Dipeptidyl Peptidase IV

On this page:

• Physical Properties and In Vivo Processing
• Physical Properties and Clinical Implications
• Specificity and Kinetics
• Enzymatic Assays
• Synthetic Substrates
• Solubility and Stability

Enzyme Commission (EC) Number: 3.4.14.5
Primary Accession Number: P27487 (DPP4_HUMAN)

Synonyms:

DPPIV, dipeptidyl aminopeptidase IV, CD26, glycoprotein GP110, glycylproline aminopeptidase, T cell triggering molecule Tp103, X-PDAP, THAM, and adenosine deaminase binding protein (ADAbp).

Physical Properties and In Vivo Processing

Native DPPIV is a ubiquitous type II transmembrane glycoprotein and a serine protease of the S9 prolyl-oligopeptidase family. In vivo, it is synthesized with a signal peptide which functions as the membrane anchoring domain.^1,2 There is an 88% sequence homology between the human and porcine kidney enzymes. ³ Both the human and porcine kidney enzymes exist as homodimers with a subunit molecular weight of approx. 30 kDa. The high mannose 100 kDa DPPIV precursor is processed in the Golgi to yield a 124 kDa heavily N-and O-linked mature glycoprotein.⁴ It is then sorted to the apical membrane through the concerted action of both N- and O-linked glycans and it’s association with lipid microdomains.⁵ The porcine enzyme contains 18.3% carbohydrates, of which the glycan composition is 0.9% fucose, 3.4% mannose, 5.1% galactose, 8.2% glucosamine, and 0.7% sialic acid.^1,2

pI: 5.2⁶ (porcine)
Extinction Coefficient: E^1% = 11.5 (280 nm)⁷ (porcine)

Physiological Properties and Clinical Implications

DPPIV is highly expressed on endothelial cells, epithelial cells and lymphocytes.^8,9,10 It is also present in plasma in its soluble form.¹¹

DPPIV is involved in the regulation of several important physiological processes: ^12,13,14

• Immune system
• Inflamation
• CNS
• Endocrine functions
• Bone marrow mobilization
• Cancer growth
• Cell adhesion
• Glucose hemostasis
• Sepsis/severe infection

Natural DPPIV substrates include: ^12,13

• Glucagon-like peptides-1 & 2
• Glucose–dependent insulinotropic peptide
• Neuropeptide Y
• Substance P
• Peptide YY
• IGF-1
• Prolactin
• hCGα
• Growth Hormone Releasing Factor
• LHα
• Thyrotropinα
• Enkephalins
• Vasostatin
• Eotaxin
• Interferon-γ inducible protein
• IFN-inducible T-cell alpha-chemoattractant
• Procalcitonin15
• Macrophage-derived chemokine
• Monokine induced by Interferon-γ

Natural DPPIV ligands include:^{12,16,17,18,19}

• Adenosine deaminase-I and II
• Renal Na+/H+ ion exchanger NHE³ isoform
• Fibronectin

DPPIV inhibitors have been found to improve glucose tolerance and preserve islet function in mice.²⁰ DPPIV inhibitors were found to block entry of HIV-1 or HIV-2 into T lymphoblastoid and monocytoid cell lines.²¹

Specificity and Kinetics

DPPIV has a post-proline dipeptidyl aminopeptidase activity that hydrolyzes N-terminal dipeptides from the unsubstituted N-terminus of peptides with the sequence of X-Pro-Z and X-Ala-Z.

The optimum pH for activity is 7.4-8.7.^13,22 At pH 7.0 DPPIV exhibits about 45% of maximal activity and at pH 9.6 it exhibits about 90% of maximal activity. Below pH 5.0 the enzyme is essentially inactive.^11,23

We determine the enzymatic activity of DPPIV using the chromogenic substrate Gly-Pro-p-nitroanilde. Reported K_M values are 0.66 mM for Gly-Pro-2-naphthylamide²⁴ and 1 mM for Ala-Ala-2-naphthylamide.²³

Enzymatic Assay

Unit Definition: One unit will produce 1.0 µmole of p-nitroaniline from Gly-L-Pro p-nitroanilide per min in 100 mM Tris-HCl at pH 8.0 at 37 °C.

