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ID 47022
file
creator
Ishii, Kaori
subject
HRT
MGL_1304
Pichia pastoris
Sweat allergy
β-hexosaminidase release
NDC
Medical sciences
abstract
Background: We previously identified MGL_1304 secreted by Malassezia globosa as a sweat antigen for patients with atopic dermatitis (AD) and cholinergic urticaria (ChU). However, purifying native MGL_1304 from human sweat or culture supernatant of M. globosa (sup-MGL_1304) is costly and timeconsuming. Moreover, recombinant MGL_1304 expressed by using Escherichia coli (TF-rMGL_1304) needs a large chaperon protein and lacks the original glycosylation of yeasts. Thus, we generated a recombinant MGL_1304 by Pichia pastoris (P-rMGL_1304) and investigated its characteristic features.
Methods: Recombinant MGL_1304 proteins expressed by E. coli and P. pastoris were generated. Properties of these recombinants and native antigens were compared by western blot analysis, histamine release tests (HRT) of patients with AD and ChU, and b-hexosaminidase release tests with RBL-48 cells. PrMGL_ 1304-specific IgE in sera of patients with AD were measured by sandwich ELISA.
Results: Western blot analysis revealed that IgE of patients with AD bound to all MGL_1304 recombinants and native antigens. The histamine releasing ability of P-rMGL_1304 was 100 times higher than that of TF-rMGL_1304, and was comparable to that of sup-MGL_1304. Degranulation rates of RBL-48 cells, sensitized with sera of patients with AD in response to the stimulation of P-rMGL_1304, were comparable to those of sup-MGL_1304, whereas those of TF-rMGL_1304 were relatively weak. The levels of P-rMGL_1304-specific IgE in sera of patients with AD were correlated with their disease severities.
Conclusions: P-rMGL_1304 has an antigenicity comparable to the native antigen, and is more useful than TF-rMGL_1304, especially in HRT and degranulation assay of RBL-48 cells.
description
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT/ JSPS KAKENHI, Grant numbers 19790789, 22591239, 25461669), Grant-in-Aid for Young Scientists (B), and Scientific Research (C); and Health and Labour Sciences Research Grant; A-STEP program of Japan Science and Technology Agency.
journal title
Allergology International
volume
Volume 64
issue
Issue 3
start page
266
end page
271
date of issued
2015-07
publisher
Japanese Society of Allergology
issn
1323-8930
publisher doi
pubmed id
language
eng
nii type
Journal Article
HU type
Journal Articles
DCMI type
text
format
application/pdf
text version
publisher
rights
Copyright © 2015, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/)
relation url
department
Graduate School of Biomedical & Health Sciences
University Medical Hospital