Rodriguez G M, Diment S
Michael Heidelberger Division of Immunology, Department of Pathology, New York University Medical Center, NY 10016.
J Immunol. 1992 Nov 1;149(9):2894-8.
Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.
通过蛋白水解对蛋白质抗原进行修饰是将抗原呈递给Th细胞的主要步骤之一。然而，对于参与这些过程的酶、它们的特异性或它们产生的天然片段的特征知之甚少。组织蛋白酶D（CD）是一种在抗原呈递细胞（APC）的内体中发现的天冬氨酸蛋白酶。在本报告中，研究了CD在卵清蛋白（OVA）加工过程中的作用。当用固定的APC呈递时，用纯化的CD体外消化的OVA能够刺激三种不同的OVA特异性I-Ad限制性Th细胞杂交瘤分泌白细胞介素-2。OVA的消化产物在I-Ad的背景下被识别，但不被I-Ak限制性OVA特异性Th细胞识别。在不存在胎牛血清（FCS）或存在蛋白酶抑制剂的情况下，用CD消化的OVA刺激Th细胞的能力没有差异，这表明细胞外蛋白酶不太可能参与OVA的加工。综上所述，这些结果表明CD对于从OVA产生抗原表位是必要且充分的。通过反相高效液相色谱从OVA消化产物中分离出含有该表位的片段。该片段在SDS-PAGE中作为10 kDa多肽迁移，是一种有效的表位。将其激活Th细胞的能力与胰蛋白酶肽OVA323-339的能力进行了比较。
