Site-specific protein O-glycosylation modulates proprotein processing — Deciphering specific functions of the large polypeptide GalNAc-transferase gene family

Biochimica et Biophysica Acta 1820 (2012) 2079–2094

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Review

Site-specific protein O-glycosylation modulates proprotein processing — Deciphering specific functions of the large polypeptide GalNAc-transferase gene family Katrine T.-B. G. Schjoldager ⁎, Henrik Clausen Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Institute of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen-N, Denmark

a r t i c l e

i n f o

Article history: Received 14 May 2012 Received in revised form 17 September 2012 Accepted 19 September 2012 Available online 26 September 2012 Keywords: O-glycosylation GalNAc-transferase (UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase) Zinc finger nuclease gene targeting Pro-protein convertase (PC) processing Congenital disorder of glycosylation (CDG)

a b s t r a c t Background: Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis is initiated in the Golgi by up to twenty distinct UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). These GalNAc-Ts are differentially expressed in cells and have different (although partly overlapping) substrate specificities, which provide for both unique functions and considerable redundancy. Recently we have begun to uncover human diseases associated with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3) and dysregulated lipid metabolism (GALNT2). These phenotypes appear to be caused by deficient site-specific O-glycosylation that co-regulates proprotein convertase (PC) processing of FGF23 and ANGPTL3, respectively. Scope of review: Here we summarize recent progress in uncovering the interplay between human O-glycosylation and protease regulated processing and describes other important functions of site-specific O-glycosylation in health and disease. Major conclusions: Site-specific O-glycosylation modifies pro-protein processing and other proteolytic events such as ADAM processing and thus emerges as an important co-regulator of limited proteolytic processing events. General significance: Our appreciation of this function may have been hampered by our sparse knowledge of the O-glycoproteome and in particular sites of O-glycosylation. New strategies for identification of O-glycoproteins have emerged and recently the concept of SimpleCells, i.e. human cell lines made deficient in O-glycan extension by zinc finger nuclease gene targeting, was introduced for broad O-glycoproteome analysis. © 2012 Elsevier B.V. All rights reserved.

1. Introduction 1.1. Protein glycosylation — a major PTM regulating protein functions Posttranslational modifications (PTMs) of proteins increase the size of the proteome and diversify functions of proteins (Fig. 1). Several hundred different PTMs have been described to date and the important roles of these modifications of proteins in health and disease are becoming increasingly apparent [1,2]. A large number of enzymes, proteases and kinases, orchestrate PTMs and thereby diversify and regulate the functions of proteins. Defects or dysregulation of genes involved in the PTMs of proteins are known to cause a number of diseases. However, as PTMs are produced by complex and (partially) redundant biosynthetic pathways and often involve many different proteins, it is not straightforward to identify defects causing disease or the mechanisms underlying disease phenotypes. Thus, it ⁎ Corresponding author. E-mail address: [email protected] (K.T.-B.G. Schjoldager). 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.09.014

is likely that our appreciation of the role of PTMs in disease is greatly underestimated. This is particularly true for protein glycosylation being the most abundant and diversified PTM known. Protein glycosylation in man is a complex process involving several hundred distinct genes, glycogenes, and the human glycogenomes comprise more than 400 genes. More than 50% of the proteome are glycosylated [3] and this occurs in a number of different ways. Protein N-linked glycosylation of asparagine (Asn) [4] and O-linked glycosylation of serine (Ser) and threonine (Thr) amino acids are the most abundant forms [5]. O-glycosylation may also occur on tyrosine (Tyr) [6,7] and hydroxylysine (Hyl) [8]. Several different types of O-glycosylation exist in man including O-N-acetylgalactosamine (O-GalNAc or mucin-type) [9], O-mannose [10], O-xylose [11], O-fucose [12], O-glucose [13], and O-galactose (on Hyl) [8] found on proteins passing through the secretory pathway, and O-GlcNAc [14] found primarily on cytosolic proteins (recently extended O-GlcNAc modifications of extracellular proteins have been described [15–17]). All types of protein glycosylation involve two fundamental steps (except for cytosolic-type O-GlcNAc, which only includes the

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mRNA editing Alternative splicing Alternative promoters

~25.000 genes

~100.000 transcripts

Sulfation Amidation Lipidation Processing Acetylation Methylation Sumoylation Citrullination Octanolyation Hydroxylation palmitoylation Ubiquitinylation Phosphorylation N-linked glycosylation O-linked glycosylation

