title: Protein and site specificity of fucosylation in liver-secreted glycoproteins.
abstract: Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.
authors.affiliation: Institute of Microbiology v.v.i., Czech Academy of Sciences , Videnska 1083, Prague 142 20, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: David J
authors.initials: DJ
authors.lastname: Ashline
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Brnakova
authors.forename: Julius
authors.initials: J
authors.lastname: Benicky
authors.forename: Miloslav
authors.initials: M
authors.lastname: Sanda
authors.forename: Radoslav
authors.initials: R
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000097:blood* / Q000378:metabolism; D006031:Glycosylation; D006526:Hepatitis C / Q000097:blood; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000097:blood / Q000821:virology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational
chemical_list: D006023:Glycoproteins
keywords: MSn structural analysis; N-glycans; exoglycosidase treatment; fucosylation; glycopeptides; hepatocellular carcinoma; mass spectrometry; microheterogeneity; permethylation; site specificity