A recombinant bait region mutant of human alpha 2-macroglobulin exhibitingan altered proteinase-inhibiting spectrum

Alpha 2-macroglobulin (alpha 2M), a plasma glycoprotein produced in the liv er, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been exp lained by the trapping theory by which a proteinase recognizes a region of 25-30 amino acid peptide in alpha 2M called bait region and cleaves it, lea ding to the conformational change of alpha 2M, and to the subsequent entrap ment and inhibition of the proteinase. We constructed alpha 2M cDNAs with mutated DNA sequences in the bait region , and obtained recombinant CHO cell lines producing either wild type alpha 2M, or mutant alpha 2Ms, i.e., alpha 2M/K692 and alpha 2M/K696, each with s ubstitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type alpha 2M, but could be inhibited by these engineered mutant alpha 2Ms. Thus, recombinant alpha 2M /K696 protein successfully inhibited lysyl endopeptidase activity, while re combinant alpha 2M/K692 protein was not sensitive to lysyl endopeptidase, s uggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the t rapping theory with additional supportive evidence, but the first experimen tal evidence for the value of engineered alpha 2M-derived proteinase inhibi tor with an artificial proteinase inhibitory spectrum of potential industri al and/or therapeutic usefulness.