Free‐thiol Cys331 exposed during activation process is critical for native tetramer structure of cathepsin C (dipeptidyl peptidase I)

Abstract

The mature bovine cathepsin C (CC) molecule is composed of four identical monomers, each proteolytically processed into three chains. Five intrachain disulfides and three nonpaired cysteine residues per monomer were identified. Beside catalytic Cys234 in the active site, free-thiol Cys331 and Cys424 were characterized. Cys424 can be classified as inaccessible buried residue. Selective modification of Cys331 results in dissociation of native CC tetramer into dimers. The 3D homology-based model of the CC catalytic core suggests that Cys331 becomes exposed as the activation peptide is removed during procathepsin C activation. The model further shows that exposed Cys331 is surrounded by a surface hydrophobic cluster, unique to CC, forming a dimer-dimer interaction interface. Substrate/inhibitor recognition of the active site in the CC dimer differs significantly from that in the native tetramer. Taken together, a mechanism is proposed that assumes that the CC tetramer formation results in a site-specific occlusion of endopeptidase-like active site cleft of each CC monomeric unit. Thus, tetramerization provides for the structural basis of the dipeptidyl peptidase activity of CC through a substrate access-limiting mechanism different from those found in homologous monomeric exopeptidases cathepsin H and B. In conclusion, the mechanism of tetramer formation as well as specific posttranslational processing segregates CC in the family of papain proteases.

Hemoglobin Digestion in Blood-Feeding Ticks: Mapping a Multipeptidase Pathway by Functional Proteomics

“…The competition experiments with CA-074 and Z-Phe-Phe-DMK clearly distinguished cathepsin B and cathepsin L. The molecular masses determined for cathepsin B and L (MW ~32 and 30 kDa) and legumain (MW ~38–40 kDa) are in accord with values calculated from the cDNA sequences of the respective mature enzymes (Sojka et al, 2008). The observed mass of cathepsin C (MW ~23–25 kDa) suggests that the enzyme precursor of MW ~50 kDa (Sojka et al, 2008) is proteolytically processed; the same chain pattern was found for mammalian cathepsin C (Horn et al, 2002). …”

Cotranslational and Posttranslational N-Glycosylation of Polypeptides by Distinct Mammalian OST Isoforms

“…Cysteine residues within the N29 and N53 sequons form disulfides with cysteines flanking the third sequon (Horn et al, 2002). We tested the hypothesis that N29 is hypoglycosylated in STT3B-depleted cells by expressing HA-tagged derivatives of pCatC (Figure 4E).…”

“…As a control, we examined pegylation of pCatC, a protein with five disulfide bonds and three free cysteine residues (Figure 4A). Only one cysteine (C355) in pCatC is accessible to a bulky reagent like PEG-maleimide in the folded state (Turk et al, 2001; Horn et al, 2002). Pegylation of pulse-labeled pCatC yields a ladder (Figure 6A) as observed previously for other proteins (Lu and Deutsch, 2001), where each PEG adduct increases the apparent molecular weight of pCatC by 10-15 kD.…”

Papain-like peptidases: structure, function, and evolution

“…The exclusion domain, which also determines the activity of the enzyme as discussed above, appears to be one of the major driving forces in the maturation of the enzyme (71) , together with a free Cys residue exposed on the surface of the peptidase domain (72) . Another unique functional characteristic is the need for chloride ion in catalysis (12) .…”