How Immune Peptidases Change Specificity: Cathepsin G Gained Tryptic Function but Lost Efficiency during Primate Evolution

Abstract

Cathepsin G is a major secreted serine peptidase of neutrophils and mast cells. Studies in Ctsg-null mice suggest that cathepsin G supports antimicrobial defenses but can injure host tissues. The human enzyme has an unusual “Janus-faced” ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu²²⁶ in the primary specificity pocket and underlies proposed important functions, such as activation of prourokinase. However, most mammals, including mice, substitute Ala²²⁶ for Glu²²⁶, suggesting that human tryptic activity may be anomalous. To test this hypothesis, human cathepsin G was compared with mouse wild-type and humanized active site mutants, revealing that mouse primary specificity is markedly narrower than that of human cathepsin G, with much greater Tyr activity and selectivity and near absence of tryptic activity. It also differs from human in resisting tryptic peptidase inhibitors (e.g., aprotinin), while favoring angiotensin destruction at Tyr⁴ over activation at Phe⁸. Ala²²⁶Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate prourokinase. Phylogenetic analysis reveals that the Ala²²⁶Glu missense mutation appearing in primates 31–43 million years ago represented an apparently unprecedented way to create tryptic activity in a serine peptidase. We propose that tryptic activity is not an attribute of ancestral mammalian cathepsin G, which was primarily chymotryptic, and that primate-selective broadening of specificity opposed the general trend of increased specialization by immune peptidases and allowed acquisition of new functions.