Ethanol dependence of α₁-antitrypsin C-terminal Lys truncation mediated by basic carboxypeptidases

BACKGROUND:Patients with hereditary emphysema are treated with alpha(1)-antitrypsin (alpha(1)-proteinase inhibitor [A1PI]) concentrates. High-resolution isoelectric focusing (IEF) analysis of A1PI shows that commercial A1PI products have different glycoisoform band patterns predominantly caused by varying degrees of C-terminal Lys truncation at position 394 from the A1PI molecule. Basic carboxypeptidases (CPs) are a group of enzymes that specifically cleave C-terminal basic amino acids (Arg or Lys) from peptides and proteins. STUDY DESIGN AND METHODS: In this study, whether A1PI is a substrate for basic CPs was investigated. CPN and CPU, two CPs present in plasma, and CPM, a GPI-anchored membrane protein highly expressed in lung tissues, were included. RESULTS: Basic CPs are able to mediate the C-terminal Lys truncation of A1PI although with a very low efficiency. However, presence of ethanol, for example, during Cohn fractionation, renders A1PI highly susceptible to cleavage by CP with the extent of Lys truncation depending on the ethanol concentration. This ethanol concentration dependence elegantly explains the varying amounts of des-Lys A1PI present in commercial preparations purified from different Cohn fractions. CONCLUSIONS: The cause of C-terminal truncation of A1PI present in products used for augmentation therapy has been identified, and it has been shown that A1PI becomes a substrate for CPs, specifically CPN, because of the presence of ethanol during Cohn fractionation.