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 ve rified that pig flavin-containing monooxygenase (FMO1) was N-glycosyla ted. By using mass spectrometry approaches [i.e., peptide mapping, gas chromatography/mass spectrometry, microbore HPLC/electrospray ionizat ion mass spectrometry (LC/ESI/MS), chemical ionization gas chromatogra phy/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-glycosy lation. Pig FMO1 contains two putative consensus sites of N-glycosylat ion. The results showed that pig FMO1 amino acid Asn120 was selectivel y N-glycosylated. Highly purified pig FMO1 avidly bound concanavalin A and reacted positively for carbohydrates by the periodic acid/Schiffs 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 p rotein as determined by sodium dodecyl sulfate-polyacrylamide gel elec trophoresis (SDS-PAGE) and immunoblot experiments. Peptide mapping of pig FMO1 showed that the protein used in the study was not contaminate d with another glycoprotein. MALDI/MS experiments showed that pig FMO1 was present with the expected molecular mass but that higher-molecula r mass forms consistent with the presence of N-linked high-mannose oli gosaccharide structures were also covalently attached to the enzyme. T he 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 wa s N-glycosylated in pig FMO1 was Asn120. Knowledge of the structural a spects of FMO may be useful in understanding the membrane association properties of the enzyme.