N-Glycosylation of pig flavin-containing monooxygenase form 1: determination of the site of protein modification by mass spectrometry.
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.
Department of Pharmacology and Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.
K K
KK
Korsmeyer
S
S
Guan
Z C
ZC
Yang
A M
AM
Falick
D M
DM
Ziegler
J R
JR
Cashman
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013552:Swine
D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)