van Lierop M J, van Noort J M, Wagenaar J P, Rutten V P, Langeveld J, Meloen R H, Hensen E J
Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands.
J Gen Virol. 1994 Nov;75 ( Pt 11):2937-46. doi: 10.1099/0022-1317-75-11-2937.
Cathepsin D and cathepsin B are endosomal/lysosomal proteases that are thought to play a role during in vivo antigen processing, releasing fragments for binding to major histocompatibility complex class II products and subsequent presentation to T cells. Here we treated purified foot-and-mouth disease virus (FMDV) strain A10Holland with both enzymes. Cathepsin D, but not cathepsin B, was shown to release fragments from reduced or non-reduced FMDV under mild conditions in vitro. Twenty-eight predominant cathepsin D-released fragments were purified by HPLC and identified by amino acid composition analysis and sequencing. The unseparated set of fragments produced (the digest) was able to stimulate T cells from eight vaccinated cattle. With respect to the response to intact virus the extent of the response to the digest differed between animals: four animals could be classified as good responders, three as intermediate responders and one as a low responder. Subsequently, we investigated the proliferative T cell response to a large set of synthetic peptides in detail for two animals, one belonging to the group of good responders, the other being the low responder. The peptides covered all 28 cathepsin D-released fragments analysed and also several sequences not recovered from the digest. In this way seven T cell sites could be identified, five of which coincided with cathepsin D-released fragments. The other two T cell sites were VP2[54-72], being a homologue of a T cell site identified for FMDV strain O1K and the N terminus of VP4. Whether the most dominantly recognized T cell site was recovered from the digest or not was shown to be related to the good or low response to the digest. These findings suggest a role for cathepsin D in the release of some but not all T cell-stimulatory fragments from FMDV.
组织蛋白酶D和组织蛋白酶B是内体/溶酶体蛋白酶，被认为在体内抗原加工过程中发挥作用，释放片段以结合主要组织相容性复合体II类产物并随后呈递给T细胞。在此，我们用这两种酶处理纯化的口蹄疫病毒（FMDV）A10荷兰株。结果显示，在温和条件下，组织蛋白酶D而非组织蛋白酶B能在体外从还原或非还原的FMDV中释放片段。通过高效液相色谱法纯化了28个主要的组织蛋白酶D释放片段，并通过氨基酸组成分析和测序进行了鉴定。所产生的未分离片段组（消化产物）能够刺激8头接种疫苗牛的T细胞。就对完整病毒的反应而言，不同动物对消化产物的反应程度有所不同：4只动物可归类为良好反应者，3只为中等反应者，1只为低反应者。随后，我们详细研究了两只动物对大量合成肽的增殖性T细胞反应，一只属于良好反应者组，另一只为低反应者。这些肽涵盖了分析的所有28个组织蛋白酶D释放片段，以及一些未从消化产物中回收的序列。通过这种方式，确定了7个T细胞位点，其中5个与组织蛋白酶D释放片段一致。另外两个T细胞位点是VP2[54 - 72]，它是FMDV O1K株鉴定的T细胞位点的同源物以及VP4的N端。结果表明，最主要被识别的T细胞位点是否从消化产物中回收与对消化产物的良好或低反应有关。这些发现表明组织蛋白酶D在从FMDV释放一些但不是所有T细胞刺激片段中发挥作用。
