Construction of hevein (Hev b 6.02) with reduced allergenicity for immunotherapy of latex allergy by comutation of six amino acid residues on the conformational IgE epitopes.
Recently we have established that IgE Abs bind to conformational epitopes in the N- and C-terminal regions of the major natural rubber latex allergen, hevein (Hev b 6.02). To identify the critical amino acid residues that interact with IgE, the hevein sequence was scanned by using site-specific mutations. Twenty-nine hevein mutants were designed and produced by a baculovirus expression system in insect cells and tested by IgE inhibition-ELISA using sera from 26 latex allergic patients. Six potential IgE-interacting residues of hevein (Arg(5), Lys(10), Glu(29), Tyr(30), His(35), and Gln(38)) were identified and characterized further in detail. Based on these six residues, two triple mutants (Hdelta3A, Hdelta3B) and hevein mutant where all six residues were mutated (Hdelta6), were designed, modeled, and produced. Structural and functional properties of these combinatory mutants were compared experimentally and in silico with those of recombinant hevein. The IgE-binding affinity of the mutants decreased by three to five orders of magnitude as compared with that of recombinant hevein. Skin prick test reactivity of the triple mutant HDelta3A was drastically reduced and that of the six-residue mutant Hdelta6 was completely abolished in all patients examined in this study. The approach presented in this paper offers tools for identification and modification of amino acid residues on conformational epitopes of allergens that interact with IgE. Hevein with a highly reduced ability to bind IgE should provide a valuable candidate molecule for immunotherapy of latex allergy and is anticipated to have a low risk of systemic side effects.
Karisola
Piia
P
Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland.
Mikkola
Jari
J
Kalkkinen
Nisse
N
Airenne
Kari J
KJ
Laitinen
Olli H
OH
Repo
Susanna
S
Pentikäinen
Olli T
OT
Reunala
Timo
T
Turjanmaa
Kristiina
K
Johnson
Mark S
MS
Palosuo
Timo
T
Kulomaa
Markku S
MS
Alenius
Harri
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D019943:Amino Acid Substitution / Q000235:genetics* / Q000276:immunology; D023181:Antimicrobial Cationic Peptides; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D002648:Child; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D003888:Desensitization, Immunologic / Q000379:methods; D000939:Epitopes / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D020315:Latex Hypersensitivity / Q000276:immunology / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000378:metabolism / Q000627:therapeutic use
D000485:Allergens; D023181:Antimicrobial Cationic Peptides; D000939:Epitopes; D037121:Plant Lectins; D011994:Recombinant Proteins; C045875:hevein; D007073:Immunoglobulin E