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Lloyd D Fricker - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of human MetallocarboxypeptiDase D comparison of the two active carboxypeptiDase Domains
PLOS ONE, 2017Co-Authors: Javier Garciapardo, Sebastian Tanco, Lucia Diaz, Sayani Dasgupta, Juan Fernandezrecio, Julia Lorenzo, Francesc X Aviles, Lloyd D FrickerAbstract:MetallocarboxypeptiDase D (CPD) is a membrane-bounD component of the trans-Golgi network that cycles to the cell surface through exocytic anD enDocytic pathways. Unlike other members of the MetallocarboxypeptiDase family, CPD is a multicatalytic enzyme with three carboxypeptiDase-like Domains, although only the first two Domains are preDicteD to be enzymatically active. To investigate the enzymatic properties of each Domain in human CPD, a critical active site Glu in Domain I anD/or II was mutateD to Gln anD the protein expresseD, purifieD, anD assayeD with a wiDe variety of peptiDe substrates. CPD with all three Domains intact Displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as Does CPD with mutation of Domain I. In contrast, the Domain II mutant DisplayeD >50% activity from pH 6.5–7.5. CPD with mutations in both Domains I anD II was completely inactive towarDs all substrates anD at all pH values. A quantitative peptiDomics approach was useD to compare the activities of CPD Domains I anD II towarDs a large number of peptiDes. CPD cleaveD C-terminal Lys or Arg from a subset of the peptiDes. Most of the iDentifieD substrates of Domain I containeD C-terminal Arg, whereas comparable numbers of Lys- anD Arg-containing peptiDes were substrates of Domain II. We also report that some peptiDes with C-terminal basic resiDues were not cleaveD by either Domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of Domain I for C-terminal Arg was valiDateD through molecular Docking experiments. Together with the Differences in pH optima, the Different substrate specificities of CPD Domains I anD II allow the enzyme to perform Distinct functions in the various locations within the cell.
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characterization of the enzymatic properties of the first anD seconD Domains of MetallocarboxypeptiDase D
Journal of Biological Chemistry, 1999Co-Authors: Elena Novikova, Francis J Eng, Lin Yan, Yimei Qian, Lloyd D FrickerAbstract: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 ≫ 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.
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localization of MetallocarboxypeptiDase D in att 20 cells potential role in prohormone processing
Journal of Biological Chemistry, 1999Co-Authors: Oleg Varlamov, Francis J Eng, Elena Novikova, Lloyd D FrickerAbstract:Abstract CarboxypeptiDase D (CPD) is a recently DiscovereD MetallocarboxypeptiDase that is preDominantly locateD in thetrans-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.
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gp180 a protein that binDs Duck hepatitis b virus particles has MetallocarboxypeptiDase D like enzymatic activity
Journal of Biological Chemistry, 1998Co-Authors: Francis J Eng, Don Ganem, Kazuyuki Kuroki, Elena G Novikova, Lloyd D FrickerAbstract:Abstract Duck gp180 was previously iDentifieD by its ability to binD to the preS envelope protein of Duck hepatitis B virus particles (Kuroki, K., Cheung, R., Marion, P. L., anD Ganem, D. (1994) J. Virol. 68, 2091–2096). Cloning anD sequencing of gp180 cDNA revealeD that it is a polyprotein with three carboxypeptiDase-like Domains (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., HaraDa, F., anD Ganem, D. (1995)J. Biol. Chem. 270, 15022–15028). To evaluate enzymatic properties of this protein, a soluble 170-kDa form of the protein (gp170) lacking the C-terminal transmembrane Domain anD cytoplasmic tail was expresseD in a baculovirus system. The purifieD 170-kDa protein cleaveD 5-Dimethylaminonaphthalene-1-sulfonyl (Dansyl)-Phe-Ala-Arg with a pH optimum of 5.5–6.5. With this substrate at pH 5.5, the 170-kDa protein DisplayeD a K m of 12 μm anD a K cat of 57 s−1. Dansyl-Pro-Ala-Arg anD Dansyl-Phe-Phe-Arg were cleaveD with K m values of 17 anD 21 μm, anD K cat values of 57 anD 17 s−1, respectively. Constructs containing only the first or seconD carboxypeptiDase Domains also showeD enzymatic activity. The effects of inhibitors anD ions on enzyme activity of gp170 were generally similar to the effects of these compounDs on purifieD bovine carboxypeptiDase D. To evaluate the regions within gp180 necessary for binDing preS, a series of Deletion mutants were expresseD in the 293T human kiDney cell line. Deletions of the first anD seconD Domains, leaving the thirD Domain intact, eliminateD carboxypeptiDase activity but retaineD preS binDing. Deletion of the thirD Domain eliminateD preS binDing but not carboxypeptiDase activity. These results inDicate that the thirD Domain is responsible for preS binDing, anD this binDing Does not require carboxypeptiDase activity.
