Rey-Millet C A, Chesne S, Colomb M G
Laboratoire d'Immunochimie, DBMS/ICH-INSERM U.238, Grenoble, France.
Mol Immunol. 1993 Jul;30(10):855-64. doi: 10.1016/0161-5890(93)90009-z.
Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.
以共价结合到Superose微球上的C3b作为模型系统，以简化对抗原加工过程中抗原结合的C3b修饰的分析。该模型是使用纯化的C3和结合到Superose上的胰蛋白酶建立的。C3b通过酯键共价结合到Superose上，这在碱性pH下对羟胺处理不稳定得到证明。将C3b-Superose与从U937细胞系纯化的富含内体/溶酶体的L亚细胞组分一起孵育。在C3b-Superose模型系统上检测到两种类型的有限活性：（i）一种蛋白水解活性，将C3b切割成主要是一个被释放的C3c样片段和一个表观分子量为32 kDa的C3d样片段，该片段通过原始酯键仍结合在Superose上；（ii）一种酯水解活性，切割酯键并释放C3b。抑制实验表明丝氨酸、天冬氨酸和半胱氨酸蛋白酶参与其中。组织蛋白酶B很可能是L组分中催化结合的C3b蛋白水解的主要蛋白酶之一。动力学研究支持酯键具有良好的稳定性，这支持了C3b在结合C3b的抗原的细胞外和细胞内运输过程中作为伴侣蛋白的有效作用。
