Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans
Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans
复制标题
白色念珠菌在胚管形成过程中合成的壁特异性抗原的鉴定
DOI：
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发表时间：
1989
影响因子：
3.1
通讯作者：
Rafael SENTANDREUl
中科院分区：
文献类型：
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作者：
M. Casanova;'. M. L. Gil;L. Cardeñoso;José P. Martínez;Rafael SENTANDREUl
用14c蛋白水解物和[3H]苏氨酸代谢标记（6 h脉冲）的细胞获得了白色念珠菌ATCC 26555的两种细胞形式（胚孢子和菌丝）的细胞壁。用缓冲溶液和十二烷基硫酸钠溶液彻底清洗壁，并用酶解酶20T消化。酶处理从酵母细胞中释放出四种主要的高分子量甘露蛋白（HMWM），表观分子质量分别为650、500、340和200千道尔顿（HMWM-650、HMWM-500、HMWM-340和HMWM-200），而从菌丝细胞中溶解出两种高分子量甘露蛋白（HMWM-260和HMWM-180）。在这两种类型细胞的细胞壁酶解中也检测到一些较小的低分子量物种。单脉冲和双脉冲追踪实验表明，HMWM-260和HMWM-180反映了菌丝特异性新蛋白的从头合成，而不代表胚分生孢子壁成分的拓扑重排。制备了针对HMWM-260菌株（菌丝壁上的主要成分）的单克隆抗体，获得了针对酵母或菌丝细胞壁的兔多克隆抗体。将抗菌丝细胞壁多克隆抗体吸附在全灭活胚分生孢子上，去除针对常见胚分生孢子和菌丝细胞壁抗原的抗体。酶联免疫吸附法滴定表明，单克隆抗体能够识别HMWM-260甘露糖蛋白片段的一个表位。利用这些单克隆和多克隆抗体进行免疫印迹和免疫荧光技术证实，HMWM-260和HMWM-180是菌丝细胞壁包膜的特异性成分。
Walls of the two cellular forms (blastoconidia and mycelia) of Candida albicans ATCC 26555 were obtained from cells metabolically labeled (6-h pulse) with 14C-protein hydrolysate and [3H]threonine. Walls were purified by thorough washings with buffered and sodium dodecyl sulfate solutions and digested with Zymolyase 20T. The enzymatic treatment released four major high-molecular-weight mannoproteins (HMWM), with apparent molecular masses of 650, 500, 340, and 200 kilodaltons (HMWM-650, HMWM-500, HMWM-340, and HMWM-200, respectively), from yeast cells, whereas two high-molecular-mass mannoproteins (HMWM-260 and HMWM-180) were solubilized from mycelial cells. Some additional minor low-molecular-weight species were also detected in the enzymatic digests of walls from both types of cell. Single and dual pulse-chase experiments indicated that the HMWM-260 and HMWM-180 species reflect de novo synthesis of new proteins specific for the mycelia and do not represent a topological rearrangement of blastoconidium wall components. Monoclonal antibodies were raised against the HMWM-260 species (quantitatively the predominant component in the mycelial walls), and polyclonal rabbit antibodies were obtained against yeast or mycelial cell walls. Anti-mycelial cell wall polyclonal antibodies were adsorbed to whole killed blastoconidia to remove antibodies against common blastoconidium and mycelial wall antigens. Titration by enzyme-linked immunosorbent assay revealed that the monoclonal antibodies could recognize an epitope of the protein moiety of the HMWM-260 mannoprotein. Immunoblotting and immunofluorescence techniques using these monoclonal and polyclonal antibodies confirmed that the HMWM-260 and HMWM-180 species are specific components of the envelope of the mycelial cell walls.