~1.000.000 proteins

Fig. 1. Posttranslational modifications diversify the human proteome. The number of genes encoded in the human proteome is greatly exceeded by the number of distinct covalent forms of existing proteins. This is in part due to post-transcriptional modifications as mRNA-editing and alternative splicing but even more to the posttranslational modifications giving rise to the large human proteome. The figure illustrates the expansion from number of genes to number of transcripts to number of proteins. A segment of the more than 200 known post-translational modifications is listed at the far right and the order is based on the number of letters in the name of the modification.

first step): i) an initiation step, where glycosyltransferases recognize motifs in acceptor proteins and transfer the first monosaccharide (or for N-glycosylation preformed oligosaccharide); and ii) a processing step, where a large number of glycosyltransferases (and glycosidases for N-glycosylation) by sequential actions build diverse glycan structures (See Fig. 2 for illustration of principles of the GalNAc O-glycosylation pathway). Protein glycosylation is one of the only PTM that involves such a processing step where orchestrated sequential reactions by multiple enzymes build diverse oligosaccharide structures with independent biological functions. 2. GalNAc-type protein O-glycosylation GalNAc-type (or mucin-type) protein O-glycosylation (hereafter simply O-glycosylation unless otherwise specified) differs from other types of protein glycosylation in that the initiation step is controlled by a large number of isoenzymes. GalNAc-type O-glycosylation in man is initiated by up to 20 UDP-N-acetyl-α-D-galactosamine polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that all catalyze the initiation step where GalNAc is attached to Ser and Thr residues in proteins. The GalNAc-T gene family is old in evolutionary terms and found throughout the animal kingdom, but not in yeast, plants and prokaryotes. We have recently reviewed the current state of this large gene family [18]. These GalNAc-T isoenzymes have different albeit partly overlapping peptide substrate specificities [18–27], such that they are expected to decorate proteins at different Ser and Thr sites. Since GalNAc-Ts are differentially expressed in cells and tissues the capacity for GalNAc O-glycosylation of proteins in a cell is strictly dependent on the repertoire of enzymes expressed, and a given O-glycoprotein expressed in one cell type may be decorated with a different pattern of glycans or may not even be an O-glycoprotein in another [28,29]. In a striking contrast all other types of protein glycosylation in man are initiated by one or two enzymes, or in the case of N-glycosylation by an oligomeric enzyme complex of eight subunits, which leaves little room for differential regulation of these types of protein glycosylation. In agreement with this, the genes controlling the initiation of these are generally essential for animal development [30], while deficiencies in a single GalNAc-T of the twenty isoforms controlling GalNAc-type O-glycosylation have shown more subtle phenotypes as will be discussed later.

The large potential for functional redundancy in GalNAc O-glycosylation opens a new dimension in protein glycosylation, where site-specific O-glycosylation can be differentially regulated by a repertoire of GalNAc-T isoforms expressed in a cell — not much different from how we view the differential regulation of phosphorylation in signaling by the > 500 kinases. 3. Site-specific O-glycosylation controls proprotein processing Site-specific protein GalNAc O-glycosylation is emerging as a differentially regulated PTM that co-regulates the important proprotein processing process [26,28,31,32]. Traditionally O-glycosylation has been considered a form of protein glycosylation occurring in dense clusters on mucin proteins and in the mucin-domains of proteins, hence its designation mucin-type O-glycosylation [33]. However, it is becoming increasingly clear that this type of O-glycosylation is widely distributed in isolated sites of many hundreds of different proteins without mucin-like features [6,7,29,34], and as the existence of the large GalNAc-T family has emerged during the last decade [35,36], it has also become clear that discrete isoform-specific O-glycosylation at isolated sites in different types of proteins exert important functions [26,32,37]. The true impact of site-specific O-glycosylation was first realized with the discovery of a disease associated with deficiency of a GalNAc-T [38]. Deciphering the molecular disease mechanism revealed the surprising finding that the phenotype was primarily induced by enhanced proprotein convertase (PC) processing of a growth factor due to the loss of site-specific O-glycosylation [32]. PC processing is an important post-translational modification and a fundamental step in protein maturation where (limited) targeted proteolysis activates, or in some cases inactivates, many proteins of different classes e.g. hormones, growth factors, cytokines, proteases, and receptors [39]. The synthesis of insulin was one of the first examples of proprotein processing described where sequential actions of PCs cleave pro-insulin to the C-peptide chain and insulin which is trimmed by carboxypeptidase E to generate mature insulin [40–42]. Today 9 members of the subtilisin-like proprotein convertase family (PCSKs; PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P, and PCSK9) are known, and these differentially regulate PC processing in a tissue- and protein-specific manner [39]. The majority of PCs cleave

