Substrate Specificity Of Human Metallocarboxypeptidase D: Comparison Of The Two Active Carboxypeptidase Domains

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The primary goal of the present study was the detailed comparison of the substrate specificity of the first and second catalytic domains of CPD, as well as testing the third carboxypeptidase-like domain for catalytic activity using a broad array of peptides. CPD is the only member of the MCP family that contains multiple carboxypeptidase domains [11,15,16]. Proteolytic enzymes with multiple catalytic sites are rare in nature, especially enzymes with catalytic sites that perform generally similar roles such as domains I and II of CPD. Angiotensin-converting enzyme (ACE) is another peptidase with two active domains in most species and tissues (the form produced in sperm contains a single catalytic domain). The two domains of ACE have similar catalytic activities that differ mainly in their sensitivity to chloride and substrate preferences [56,57]. Another group of enzymes with multiple domains that perform related catalytic functions are the polyserases. As with CPD, the polyserases contain enzymatically active and inactive domains of unknown function. Polyserase-1 is post-translationally cleaved into distinct subunits that contain a single catalytic activity, but polyserase-2 and -3 remain intact as multi-domain-containing proteins [5860]. The presence of multiple catalytically active domains can increase the diversity of substrates cleaved, or the efficiency of the enzyme under different cellular conditions. Both appear to be the case for the two active domains of CPD.

One important contribution of the present study is that human CPD domain I and II have distinct pH optima, with domain I working best at neutral pH while domain II is optimal at mildly acidic pH values. This observation is consistent with the properties previously reported for duck and Drosophila CPD [16,22]. indicating that this feature is highly conserved through hundreds of millions of years of evolution. Although CPD is primarily detected in the trans Golgi network, its presence there is not static. CPD is present in vesicles that bud from the trans Golgi, but is retrieved from these vesicles and sorted back to the trans Golgi. CPD is also transiently found on the cell surface, where it is internalized and a fraction is transported through the endocytic pathway back to the trans Golgi network [17]. The pH of each of these compartments is different, ranging from neutral (the cell surface) to slightly acidic (the trans Golgi network) to acidic (endosomes). The broad pH range of CPD domain II implies that this activity is functional in the various cellular compartments, while CPD domain I is optimally active on the cell surface where the pH is neutral.

Another important finding of the present study is that human CPD domain I and II have differences in their substrate specificities. Both are specific for C-terminal basic residues, with no detectable cleavage of peptides with non-basic residues on the C-terminus. The present study tested dozens of peptides in a peptidomic assay, and extended the previous studies performed with duck and Drosophila CPD which tested a small number of substrates [16,22]. The preference of CPD domain I for Arg over Lys, and the broad ability of domain II to cleave both Arg and Lys with comparable efficiency was previously noted in studies that tested duck and Drosophila CPD with a single pair of peptides that differed only in the C-terminal residue [16,22]. The present study tested more than 100 peptides, and while each domain was able to cleave either Lys or Arg from some peptides, there was a clear preference for domain I to cleave Arg and not Lys. Interestingly, despite this strict specificity towards basic residues of both domains, in our study we found some peptides containing this residue that were not cleaved by domain I or II of CPD. These peptides contained Pro, Asp or Ile in P1 position, confirming that other residues different from the P1’ affect the efficiency of this human enzyme. Taken together with the pH optima, it appears that when present on the cell surface, CPD domain I will be active and cleave peptides/proteins with C-terminal Arg, while CPD present in the trans Golgi and endocytic pathways will have domain II active and cleave both C-terminal Lys and Arg.

Previous studies using small numbers of substrates have found that other members of the M14B subfamily of MCPs are more efficient at cleaving C-terminal Arg than Lys. For example, CPM cleaves Met5-Arg6-enkephalin with a kcat of 934 min-1 and Km of 46 μM, whereas Met5-Lys6-enkephalin is cleaved with kcat of 663 min-1 and Km of 375 μM [61]. Other studies evaluated the specificity of MCPs using peptides with C-terminal Arg or Lys residues as competitive inhibitors. For example, CPE is inhibited by Leu5-Lys6-enkephalin with a ki of 174 μM and by Leu5-Arg6-enkephalin with a ki of 83 μM [62]. CPZ is inhibited by hippuryl-Arg but not by hippuryl-Lys [63]. Taken together with the present studies, CPZ and CPM are like CPD domain I with a strong preference for C-terminal Arg over Lys, while CPE and CPN are more like CPD domain II which does not have a marked preference for one basic residue over another. One residue within the substrate binding pocket that correlates with selectivity is the residue in position equivalent to Leu203 in CPA1. In CPD domain I Ser264 is present in this position, while in CPD domain II an Asn678 is present (

