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 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 pro-urokinase. However, most mammals, including mice, substitute Ala 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 pro-urokinase. 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.

Identification and Characterization of Circulating Variants of CXCL12 from Human Plasma: Effects on Chemotaxis and Mobilization of Hematopoietic Stem and Progenitor Cells

“…The absence of CXCL12 might be related to species-specific differences in the neutrophil serine protease activities [42,43].…”

Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein

“…12, 45) show that LCV-infected B cells have a central pathogenic role in the activation of highly pathogenic MHC-E-restricted effector memory cytotoxic T cells that, upon activation with the MOG34-56 peptide (core epitope [40][41][42][43][44][45][46][47][48], can elicit MS-like pathology and disease. Notably, similar autoaggressive CTLs were found in MS lesions, where they seemed to be engaged in cytotoxic interactions with HLA-E + oligodendrocytes (5).…”

“…Although CatG of marmosets has a mouse-like narrow specificity for aromatic (chymotryptic) cleavage sites (Trp, Phe, Tyr), a double mutation in the specificity-determining residues (Ala-189-Ser and Glu-226-Ala) broadens the activity of rhesus monkey and human CatG to basic (tryptic) cleavage sites (e.g., Arg and Lys) (42). To test the proteolytic degradation of the pathogenically dominant Mm-MOG34-56 peptide we used the shorter peptide MOG35-51 to avoid interference of the Arg residue at position 52, which is outside the epitope of interest (residues [40][41][42][43][44][45][46][47][48]. The reported in vitro data show that native Mm-MOG35-51 peptide is rapidly degraded in cellfree lysates of noninfected and LCV infected B cells of rhesus monkeys as well as in LCV-infected B cells of marmosets.…”

“…Again, a dominant role of the endolysosomal protease CatG in the degradation of Mm-MOG35-51 was observed. Interestingly, substitution of the Pro 42 residue for Ser and the Arg46 residues for Cit conferred complete resistance of the MOG35-51 peptide to proteolytic degradation in the rhesus monkey cell lysates. Substitution of the Arg41 residue for Cit had no detectable effect.…”

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis

“…However, cathepsin G inhibition using an inhibitor of the human enzyme failed to block PCMC-mediated IL-13 degradation. Notably though, human and murine cathepsin G differ substantially within their respective active sites, human cathepsin G having dual trypsin-and chymotrypsin-like activity, whereas murine cathepsin G has sole chymotrypsin-like activity (Raymond et al , 2010 ). Thus, we cannot exclude that the available inhibitor of human cathepsin G has poor efficacy towards the murine counterpart, To ascertain that mMCP-5 was enzymatically active, recombinant mMCP-5 (0.08 μ g) was incubated with four different recombinant substrates for 3 h followed by SDS-PAGE and Coomassie brilliant blue staining.…”