Genomics Meets Glycomics—The First GWAS Study of Human N-Glycome Identifies HNF1α as a Master Regulator of Plasma Protein Fucosylation

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

Protein kinase B/Akt activity is involved in renal TGF-β1-driven epithelial-mesenchymal transition in vitro and in vivo

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

Glycomics

Paramount to our understanding of carbohydrates in biology has been the development of methods for analyzing and characterizing the essential features of glycans. This has resulted in a comprehensive appreciation for the various roles of glycans and the pathways associated with their synthesis. Indeed, the importance of glycosylation has been recognized by the biopharmaceutical industry. Therapeutics for a multitude of formerly untreatable diseases are coming on stream and this is largely due to the ability of the biopharmaceutical industry to produce complex proteins carrying appropriate posttranslational modifications. Importantly, glycosylation as a posttranslational modification has taken a prominent role in the focus of bioproduction, as subtle changes in protein glycosylation can significantly impact the safety, efficacy, and biological activity of a therapeutic. In response, the biopharmaceutical industry is dedicated to the generation of expression systems that provide humanized glycosylation to maximize the potency, safety, and pharmacokinetic behavior of glycoprotein therapies. Central to this strategy is the ability to manipulate glycosylation, which is largely dependent on the development of techniques that allow the assessment and characterization of glycans. To be of benefit to the biopharmaceutical industry, methods for glycan analysis will be heavily reliant on platforms that offer robust, rapid, quantifiable, sensitive, and cheap solutions, which can be performed at-line in a high-throughput format. This article provides an introduction to the field of glycomics and how specific aspects of glycosylation have been employed by the biopharmaceutical industry to generate therapeutics with enhanced biological attributes. Methods for the analysis of glycans and associated curation and access of glycan data through bioinformatics are presented.20 page(s

Protein glycosylation

Protein glycosylation is a complex posttranslational modification that manipulates the biological activity and function of therapeutic glycoproteins. To date, a plethora of functions related to glycan composition have been described in the literature: from solubility, stability, cellular localization, molecular trafficking, self-recognition, clearance, transport, immunogenicity, and circulating half-life. Most recently, glycan composition has been the target of modification to enhance the safety and efficacy of glycoprotein therapeutics. These links between the complex nature of the glycans and their associated functions have raised serious concerns within the drug regulatory authorities across the world. Glycans are the most complex and heterogeneous class of molecules due to their non-template-driven biosynthetic process, consequently making glycan characterization difficult. There is no universal method available that can characterize the complete intact glycoprotein structure; it is essential to apply several orthogonal methods to measure individual parameters such as glycosylation site analysis, oligosaccharide sequence, and monosaccharide content of a therapeutic glycoprotein. In this article, we discuss various state-of-the-art glycoanalytical approaches and strategies for evaluating total glycoprotein structure and function.20 page(s

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors.

Mucins 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.In 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.The 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).Mucinous 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

Localization of MUC5AC and MUC16 in mucinous adenocarcinoma.

<p>Serial sections from low-grade mucinous adenocarcinoma stage IV (¹⁰M-Low-IV) from (left-right) stained against MUC5AC and MUC16. The histological staining indicated that MUC5AC and MUC16 were expressed on discrete areas in the tumor.</p

Clinical and biochemical characteristics of ovarian cyst fluid and tissue samples obtained from patients with mucinous and serous ovarian tumors.

<p>^a The O-linked oligosaccharides from cyst fluid were analyzed by LC-ESI-MSⁿ.</p><p>^b The cyst fluid sample was analyzed by proteomic analysis.</p><p>NR- means not relevant</p><p>^“–”means not determined</p><p>Clinical and biochemical characteristics of ovarian cyst fluid and tissue samples obtained from patients with mucinous and serous ovarian tumors.</p

Hierarchical cluster analyses of <i>O</i>-glycans from serous and mucinous ovarian tumors.

<p>The clustering based on three variables Fuc, NeuAc and S (A) and also only the two variables NeuAc and S (B). The variables were evaluated using ROC AUC (C). The dendrograms (A, B) identify mucinous benign, LMPs and low-grade ovarian tumors as a distinct group (the group is marked by red color) separate from serous and high grade mucinous samples (the group marked in black). The exception is the LMP mucinous cyst fluid sample obtained from a non-secretor patient (green color). A separate cluster is generated by the two serous benign samples (violet color).</p