Rowan A D, Mason P, Mach L, Mort J S
Joint Diseases Laboratory, Shriners Hospital for Crippled Children, Montreal, Quebec, Canada.
J Biol Chem. 1992 Aug 5;267(22):15993-9.
Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner.
大鼠组织蛋白酶B前体在酵母中的表达导致该酶的潜伏形式和成熟形式均被分泌。在半胱氨酸蛋白酶抑制剂存在的情况下进行培养可阻止这种加工过程。我们已经表达并纯化了大鼠组织蛋白酶B前体的一种突变形式，其活性位点的半胱氨酸残基已被替换为丝氨酸，并且该蛋白成熟区域中的糖基化位点也已缺失。这种无活性的突变蛋白基本上以未加工的形式被分泌。已将纯化的蛋白与多种蛋白酶一起孵育，结果表明组织蛋白酶D和L以及成熟的组织蛋白酶B本身都可以从该前体产生加工后的（单链）组织蛋白酶B形式。对这些加工形式进行氨基末端测序表明，相对于体内发现的成熟形式，它们都延长了几个残基。组织蛋白酶B与二肽基肽酶I联合作用产生了具有正确氨基末端的单链组织蛋白酶B形式。这项工作还表明，组织蛋白酶B前体加工成单链形式至少可以以分子间的方式成为一个自催化过程。