K.T.-B. G. Schjoldager, H. Clausen / Biochimica et Biophysica Acta 1820 (2012) 2079–2094

Cytosol

ER

ER OGT

EOGT

2081

ER

GL25D1/2

S/T

O-GlcNAc

S/T

O-GlcNAc

XXYLT1

K(OH)

O-Gal

ER ST3Gals ST6Gals

ER

GXYLT1/2 POGLUT1/2 S/T

β4GalT-1

O-Glc ST3Gals ST6Gals

β4Gal-Ts β3Glc-T

Golgi

FRINGEs POFUT1

POFUT2 S/T

β3Gal-T6

O-Fuc β4Gal-Ts

β3Gn-Ts

PomGnT1

β4Gal-T7 XT1/2

PomT1/2 S/T

S/T

O-Man

O-Xyl

ST6GalNAc-III/IV

ST3Gal-I

ST3Gal-Ts ST6Gal-Ts

ST3Gal-Ts ST6Gal-Ts

β3Gn-Ts

β4Gal-Ts

β4Gal-Ts

β4Gal-Ts

Core 2

β3Gn-T3

Core 4 C2/C4GnT

C2GnT1 C2/C4GnT C2GnT3

β3Gal-T5 β4Gal-T4

Core 3

Core 1 β3Gn-T6

C1GalT1 (Cosmc) ST6GalNAc-I

GalNAc-T1-20

S/T

Golgi

ST3Gals

O-GalNAc

GalNAc

GlcNAc

NeuAc

Xyl

Gal

Glc

Man

Fuc

Fig. 2. Schematic depiction of the main biosynthetic pathways of animal O-linked protein glycosylation. Overview of the most common types of mammalian O-linked protein glycosylation: O-mannose, O-fucose, O-GlcNAc, O-galactose, O-glucose, O-xylose and O-GalNAc. O-linked mannose is found on dystroglycan, CD24, receptor tyrosine phosphatase b and brain proteoglycans and is initiated in the ER by O-mannosyltransferase POMT1 [194]. Recently phosphorylated O-mannose with β1-4 substituted O-GlcNAc was reported [195]. O-fucose is found on EGF-repeats of several proteins and synthesis is initiated in the ER by the addition of fucose to Ser/Thr by the O-fucosyltranseferase POFUT1 (elongated by FRINGE) or POFUT2 (elongated by β3Glc-T) [196]. Recently elongated O-GlcNAc has been found on secreted proteins where synthesis is initiated by EOGT in the ER [17]. O-galactose is specifically added to hydroxylysines (K(OH) in collagens by the hydroxylysine galactosyltransferase 1 (GL25D1)) in the ER [197]. O-glucose is initiated by the O-glucosyltransferase 1 [198] in the ER and is found on e.g. Notch. The original O-GlcNAc modification is exclusively found in the nucleus or cytoplasm. Synthesis is initiated by the O-GlcNAc transferase OGT [199] and is not further extended than GlcNAc. O-GalNAc glycosylation is initiated by up to 20 different GalNAc-transferases. The addition of GalNAc to serines or threonines (or tyrosines) forms the Tn structure that is either sialylated or further elongated to form up to 4 core structures. The core structures can be further elongated.

proprotein substrates C-terminal of a polybasic consensus cleavage motif (K/R)-(X)n-(K/R) (n = 0, 2, 4, or 6 and X being any amino acid). It is believed that a substantial fraction of the 3500 secreted mammalian proteins undergo PC-mediated maturation, and several co-regulatory mechanisms of processing have been described including pH dependent activation [43], membrane topology [44], and endogenous protein, or peptide inhibitors [45,46]. More recently sitespecific O-glycosylation has offered a co-regulatory mechanism of PC processing, which is complex and differentially regulated by the

large number of GalNAc-T isoforms directly orchestrating O-GalNAc attachment sites (for an overview see Table 1). PC processing of pro-opiomelanocortin (POMC), the precursor of the peptide hormones adeno corticotropic hormone (ACTH), α-, β- and γ-MSH (melanocyte stimulating hormone) as well as β-endorphin, was one of the first examples where O-glycosylation was proposed to be related to differential tissue specific PC processing [28]. During synthesis POMC is differentially processed by PC1/3 and PC2 in the anterior lobe of the pituitary and intermediate lobes of the pituitary gland

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Table 1 Examples of O-linked glycosylation affecting protein processing. Protein

