Cloning and Sequence Analysis of cDNA Encoding Rat Carboxypeptidase D

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Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala > Met > Ser, Phe > Tyr > Trp > Thr > Gln, Asp, Leu, Gly Ͼ Ͼ Ͼ Ͼ Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5.0 -6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

Novikova

Eng

Lin

et al. 1999

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“…This protein was independently discovered as gp180, a duck protein that binds duck hepatitis B virus particles (15). In duck and rat, CPD is present in many tissues (15)(16)(17)(18)(19) suggesting a broad function. CPD cDNA has been cloned and sequenced from human, rat, duck, Drosophila, and Aplysia (16, 18, 20 -22).…”

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confidence: 99%

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Varlamov

Eng

Novikova

et al. 1999

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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.

“…These family members are all enzymatically active, and are 30 -40-kDa proteins. The other group includes CPE, carboxypeptidase M (CPM), carboxypeptidase N (CPN), carboxypeptidase D (CPD), carboxypeptidase Z (CPZ), and three proteins designated CPX1, CPX2, and AEBP1 (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Except for CPD, which is 180 kDa and contains 3 distinct carboxypeptidase-like domains, the members of this family contain a single carboxypeptidase domain of approximately 400 amino acids.…”

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confidence: 99%

Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway

Qian

Varlamov

Fricker

1999

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Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu 300 (equivalent to Glu 270 of carboxypeptidase A and B). To investigate the importance of this position, Glu 300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln 300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wildtype CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln 300 and Lys 300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu 300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway.