Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Simple item page
dc.contributor.authorYu, X Christopher
dc.contributor.authorSturrock, Edward D
dc.contributor.authorWu, Zhuchun
dc.contributor.authorBiemann, Klaus
dc.contributor.authorEHLERS, Mario R W
dc.contributor.authorRiordan, James F
dc.date.accessioned2021-10-08T07:20:45Z
dc.date.available2021-10-08T07:20:45Z
dc.date.issued1997
dc.description.abstractThe sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.
dc.identifier.apacitationYu, X. C., Sturrock, E. D., Wu, Z., Biemann, K., EHLERS, M. R. W., & Riordan, J. F. (1997). Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form. <i>The Journal of Biological Chemistry</i>, 272(6), 3511 - 3519. http://hdl.handle.net/11427/34997en_ZA
dc.identifier.chicagocitationYu, X Christopher, Edward D Sturrock, Zhuchun Wu, Klaus Biemann, Mario R W EHLERS, and James F Riordan "Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form." <i>The Journal of Biological Chemistry</i> 272, 6. (1997): 3511 - 3519. http://hdl.handle.net/11427/34997en_ZA
dc.identifier.citationYu, X.C., Sturrock, E.D., Wu, Z., Biemann, K., EHLERS, M.R.W. & Riordan, J.F. 1997. Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form. <i>The Journal of Biological Chemistry.</i> 272(6):3511 - 3519. http://hdl.handle.net/11427/34997en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Yu, X Christopher AU - Sturrock, Edward D AU - Wu, Zhuchun AU - Biemann, Klaus AU - EHLERS, Mario R W AU - Riordan, James F AB - The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies. DA - 1997 DB - OpenUCT DP - University of Cape Town IS - 6 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 1997 SM - 0021-9258 SM - 1083-351X T1 - Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form TI - Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form UR - http://hdl.handle.net/11427/34997 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/34997
dc.identifier.vancouvercitationYu XC, Sturrock ED, Wu Z, Biemann K, EHLERS MRW, Riordan JF. Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form. The Journal of Biological Chemistry. 1997;272(6):3511 - 3519. http://hdl.handle.net/11427/34997.en_ZA
dc.language.isoeng
dc.publisher.departmentDivision of Medical Biochemistry
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue6
dc.source.journalvolume272
dc.source.pagination3511 - 3519
dc.source.urihttps://dx.doi.org/10.1074/jbc.272.6.3511
dc.subject.otherAnimals
dc.subject.otherCHO Cells
dc.subject.otherChromatography, High Pressure Liquid
dc.subject.otherCricetinae
dc.subject.otherCricetulus
dc.subject.otherFemale
dc.subject.otherGlycosylation
dc.subject.otherHumans
dc.subject.otherMale
dc.subject.otherOvary
dc.subject.otherPeptidyl-Dipeptidase A
dc.subject.otherSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
dc.subject.otherTestis
dc.subject.otherPeptidyl-Dipeptidase A
dc.titleIdentification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
YuXChristopher_IdentificationN_1997.pdf
Size:
327.31 KB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Journal Articles