Sequencea

Processing event

Effectb

Ref

ANGPTL3 APOER2 APP β1AR C1qR/CD93 FGF23 LDLR IGF2 IGFBP6 hCTR1 Meprinβ MT1-MMP POMC pro-BNP TfR TNFα TNFβ VLDLR

221

PC Extracellular protease β-secretase Unknown Extracellular protease PC Unknown PC Chymotrypsin & trypsin Unknown Extracellular protease Autocatalytic PC PC Unknown ADAM-17 Unknown Extracellular protease

↑ ↓↑ ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↓ ↔ ↓ ↑ ↑ ↓ ↓↑ ↔ ↓

[26] [95] [6,112] [103] [209] [32] [94] [49] [210] [110] [98] [211] [28] [31] [212] [102] [213] [96]

RAPRATTPFLQL231 740 STSTTTLASTMTRTVPATTRAPGTT N 800 665 EISEVKMADAEFRHDSGYEVHHQAKALL N GGVVAIA713 28 TAARALLVPASPPASLLPPASESPEPALS54 491 N SPTRGPEGTPKATPTTSRPS N 570 168 HFNTPIPRRHTRASAED183 721 TQETSTVRLKVSSTAVRTQHTTTRPVPDTSR751 86 TPAKSERADVSTPPTVLP102 124 AGTARPQDVNRRDQQRNPGTSTTPSQPNSAGVQD157 22 HHHPTTSAAASAHASAHAG34 594 TQIQLATPAPSVQDLC608 285 GAESGFPTKMPPQPRTTSRPSVP306 66 DEQPLTENPRKAY76 66 KMVLYTLRAPRSPKMVQ82 100 RALAGTESPVR109 75 QAAVARASSSR82 31 GAQGLPGVGLTPSAAQTARQ50 751 STATTVTYSETKDTNTTEISATSGLVPGGINVTT783

a Protein sequences covering cleavage sites indicated by downward arrow (↓) and sites of O-glycosylation underlined (S or T). In some sequences actual site(s) of O-glycosylation has not been determined (italicized) and in some cases the actual sequences are longer than presented here (indicated by dotted lines). b ↑Processing activates protein function, ↓processing inhibits or deactivates protein function and ↔ no effect is observed.

leading to different peptide hormones and N-terminal POMC peptides of varying lengths [47]. N-POMC1–77, N-POMC1–49 and Lysγ3MSH are found in the intermediate pituitary, whereas only N-POMC1–77 is found in the anterior. However, only the longest peptide, N-POMC1–77, is O-glycosylated at Thr45 suggesting that this structure interferes with the PC processing of N-terminal POMC (PLTENPRK Y51) [28] (Fig. 3). The human pro-insulin-like growth factor (pro-IGF-II) is a potent mitogen that is sequentially processed to generate a mature 67 amino acid IGF-II (Fig. 3). The first report describing O-glycosylation of pro-IGF-II identified Thr75 in pro-IGF-II isolated from Chon fraction IV1 [48], and subsequently O-glycans on Ser71, Thr72 and Thr139 were also identified in recombinant pro-IGF-II expressed in HEK293 cells [49]. Interestingly, the analysis of IGF-II from human cerebrospinal fluid also identified one O-glycan at Thr139 [50]. Furthermore, two reports (although contradictory) describe aberrant IGF-II glycosylation in non-islet cell tumor hypoglycemia [51,52]. One of these associates the lack of O-glycosylation with aberrant processing and a third report describes recombinant non-glycosylated pro-IGF-II E-domain as a more potent stimulator of growth compared to the glycosylated, thus linking IGF-II O-glycosylation to biological function and activation [53]. O-glycosylation of other proteins has also been shown to affect their processing. Pro-brain natriuretic peptide (pro-BNP) is synthesized by ventricles of the heart as a 108 amino acid proprotein that is activated by PCs in response to cardiomyocyte stress. PC processing in RAPR↓SP78 activates pro-BNP and releases a 32 amino acid C-terminal peptide hormone that leads to natriuresis and vasodilation. Pro-BNP is a glycoprotein that carries 4-5 O-glycans [54] of which one, at Thr71, occupies the P6 position of the furin cleavage site LYTLRAPR↓SP78 (Fig. 3). Mutagenesis studies in human and murine cell lines have suggested that O-glycosylation of Thr71 protects against proteolytic processing by furin and possibly corin [31,55], and intriguingly increased plasma concentrations of pro-BNP was associated with heart failure. We recently identified Thr71 in pro-BNP as a specific substrate of the GalNAc-T3 isoform [27]. Our studies suggest that GalNAc-T3 is not expressed in the heart [18], which appears to be in agreement with the finding that a T71A pro-BNP mutant is processed to the same degree as wild type in murine atrial HL-1 cardiomyocytes [55]. The first direct evidence for the function of isoform- and sitespecific O-glycosylation was found with the discovery that deficiency in the GALNT3 gene causes familial tumoral calcinosis (FTC). FTC and

