Characterization and Evaluation of the Activity of Dipeptidyl Peptidase IV from the Black-Bellied Hornet Vespa basalis
Characterization and evaluation of the activity of Vespa basalis DPP-IV, which expressed in Spodoptera frugiperda 21 cells. The expression of rDPP-IV was confirmed by SDS–PAGE, Western blot analyses, LC-MS/MS and measurement of its peptidase specificity. One-step purification by Ni-NTA affinity chromatography and the total amount of rDPP-IV recovered was approximately 6.4mg per liter from infected culture medium; an equivalent amount would be produced by 1x109 infected Sf21 insect cells. Through the affinity purification led to highly stable rDPP-IV enzyme was recovered and with significant peptidase activity. The rDPP-IV exhibited classical Michaelis–Menten kinetics, with kcat/Km in the range of 10-500 mM-1×S-1 for the five synthetic substrates and optimum substrate is Ala-Pro-pNA. As expected in inhibition assay, the enzymatic activity of rDPP-IV was significantly reduced by 80 or 60% in the presence of sitagliptin (a DPP-IV inhibitor) or PMSF (a serine protease inhibitor), but was not apparently affected by iodoacetamide (a cysteine protease inhibitor).
[1] Krepela E. Dipeptidyl peptidase IV- a serine exopeptidase of the cellular
membrane. Cest Fysiol. 1982; 31(1): 27-53.
[2] Gorrell, MD. Dipeptidyl peptidase IV and related enzymes in cell biology
and liver disorders. Clin Sci. (Lond) 2005; 108: 277-292.
[3] Kikkawa, F., Kajiyama, H., Shibata, K., Ino, K., Normura, S. and
Mizutani, S. Dipeptidyl peptidase IV in tumor progression. Biochim
Biophys Acta. 2005;1571: 45-51.
[4] Molly, C., Vilas U., Hutticher A. Kreil G. Isolation of a dipeptidyl
aminopeptidase, a putative processing enzyme from skin secretion of
Xenopus laevis. Eur J Biochem. 1986; 160: 31-35.
[5] Tsakalidou E., Anastasiou, R., Papadimitriou K., Manolopoulou E.,
Kalantzopoulos G. Purification and characterization of an intracellular
X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus
ACA-DC 4. J Biotechnol. 1997; 59: 203-211.
[6] Chihara CJ., Song C, LaMonte G, Fetalvero K, Hinchman K, Phan H,
Pineda M, Robinson K, Scheider GP. Identification and partial
characterization of the enzyme of omega: one of five putative DPPIV
genes in Drosophila melanogaster. J Insect Sci. 2005:1-16.
[7] Kreil, G., Haiml L., Suchanek G. Stepwise cleavage of the pro part of
promelittin by dipeptidylpeptidase IV. Eur J Biochem. 1980; 111: 49-58.
[8] Lee,V.S.Y., Tu, W.C., Jinn, T.R., Peng, C. C., Lin, L. J. and Tzen, J.T.C.
Molecular cloning of the precursor polypeptide of mastoparan B and its
putative processing enzyme, dipeptidyl peptidase IV, from the
black-bellied hornet, Vespa basalis. Insect Mol Bio. 2007;16:231-237.
[9] Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U.,
Kiefersauer, r., Huber, R., Bode, W., Demuth, H.U. and Brandstetter, H.
The crystal structure of dipeptidyl peptidase IV (CD26) reveals its
functional regulation and enzymatic mechanism. Proc Natl Acad Sci.
USA 2003; 100: 5063-5068.
[10] Aertgeerts K., Sheng S., Shi L., Prasad SG., Witmer D., Chi E., Wijnands
RA., Webb DR., and Swanson RV. N-linked glycosylation of dipeptidyl
peptidase IV (CD26): Effects on enzyme activity, homodimer formation,
and adenosine deaminase binding. Protein Sci. 2004; 13:145-154.
[11] Luque T. O-Reilly DR. Generation of baculovirus expression vectors.
Mol Biotechnol. 1999; 13(2):153-163.
[12] Lin J. Toscano PJ. Welch JT. Inhibition of dipeptidyl peptidase IV by
fluoroolefin-containing N-peptidyl -O-hydroxylamine peptidomimetics.
