Cytotechnology. 1999 Sep;31(1-2):53-60. doi: 10.1023/A:1008011919876.
Alpha 2-macroglobulin (alpha2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25-30 amino acid peptide in alpha2M called bait region and cleaves it, leading to the conformational change of alpha2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed alpha2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type alpha2M, or mutant alpha2Ms, i.e., alpha2M/K692 and alpha2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type alpha2M, but could be inhibited by these engineered mutant alpha2Ms. Thus, recombinant alpha2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant alpha2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered alpha2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness.
α2-巨球蛋白（α2M）是一种在肝脏中产生的血浆糖蛋白，可抑制多种蛋白酶，因此被认为在体内发挥着重要的动态平衡作用。这种广泛的抑制谱可以用捕获理论来解释，该理论认为蛋白酶识别α2M 中称为诱饵区域的 25-30 个氨基酸肽区域，并切割它，导致α2M 的构象变化，随后捕获并抑制蛋白酶。我们构建了诱饵区域中突变 DNA 序列的α2M cDNA，并获得了产生野生型α2M 或突变型α2M 的重组 CHO 细胞系，即α2M/K692 和 α2M/K696，每个突变都在密码子 692 和 696 处用赖氨酸取代精氨酸。我们测试了赖氨酸内肽酶是否不受野生型α2M 的抑制，但可以被这些工程突变的α2M 抑制。因此，重组α2M/K696 蛋白成功抑制了赖氨酸内肽酶活性，而重组α2M/K692 蛋白对赖氨酸内肽酶不敏感，表明并非所有的诱饵区域肽键都能同等地被蛋白酶接近和敏感。这些结果不仅为捕获理论提供了额外的支持证据，而且还为工程化的α2M 衍生蛋白酶抑制剂提供了第一个实验证据，该抑制剂具有潜在的工业和/或治疗用途的人工蛋白酶抑制谱。