hyperphosphatemia–hyperstosis (HHS) are rare syndromes associated with ectopic calcifications and elevated serum phosphate levels. Initially genetic linkage-analysis of affected individuals identified bi-allelic deleterious mutations in GALNT3 leading to loss of the GalNAc-T3 enzyme [38]. Later multiple studies reported findings of biallelic inactivated GALNT3 in patients suffering from FTC and HHS [56–68]. Furthermore locus heterogeneity exists in FTC as inactivating mutations in FGF23 and the FGF receptor-modulating gene, KLOTHO, both cause FTC, pointing to a common biological pathway [69–71]. In the first report identifying mutations in GALNT3 in FTC patients, the authors reported that patients had ~ 20 fold elevated levels of FGF23 in the serum suggesting a compensatory mechanism [38]. However, the assay used to detect serum FGF23 only detected the presence of the C-terminal region of the protein, and FGF23 was known to undergo inactivating proprotein processing [72]. FGF23 is a circulating peptide hormone secreted by bone cells that reduces phosphate reabsorption in the kidney, reduces systemic 1,25 dihydroxyvitamin D (1,25-(OH)2 D) levels through decreased production and enhanced metabolism of 1,25-(OH)2 D, and suppresses parathyroid hormone transcription and protein secretion [73]. FGF23 was originally shown to be O-glycosylated [56], but the actual sites of glycosylation were not identified. We speculated that the function of GalNAc-T3 was related to O-glycosylation of FGF23, and found that GalNAc-T3 in fact was the only GalNAc-T capable of glycosylating Thr 178 in the furin processing site (RHTR↓SA181) of FGF23 (Fig. 3) [32]. Furthermore, we found that glycosylation of this site in FGF23 blocked furin processing and markedly enhanced intact FGF23 secreted from CHO cells. More recently it was found that the released C-terminal fragment of FGF23 functions as a potent inhibitor of FGF23 mediated signaling and renal phosphate reabsorption [74]. Importantly, mutations of the Arg residues in the RHTR179 cleavage site of FGF23, thereby blocking PC processing, leads to the mirror disease HHS autosomal dominant hypophosphatemic rickets [75]. Thus it appears that PC processing and inactivation of FGF23 is a delicately regulated event that requires balanced processing pointing to a dynamic co-regulation by PCs and site-specific O-glycosylation. Recently, we have identified another example, where site-specific O-glycosylation in a PC processing site by a specific GalNAc-T isoform, may be involved in dysregulated lipid metabolism [26]. Several genome-wide association studies (GWAS) have identified GALNT2 to be associated with high serum levels of triglyceride and highdensity lipoprotein cholesterol (HDL-C) [76,77]. Moreover, GalNAcT2 has been demonstrated to have a direct functional role by

K.T.-B. G. Schjoldager, H. Clausen / Biochimica et Biophysica Acta 1820 (2012) 2079–2094

2083

267 α

POMC

γ-MSH PLTENPRK

50

JP

YV

β-lipotropin

ACTH

180

IGFII

Insulin Growth Factor

α

E peptide

α

α

AKSER DVSTPPTVLPDNFPRYPVGK FFQYDTWKQSTQRLRR GL 67

134 α

Pro-BNP

NT-pro-BNP

BNP

LYTLRAPR SP 76

251

FGF23

α

FGF-like domain

FGFR inhib.

