TY - JOUR

T1 - Molecular characterization of major cat allergen Fel d 1

T2 - Expression of heterodimer by use of a baculovirus expression system

AU - Seppälä, Ulla

AU - Hägglund, Per

AU - Wurtzen, Peter A.

AU - Ipsen, Henrik

AU - Thorsted, Peter

AU - Lenhard, Thomas

AU - Roepstorff, Peter

AU - Spangfort, Michael D.

PY - 2005/2/4

Y1 - 2005/2/4

N2 - 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 α70-β7, α44-β48, and α3-β373 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 α1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histanaine 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.

AB - 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 α70-β7, α44-β48, and α3-β373 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 α1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histanaine 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.

UR - http://www.scopus.com/inward/record.url?scp=13544249596&partnerID=8YFLogxK

U2 - 10.1074/jbc.M410668200

DO - 10.1074/jbc.M410668200

M3 - Journal article

C2 - 15546862

AN - SCOPUS:13544249596

VL - 280

SP - 3208

EP - 3216

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 5

ER -