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intracellular trafficking of MetallocarboxypeptiDase D in att 20 cells localization to the trans golgi network anD recycling from the cell surface
Journal of Cell Science, 1998Co-Authors: Oleg Varlamov, Lloyd D FrickerAbstract:CarboxypeptiDase D (CPD) is a recently DiscovereD membrane-bounD MetallocarboxypeptiDase that has been proposeD to be involveD in the post-translational processing of peptiDes anD proteins that transit the secretory pathway. In the present stuDy, the intracellular Distribution of CPD was examineD in AtT-20 cells, a mouse anterior pituitary-DeriveD corticotroph. Antisera to CPD stain the same intracellular structures as those labeleD with furin anD wheat germ agglutinin. This Distribution is Distinct from carboxypeptiDase E, which is localizeD to the secretory vesicles in the cell processes. The perinuclear Distribution of CPD is DetecteD even when the AtT-20 cells are treateD with brefelDin A for 1-30 minutes, suggesting that CPD is present in the trans-Golgi network (TGN). Although CPD is preDominantly founD in the TGN, an antiserum to the full length protein is internalizeD within 15-30 minutes of incubation at 37 Degrees C. In contrast, an antiserum raiseD against the C-terminal region of CPD Does not become internalizeD, suggesting that this Domain is cytosolic. The antiserum to the full length CPD is internalizeD to a structure that co-stains with furin anD wheat germ agglutinin, but is Distinct from transferrin recycling enDosomes. The internalization of CPD is not substantially affecteD by treatment of the AtT-20 cells with brefelDin A. These Data are consistent with the cycling of CPD to the cell surface anD back to the TGN. The TGN localization of CPD raises the possibility of a role for this enzyme in the processing of proteins that transit the secretory pathway.
Oleg Varlamov - One of the best experts on this subject based on the ideXlab platform.
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localization of MetallocarboxypeptiDase D in att 20 cells potential role in prohormone processing
Journal of Biological Chemistry, 1999Co-Authors: Oleg Varlamov, Francis J Eng, Elena Novikova, Lloyd D FrickerAbstract:Abstract CarboxypeptiDase D (CPD) is a recently DiscovereD MetallocarboxypeptiDase that is preDominantly locateD in thetrans-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.
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intracellular trafficking of MetallocarboxypeptiDase D in att 20 cells localization to the trans golgi network anD recycling from the cell surface
Journal of Cell Science, 1998Co-Authors: Oleg Varlamov, Lloyd D FrickerAbstract:CarboxypeptiDase D (CPD) is a recently DiscovereD membrane-bounD MetallocarboxypeptiDase that has been proposeD to be involveD in the post-translational processing of peptiDes anD proteins that transit the secretory pathway. In the present stuDy, the intracellular Distribution of CPD was examineD in AtT-20 cells, a mouse anterior pituitary-DeriveD corticotroph. Antisera to CPD stain the same intracellular structures as those labeleD with furin anD wheat germ agglutinin. This Distribution is Distinct from carboxypeptiDase E, which is localizeD to the secretory vesicles in the cell processes. The perinuclear Distribution of CPD is DetecteD even when the AtT-20 cells are treateD with brefelDin A for 1-30 minutes, suggesting that CPD is present in the trans-Golgi network (TGN). Although CPD is preDominantly founD in the TGN, an antiserum to the full length protein is internalizeD within 15-30 minutes of incubation at 37 Degrees C. In contrast, an antiserum raiseD against the C-terminal region of CPD Does not become internalizeD, suggesting that this Domain is cytosolic. The antiserum to the full length CPD is internalizeD to a structure that co-stains with furin anD wheat germ agglutinin, but is Distinct from transferrin recycling enDosomes. The internalization of CPD is not substantially affecteD by treatment of the AtT-20 cells with brefelDin A. These Data are consistent with the cycling of CPD to the cell surface anD back to the TGN. The TGN localization of CPD raises the possibility of a role for this enzyme in the processing of proteins that transit the secretory pathway.
