Schjoldager Group

The major focus is to explore the biological role of O-glycans in health and disease.

Katrine Schjoldager is a co-PI at the DNRF Copenhagen Center for Glycomics where she lead the GlycoCell projects on O-glycans in peptide hormone biology, lipid metabolism, LDLR function and protein stability.

Current project areas

O-glycans in Protein and Peptide Stability and Function
Traditionally, O-glycans have been found and associated with large mucins containing P/T/S rich domains with high density of O-glycans, where they serve important functions for the structure and stability of mucins required for lubrication and protective effects on lining mucosa and in body fluids. However, recent proteome-wide discovery strategies for O-glycosylation demonstrate that O-glycans are abundantly found on all types of proteins (>80% of all proteins trafficking the secretory pathway are O-glycosylated, Steentoft et al., 2013), and in particular found in more isolated sites often in close proximity to other important PTMs such as PC processing sites, phosphorylation sites (not published), mannosylation sites, ectodomain shedding sites, and most recently activation of G-coupled protein receptors (GPCRs), where they may serve important (co-)regulatory roles in fine-tuning protein functions (Schjoldager et al.,2010, 2011, 2012, Goth et al., 2015, 2012). Our breakthrough in O-glycoproteomics has opened for wide discovery of O-glycoproteins and is now driving a plethora of exciting novel hypotheses of the role of O-glycosylation for diverse types of protein including GPCRs and LDLR related proteins.

Participants
Christoffer Goth
Zilu Ye
Sergey Y Vakhrushev
Yang Mao
Christina Christoffersen (RH, Department of Clinical Biochemistry)
Nabil Seidah (University of Montreal, CA)

figure - Schematic depiction of site specific protein O-glycosylation and suggested biological function — Figure 1. Schematic depiction of site specific protein O-glycosylation and suggested biological function. Site-specific O-glycosylation is catalysed by selected isoforms in the large GalNAc-T family and is often found between protein domains in linker regions of varying length. We have demonstrated that O-glycans can modulate limited proteolytic processing by a number of proteases and thereby regulate protein activation, stability and solubilisation. We have also demonstrated that O-glycans are found in shorter linker regions in lipid binding domains in members of the LDLR family where deficient glycosylation impair lipoprotein particle binding and uptake.

Exploring dose-dependent GalNAc-T activity
Numerous studies, from our lab and others, have revealed non-redundant and isoform specific functions of GalNAc-transferases (Kato et al. 2006, Schjoldager et al. 2010, Pedersen et al. 2014, Khetarpal and Schjoldager et al. 2016). These results were generated in knock-out/knock-in systems displaying the abstract effects of complete loss or gain of GalNAc-transferase function. The GALNT genes are often found as candidate genes associated to complex disease traits in Genome-Wide-Association Studies (GWAS). Recently, we validated one such candidate genes, GALNT2, with a GWAS predicted role in regulating HDL and TG (Khetarpal and Schjoldager et al. 2016). We furthermore demonstrate that the GWAS signal for GALNT2 and low HDL is located in the first large intron of the gene close to a liver-specific regulatory element, and several studies have demonstrated that the GWAS SNP signal induced allele-specific transcription differences (Roman et al. 2015, Cavalli et al. 2016). We hypothesize that within the subset of non-redundant GalNAc-T functions hide substrates that are far more sensitive to changes in expression of a specific GalNAc-T, substrates that are selectively regulated in a “fine-tune” manner. To address this, we combined the use of precise genome editing tools and last generation Tet-On system to generate isogenic HEK293 cells in which GalNAc-T2, GalNAc-T3 and GalNAc-T11 can be induced within wild-type range. By subjecting differentially induced isogenic cells to quantitative O-glycoproteomics explore dose-dependent functions of GalNac-Ts ex vivo.

