Zargari A, Emilson A, Halldén G, Johansson S, Scheynius A
Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden.
Clin Exp Allergy. 1997 May;27(5):584-92.
We have previously identified two major allergens of Pityrosporum orbiculare and characterized these as 37 kDa and 67 kDa proteins.
In the present study we have investigated the presence and subcellular location of the 37 kDa and 67 kDa allergen components in various members of the genus Pityrosporum as well as in Candida albicans, Candida parapsilosis and Saccharomyces cerevisiae.
To detect both cell surface and intracellular expression of the allergens, flow cytometry and confocal laser scanning microscopy (CLSM) were used. The cells were stained with indirect immunofluorescent (IIF) or alkaline phosphatase anti-alkaline phosphatase (APAAP) methods using mouse monoclonal antibodies (MoAbs).
Ninety-five per cent of the P. orbiculare (P. ovale) cells cultured for 4 days showed cell surface-binding of the anti-37 kDa MoAb and 88% of the cells bound the anti-67 kDa MoAb when analysed with IIF and flow cytometry. It was found that the members of the genus Pityrosporum (Malassezia), P. pachydermatis and M. sympodialis, expressed the 37 kDa and 67 kDa allergens to a similar extent as did P. orbiculare. Less than 5% of the cells of the genus Candida and S. cerevisiae showed positive staining with the MoAbs. The CLSM revealed that the 37 kDa and the 67 kDa components were located to the cell wall and could not be detected inside the acetone fixed and APAAP stained yeast cells of the genus Pityrosporum. When the yeast cells were cultured for more than 4 days the expression of both allergens decreased significantly.
All three members of the genus Pityrosporum express the 37 kDa and 67 kDa major allergens on the cell surface, whereas these proteins could virtually not be detected in the Candida genus and S. cerevisiae.
我们之前已鉴定出圆形糠秕孢子菌的两种主要过敏原，并将其表征为37 kDa和67 kDa蛋白。
在本研究中，我们调查了糠秕孢子菌属各成员以及白色念珠菌、近平滑念珠菌和酿酒酵母中37 kDa和67 kDa过敏原成分的存在情况及亚细胞定位。
为检测过敏原在细胞表面和细胞内的表达，使用了流式细胞术和共聚焦激光扫描显微镜（CLSM）。细胞用间接免疫荧光（IIF）法或碱性磷酸酶抗碱性磷酸酶（APAAP）法，采用小鼠单克隆抗体（MoAbs）进行染色。
用IIF和流式细胞术分析时，培养4天的圆形糠秕孢子菌（卵形糠秕孢子菌）细胞中有95%显示抗37 kDa MoAb的细胞表面结合，88%的细胞结合抗67 kDa MoAb。发现糠秕孢子菌属（马拉色菌属）的成员，厚皮马拉色菌和合轴马拉色菌，表达37 kDa和67 kDa过敏原的程度与圆形糠秕孢子菌相似。念珠菌属和酿酒酵母的细胞中不到5%显示MoAbs阳性染色。CLSM显示，37 kDa和67 kDa成分位于细胞壁，在丙酮固定并用APAAP染色的糠秕孢子菌属酵母细胞内未检测到。当酵母细胞培养超过4天时，两种过敏原的表达均显著下降。
糠秕孢子菌属的所有三个成员在细胞表面表达37 kDa和67 kDa主要过敏原，而在念珠菌属和酿酒酵母中几乎检测不到这些蛋白。
