Mechanisms in Protein O-Glycan Biosynthesis and Clinical and Molecular Aspects of Protein O-Glycan Biosynthesis Defects: A Review

Abstract

Background: Genetic diseases that affect the biosynthesis of protein O-glycans are a rapidly growing group of disorders. Because this group of disorders does not have a collective name, it is difficult to get an overview of O-glycosylation in relation to human health and disease. Many patients with an unsolved defect in Nglycosylation are found to have an abnormal O-glycosylation as well. It is becoming increasingly evident that the primary defect of these disorders is not necessarily localized in one of the glycan-specific transferases, but can likewise be found in the biosynthesis of nucleotide sugars, their transport to the endoplasmic reticulum (ER)/Golgi, and in Golgi trafficking. Already, disorders in O-glycan biosynthesis form a substantial group of genetic diseases. In view of the number of genes involved in O-glycosylation processes and the increasing scientific interest in congenital disorders of glycosylation, it is expected that the number of identified diseases in this group will grow rapidly over the coming years. Content: We first discuss the biosynthesis of protein O-glycans from their building blocks to their secretion from the Golgi. Subsequently, we review 24 different genetic disorders in O-glycosylation and 10 different genetic disorders that affect both N-and O-glycosylation. The key clinical, metabolic, chemical, diagnostic, and genetic features are described. Additionally, we describe methods that can be used in clinical laboratory screening for protein O-glycosylation biosynthesis defects and their

The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

“…PolyHis-tagged ppGalNAc T3, T6, and T13, expressed from Xpress SF ϩ insect cells (Protein Sciences Corp., Meriden, CT), were used directly bound to Ni-NTA affinity beads (Thermo Fisher) after extensive washing. 5 Soluble N-terminal poly-His-tagged ppGalNAc T5 and T16 were expressed from HEK293f cells and purified by Ni-NTA superflow (Qiagen) nickel affinity chromatography by procedures analogous to those for the production of rat ST6GAL1 (57). 5 Soluble N-terminal poly-His-tagged ppGalNAc T14 and T16 were expressed from High Five insect cells and purified on Ni-NTA-agarose (Invitrogen) and MonoQ 5/50 GL ion exchange chromatography (GE Healthcare) essentially as described previously (58).…”

“…5 Soluble N-terminal poly-His-tagged ppGalNAc T5 and T16 were expressed from HEK293f cells and purified by Ni-NTA superflow (Qiagen) nickel affinity chromatography by procedures analogous to those for the production of rat ST6GAL1 (57). 5 Soluble N-terminal poly-His-tagged ppGalNAc T14 and T16 were expressed from High Five insect cells and purified on Ni-NTA-agarose (Invitrogen) and MonoQ 5/50 GL ion exchange chromatography (GE Healthcare) essentially as described previously (58). Glycosylation of Random Lectin Glycopeptide SubstratesTypical reaction conditions utilizing random lectin glycopeptides substrates GPIV, GPIV-C, GPV, and GPV-C were as follows: 100 mM HEPES, pH 7.5 (ppGalNAc T1, T2, and T3) or 68 mM sodium cacodylate, pH 6.5 (ppGalNAc T3, T5, T6, T13, T14, and T16), 1.8 mM 2-mercaptoethanol, 10 mM MnCl 2 , 50 M 3 H-radiolabeled UDP-GalNAc (ϳ6 ϫ 10 8 dpm/mol), and 1.5-1.7 mM (5 mg/ml) of glycopeptide substrates and up to 50 l of soluble transferase or transferase-bound affinity beads.…”

“…Glycoproteins containing O-glycosylated mucin domains commonly function in the protection of the cell surface and the modulation of cell-cell interactions and hence play important roles, for example in inflammation, the immune response, metastasis, and tumorigenesis (2)(3)(4)(5)(6). Because the role of the mucin domain in these glycoproteins has been thought to produce a stable extended hydrophilic motif, the actual glycosylation pattern has usually been thought to be of relatively low importance.…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…Seven types of O-glycosylation are known: the most common mucin type of secretory proteins requires a Ser or Thr-Oγ atom linked to N-acetylgalactosamine (GalNAc), which can be either connected to another galactose or N-acteylglucosamine in an α/β1-3 or α/β1-6 bond and extended by sialic acid or various branches (Figure 1) (Wopereis et al, 2006). The already mentioned GAG trees can be O-linked via a tetrasaccharide with an O-xylose-Ser link, while O-GlcNAc, -Glu, -Fuc, and -Man links might not be relevant for KLKs (Steen et al, 1998;Lommel and Strahl, 2009).…”

The Emerging Field of Diagnostics in Glycosylation Disorders

“…There are seven different types of O-linked glycans known in humans and are classified according to the first sugar attached to a Serine, or Threonine residue of a protein [8]. Although there is a consensus sequon for determining N-glycosylation, there is no sequon for determining O-glycosylation, however there are some bioinformatics websites such as the NetOGlyc 3.1 server (http:// www.cbs.dtu.dk/services/NetOGlyc/) that predict sites of potential mucin type O-glycans, based on information of known and documented O-glycosylated proteins [9].…”