Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme.
Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DP IV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.
Bär
Joachim
J
Probiodrug AG, Department of Enzymology, D-06120 Halle, Germany.
Weber
Anja
A
Hoffmann
Torsten
T
Stork
Jörg
J
Wermann
Michael
M
Wagner
Leona
L
Aust
Susanne
S
Gerhartz
Bernd
B
Demuth
Hans-Ulrich
HU
D000243:Adenosine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004151:Dipeptides / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005934:Glucagon; D052216:Glucagon-Like Peptide 1; D004763:Glucagon-Like Peptides; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments; D010455:Peptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
D004151:Dipeptides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006023:Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D004763:Glucagon-Like Peptides; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4; D000243:Adenosine Deaminase; C051322:glucagon-like peptide 1 (7-37)