Participants
John Hintze
Eric P. Bennett

figure - Generation of GalNAc-T2 inducing Hek293 cells by precise integration of inducible transcriptional elements — Figure 1. Generation of GalNAc-T2 inducing Hek293 cells by precise integration of inducible transcriptional elements. Precise integration is achieved using a heterodimeric zinc-finger nuclease (ZFN) pair that simultaneously; targets the genome and in situ linearizes the donor plasmid enabling it to be “ligated” into the genome6. A) AAVS1 Tet-On®3G plasmid was co-transfected with AAVS1 zinc-finger (ZFN) plasmids into Hek293 T2 KO cells. The ZFNs are tagged with GFP or Crimson enabling FACS enrichment of cells expressing both ZFNs. The recovered cell bulk was single cell sorted and positive clones identified by junction PCR. Positive clones with correct integration of AAVS1Tet-On®3G contain the CHO safe harbor landing pad (CHO SH landing pad) downstream of the Tet-On®3G gene. B) The CHO SH landing pad can be targeted for integration using a second ZFN pair analogous to AAVS1 Tet-On®3G integration. Hek293 T2 KO Tet-On®3G positive clones were transfected with CHO SH pTRE3G-GALNT2 together with CHO SH ZFN plasmids, enriched by FACS and subsequently single cell sorted. Positive clones were identified by junction PCR and ability to express GalNAc-T2 when induced with doxycycline. C) Upon addition of doxycycline (dox) to cell culture media the Tet-On 3G transactivator binds to and activates expression from TRE promoters.

Experimental and Clinical Study of GALNT2-Induced Dyslipidemia – Protein O-Glycosylation plays a central role in proprotein processing and lipid metabolism
In a large international collaboration, we have validated a genome-wide association study (GWAS) linkage between O-glycosylation (the GALNT2 gene) and dyslipidemia in multiple species, including rare individuals we identified with homozygous loss of the gene. Using genetic engineering (ZFNs and Crispr/Cas9) and a novel strategy for quantitative differential glycoproteomics, we demonstrated that loss of the GALNT2 gene specifically affects high-density lipoprotein cholesterol through non-redundant O-glycosylation of several known important regulators of lipoprotein metabolism including ANGPTL3 and phospholipid transfer protein (Khetarpal and Schjoldager et al., 2016 and Schjoldager et al. 2015). Now ongoing studies focus to dissect molecular mechanisms using our animal models and further demonstrate that the GALNT2 gene is a common regulator of human metabolism.

Participants
Daniel Rader, University of Pennsylvania
Rami Abou Jamra, University of Leipzig
Eric Leguern, Sorbonne Université
Christina Christoffersen, RH Department of Clinical Biochemistry

overview of model to demonstrate that loss of function of GALNT2 reduces HDL-C in humans, rodents, and nonhuman primates — Figure 1. SNPs in GALNT2 are associated with HDL-C metabolism, but whether GALNT2 causes HDL-C to go up or down has been debated. In this project, we have demonstrated that loss of function of GALNT2 reduces HDL-C in humans, rodents, and nonhuman primates. Using these models, precise genetic engineering to produce isogenic cell lines, state-of-art glycoprotein mass spectrometry and RNA seq expression analysis we identify and explore GalNAc-T2 specific glycosylation targets.

Novel Proteoforms of Peptide Hormones Provide Exciting Options for Improving Drug Design
Peptide hormones, neuropeptides or bioactive peptides are small polypeptides with important biological functions. O-glycosylation is a post-translational modification that takes place in the Golgi where up to 20 different isoenzymes glycosylates proteins travelling through the secretory pathway. Through O-glycoproteomics analysis of tissues and biological fluids we recently identified O-glycosylation on mature peptide hormones in biologically important regions. We have identified O-glycans on a number of major therapeutic targets for drug development in endocrinology and in preliminary data, we have shown that O-glycans on peptide hormones can alter receptor signalling and give resistance to proteolytic degradation in vitro. In this project, we join experts from glycobiology and endocrinology to explore and characterize the biology of O-glycans on peptide hormones.

Participants
Thomas Daugbjerg Madsen
Lasse Holst Hansen (RH, Department of Clinical Biochemistry)
Jens Juul Holst (The NNF Center for Basic Metabolic Research)
Jens Peter Gøtze (RH, Department of Clinical Biochemistry)

