N-Glycosylation of pig flavin-containing monooxygenase form 1: determination of the site of protein modification by mass spectrometry.

Abstract:

:By using a combination of biochemical methods (i.e., endoglycosidase H digestion and immunoblot and plant lectin binding studies), it was verified that pig flavin-containing monooxygenase (FMO1) was N-glycosylated. By using mass spectrometry approaches [i.e., peptide mapping, gas chromatography/mass spectrometry, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), chemical ionization gas chromatography/mass spectrometry (CI/GC/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS)], we were able to confirm that pig FMO1 was N-glycosylated and we were able to identify the site of N-glycosylation. Pig FMO1 contains two putative consensus sites of N-glycosylation. The results showed that pig FMO1 amino acid Asn120 was selectively N-glycosylated. Highly purified pig FMO1 avidly bound concanavalin A and reacted positively for carbohydrates by the periodic acid/Schiff's base method of analysis. In addition, treatment of pig FMO1 with endo-N-acetylglucosaminidase converted the enzyme to another species with a molecular mass approximately 5000 Da lower than that of the parent protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments. Peptide mapping of pig FMO1 showed that the protein used in the study was not contaminated with another glycoprotein. MALDI/MS experiments showed that pig FMO1 was present with the expected molecular mass but that higher-molecular mass forms consistent with the presence of N-linked high-mannose oligosaccharide structures were also covalently attached to the enzyme. The presence of N-acetylglucosamine isolated from acid hydrolysates of the N-linked high-mannose oligosaccharide of pig FMO1 was confirmed by high-pH anion exchange HPLC studies and verified by CI/GC/MS studies of derivatized monosaccharide fractions. Further analysis of pig FMO1 proteolytic peptides by LC/ESI/MS showed that the only residue that was N-glycosylated in pig FMO1 was Asn120. Knowledge of the structural aspects of FMO may be useful in understanding the membrane association properties of the enzyme.

journal_name

Chem Res Toxicol

authors

Korsmeyer KK,Guan S,Yang ZC,Falick AM,Ziegler DM,Cashman JR

doi

10.1021/tx980117p

subject

Has Abstract

pub_date

1998-10-01 00:00:00

pages

1145-53

issue

10

eissn

0893-228X

issn

1520-5010

pii

tx980117p

journal_volume

11

pub_type

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