PIPRRHTR SA 179

460 α

ANGPTL3

LPL inhibitor

Fibrinogen

SSKPRAPR TT 224

860 α

LDL receptor

LDLR

α

α

TSTVRLKVSSTAVR

TM 701

α

Meprin Aβ

Metalloprotease

α

QIQL TPAPS

TM

598 233 α

TNFα

TNFα soluble

AQA V R SSSRTPSDKPVA

TM

73 770

APP

Amyloid beta A4

TM

α

α

EISEVKM DAEFRHDSGYEVHHQ K LVFFAEDVGSNKGAIIGLMVGGVV IA 671

713 477

β1AR

α

α

α

α

α

TAAR LLVPASPPASLLPPASESPEPL S

Multi pass TM

53 Fig. 3. Examples of O-glycosylation regulating the processing of secreted proteins. Schematic depiction of selected secreted and transmembrane proteins where O-glycosylation and proteolytic processing affect function. Cytosolic domains are in orange and extracellular domains are in gray. Yellow squares indicate position of GalNAc O-glycan and downward arrow indicates position of cleavage. The individual examples are described in the main text but the depiction illustrates how sites of O-glycosylation and cleavage are located between the protein domains in secreted proteins and close to the transmembrane domain (TM) in the transmembrane proteins. Different types of the transmembrane proteins are represented; type I (LDLR and meprin Aβ), type II (TNFα, and APP) and multipass 7TM (β1AR). Italicized sequence and stippled lines indicate that exact positions of O-glycans and cleavage are not known.

transient knock-down and overexpression of galnt2 in mouse liver resulting in respectively increased and lowered plasma HDL-C [78]. In search for a mechanism, we screened for proteins known to have a role in lipid metabolism and validated or potential O-glycosylation sites selectively utilized by GalNAc-T2, and identified Angiopoeitinlike-3 (ANGPTL3) as a specific substrate for the GalNAc-T2 isoenzyme. We found that GalNAc-T2 glycosylated Thr226 adjacent to a known furin-processing site of ANGPTL3 (RAPR↓TT226), and that glycosylation inhibited furin processing in vitro and ex vivo (Fig. 3) [26,79]. Furin processing releases the N-terminal coiled-coil domain of ANGPTL3 that is a potent inhibitor of the endothelial lipase (EL) and lipoprotein lipase (LPL) [80,81]. The majority of studies on the lipase inhibitor function of ANGPTL3 have been conducted in mice [80–83]. However, new data suggests that human ANGPTL3 exclusively functions as an inhibitor of hepatic triacylglycerol lipase (HTGL), and not as an inhibitor of EL in vivo measured in overweight

and obese healthy volunteers [84]. How ANGPTL3 processing affects inhibition of HTGL is currently not known. In order to evaluate the function of site-specific O-glycosylation for PC processing on a proteome-wide basis, we recently systematically explored the potential effect of O-glycosylation in or adjacent to a minimal furin RXXR cleavage motif (where X is any amino acid) [27]. Furin is the most extensively studied PC and its stringent specificity for the RXXR motif relies on a wide negatively charged catalytic pocket as evidenced from crystallization studies [85]. We found that small hydrophilic O-GalNAc moieties were sufficient to inhibit processing when located in positions +/− 3 amino acid residues to furin cleavage site. Modeling the structures of the other family members to that of furin reveals, especially for PC1/3 and PC2, a less stringent requirement for basic residues besides P1 and P2 [86]. Thus it is likely that the inhibitory effect of glycans is more restricted on substrates of these PCs. In the study, we found that as much as 700

