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.

Changes in the Glycosylation of Kininogen and the Development of a Kininogen-Based Algorithm for the Early Detection of HCC

“…Kininogen contains four potential N-linked glycosylation sites, and we performed glycoproteomics to identify the site specific glycoforms on the kininogen molecule (47). Table 3 presents the detected glycoforms on the four N-linked glycosylation sites and shows that complex N-linked glycan structures were detectable at all the sites of N-linked glycan attachment.…”

Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

“…The ultra-high resolution MS survey scan without data dependent acquisition (DDA) performed on such a high magnetic field instrument enables precise mass determination and quantification of peptides or glycopeptides [50]. Here, we demonstrate, for the first time, quantification of glycopeptides from partially depleted human sera by ultra-high resolution full-MS scan analysis performed by nanocapillary reversed phase chromatography coupled with the FT-ICR mass spectrometer and compare these data with results obtained by our previously optimized LC-MS/MS-MRM workflow [11,13,27]. …”

“…Formations of Lewis type epitopes such as SLeX, LeX, or LeY were recently observed on liver secreted glycoproteins [17–21], in addition to the well-known increase in core fucosylated α- fetoprotein in hepatocellular carcinoma [22–24]. These alterations in the glycosylation of proteins were extensively studied in human tissues and especially in serum [13,15,25–27]. Other described alterations of glycans include increased branching, sialylation, agalactosylated glycans or polylactosamine chains [16,28,29].…”

“…Each of the approaches has its benefits and the methods offer complementary view of protein glycosylation. Major benefit of the quantification of glycopeptides is its ability to quantify site-specific glycoforms because the knowledge of glycan distribution within a protein is important for its function [40–42]. We therefore describe two LC-MS workflows for quantitative analysis of glycopeptides and document their applicability to serologic analysis of glycoproteins in cancer diseases.…”

Association between Serum Sialic Acid Levels and Nonalcoholic Fatty Liver Disease: A Cross-Sectional Study

“…In this present study, we found that serum SA levels were negatively correlated with FIB-4 score and lower serum SA level was the risk factor of advanced fibrosis in subjects with NAFLD. The liver released a large number of glycoproteins and glycolipids into the circulation, most of which are sialylated on the termini of their glycans [39]. The decline in serum SA may be due to the downregulation of sialyltransferase participating in SA synthesis in the liver or the synthesis of proteins decreased in the liver cirrhosis.…”