Pro Natl Acad Sci. USA. 1998; 95: 14020-14024.
[13] Rasmussen, H.B., Branner, S., Wiberg, F.C. and Wagtmann, N. Crystal
structure of human dipeptidyl peptidase IV/CD26 in complex with a
substrate analog. Nat Struct Biol. 2003; 10: 19-25.
[14] Thoma, R., Loffler, B., Stihle, M., Huber, W., Ruf, A. and Henning, M.
Structural basis of proline-specific exopeptidase activity as observed in
human dipeptidyl peptidase-IV. Structure. 2003; 11:947-959.
[15] Huang, Y.W., Lu, M.L., Qi, H., Lin, S.X. Membrane-bound human
3β-hydroxysteroid dehydrogenase: overexpression with His-tag using a
baculovirus system and single-step purification, Protein Expr Purif. 2000;
18: 169-174.
[16] Frohman, L.A., Downs, T.R., Heimer, E.P., and Felix, A. M.
Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human
growth hormone-releasing hormone in plasma. J. Clin. Invest. 1989; 83:
1533-1540.
[17] Nausch, I., and Heymann, E. Substance P in human plasma is degraded
by dipeptidyl peptidase IV, not by cholinesterase. J. Neurochem. 1985;
44: 1354-1357.
[18] Ahmad , S., Wang, L., and Ward, P.E. Dipeptidyl(amino)peptidase IV
and aminopeptidase M metabolize circulating substance P in vivo. J.
Pharmacol. Exp. Ther. 1992; 260: 1257-1261.
[19] Mentlein, R. Dipeptidyl-peptidase IV (CD26): Role in the inactivation of
regulatory peptides. Regul. Pept. 1999; 85:9-24.
[20] J. Dobers, S. Grams, W. Reutter, h. Fan, Roles of cysteines in rat
dipeptidyl peptidase IV/CD26 in processing and proteolytic activity, Eur.
J. Biochem. 2000; 267: 5093-5100.
[1] Krepela E. Dipeptidyl peptidase IV- a serine exopeptidase of the cellular
membrane. Cest Fysiol. 1982; 31(1): 27-53.
[2] Gorrell, MD. Dipeptidyl peptidase IV and related enzymes in cell biology
and liver disorders. Clin Sci. (Lond) 2005; 108: 277-292.
[3] Kikkawa, F., Kajiyama, H., Shibata, K., Ino, K., Normura, S. and
Mizutani, S. Dipeptidyl peptidase IV in tumor progression. Biochim
Biophys Acta. 2005;1571: 45-51.
[4] Molly, C., Vilas U., Hutticher A. Kreil G. Isolation of a dipeptidyl
aminopeptidase, a putative processing enzyme from skin secretion of
Xenopus laevis. Eur J Biochem. 1986; 160: 31-35.
[5] Tsakalidou E., Anastasiou, R., Papadimitriou K., Manolopoulou E.,
Kalantzopoulos G. Purification and characterization of an intracellular
X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus
ACA-DC 4. J Biotechnol. 1997; 59: 203-211.
[6] Chihara CJ., Song C, LaMonte G, Fetalvero K, Hinchman K, Phan H,
Pineda M, Robinson K, Scheider GP. Identification and partial
characterization of the enzyme of omega: one of five putative DPPIV
genes in Drosophila melanogaster. J Insect Sci. 2005:1-16.
[7] Kreil, G., Haiml L., Suchanek G. Stepwise cleavage of the pro part of
promelittin by dipeptidylpeptidase IV. Eur J Biochem. 1980; 111: 49-58.
[8] Lee,V.S.Y., Tu, W.C., Jinn, T.R., Peng, C. C., Lin, L. J. and Tzen, J.T.C.
Molecular cloning of the precursor polypeptide of mastoparan B and its
putative processing enzyme, dipeptidyl peptidase IV, from the
black-bellied hornet, Vespa basalis. Insect Mol Bio. 2007;16:231-237.
[9] Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U.,
Kiefersauer, r., Huber, R., Bode, W., Demuth, H.U. and Brandstetter, H.
The crystal structure of dipeptidyl peptidase IV (CD26) reveals its
functional regulation and enzymatic mechanism. Proc Natl Acad Sci.
USA 2003; 100: 5063-5068.
