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


METHODS
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.


RESULTS
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 activation process. The mature C35S/N53Q showed low protease activity.


CONCLUSIONS
N-glycosylation is essential for secretion in insect SF9 cells but not in P. pastoris. Disulfide bonds are essential for secretion in P. pastoris. A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity. The findings are useful for future design of recombinant products for application in immunotherapy.
Takahashi
K
K
Bioscience Research and Development Laboratory, Asahi Breweries, Ltd., Kitasoma-gun, Ibaraki, Japan.
Takai
T
T
Yasuhara
T
T
Yokota
T
T
Okumura
Y
Y
D020816:Amino Acid Motifs; D000818:Animals; D039741:Antigens, Dermatophagoides; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D018411:Spodoptera / Q000235:genetics; D014162:Transfection
D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D003545:Cysteine