Abstract
Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1′ position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.
Highlights
Metallocarboxypeptidases (MCPs) are a group of zinc-containing exopeptidases that cleave single C-terminal amino acids from proteins and peptides [1]
Carboxypeptidase D (CPD) is necessary for the formation of the active insulin-like growth factor 1 receptor (IGF1R); deletion of the CPD gene inhibited signaling through IGF1R and reduced the three-dimensional growth of a lung adenocarcinoma cell line [13]
A previous study showed that this enzyme has a strong preference for substrates with C-terminal Arg, as deduced from a single assay using a pair of synthetic substrates [15]
Summary
Metallocarboxypeptidases (MCPs) are a group of zinc-containing exopeptidases that cleave single C-terminal amino acids from proteins and peptides [1]. We performed structural modeling of the catalytic domain, as well as the Fz domain of human CPZ, to dissect its structural features From these studies, we found that CPZ is a metallocarboxypeptidase that cleaves substrates and peptides with C-terminal basic amino acids, especially those containing C-terminal Arg residues. Four peptides were identified as products of CPZ; these peptides resulted from cleavage of Lys or Arg from the C-terminus and with Ala, Phe, Val, or Leu at the P1 position of the cleavage site (Table 3) Taken together, these data suggest that that CPZ only cleaves substrates with either C-terminal Arg or Lys amino acids. No peptides identified as CPZ substrates contained Pro, Asp, Gln, Phe, Val, Ala, Asn, Glu, or Ile amino acids at P1 position (see Table 4). Sci. 2020, 21, x FOR PEER REVIEW not able to process a peptide with a C-terminal acidic residue (i.e., Glu) under equivalent experimental conditions (Figure 7D)
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