimental Pathology. University of Vienna, Vienna, Austria TKinderklinik CharitC, Virchow Klinikum, Berlin, Germany Type-I allergy to latex has become an important health problem worldwide. It has been reported that 29-72% of patients suffering from spina bifida (SB) are allergic to latex. The exposure through multiple surgeries and the frequent use of latex-containing instruments like catheters has been regarded as the reason for the sensitization. SB patients mount a characteristic IgE-response directed against 2 allergens in the latex extract: Hev b I and Hev b 3. The aim of this study was to characterize the T cell response to Hev b 3 in latex-allergic SB patients. We stimulated poly-, oligo- and monoclonal T cell cultures with latex extract, recombinant Hev b 3 and overlapping synthetic peptides representing the amino acid sequence of Hev b 3 and to analyze possible T cell crossreactivities - Hev b 1. First, PBMC were stimulated with titrated concentrations of rHev b 3. Lymphoproliferative responses varied considerably between the individuals, possibly because of different time intervals from the last exposure. Thereafter, allergen-specific T cell lines (TCL) were established and cloned by limiting dilution. 21 T cell clones (TCC) from 7/l 1 latex-allergic SB-patients could be established. All TCC belonged to the Th subset. i.e. they were CD3/CD4-positive and expressed the a/b TCR. According to their cytokine production pattern, l2/21 could be attributed to the Th2like subset, 2/21 produced a Thl-like type of cytokine pattern in response to stimulation with Hev b 3,7/21 produced IL-4 and IFNg in considerable amounts (ThO). I I TCL and 2 1 Hev b 3-specific TCC were tested with overlapping peptides for epitope mapping. Epitopes of 17/21 TCC could be mapped using overlapping dodekapeptides, 4/21 did not react with any of the peptides, in spite Breiteneder*.
715
Abstracts
J ALLERGY CLIN IMMUNOL JANUARY 2000
Modification of Allergenic Properties of a Major Natural ber Latex Allergen Hev b 6.02 by Site Directed Mutagenesis Karisola*, Jari Mikkolat, niusf. Nisse Kalkkinent,
Kari Airenne*, Olli Laitinen*. Harri Jari He/in-#, limo Reunalall. Krisfiina
latex profilin was assayed by in vivo and in vitro techniques in two sample populations of spina bifida children (SB) and adults allergic to latex (AL). A method was developed to obtain natural profilin fron natural latex combining affinity chromatography (poly-L-proline Sepharose column) and ammonium sulphate fractionation. The removal of Hev b 1 traces co-eluted from PLP-Sepharose column using precipitation between 70-908 of ammonium sulphate saturation was ascertained by using monoclonal and monospecific polyclonal antibodies. Recombinant latex protilin isoform (rHev b 8) was cloned by PCR amplification. The entire coding-region of Hev b 8 was subcloned into the expression vector pKNl72 and a non fusion form of Hev b 8 was expressed in E. coli BL21 (DE3). Purified recombinant protein was obtained after a single passage through PLP-Sepharose column. Natural and recombinant purified Hev b 8 were cutaneously tested by intradermoreaction in I7 SB and I4 AL patients. They were both positive in IS and 14 with average wheal areas of 290 f 41 mm2 and 215 5 32 mm*. No wheals were produced when tested in non atopic control patients. Only 50% of sera from ID test positive patients revealed specific IgE titers class I or higher by enzymoimmunoassay and only 25% of them exhibited IgE binding by SDSPAGE immunoblotting with natural and recombinant Hev b 8. In conclusion, it seems that profilin is a relevant allergen for both group of patients from a frequency point of view but with scarce presence in natural latex extracts and low IgE binding properties.