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proteins potentially could be affected by concurrent glycosylation and furin cleavage. Among these were proteins where cell/organ specific or differential processing has been demonstrated [31,87–90] and indeed in vitro cleavage of these substrates was modulated by O-GalNAc glycosylation [27]. 4. Site-specific O-glycosylation may serve more general roles in other protease processing events O-glycans have long been known to serve protective functions such as preventing proteolysis of membrane proteins in e.g. stem regions, which are important to extend functional globular domains away from the cell membrane to enhance accessibility for biological interactions. One of the earliest examples was found in a genetic mutation screen of CHO-K1 where a mutant subpopulation deficient in low-density lipoprotein (LDL) uptake by the LDL receptor (LDLR) was identified [91,92]. The mutation was mapped to the UDP-Glc/ GlcNAc-C4-epimerase leading to lack of synthesis of UDP-Gal/ GalNAc and hence deficient N- and O-glycosylations in these cells [93]. Later it was shown that the aberrant cellular LDL uptake in these cells was caused by reduced stability and cell surface expression of LDLR, and it was speculated that lack of O-glycans in the stem region of LDLR conferred susceptibility to proteolytic cleavage and ectodomain shedding [93,94] (Fig. 3). Subsequently, it was demonstrated that processing of other members of the LDL receptor family including the very low-density lipoprotein receptor (VLDLR), LDL receptor related protein 1 (LRP1), and apolipoprotein E receptor 2 (ApoER2) also are affected by loss of O-glycosylation [95,96]. Many other membrane proteins of different classes with similar requirement of O-glycosylation for proteolytic stability have been described subsequently (Table 1 and Fig. 3). E.g. the meprin metalloproteinases exist as disulphide-linked homo- or heterodimers of α and β subunits (αn, β2, α2β3 or α3β1), and function to process a vast array of substrates like gastrin, cholecystokinin, substance P, and several cytokines. When expressed as homodimers or multimers meprin α is secreted and meprin β is membrane bound, and when expressed as heterodimers meprin α is disulphide linked to β. Interestingly, there is a low steady-state shedding of meprin β [97], and inhibition of O-glycosylation of Thr599 and Ser603 by benzyl-GalNAc enhances ectodomain shedding [98]. This suggests that elongated O-glycans protect meprin β processing. In support of this, it was shown that a disintegrin and metalloprotease 17 (ADAM17) is responsible for phorbol-12-myristate-13-acetate (PMA) induced meprin β shedding where it cleaves immediately N-terminal of two sites of glycosylation QIQL↓TPAPS603 [99] (Fig. 3), however further studies are required to establish the exact biological pathway of meprin β shedding. ADAM-17 (also named TNF alpha converting enzyme (TACE)) is one of the most studied ADAMs, and it was first discovered as the enzyme that cleaves pro-TNFα (QA↓VR78) from the membrane generating the soluble form of TNFα [100,101]. Interestingly, Oglycosylated (Ser80) TNFα was found in lymphoblastic leukemia B-cells, and isolation of different species of N-terminal truncated peptides (QA↓V↓R↓SSSR82) led the authors to speculate that the O-glycan served a protective function [102]. Adding to this hypothesis we have found that Ser 80 is specifically glycosylated by one GalNAc-T isoform that protects the peptide from in vitro processing by ADAM-17 and ADAM-12 (Goth et al., unpublished). Similar roles of O-glycosylation can be found in other protein classes such as the G-protein coupled receptors (GPCRs). The extracellular N-terminal tail of the 7TM GPCR β1 adrenergic receptor is O-glycosylated at four residues (TAAR↓LLVPASPPASLLPPASESPEP ↓LS54) (Fig. 3), and these O-glycans protect against processing by a member of the ADAM family [103]. Intriguingly, several other members of the GPCR family are O-glycosylated, some with sites identified in the N-terminal tails [104–106], and others where sites are not yet

identified [107–109]. It remains to be seen if these sites serve similar protective functions. The human copper transporter CTR1 is another example of a multipass transmembrane protein where O-glycosylation of the N-terminal extracellular domain protects against proteolytic cleavage. The protein spans the membrane three times and glycosylation of Thr27 protects the protein from cleavage C-terminal to the O-glycosylation site. The cleavage, that takes place in endosomal compartments, truncates the protein's N-terminus by approximately 30 residues and furthermore reduces the copper uptake activity of the transporter by 50% [110,111]. More recent studies have suggested that the important processing events of the amyloid precursor protein (APP) also may be affected by site-specific O-glycosylation. Several groups have found O-glycosylation sites in APP, and one novel site identified at Tyr10 is particularly intriguing in that this Tyr is in close proximity to the β-secretase site, and thus might alter APP secretase processing and the amyloidogenic pathway associated with Alzheimer's disease [6]. Moreover, Taniguchi and colleagues have shown that O-glycosylation appears to affect APP processing and amyloid beta secretion in brain endothelial cells [112], perhaps suggesting functional importance of O-glycosylation in APP pathologies. All the above-discussed examples of the biological roles of sitespecific O-glycosylation clearly place O-GalNAc glycosylation as a major regulator of protein functions. The regulatory potential that lies in the multitude of isoenzymes initiating O-glycosylation is overwhelming and far from fully explored. A large task lies ahead to identify non-redundant site-specific functions of the many GalNAc-T isoforms involved in these processes, and exploring the underlying mechanism(s) should provide significant insight to important biological questions. We have recently reviewed the GALNT gene family in detail [18], but a brief summary here serves as an introduction to the role of the 20 genes in disease. 5. Cell and tissue-specific capacity for O-glycosylation is controlled by the repertoire of GalNAc-T isoforms The GalNAc-Ts are differentially expressed in cells and tissues, and the members can be classified as having more ubiquitous (GalNAc-T1, T2, T7), selected (GalNAc-T3, T4, T6, T11, T12, T14, T16, T18), and more restricted (GalNAc-T5, T8, T9, T10, T13, T15, T17, T19) expression patterns [18]. These expression patterns naturally determine the capacity for O-glycosylation in cells; however, our insight into the actual functional outcome is minimal at present. Thus the repertoire of GalNAc-Ts expressed in cells vary with cell type, differentiation, and maturation, and a number of studies have shown that malignant transformation is associated with substantial changes in expression of individual GalNAc-Ts [18]. While these changes have been documented both at the mRNA and protein levels, it is surprising how little we know about the regulatory elements of the GALNT genes. The general view has been that the expression of GalNAc-Ts in cells is static because Golgi enzymes generally have long half-lives [113] with continuous retrograde transport via COPI vesicular transport [114,115]. Release/clearance of Golgi enzymes is thought to involve endo-proteolytic cleavage in the stem region with secretions of catalytically active soluble enzymes found in body fluids. Again our knowledge of this process is limited, but two examples of specific regulation of this event have been reported and evidence for a third example presented. Thus, the Lunatic Fringe β3GlcNAc-T appears to be processed by furin in the stem region [116], the ST6Gal-I sialyltransferase is similarly processed by BACE-1 [117], and the stem region of ST6GalNAc-I has a furin processing site that at least in vitro is cleavable [27]. It is conceivable that similar specific proteolytic events ensure rapid release of other enzyme isoforms, which would allow for rapid up and down regulations of the glycosylation capacity in cells at both the gene expression/translation level and

