Synthesis in E. coli of alpha 1-antitrypsin variants of therapeutic potential for emphysema and thrombosis.
The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components. The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal. Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking. Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein. To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA. We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli. The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation. The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor. The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder.
D000595:Amino Acid Sequence; D004646:Emphysema / Q000188:drug therapy; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D009154:Mutation; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors; D013927:Thrombosis / Q000188:drug therapy; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics* / Q000627:therapeutic use
D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase; D013917:Thrombin