Fig 10). Similarly, CPM and CPZ contain a Ser in this position, and CPE and CPN contain Asn. Perhaps the smaller side chain of the Ser (versus Asn) within the binding site has a superior capability to fit and facilitate the establishment of contacts between the guanidine group of the C-terminal scissile Arg residue, leading to major preference for Arg- versus Lys-containing substrates. The analysis of the docking landscape suggests that there might be some kinetic effects involved in the processes of approaching and entering of the peptides into the active site, which are not fully explained by the structure and energetics of the best docking pose alone. These docking results are consistent with the experimental finding that peptides with a C-terminal lysine have less preference for the active site of domain I, whereas peptides with a C-terminal arginine bind both domains, with a slight preference for domain I.

Another finding of the present study is that the third carboxypeptidase-like domain of human CPD is inactive as a carboxypeptidase when tested with a fluorescent substrate at a wide range of pH values, or with a wide array of peptides at pH 6.5. In no case was carboxypeptidase activity detected for the form of CPD with mutations in a key catalytic residue in domain I and II. This observation fits with previous studies on duck and Drosophila CPD, which tested a small number of substrates [16,22]. This observation is also consistent with the lack of conserved active site, substrate-binding, and metal-binding residues in the third domain of CPD. For example, the critical active site Glu (Glu270 using bovine CPA1 numbering) is a Tyr in the third domain of human and duck CPD. Most of the changes in active site residues found in the third domain of CPD are conserved between human and duck, suggesting that these differences are not simply random events but are important for the function of this domain. One possibility is that the third domain functions in peptide binding. While Glu270 is essential for catalytic activity, it is not required for substrate binding, and substitution of Gln for Glu permits the protein to bind peptides but not hydrolyze them. Results from our peptide docking experiments with the tetrapeptide GQKR support this hypothesis.

Three other members of the M14B subfamily of MCPs are also inactive towards standard carboxypeptidase substrates. Two of these, CPX2 [64] and AEBP1/ACLP [65] also contain Tyr in place of the critical active site Glu270, suggesting that this Tyr is important. The third inactive member of this subfamily, CPX1, does contain Glu in the comparable position but is missing other critical active site and substrate-binding residues. Other families of enzymes contain members that are considered catalytically inactive, and the functions of most of these proteins are not yet known [5860,66]. Recently, AEBP1/ACLP was found to induce phosphorylation and nuclear translocation of Smad3, and this was dependent on TGFβ receptor binding and kinase activity [67]. Some of the active carboxypeptidases appear to have functions that are independent of their catalytic activity. For example, CPE was proposed to function as an extracellular trophic factor that protected neurons from hydrogen peroxide-, staurosporine- and glutamate-induced cell death, and this effect did not require CPE enzyme activity (although it did require amounts of CPE much higher than would be expected to be physiological) [68]. CPM was reported to be a positive allosteric modulator of the kinin B1 receptor, and this action is independent of CPM enzyme activity [69]. The emerging concept is that some carboxypeptidases have multiple functions, including both catalytic and non-catalytic roles, while the inactive members of this gene family like CPD domain III would only have non-catalytic roles.

Taken together, our results establish that both catalytically active domains of CPD play important physiological roles by cleaving a broad range of peptide and protein substrates with C-terminal basic residues or Arg and Lys in the trans-Golgi network, within the secretory and endocytic pathways and in the cell membrane. Although both active domains display a partial overlap of their substrate preferences, our results demonstrate that each active CPD domain has evolved to divergent substrate specificities conserved through evolution. Moreover, the third domain is catalytically inactive against a wide spectrum of peptides, and the biological function of this domain remains unknown.

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Source : http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187778

Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains
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