Selected publications

King SL, Joshi H, Schjoldager KT, Halim A, Madsen TD, Dziegiel MH, Woetmann A, Vakhrushev SY and Wandall HH (2017) Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells. Blood Advances 1: 429-442.
Goth CK, Tuhkanen HE, Khan H, Lackman JJ, Wang S, Narimatsu Y, Holst Hansen L, Overall C, Clausen H, Schjoldager KT#, Petaja-Repo UE# (2017) Site-specific O-glycosylation by Polypeptide GalNAc-transferase T2 Co-regulates Beta1-adrenergic Receptor N-terminal Cleavage. J Biol Chem. #Corresponding authors.
Khetarpal SA*, Schjoldager KT *,#, Christoffersen C, Raghavan A, Edmondson AC, Reutter HM, Ahmed B, Ouazzani R, Peloso GM, Vitali C, Zhao W, Somasundara AV, Millar JS, Park Y, Fernando G, Livanov V, Choi S, Noe E, Patel P, Ho SP, Kirchgessner TG, Wandall HH, Hansen L, Bennett EP, Vakhrushev SY, Saleheen D, Kathiresan S, Brown CD, Abou Jamra R, LeGuern E, Clausen H, Rader DJ#. (2016) Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents. Cell Metab 24: 234-45. #Corresponding authors, *First authors (This paper was subject to an Editorial comment in Current Opinion of Lipidomics 2017 Feb;28(1):81-82).
Schjoldager KT*, Joshi HJ, Kong Y, Goth CK, King SL, Wandall HH, Bennett EP, Vakhrushev SY, Clausen H* (2015) Deconstruction of O-glycosylation - GalNAc-T isoforms direct distinct subsets of the O-glycoproteome. EMBO Rep 16: 1713-22. *Corresponding authors.
Goth CK, Halim A, Khetarpal SA, Rader DJ, Clausen H, Schjoldager KT# (2015) A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation. Proc Natl Acad Sci USA 112: 14623-8. #Corresponding author.
Radhakrishnan P, Dabelsteen S, Madsen FB, Francavilla C, Kopp KL, Steentoft C, Vakhrushev SY, Olsen JV, Hansen L, Bennett EP, Woetmann A, Yin G, Chen L, Song H, Bak M, Hlady RA, Peters SL, Opavsky R, Thode C, Qvortrup K, Schjoldager KT, Clausen H, Hollingsworth MA, Wandall HH (2014) Immature truncated O-glycophenotype of cancer directly induces oncogenic features. Proc Natl Acad Sci USA 111(39):E4066-75.
Pedersen NB, Wang S, Narimatsu Y, Yang Z, Halim A, Schjoldager KT, Madsen TD, Seidah NG, Bennett EP, Levery SB, Clausen H (2014) Low density lipoprotein receptor class a repeats are o-glycosylated in linker regions. J Biol Chem 289: 17312-24.
Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT, Lavrsen K, Dabelsteen S, Pedersen NB, Marcos-Silva L, Gupta R, Bennett EP, Mandel U, Brunak S, Wandall HH, Levery SB, Clausen H (2013) Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. The EMBO journal 32: 1478-88.
Schjoldager KT, Clausen H (2012) Site-specific protein O-glycosylation modulates proprotein processing - deciphering specific functions of the large polypeptide GalNAc-transferase gene family. Biochim Biophys Acta 1820: 2079-94.
Schjoldager KT, Vakhrushev SY, Kong Y, Steentoft C, Nudelman AS, Pedersen NB, Wandall HH, Mandel U, Bennett EP, Levery SB, Clausen H (2012) Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells. Proc Natl Acad Sci USA 109: 9893-8 (This paper was subject to a comment by Jacques Baenziger entitled “Moving the Glycoproteome from form to function” in Proc Natl Acad Sci USA 25 9672-3 (2012))
Schjoldager KT, Vester-Christensen MB, Goth CK, Petersen TN, Brunak S, Bennett EP, Levery SB, Clausen H (2011) A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing. J Biol Chem 286: 40122-32.
Steentoft C, Vakhrushev SY, Vester-Christensen MB, Schjoldager KT, Kong Y, Bennett EP, Mandel U, Wandall H, Levery SB, Clausen H (2011) Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nat Methods 8: 977-82.
Schjoldager KT, Vester-Christensen MB, Bennett EP, Levery SB, Schwientek T, Yin W, Blixt O, Clausen H (2010) O-glycosylation modulates proprotein convertase activation of angiopoietin-like protein 3: possible role of polypeptide GalNAc-transferase-2 in regulation of concentrations of plasma lipids. J Biol Chem 285: 36293-303