Title : Identification of N-linked glycosylation
sites in human testis
angiotensin-converting enzyme and expression of an active deglycosylated form
Abstract :
- 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
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. glycosylation, , -, -, sites
- 1. glycosylation, , -, -, sites
- 2. glycosylated, , -, -, sites, Asn90 and Asn109
- 2. glycosylation, , -, -, sites, Asn90 and Asn109
- 3. glycopeptides, , -, -, glycopeptides
- 4. glycosylated, , -, -, Asn72
- 4. glycosylated, , -, -, sites, Asn155, Asn337, and Asn586
- 6. glycosylated, , -, -, Asn
- 6. unglycosylated, , -, -, Asn
- 7. glycosylation, , -, -, sites
Output(Part-Of) (sent_index,
protein,
site):
- 0. angiotensin-converting enzyme, sites
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 2. angiotensin-converting enzyme, -, sites, Asn90 and Asn109
- 4. angiotensin-converting enzyme, -, Asn72
- 4. angiotensin-converting enzyme, -, sites, Asn155, Asn337, and Asn586