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J. Biol. Chem., Vol. 280, Issue 5, 3208-3216, February 4, 2005

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Molecular Characterization of Major Cat Allergen Fel d 1

EXPRESSION OF HETERODIMER BY USE OF A BACULOVIRUS EXPRESSION SYSTEM^*

Ulla Seppälä, Per Hägglund¶, Peter A Wurtzen, Henrik Ipsen, Peter Thorsted, Thomas Lenhard, Peter Roepstorff, and Michael D. Spangfort||

From the Departments of Vaccine Research and Experimental Immunology, ALK-Abelló, 2970 Hørsholm, Denmark and the Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark

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 70-7, 44-48, and 3-73 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 1.3-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.

Received for publication, September 16, 2004 , and in revised form, November 15, 2004.

^* This work was part of the Therapeutic recombinant allergens from structural allergology (TRAFSA) project, funded by European Union 5th Framework Programme Grant QLK3-CT-1999-00620. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a long term postdoctoral fellowship from the Federation of European Biochemical Societies.

^|| To whom correspondence should be addressed. Tel.: 45-45-74-81-56; Fax: 45-45-74-86-42; E-mail:. MSp{at}dk.alk-abello.com.

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