Protein O-glycosylation – Structural Diversity, Biosynthesis, Regulation and Functions
Focusing on O-GalNAc, O-Man, O-Fuc/O-Xyl/O-Glc
Lectures: Henrik Clausen, Robert Haltiwanger
OVERVIEW
This course will provide insights into protein glycosylation in eukaryotes with particular emphasis on the many different types of protein O-glycosylation. Historical and up-to-date perspectives will be provided with highlights of the different approaches to identify and characterize the enzymes involved in O-glycosylation and the current state of understanding of the distinct O-glycosylation pathways in mammalian cells. State-of-the-art strategies to discover and dissect biological functions of O-glycosylation as well as an overview of the genes known to be involved in human diseases (congenital disorders of glycosylation) will be covered.
| 15:20-15:40 | Introduction (Robert Haltiwanger/Henrik Clausen) Overview of protein glycosylation in eukaryotes Biosynthetic and genetic regulation of protein glycosylation Evolutionary perspectives (yeast to man) |
| 15:40-16:30 | Henrik Clausen (Copenhagen Center for Glycomics, Denmark): The most common types of protein O-glycosylation – O-GalNAc (mucin-type) and O-Man |
| 16:30-16:50 | Questions/Discussion |
| 16:50-17:10 | Refreshment break |
| 17:10-18:00 | Robert Haltiwanger (University of Georgia, USA): O-glycosylation of cysteine-rich domains with O-Fuc, O-Glc, O-Xyl, or O-GlcNAc |
| 18.00-18.20 | Questions/Discussion |
Title: The most common types of protein O-glycosylation – O-GalNAc (mucin-type) and O-Man
Henrik Clausen
Glycosylation is arguably the most abundant and diverse type of posttranslational modification of proteins. The mucin-type (GalNAc-type) O-glycosylation stands out as being found widely on most secreted proteins without preference for classes of proteins or particular protein folds much like N-glycosylation. However, in contrast to N-glycosylation mucin-type O-glycosylation is not guided by clear peptide sequence motifs and the O-glycoproteome is therefore difficult to predict. Moreover, up to 20 isoenzymes (polypeptide GalNAc-transferases, GALNT1-20) orchestrate where O-glycans are attached on proteins providing opportunities for high degree of differential regulation of the O-glycoproteome in cells. This lecture will cover the historical developments in understanding the biosynthesis and genetic regulation of mucin-type glycosylation and the current state of the O-glycoproteome as well as highlight examples of specific biological functions of O-glycosylation such as proprotein processing and ectodomain shedding. The lecture will draw parallels to protein O-mannosylation that appear to serve equivalent functions in yeast, whereas O-mannosylation in higher eukaryotes has evolved into three distinct types each serving highly restricted proteins and protein folds. The lecture will highlight strategies (gene engineering, GlycoCRISPR, SimpleCells) and resources (cell-based glycan arrays, organotypic tissue models) currently used in studying O-glycosylation.
Title: O-glycosylation of cysteine-rich domains with O-Fuc, O-Glc, O-Xyl, or O-GlcNAc
Robert Haltiwanger
This lecture will discuss the discovery, biosynthesis, and function of O-glycans on two cysteine-rich domains: Epidermal Growth factor-like (EGF) repeats and Thrombospondin Type 1 Repeats (TSRs). Both EGF repeats and TSRs have six conserved cysteines forming three disulfide bonds, but the disulfide bonding patterns are different. EGF repeats are typically 40-50 amino acids long and are found in hundreds of cell-surface and secreted proteins. EGF repeats can be modified with up to four O-glycans, each at specific consensus sites: O-Glc (at two sites), O-Fuc, and O-GlcNAc. Some of the O-Glc sites can also be modified with O-Xyl. The presence of consensus sequences allows identifying proteins that are predicted to be modified. The enzymes that add these sugars to EGF repeats (POGLUT1/2/3, POFUT1, and EOGT) and those that elongate them (e.g. GXYLT1/2, XXYLT1, LFNG/MFNG/RFNG), have been identified. Mutations in these enzymes in humans, mice or flies cause defects in the Notch signaling pathway, indicating that the biological function of these modifications are mainly in regulation of Notch function. TSRs are somewhat larger than EGF repeats (50-70 amino acids) and occur in dozens of extracellular proteins. They also can be modified by O-Fuc at a specific consensus sequence, and the O-Fuc can be elongated to a Glc1-3Fuc disaccharide. 49 proteins in the human genome have TSRs predicted to be modified with O-Fuc, and elimination of the enzyme adding the fucose (POFUT2) results in embryonic lethality in mice, while mutations in the enzyme adding the glucose (B3GLCT) cause a Congenital Disorder of Glycosylation, Peters Plus Syndrome. The lecture will conclude with proposed mechanisms for how these O-glycans affect the function of proteins containing modified EGF repeats and TSRs. Interestingly, much like N-glycans, they have both been shown to participate in protein-protein interactions in the extracellular spaces and in quality control in the endoplasmic reticulum.
