Regulation of carboxypeptidase E. Effect of pH, temperature and Co2+ on kinetic parameters of substrate hydrolysis

Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in the trans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.

Section: Resultsmentioning

confidence: 78%

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Varlamov

Eng

Novikova

et al. 1999

“…However, and in contrast with digestive CPs, scission of the pro-segment is not necessary for expression of the activity (20). Also, in contrast with the great majority of metalloCPs, whose optimum pH value is around neutrality, CPE has its maximum activity at an acidic pH value, between 5 and 5.5 (21), coincident with the internal pH value of the secretory granules. It has also been observed that its activity is regulated by the presence of Co 2ϩ (1).…”

mentioning

confidence: 99%

The Crystal Structure of the Inhibitor-complexed Carboxypeptidase D Domain II and the Modeling of Regulatory Carboxypeptidases

Aloy

Companys

Vendrell

et al. 2001

The three-dimensional crystal structure of duck carboxypeptidase D domain II has been solved in a complex with the peptidomimetic inhibitor, guanidinoethylmercaptosuccinic acid, occupying the specificity pocket. This structure allows a clear definition of the substrate binding sites and the substrate funnel-like access. The structure of domain II is the only one available from the regulatory carboxypeptidase family and can be used as a general template for its members. Here, it has been used to model the structures of domains I and III from the former protein and of human carboxypeptidase E. The models obtained show that the overall topology is similar in all cases, the main differences being local and because of insertions in non-regular loops. In both carboxypeptidase D domain I and carboxypeptidase E slightly different shapes of the access to the active site are predicted, implying some kind of structural selection of protein or peptide substrates. Furthermore, emplacement of the inhibitor structure in the active site of the constructed models showed that the inhibitor fits very well in all of them and that the relevant interactions observed with domain II are conserved in domain I and carboxypeptidase E but not in the non-active domain III because of the absence of catalytically indispensable residues in the latter protein. However, in domain III some of the residues potentially involved in substrate binding are well preserved, together with others of unknown roles, which also are highly conserved among all carboxypeptidases. These observations, taken together with others, suggest that domain III might play a role in the binding and presentation of proteins or peptide substrates, such as the pre-S domain of the large envelope protein of duck hepatitis B virus. Carboxypeptidases (CPs)1 are enzymes that catalyze the cleavage of C-terminal peptide bonds in proteins and peptides. From a mechanistic point of view, CPs can be classified in two groups, metalloCPs and serine CPs. The metalloCPs possess a Zn 2ϩ cofactor in the active site. In mammals, this family currently contains 13 members subdivided into two subfamilies, the digestive enzymes and the regulatory enzymes (1-5). Whereas the biological function of the digestive CPs is to contribute to protein degradation, the regulatory ones are generally involved in physiological processes that require a higher specificity. Within each group, the members have 25-63% amino acid sequence identity, but it decreases to only 15-25% when comparison is performed between subfamilies. This low overall homology between subfamilies implies that they diverged early in time.The digestive CPs are soluble, non-glycosylated proteins that are synthesized as inactive precursors containing a 90 -95-amino acid N-terminal pro-segment (5, 6). The regulatory CPs have been purified and characterized from biological fluids and tissues, where they are found in soluble or in membrane-attached forms, in minor quantities. This subfamily includes CPD, CPE, CPM, CPN, CPZ, and novel proteins with ...

“…In contrast, the medium from wild type AtT-20 cells had very low levels of carboxypeptidase activity, which did not increase upon secretagogue treatment (Fig. 3C); these assay conditions are not expected to detect CPE, which is maximally active at pH 5.5 and virtually inactive at neutral pH (26).…”

Section: Resultsmentioning

confidence: 87%

Carboxypeptidase Z Is Present in the Regulated Secretory Pathway and Extracellular Matrix in Cultured Cells and in Human Tissues

Novikova¹,

Reznik²,

Varlamov³

et al. 2000

Metallocarboxypeptidases perform many functions, ranging from the digestion of food to the selective processing of neuroendocrine peptide hormones and neurotransmitters (1). Altogether, there are 12 known members of the metallocarboxypeptidase gene family, which are further divided into two subgroups based on amino acid sequence similarities. Members of each subgroup share approximately 30 -60% amino acid sequence identity with other members of the same group, but typically only 15-25% amino acid sequence identity with members of the other subgroup (1). The two groups also differ in their general function; members of the carboxypeptidase A/B subgroup are typically digestive enzymes, while members of the subgroup containing carboxypeptidase E are referred to as "regulatory" enzymes (1, 2). Other members of the carboxypeptidase E subgroup include carboxypeptidases M, N, D, and Z, and proteins designated CPX-1, CPX-2, and AEBP-1 (3-9). Except for the latter three proteins, which are not active toward standard carboxypeptidase substrates and have been hypothesized to function as binding proteins, all of the other members of this subfamily cleave C-terminal Arg from small synthetic peptides.Carboxypeptidase Z (CPZ) 1 was discovered during a search for novel carboxypeptidases that could compensate for defective carboxypeptidase E in Cpe fat /Cpe fat mice (6, 10). These mice lack carboxypeptidase E but are still capable of a reduced level of processing of neuroendocrine peptides (11, 12). However, based on the non-neuroendocrine tissue distribution of CPZ, it is unlikely that this enzyme plays a role in neuroendocrine peptide processing. For example, in brain, CPZ is primarily expressed in the leptomeningeal cells and is not detectable in neurons (13). Furthermore, the enzymatic properties of CPZ are not consistent with a role for this enzyme in the processing of peptides within the secretory pathway (6, 14). Most importantly, CPZ is essentially inactive at the acidic pH values of the regulated secretory vesicles (14). Thus, it is likely that CPZ performs another role, possibly outside the cell. CPZ is predicted to be secreted from cells based on the presence of an N-terminal signal sequence (6, 13). When expressed in insect Sf9 cells using the baculovirus system, CPZ was not detected in the medium, although enzyme activity was detected on the cell surface despite the absence of a conventional membrane-binding domain (6).CPZ is unique among the members of the metallocarboxypeptidase family in that CPZ contains a cysteine-rich domain that has amino acid sequence identity with proteins that bind Wnt/wingless, such as the frizzled receptors, frzb, sizzled, and several other proteins (15)(16)(17)(18)(19)(20)(21)(22). This cysteine-rich domain within CPZ is highly conserved between the human and rat proteins, suggesting that it is functional. Although many of the Wnt-binding proteins function as receptors with Wnt being the ligand that initiates the signal transduction events, several Wnt-binding proteins are thought to bin...