Widjaja Michael, Berry Iain J, Pont Elsa J, Padula Matthew P, Djordjevic Steven P
ithree institute, University of Technology Sydney, P.O. Box 123 Broadway, Ultimo, NSW 2007, Australia.
Proteomics Core Facility, University of Technology Sydney, Cnr Harris and Thomas St, Ultimo, NSW 2007, Australia.
Proteomes. 2015 Dec 16;3(4):512-537. doi: 10.3390/proteomes3040512.
is a significant cause of community acquired pneumonia globally. Despite having a genome less than 1 Mb in size, presents a structurally sophisticated attachment organelle that (i) provides cell polarity, (ii) directs adherence to receptors presented on respiratory epithelium, and (iii) plays a major role in cell motility. The major adhesins, P1 () and P30 (), are localised to the tip of the attachment organelle by the surface accessible cleavage fragments P90 and P40 derived from Mpn142. Two events play a defining role in the formation of P90 and P40; removal of a leader peptide at position 26 (SLA↓NTY) during secretion to the cell surface and cleavage at amino acid 455 (GPL↓RAG) generating P40 and P90. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of tryptic peptides generated by digesting size-fractionated cell lysates of identified 15 cleavage fragments of Mpn142 ranging in mass from 9-84 kDa. Further evidence for the existence of cleavage fragments of Mpn142 was generated by mapping tryptic peptides to proteins recovered from size fractionated eluents from affinity columns loaded with heparin, fibronectin, fetuin, actin, plasminogen and A549 surface proteins as bait. To define the sites of cleavage in Mpn142, neo-N-termini in cell lysates of were dimethyl-labelled and characterised by LC-MS/MS. Our data suggests that Mpn142 is cleaved to generate adhesins that are auxiliary to P1 and P30.
在全球范围内，是社区获得性肺炎的一个重要病因。尽管其基因组大小小于1 Mb，但它呈现出一种结构复杂的附着细胞器，该细胞器（i）提供细胞极性，（ii）指导与呼吸道上皮细胞上呈现的受体结合，以及（iii）在细胞运动中起主要作用。主要黏附素P1（ ）和P30（ ）通过源自Mpn142的表面可及切割片段P90和P40定位于附着细胞器的尖端。有两个事件在P90和P40的形成中起决定性作用；在分泌到细胞表面的过程中，在第26位（SLA↓NTY）去除前导肽，以及在氨基酸455位（GPL↓RAG）切割产生P40和P90。通过对经大小分级的细胞裂解物进行胰蛋白酶消化产生的胰蛋白酶肽段进行液相色谱串联质谱（LC-MS/MS）分析，鉴定出Mpn142的15个切割片段，质量范围为9 - 84 kDa。通过将胰蛋白酶肽段映射到从以肝素、纤连蛋白、胎球蛋白、肌动蛋白、纤溶酶原和A549表面蛋白为诱饵加载的亲和柱的大小分级洗脱液中回收的蛋白质上，进一步证明了Mpn142切割片段的存在。为了确定Mpn142中的切割位点，对细胞裂解物中的新N端进行二甲基标记，并通过LC-MS/MS进行表征。我们的数据表明，Mpn142被切割产生辅助P1和P30的黏附素。