Stopped Rate Spectrophotometric Determination using a Microplate Reader

CONDITIONS: T = 37 °C, pH = 8.0, A_405nm, Inc. = 15 minutes

REAGENTS:

1. 0.1 M Tris, pH 8.0 at 37 °C
2. 1 mM Gly-Pro-pNA Solution
   
3. 1 mM p-Nitroaniline Solution (pNA)
4. Dipeptidyl Peptidase Enzyme Solution

(Immediately before use prepare the following in cold reagent A. Dilute to obtain 0.04 - 0.08 units/mL.)

PROCEDURE:

1. Set up microtitre 96-well plate with the following standards:
   S1 = 20 µL 1 mM pNA (20 nmols)
   S2 = 40 µL 1 mM pNA (40 nmols)
   S3 = 60 µL 1 mM pNA (60 nmols)
   S4 = 80 µL 1 mM pNA (80 nmols)
   S5 = 100 µL 1 mM pNA (100 nmols)
   Make all wells up to 0.1 mL with Reagent A.
2. Pipette in enzyme samples as follows:
   T1 = 10 µL
   T2 = 20 µL
   T3 = 30 µL
   T4 = 40 µL
   T5 = 50 µL
   Make all wells up to 0.1 mL with Reagent A.
3. Add 0.1 mL of Reagent B to each well (including standards) to start the reaction.
4. Incubate at 37 °C for 15 minutes.
5. Read absorbance at 405 nm in a microplate reader.

CALCULATIONS:
Plot a standard curve of A_405nm versus nmoles of pNA. Calculate nmoles/minute and hence from this determine mmoles/min/mL of enzyme.

FINAL ASSAY CONCENTRATION:
In a 0.2 mL reaction mix, the final concentrations are 100 mM Tris, 0.5 mM Gly-Pro-pNA, and varying amounts of enzyme.

This procedure is for informational purposes. For a current copy of our quality control procedure, please contact our Technical Service Department.

Synthetic Substrates

Gly-Pro 4-methoxy-β-naphthylamide
Gly-Pro 4-methoxy-β-naphthylamide
Gly-Pro p-nitroanilide hydrochloride
Gly-Pro p-nitroanilide p-toluenesulfonate salt
Gly-Pro-7-amido-4-methylcoumarin hydrobromide

Inhibitors

diisopropyl fluorophosphate
phenylmethanesulfonyl fluoride
Ile-Pro-Ile (Diprotin A)
KR-62436

Enzymes

Dipeptidyl Peptidase, human
Dipeptidyl Peptidase from porcine kidney

Solubility and Stability

The porcine enzyme (Prod. No. D7052) is supplied as a lyophilized powder and is soluble in 100 mM Tris HCl buffer, pH 8.0 (1 vial/mL), yielding a clear, colorless solution. The human enzyme (Prod. No. D4943) is supplied as a solution in 10 mM Tris-HCl pH 7.6, 200 mM NaCl, 1 mM EDTA, 10 % glycerol.

As supplied, these products should be stable for a minimum of one year when stored properly at –20 °C.

References

1. Misumi Y, Hayashi Y, Arakawa F, Ikehara Y. 1992. Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1131(3):333-336. https://doi.org/10.1016/0167-4781(92)90036-y

2. Matter K, Hauri HP. 1991. Intracellular transport and conformational maturation of intestinal brush border hydrolases. Biochemistry. 30(7):1916-1923. https://doi.org/10.1021/bi00221a026

3. Weihofen WA, Liu J, Reutter W, Saenger W, Fan H. 2004. Crystal Structure of CD26/Dipeptidyl-peptidase IV in Complex with Adenosine Deaminase Reveals a Highly Amphiphilic Interface. Journal of Biological Chemistry. 279(41):43330-43335. https://doi.org/10.1074/jbc.m405001200

4. Matter K, Hauri HP. 1991. Intracellular transport and conformational maturation of intestinal brush border hydrolases. Biochemistry. 30(7):1916-1923. https://doi.org/10.1021/bi00221a026

5. Alfalah M, Jacob R, Naim HY. 2002. Intestinal Dipeptidyl Peptidase IV Is Efficiently Sorted to the Apical Membrane through the Concerted Action of N- andO-Glycans as Well as Association with Lipid Microdomains. Journal of Biological Chemistry. 277(12):10683-10690. https://doi.org/10.1074/jbc.m109357200

