Molecular characterization of major cat allergen Fel d 1: expression of heterodimer by use of a baculovirus expression system.
Fel d 1 is a major cat allergen inducing allergic rhinitis and asthma in sensitized individuals. It has a more complex structure when compared with other allergens and therefore expression of recombinant Fel d 1 has been considered a challenge. The present study shows for the first time that a Baculovirus expression system is able to produce an intact rFel d 1 molecule that is glycosylated and structurally equivalent to the natural cat allergen, nFel d 1. Enzymatic digestion of rFel d 1 and further analysis by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) resulted in a complete coverage of the amino acid sequence of rFel d 1. In addition, the three disulfide bridges at the positions alpha70-beta7, alpha44-beta48, and alpha3-beta73 were verified. The N-glycan structure of rFel d 1 was investigated by a combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAC). The N-glycosylation analyses of rFel d 1 refer to a pattern of glycoforms including core alpha1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histamine release, and lymphocyte proliferation assays demonstrated that the immunological characteristics of rFel d 1 are similar to those of nFel d 1. Detailed characterization of both natural and recombinant allergens provides tools to explore immunological mechanisms associated with allergen sensitization and desensitization.
Seppälä
Ulla
U
Department of Vaccine Research, ALK-Abelló, 2970 Hørsholm, Denmark.
Hägglund
Per
P
Wurtzen
Peter A
PA
Ipsen
Henrik
H
Thorsted
Peter
P
Lenhard
Thomas
T
Roepstorff
Peter
P
Spangfort
Michael D
MD
D000485:Allergens / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D001667:Binding, Competitive / Q000276:immunology; D002240:Carbohydrate Sequence; D002415:Cats; D002460:Cell Line; D019281:Dimerization; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D006031:Glycosylation; D006632:Histamine / Q000378:metabolism; D007073:Immunoglobulin E / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
D000485:Allergens; D006023:Glycoproteins; D007073:Immunoglobulin E; D006632:Histamine; C059296:Fel d 1 protein, Felis domesticus