Heterologous Expression and Characterization of Human Glutaminyl Cyclase: Evidence for a Disulfide Bond with Importance for Catalytic Activity
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- 作者：Stephan Schilling ; Torsten Hoffmann ; Fred Rosche ; Susanne Manhart ; Claus Wasternack ; and Hans-Ulrich Demuth
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：September 3, 2002
- 年：2002
- 卷：41
- 期：35
- 页码：10849 - 10857
- 全文大小：134K
- 年卷期：v.41,no.35(September 3, 2002)
- ISSN：1520-4995
文摘
Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues fromglutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionallyexpressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purificationfrom the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC usingMALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at bothpotential sites with similar 2 kDa extensions. CD spectral analysis indicated a high 
-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 purifiedbovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, andH-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularlyin E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain adisulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistentlyinactivated 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 aconformational change of the protein upon treatment with DTT. In terms of the different enzymaticproperties, the consequences of QC expression in different environments are discussed.
-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 purifiedbovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, andH-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularlyin E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain adisulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistentlyinactivated 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 aconformational change of the protein upon treatment with DTT. In terms of the different enzymaticproperties, the consequences of QC expression in different environments are discussed.