6. Fukasawa KM, Fukasawa K, Hiraoka BY, Harada M. 1981. Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney. Biochimica et Biophysica Acta (BBA) - Enzymology. 657(1):179-189. https://doi.org/10.1016/0005-2744(81)90141-8

7. Kenny AJ, Booth AG, George SG, Ingram J, Kershaw D, Wood EJ, Young AR. 1976. Dipeptidyl peptidase IV, a kidney brush-border serine peptidase. 157(1):169-182. https://doi.org/10.1042/bj1570169

8. Hegen M, Kameoka J, Dong R, Morimoto C, Schlossman SF. 1997. Structure of CD26 (Dipeptidyl Peptidase IV) and Function in Human T Cell Activation.109-116. https://doi.org/10.1007/978-1-4757-9613-1_15

9. De Meester I, Korom S, Van Damme J, Scharpé S. 1999. CD26, let it cut or cut it down. Immunology Today. 20(8):367-375. https://doi.org/10.1016/s0167-5699(99)01486-3

10. Andersen TF. Kirsten Drotner et al.: Øjenåbnere / Kirsten Drotner et al.: Tankestreger / Torben Fridberg et al.: Mønstre i mangfoldigheden. MedieKultur. 15(29):4. https://doi.org/10.7146/mediekultur.v15i29.1137

11. Durinx C, Lambeir A, Bosmans E, Falmagne J, Berghmans R, Haemers A, Scharpé S, De Meester I. 2000. Molecular characterization of dipeptidyl peptidase activity in serum. 267(17):5608-5613. https://doi.org/10.1046/j.1432-1327.2000.01634.x

12. GORRELL M. 2005. Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders. 108(4):277-292. https://doi.org/10.1042/cs20040302

13. Aertgeerts K. 2004. Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation. Protein Science. 13(2):412-421. https://doi.org/10.1110/ps.03460604

14. Busek P. 2004. Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer. The International Journal of Biochemistry & Cell Biology. 36(3):408-421. https://doi.org/10.1016/s1357-2725(03)00262-0

15. Wrenger S, Kähne T, Bohuon C, Weglöhner W, Ansorge S, Reinhold D. 2000. Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV). 466(1):155-159. https://doi.org/10.1016/s0014-5793(99)01779-2

16. Shibuya-Saruta, H., et al.,. 1996. 1996 Human serum dipeptidyl peptidase IV (DPPIV) and its unique properties. Clin. Lab. Anal. . 10 435-440

17. Morrison ME, Vijayasaradhi S, Engelstein D, Albino AP, Houghton AN. 1993. A marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase.. 177(4):1135-1143. https://doi.org/10.1084/jem.177.4.1135

18. Girardi AC, Degray BC, Nagy T, Biemesderfer D, Aronson PS. 2001. Association of Na+-H+ Exchanger Isoform NHE3 and Dipeptidyl Peptidase IV in the Renal Proximal Tubule. Journal of Biological Chemistry. 276(49):46671-46677. https://doi.org/10.1074/jbc.m106897200

19. Cheng H, Abdel-Ghany M, Pauli BU. 2003. A Novel Consensus Motif in Fibronectin Mediates Dipeptidyl Peptidase IV Adhesion and Metastasis. Journal of Biological Chemistry. 278(27):24600-24607. https://doi.org/10.1074/jbc.m303424200

20. Reimer M, Holst J, Ahren B. 2002. Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice.717-727. https://doi.org/10.1530/eje.0.1460717

21. Callebaut C, Krust B, Jacotot E, Hovanessian A. 1993. T cell activation antigen, CD26, as a cofactor for entry of HIV in CD4+ cells. Science. 262(5142):2045-2050. https://doi.org/10.1126/science.7903479

22. 1992. Internationales Symposium für Kinderchirurgie. Acta Chir Austriaca. 24(4):210-210. https://doi.org/10.1007/bf02601745

23. PUSCHEL G, MENTLEIN R, HEYMANN E. 1982. Isolation and Characterization of Dipeptidyl Peptidase IV from Human Placenta. Eur J Biochem. 126(2):359-365. https://doi.org/10.1111/j.1432-1033.1982.tb06788.x

24. Caporale C, Fontanella A, Petrilli P, Pucci P, Molinaro MF, Picone D, Auricchio S. 1985. Isolation and characterization of dipeptidyl peptidase IV from human meconium. 184(2):273-277. https://doi.org/10.1016/0014-5793(85)80621-9