Sequence analysis of wheat grain allergens separated by two-dimensional electrophoresis with immobilized pH gradients.
Micropreparative two-dimensional (2-D) gel electrophoresis with immobilized pH gradients (4-8) in the first dimension (IPG-DALT) was optimized for the separation of salt-soluble wheat grain proteins, associated with bakers' asthma disease. The resolved polypeptides were electroblotted onto a polyvinylidene difluoride (PVDF) membrane and incubated with the pooled sera from four asthmatic bakers. Bound IgE was demonstrated by alkaline phosphatase conjugated anti-human IgE. Major IgE binding was detected in the 27 kDa, 37 kDa and, to a lesser extent, in the 14-18 kDa area of the 2-D immunoblots, respectively. Since the main purpose of our study was to determine the N-terminal amino acid sequences of the major wheat grain allergens, N-terminal sequencing was performed for six out of a total of eleven major allergens located in the 27 kDa area, for one out of two 37 kDa allergens, and for two out of four 14-18 kDa allergens. Our results revealed that two of the 27 kDa polypeptides are clearly related to several Acyl-CoA oxidase variants of barley and rice, whereas no significant homologies were found for the remaining four 27 kDa allergens analyzed. The N-terminus of the 37 kDa allergen appeared to be blocked so that no sequence information was obtained, while the two 14-18 kDa allergens analyzed were identified as members of the wheat alpha-amylase-inhibitor family.
D000485:Allergens / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation*; D006863:Hydrogen-Ion Concentration; D008832:Microchemistry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D017455:Receptors, IgE / Q000737:chemistry; D012639:Seeds / Q000737:chemistry* / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000737:chemistry*
D000485:Allergens; D010940:Plant Proteins; D017455:Receptors, IgE