A recombinant bait region mutant of human alpha2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum.

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.