Hogan L H, Josvai S, Klein B S
Department of Pediatrics, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics, Madision 53792, USA.
J Biol Chem. 1995 Dec 22;270(51):30725-32. doi: 10.1074/jbc.270.51.30725.
WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We isolated and analyzed a clone of genomic WI-1 to characterize this key adherence mechanism of the yeast. The 9.3-kilobase insert contains an open reading frame of 3438 nucleotides and no introns. The amino acid sequence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned gene is authentic WI-1. The complete coding sequence has 30 highly conserved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersiniae adhesin invasin, the C terminus displays an epidermal growth factor-like domain, and the N terminus has a short hydrophobic sequence that may be a membrane-spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the adhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nucleotide positions 2287-2290), TATA box (2380-2385), and CT motif (2399-2508). Transcription is initiated within the CT motif at nucleotide 2431. A 5.5-kilobase subclone containing the full coding sequence of WI-1 was expressed as a histidine-tagged fusion protein in Escherichia coli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is strongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. This work clarifies a key adherence mechanism of B. dermatitidis and will permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.
WI-1是皮炎芽生菌酵母上一种120千道尔顿的表面蛋白黏附素，它能与人巨噬细胞上的CD18和CD14受体结合。我们分离并分析了基因组WI-1的一个克隆，以表征酵母的这一关键黏附机制。这个9.3千碱基的插入片段包含一个3438个核苷酸的开放阅读框，没有内含子。天然WI-1的氨基酸序列在第757 - 769位、901 - 913位和1119 - 1138位与基因组WI-1的推导序列匹配，表明克隆的基因是真实的WI-1。完整的编码序列有30个24个氨基酸的高度保守重复序列，串联排列在蛋白质的两个不连续区域。该重复序列与耶尔森菌黏附素侵袭素同源，C末端显示一个表皮生长因子样结构域，N末端有一个短的疏水序列，可能是一个跨膜结构域。预计串联重复序列位于蛋白质的暴露表面，从而解释了WI-1的黏附特性。WI-1启动子包含一个CAAT框（核苷酸位置2287 - 2290）、TATA框（2380 - 2385）和CT基序（2399 - 2508）。转录在CT基序内的核苷酸2431处起始。一个包含WI-1完整编码序列的5.5千碱基亚克隆在大肠杆菌中表达为组氨酸标签融合蛋白。重组WI-1具有预期的120千道尔顿分子量，在蛋白质印迹中被兔抗WI-1抗血清强烈识别，并以与天然WI-1相同的方式结合人巨噬细胞受体。这项工作阐明了皮炎芽生菌的一个关键黏附机制，并将允许进一步分析WI-1介导的与宿主细胞、受体和细胞外基质的附着。
