Protein glycosylation is an important post-translational modification. It is a feature that enhances the functional diversity of proteins and influences their biological activity. A wide range of functions for glycans have been described, from structural roles to participation in molecular trafficking, self-recognition and clearance. Understanding the basis of these functions is challenging because the biosynthetic machinery that constructs glycans executes sequential and competitive steps that result in a mixture of glycosylated variants (glycoforms) for each glycoprotein. Additionally, naturally occurring glycoproteins are often present at low levels, putting pressure on the sensitivity of the analytical technologies. No universal method for the rapid and reliable identification of glycan structure is currently available; hence, research goals must dictate the best method or combination of methods. To this end, we introduce some of the major technologies routinely used for structural N- and O-glycan analysis, describing the complementary information that each provides.

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-β (TGF-β1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-β1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-β1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3β phosphorylation. TGF-β1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and α-smooth muscle actin expression, consistent with the fibrotic actions of TGF-β1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-β1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3β, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-β1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high-throughput high-performance liquid chromatography (HPLC) analysis method, we have reported, in a pilot genome-wide association study of 13 glycan features in 2705 individuals from three European populations, that polymorphisms at three loci (FUT8, FUT6/FUT3 and HNF1A) affect plasma levels of N-glycans. Here, we extended the analysis to 33 directly measured and 13 derived glycosylation traits in 3533 individuals and identified three novel gene association (MGAT5, B3GAT1 and SLC9A9) as well as replicated the previous findings using an additional European cohort. MGAT5 (meta-analysis association P-value = 1.80 × 10(-10) for rs1257220) encodes a glycosyltransferase which is known to synthesize the associated glycans. In contrast, neither B3GAT1 (rs7928758, P = 1.66 × 10(-08)) nor SLC9A9 (rs4839604, P = 3.50 × 10(-13)) had previously been associated functionally with glycosylation of plasma proteins. Given the glucuronyl transferase activity of B3GAT1, we were able to show that glucuronic acid is present on antennae of plasma glycoproteins underlying the corresponding HPLC peak. SLC9A9 encodes a proton pump which affects pH in the endosomal compartment and it was recently reported that changes in Golgi pH can impair protein sialylation, giving a possible mechanism for the observed association.

OBJECTIVEGremlin (grem1) is an antagonist of the bone morphogenetic protein family that plays a key role in limb bud development and kidney formation. There is a growing appreciation that altered grem1 expression may regulate the homeostatic constraints on damage responses in diseases such as diabetic nephropathy.RESEARCH DESIGN AND METHODSHere we explored whether knockout mice heterozygous for grem1 gene deletion (grem1+/−) exhibit protection from the progression of diabetic kidney disease in a streptozotocin-induced model of type 1 diabetes.RESULTSA marked elevation in grem1 expression was detected in the kidneys and particularly in kidney tubules of diabetic wild-type mice compared with those of littermate controls. In contrast, diabetic grem1+/− mice displayed a significant attenuation in grem1 expression at 6 months of diabetes compared with that in age- and sex-matched wild-type controls. Whereas the onset and induction of diabetes were similar between grem1+/− and wild-type mice, several indicators of diabetes-associated kidney damage such as increased glomerular basement membrane thickening and microalbuminuria were attenuated in grem1+/− mice compared with those in wild-type controls. Markers of renal damage such as fibronectin and connective tissue growth factor were elevated in diabetic wild-type but not in grem1+/− kidneys. Levels of pSmad1/5/8 decreased in wild-type but not in grem1+/− diabetic kidneys, suggesting that bone morphogenetic protein signaling may be maintained in the absence of grem1.CONCLUSIONSThese data identify grem1 as a potential modifier of renal injury in the context of diabetic kidney disease.

BackgroundMucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.MethodsIn this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant) and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC) coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSn). The LC-ESI-MSn of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.ResultsThe obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline) and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC).ConclusionMucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from serous and high-grade mucinous carcinomas based on their O-glycan profiles.

Glycosylation is highly variable depending on many environmental factors. Using our fully quantitative high-throughput normal phase hydrophilic interaction liquid chromatography platform we have identified glycosylation changes associated with medication in the plasma N-glycome from three different population cohorts: ORCADES from the Orkney Islands in Scotland and CROATIA-Vis and CROATIA-Korcula from the Croatian islands of Vis and Korcula. Associations between glycosylation and the use of hormones (oral contraceptives, hormone replacement therapy), nonsteroidal anti-inflammatory drugs (aspirin and other NSAIDs), oral steroids (prednisolone) and steroid inhalers (beclomethasone) were investigated. Significant differences associated with usage of oral contraceptives were found with increased core-fucosylated biantennary glycans. Decreases in core-fucosylated biantennary glycans, core-fucosylated triantennary glycans with outer-arm fucose, and high mannosylated glycans were associated with the use of anti-inflammatory drugs. All of the changes in glycosylation were independent of blood group status. In conclusion, hormones and anti-inflammatory medication were associated with changes in glycosylation, possibly as a result of the modulatory effect of these drugs on the inflammatory response. In general, cancer is associated with inflammation, and many glycoproteins in the plasma are acute phase related to the host response. These preliminary data indicate the importance of correcting the levels of glycans used as biomarkers for the effects of medication.