[10] Aertgeerts K., Sheng S., Shi L., Prasad SG., Witmer D., Chi E., Wijnands
RA., Webb DR., and Swanson RV. N-linked glycosylation of dipeptidyl
peptidase IV (CD26): Effects on enzyme activity, homodimer formation,
and adenosine deaminase binding. Protein Sci. 2004; 13:145-154.
[11] Luque T. O-Reilly DR. Generation of baculovirus expression vectors.
Mol Biotechnol. 1999; 13(2):153-163.
[12] Lin J. Toscano PJ. Welch JT. Inhibition of dipeptidyl peptidase IV by
fluoroolefin-containing N-peptidyl -O-hydroxylamine peptidomimetics.
Pro Natl Acad Sci. USA. 1998; 95: 14020-14024.
[13] Rasmussen, H.B., Branner, S., Wiberg, F.C. and Wagtmann, N. Crystal
structure of human dipeptidyl peptidase IV/CD26 in complex with a
substrate analog. Nat Struct Biol. 2003; 10: 19-25.
[14] Thoma, R., Loffler, B., Stihle, M., Huber, W., Ruf, A. and Henning, M.
Structural basis of proline-specific exopeptidase activity as observed in
human dipeptidyl peptidase-IV. Structure. 2003; 11:947-959.
[15] Huang, Y.W., Lu, M.L., Qi, H., Lin, S.X. Membrane-bound human
3β-hydroxysteroid dehydrogenase: overexpression with His-tag using a
baculovirus system and single-step purification, Protein Expr Purif. 2000;
18: 169-174.
[16] Frohman, L.A., Downs, T.R., Heimer, E.P., and Felix, A. M.
Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human
growth hormone-releasing hormone in plasma. J. Clin. Invest. 1989; 83:
1533-1540.
[17] Nausch, I., and Heymann, E. Substance P in human plasma is degraded
by dipeptidyl peptidase IV, not by cholinesterase. J. Neurochem. 1985;
44: 1354-1357.
[18] Ahmad , S., Wang, L., and Ward, P.E. Dipeptidyl(amino)peptidase IV
and aminopeptidase M metabolize circulating substance P in vivo. J.
Pharmacol. Exp. Ther. 1992; 260: 1257-1261.
[19] Mentlein, R. Dipeptidyl-peptidase IV (CD26): Role in the inactivation of
regulatory peptides. Regul. Pept. 1999; 85:9-24.
[20] J. Dobers, S. Grams, W. Reutter, h. Fan, Roles of cysteines in rat
dipeptidyl peptidase IV/CD26 in processing and proteolytic activity, Eur.
J. Biochem. 2000; 267: 5093-5100.
@article{"International Journal of Chemical, Materials and Biomolecular Sciences:54577", author = "Feng Chia Hsieh and Sheng Kuo Hsieh and Tzyy Rong Jinn", title = "Characterization and Evaluation of the Activity of Dipeptidyl Peptidase IV from the Black-Bellied Hornet Vespa basalis", abstract = "Characterization and evaluation of the activity of Vespa basalis DPP-IV, which expressed in Spodoptera frugiperda 21 cells. The expression of rDPP-IV was confirmed by SDS–PAGE, Western blot analyses, LC-MS/MS and measurement of its peptidase specificity. One-step purification by Ni-NTA affinity chromatography and the total amount of rDPP-IV recovered was approximately 6.4mg per liter from infected culture medium; an equivalent amount would be produced by 1x109 infected Sf21 insect cells. Through the affinity purification led to highly stable rDPP-IV enzyme was recovered and with significant peptidase activity. The rDPP-IV exhibited classical Michaelis–Menten kinetics, with kcat/Km in the range of 10-500 mM-1×S-1 for the five synthetic substrates and optimum substrate is Ala-Pro-pNA. As expected in inhibition assay, the enzymatic activity of rDPP-IV was significantly reduced by 80 or 60% in the presence of sitagliptin (a DPP-IV inhibitor) or PMSF (a serine protease inhibitor), but was not apparently affected by iodoacetamide (a cysteine protease inhibitor).
", keywords = "Dipeptidyl-Peptidase IV, Phenylmethylsulfonyl
fluoride; Serine protease, Sitagliptin, Vespa basalis", volume = "6", number = "5", pages = "442-4", }
