Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity.
Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high alpha-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.
Laboratory of Biochemistry, Probiodrug AG, Weinbergweg 22, 06120 Halle/Saale, Germany.
Stephan
S
Schilling
Torsten
T
Hoffmann
Fred
F
Rosche
Susanne
S
Manhart
Claus
C
Wasternack
Hans-Ulrich
HU
Demuth
D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005285:Fermentation / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase