Secretion of cryptococcal phospholipase B1 (PLB1) is regulated by a glycosylphosphatidylinositol (GPI) anchor
Secretion of cryptococcal phospholipase B1 (PLB1) is regulated by a glycosylphosphatidylinositol (GPI) anchor
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DOI：
10.1042/bj20050063
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发表时间：
2005-08-01
影响因子：
4.1
通讯作者：
Wright, LC
中科院分区：
文献类型：
--
作者：
Djordjevic, JT;Del Poeta, M;Wright, LC
分泌的多功能酶PLB 1（PLB 1基因编码的磷脂酶B1蛋白）是致病性真菌新生隐球菌的毒力决定因子，但其分泌机制尚不清楚。隐球菌PLB 1基因编码假定的N-末端LP（前导肽）和C-末端GPI（糖基磷脂酰肌醇）锚附着基序，表明PLB 1是GPI锚定分泌前。为了研究这些基序在PLB 1分泌中的作用，创建了四个cDNA构建体，编码全长构建体（PLB 1）和三个不含LP和/或GPI锚附着基序的截短版本[（LP-）PLB 1（PLB 1表达无LP共有基序），（LP-）PLB 1（GPI-）（PLB 1在没有LP和GPI共有基序的情况下表达）和PLB 1（GPI-）（PLB 1在没有GPI锚连接基序的情况下表达）]。将构建体连接到pYES 2中，并在酿酒酵母中实现半乳糖诱导的表达。LP是分泌PLB 1蛋白及其三种活性（PLB，溶血磷脂酶和溶血磷脂酶转酰酶）所必需的。缺失GPI基序以产生PLB 1（GPI-）导致活性从细胞壁和膜重新分配到分泌和胞质组分，与PLB 1的< 5%相比，总活性的36-54%被分泌。PLB 1产生最大的细胞相关活性（比PLB 1（GPI-）多> 2倍），其中75-86%在细胞壁部分中，6-19%在膜部分中，3-7%在胞质部分中。细胞壁定位证实释放的活性与β-葡聚糖酶在两个S。酿酒酵母重组体和野生型C.新人类通过GPI锚定PLB 1在细胞壁中的优势位置可能允许酶响应于变化的环境条件而立即释放，并且可能代表用于调节真菌毒力决定子分泌的新机制的一部分。
The secreted, multifunctional enzyme PLB1 (phospholipase B1 protein encoded by the PLB1 gene) is a virulence determinant of the pathogenic fungus Cryptococcus neoformans, but the mechanism of its secretion is unknown. The cryptococcal PLB1 gene encodes putative, N-terminal LP (leader peptide) and C-terminal GPI (glycosylphosphatidylinositol) anchor attachment motifs, suggesting that PLB1 is GPI-anchored before secretion. To investigate the role of these motifs in PLB1 secretion, four cDNA constructs were created encoding the full-length construct (PLB1) and three truncated versions without the LP and/or the GPI anchor attachment motifs [(LP-)PLB1 (PLB1 expressed without the LP consensus motif), (LP-)PLB1(GPI-) (PLB1 expressed without the LP and GPI consensus motifs) and PLB1(GPI-) (PLB1 expressed without the GPI anchor attachment motif) respectively]. The constructs were ligated into pYES2, and galactose-induced expression was achieved in Saccharomyces cerevisiae. The LP was essential for secretion of the PLB1 protein and its three activities (PLB, lysophospholipase and lysophospholipase transacylase). Deletion of the GPI motif to create PLB1(GPI-) resulted in a redistribution of activity from the cell wall and membranes to the secreted and cytosolic fractions, with 36-54% of the total activity being secreted as compared with < 5% for PLB1. PLB1 produced the maximum cell-associated activity (> 2-fold more than that for PLB1(GPI-)), with 75-86% of this in the cell-wall fraction, 6-19% in the membrane fraction and 3-7% in the cytosolic fraction. Cell-wall localization was confirmed by release of activity with beta-glucanase in both S. cerevisiae recombinants and wild-type C. neoformans. The dominant location of PLB1 in the cell wall via GPI anchoring may permit immediate release of the enzyme in response to changing environmental conditions and may represent part of a novel mechanism for regulating the secretion of a fungal virulence determinant.