Djordjevic Julianne T, Del Poeta Maurizio, Sorrell Tania C, Turner Kylie M, Wright Lesley C
Centre for Infectious Diseases and Microbiology, ICPMR and Westmead Millennium Institute, Westmead Hospital, Westmead 2145, NSW, Australia.
Biochem J. 2005 Aug 1;389(Pt 3):803-12. doi: 10.1042/BJ20050063.
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.
分泌型多功能酶PLB1（由PLB1基因编码的磷脂酶B1蛋白）是致病真菌新生隐球菌的一种毒力决定因素，但其分泌机制尚不清楚。隐球菌PLB1基因编码推定的N端LP（前导肽）和C端GPI（糖基磷脂酰肌醇）锚定附着基序，这表明PLB1在分泌前是GPI锚定的。为了研究这些基序在PLB1分泌中的作用，构建了四个cDNA构建体，分别编码全长构建体（PLB1）和三个没有LP和/或GPI锚定附着基序的截短版本[（LP-）PLB1（无LP共有基序表达的PLB1）、（LP-）PLB1（GPI-）（无LP和GPI共有基序表达的PLB1）和PLB1（GPI-）（无GPI锚定附着基序表达的PLB1）]。将构建体连接到pYES2中，并在酿酒酵母中实现半乳糖诱导表达。LP对于PLB1蛋白的分泌及其三种活性（磷脂酶B、溶血磷脂酶和溶血磷脂酶转酰基酶）至关重要。删除GPI基序以产生PLB1（GPI-）导致活性从细胞壁和细胞膜重新分布到分泌和胞质部分，总活性的36-54%被分泌，而PLB1的这一比例小于5%。PLB1产生的细胞相关活性最高（比PLB1（GPI-）高2倍以上），其中75-86%在细胞壁部分，6-19%在膜部分，3-7%在胞质部分。在酿酒酵母重组体和野生型新生隐球菌中，通过β-葡聚糖酶释放活性证实了细胞壁定位。PLB1通过GPI锚定在细胞壁中的主要定位可能允许该酶在环境条件变化时立即释放，并且可能代表调节真菌毒力决定因素分泌的新机制的一部分。