RubPiia AleTur-
Time Palosuo$, Markku S. Kulomaa* *Department of Biological and Environmental Science, University of Jyvlskyl. Finland tFinnish Institute of Occupational Health, Helsinki, Finland SInstitute of Biotechnology. University of Helsinki, Finland [IDepartment of Dermatology, University Hospital of Tampere, Finland mational Public Health Institute, Helsinki, Finland Natural rubber latex (NRL) allergy is a growing worldwide problem particularly among health care employees as well as in patients. We have produced recombinant major latex allergen, hevein (Hev b 6.02). and its several mutants in baculovitus infected insect cells as a fusion protein with avidin (AvHev). Nine point mutations were designed by rotamer modeling with Insight97 program to search for the conformational IgE-epitopes. AvHev could be purified by affinity chromatography on a 2-iminobiotin column. The avidin tag formed the natural tetrameric structure, since AvHev bound tightly to a biotin surface. Its functional properties could thus be studied by an optical biosensor (IASyS, Affinity Sensor). Hevein was cleaved from AvHev by enterokinase and purified with reverse phase chromatography. Four extra amino acids (DDDK) were to our surprise detected at the N-terminus. Using another protease thrombin only two extra N-terminal amino acids were detected. MALDI-TOF mass spectrometric analysis indicated that like native hevein, both these recombinant heveins had four disulfide bonds. Site-directed mutagenesis (Megaprimer) was used to produce single amino acid or deletion mutants of hevein. An IgE-ELISA inhibition assay with sera from 23 allergic patients showed a significant decrease in the inhibition capacity of two mutants (HevD2 and HevD7). Amino acid substitution was directed to same amino acid in both of these mutants. Average inhibition percentages for HevD2, HevD7, recombinant hevein and native hevein (I mg inhibitor/ml) were 67%, 69%. 90% and 97%, respectively, when native hevein was the solid phase antigen. In skin prick tests, HevD2 was capable to induce clear-cut reaction only in two of five NRL-allergic patients tested, while HevD7, recombinant hevein and native hevein induced strong reactions in all five patients. More detailed analyses of the mutant heveins are in progress. Other mutated heveins showed no significant difference as compared to recombinant or native hevein in immunoreactivity with the patient sera. In conclusion, we have been able to detect one conformational IgE-epitope on the surface of hevein. Specific modification of this epitope eliminates the ability of modified hevein to cause reaction for a considerable proportion of NRL-allergic patients in skin prick tests and reduces significantly its inhibition capacity in IgE-ELISA inhibition assay. This epitope has not been recognized when studying overlapping synthetic peptides to detect linear IgG or IgE epitopes.
janmaall,
716 Assessment Patients Asturiasf,
of Protllin
as an Allergen
for
Latex
Sensitized
A Nieto*, A Mazdn*, M Boquetet. F Carballadaf. JA F Pinedat, J Martonezg, A Martonezf *Hospital Infan-
til La Fe. Valencia, Spain tComplexo Hospitalario tBial-Aristegui. Bilbao, Spain $Fac Farmacia, Univ Pais Vasco, Vitoria, Spain The presence of the actin-binding protein, profilin, has been demonstrated in natural latex extracts; but the significance of this molecule as an allergen for latex allergic patients still remains undocumented. In the present study, the allergenic reactivity of
717
Hev b 3, an Allergen Associated with Latex Bifida Patients, Harbours Multiple T Cell Bohle*, mannf,
Birgit .&o/t
Allergy Epitopes
in Spina-
Barbara Wagner*. Ute Vollmann*, Dietke Buckf, Bodo NiggeSzPpfalusi*. Otto Scheiner*. Dietrich Kraft, Heirno
Christof Ebner * *Department of General and Experimental Pathology. University of Vienna, Vienna, Austria TKinderklinik CharitC, Virchow Klinikum, Berlin, Germany Type-I allergy to latex has become an important health problem worldwide. It has been reported that 29-72% of patients suffering from spina bifida (SB) are allergic to latex. The exposure through multiple surgeries and the frequent use of latex-containing instruments like catheters has been regarded as the reason for the sensitization. SB patients mount a characteristic IgE-response directed against 2 allergens in the latex extract: Hev b I and Hev b 3. The aim of this study was to characterize the T cell response to Hev b 3 in latex-allergic SB patients. We stimulated poly-, oligo- and monoclonal T cell cultures with latex extract, recombinant Hev b 3 and overlapping synthetic peptides representing the amino acid sequence of Hev b 3 and to analyze possible T cell crossreactivities - Hev b 1. First, PBMC were stimulated with titrated concentrations of rHev b 3. Lymphoproliferative responses varied considerably between the individuals, possibly because of different time intervals from the last exposure. Thereafter, allergen-specific T cell lines (TCL) were established and cloned by limiting dilution. 21 T cell clones (TCC) from 7/l 1 latex-allergic SB-patients could be established. All TCC belonged to the Th subset. i.e. they were CD3/CD4-positive and expressed the a/b TCR. According to their cytokine production pattern, l2/21 could be attributed to the Th2like subset, 2/21 produced a Thl-like type of cytokine pattern in response to stimulation with Hev b 3,7/21 produced IL-4 and IFNg in considerable amounts (ThO). I I TCL and 2 1 Hev b 3-specific TCC were tested with overlapping peptides for epitope mapping. Epitopes of 17/21 TCC could be mapped using overlapping dodekapeptides, 4/21 did not react with any of the peptides, in spite Breiteneder*.