Jasmer Douglas P, Rosa Bruce A, Mitreva Makedonka
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America.
The Genome Institute, Washington University School of Medicine, St. Louis, Missouri, United States of America.
PLoS Negl Trop Dis. 2015 Jan 8;9(1):e3375. doi: 10.1371/journal.pntd.0003375. eCollection 2015 Jan.
The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.
线虫肠道是开发治疗和控制寄生线虫新方法所关注的组织。然而，肠道细胞功能的生物学细节仍然不清楚，线虫顶端肠膜（AIM）和肠腔（IL）中的蛋白质及分子功能也是如此。因此，人们开发了一些方法来全面鉴定在成年雌性猪蛔虫肠道中发挥作用的肽酶。在pH值范围为5.0至8.0的条件下，在猪蛔虫肠道的多个组分中检测到了肽酶活性。使用肽酶类抑制剂来表征这些活性。这些组分包括全裂解物、富含膜的组分以及肠腔的生理灌注液和4摩尔尿素灌注液。证实伴刀豆球蛋白A（ConA）与AIM结合，并通过质谱将在ConA磁珠上亲和分离的肠道蛋白与膜组分和灌注液组分中的蛋白进行比较。鉴定出了29种预测的肽酶，包括天冬氨酸、半胱氨酸和丝氨酸肽酶，以及数量出乎意料的多（16种）金属肽酶。其中许多蛋白质共定位于多个组分，为定位于特定肠道区室（包括IL和AIM）提供了独立的支持。这种独特的灌注模型对可能在猪蛔虫或任何线虫的这些肠道区室中发挥作用的消化肽酶给出了最全面的视图。该模型提供了一种直接确定这些蛋白质在猪蛔虫肠道中的功能的方法，更广泛地说，可推断出线虫肠道这些细胞区室中存在的广泛功能。