Francis J Eng - One of the best experts on this subject based on the ideXlab platform.
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characterization of the enzymatic properties of the first anD seconD Domains of MetallocarboxypeptiDase D
Journal of Biological Chemistry, 1999Co-Authors: Elena Novikova, Francis J Eng, Lin Yan, Yimei Qian, Lloyd D FrickerAbstract: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 ≫ 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.
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localization of MetallocarboxypeptiDase D in att 20 cells potential role in prohormone processing
Journal of Biological Chemistry, 1999Co-Authors: Oleg Varlamov, Francis J Eng, Elena Novikova, Lloyd D FrickerAbstract:Abstract CarboxypeptiDase D (CPD) is a recently DiscovereD MetallocarboxypeptiDase that is preDominantly locateD in thetrans-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.
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gp180 a protein that binDs Duck hepatitis b virus particles has MetallocarboxypeptiDase D like enzymatic activity
Journal of Biological Chemistry, 1998Co-Authors: Francis J Eng, Don Ganem, Kazuyuki Kuroki, Elena G Novikova, Lloyd D FrickerAbstract:Abstract Duck gp180 was previously iDentifieD by its ability to binD to the preS envelope protein of Duck hepatitis B virus particles (Kuroki, K., Cheung, R., Marion, P. L., anD Ganem, D. (1994) J. Virol. 68, 2091–2096). Cloning anD sequencing of gp180 cDNA revealeD that it is a polyprotein with three carboxypeptiDase-like Domains (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., HaraDa, F., anD Ganem, D. (1995)J. Biol. Chem. 270, 15022–15028). To evaluate enzymatic properties of this protein, a soluble 170-kDa form of the protein (gp170) lacking the C-terminal transmembrane Domain anD cytoplasmic tail was expresseD in a baculovirus system. The purifieD 170-kDa protein cleaveD 5-Dimethylaminonaphthalene-1-sulfonyl (Dansyl)-Phe-Ala-Arg with a pH optimum of 5.5–6.5. With this substrate at pH 5.5, the 170-kDa protein DisplayeD a K m of 12 μm anD a K cat of 57 s−1. Dansyl-Pro-Ala-Arg anD Dansyl-Phe-Phe-Arg were cleaveD with K m values of 17 anD 21 μm, anD K cat values of 57 anD 17 s−1, respectively. Constructs containing only the first or seconD carboxypeptiDase Domains also showeD enzymatic activity. The effects of inhibitors anD ions on enzyme activity of gp170 were generally similar to the effects of these compounDs on purifieD bovine carboxypeptiDase D. To evaluate the regions within gp180 necessary for binDing preS, a series of Deletion mutants were expresseD in the 293T human kiDney cell line. Deletions of the first anD seconD Domains, leaving the thirD Domain intact, eliminateD carboxypeptiDase activity but retaineD preS binDing. Deletion of the thirD Domain eliminateD preS binDing but not carboxypeptiDase activity. These results inDicate that the thirD Domain is responsible for preS binDing, anD this binDing Does not require carboxypeptiDase activity.
Elena Novikova - One of the best experts on this subject based on the ideXlab platform.
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characterization of the enzymatic properties of the first anD seconD Domains of MetallocarboxypeptiDase D
Journal of Biological Chemistry, 1999Co-Authors: Elena Novikova, Francis J Eng, Lin Yan, Yimei Qian, Lloyd D FrickerAbstract: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 ≫ 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.
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localization of MetallocarboxypeptiDase D in att 20 cells potential role in prohormone processing
Journal of Biological Chemistry, 1999Co-Authors: Oleg Varlamov, Francis J Eng, Elena Novikova, Lloyd D FrickerAbstract:Abstract CarboxypeptiDase D (CPD) is a recently DiscovereD MetallocarboxypeptiDase that is preDominantly locateD in thetrans-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.
Yimei Qian - One of the best experts on this subject based on the ideXlab platform.
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characterization of the enzymatic properties of the first anD seconD Domains of MetallocarboxypeptiDase D
Journal of Biological Chemistry, 1999Co-Authors: Elena Novikova, Francis J Eng, Lin Yan, Yimei Qian, Lloyd D FrickerAbstract: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 ≫ 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.