Glycosylation of recombinant proforms of major house dust mite allergens Der p 1 and Der f 1 decelerates the speed of maturation.
BACKGROUND
The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared.


METHODS
Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms.


RESULTS
Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain.


CONCLUSIONS
Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.
Takai
Toshiro
T
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan. t-takai@med.juntendo.ac.jp
Mizuuchi
Eri
E
Kikuchi
Yuko
Y
Nagamune
Teruyuki
T
Okumura
Ko
K
Ogawa
Hideoki
H
D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039981:Pyroglyphidae / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1; C451162:Dermatophagoides pteronyssinus antigen p 1