Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity

International Archives of Allergy and Immunology
K TakahashiY Okumura

Abstract

The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens. In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1). The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells. Secretion of their proforms was checked by SDS-PAGE or immunoblotting. Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1. The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P. pastoris. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain. A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P. pastoris. The prosequence of pro-C35S/N53Q was removed by an in vitro activa...Continue Reading

Citations

Oct 6, 2007·Journal of Molecular Biology·Andy ChevignéMoreno Galleni
Nov 26, 2009·The Journal of Immunology : Official Journal of the American Association of Immunologists·Toshiro TakaiKo Okumura
Sep 9, 2005·The Journal of Immunology : Official Journal of the American Association of Immunologists·Kåre MenoKaare Lund
Apr 13, 2010·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Jannel AcostaMario P Estrada

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