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at the protein level. Such a dynamic scenario for regulation of glycosyltransferases may be required for glycosylation events regulating other dynamic biological functions. Thus, as an example the Lunatic Fringe modulates the ligand specificity and function of the Notch receptor through extension of the O-Fuc O-glycans in EGF repeats, and tight regulation of this step may require quite rapid changes in the Fringe expression with both up and down regulations. Use of the PC processing machinery may be one way to obtain rapid down regulation of glycosylation by cleavage. Interesting, the fringe glycosyltransferases were originally described as secreted neurogenic factors because they are mainly found as secreted proteins [118]. However, we showed that it is the membrane bound form that functions in modulating Notch signaling [119]. Interestingly we have recently identified multiple glycosylation sites in the stem regions of glycosyltransferases using our SimpleCell strategy ([7,29], Vakhrushev et al. submitted]) and it remains to be seen if these O-glycans serve similar functions. There are a number of examples of glycosyltransferase genes that respond to external stimuli, however the molecular mechanisms behind are rarely understood. One example is the apparent regula tion of GalNAc-T3 expression in response to inorganic phosphate (upregulation) and calcium and vitamin D (down regulation) in HEK293 and human fibroblasts [120], the same substances that modulate expression of the phosphoturic factor FGF23, which in turn is regulated by GalNAc-T3 O-glycosylation. Another example describes how O-glycosylation of MT1-MMP is upregulated or induced in PTEN −/− mice with activated PI3K/AKT pathway [121]. A more recent finding that could have a profound impact on our understanding of site-specific O-glycosylation relates to plasticity in the subcellular localization of the GalNAc-Ts [114]. Thus, Src activation was found to selectively relocate the GalNAc-Ts from Golgi to ER compartments, which appear to result in denser O-glycosylation with immature O-glycan structures. We have previously demonstrated that GalNAc-Ts and O-glycosylation are not normally found in ER in human cell lines (HeLa), but that the capacity i.e. sugar nucleotide donors and conditions for O-glycosylation exist in ER if a recombinant GalNAc-T and an appropriate acceptor protein are located to ER [122]. The perspectives of subcellular topology as another regulatory level for O-glycosylation challenges our view of an ordered glycosylation machinery with enzymes placed in a fixed order of their function [123]. Moving GalNAc-Ts into ER isolates O-GalNAc glycosylation from competing O-glycan extension enzymes, which may in fact serve to control efficiency and density of O-glycosylation on e.g. mucins with requirements for O-glycosylation of up to 50% of the amino acids in tandem repeated regions. It is believed that the topological feature of GalNAc-Ts with their C-terminal lectin domains provides for efficient competition with elongation enzymes during the initiation process. These lectins all appear to bind GalNAc and therefore provide additional adhesive forces to those of the catalytic domain to unused substrate sites [124,125]. The current hypothesis is that this dual binding mode allows for the completion of the initiation process before the elongation process extends O-glycans and sterically blocks further initiation. However, complete segregation of the initiation step and the elongation step may provide even better potential for complete O-glycosylation. The capability to relocate GalNAc-Ts to ER potentially also opens for competition with other types of protein O-glycosylation including O-mannosylation. Studies have shown that there are at least partly overlapping peptide substrate specificities among the polypeptide O-mannosyltransferases and GalNAc-Ts [126–128]. Moreover, several studies have recently demonstrated that O-GalNAc and O-Man glycosylation sites on the only well characterized O-mannosylated mammalian glycoprotein dystroglycan are in fact partly overlapping [129]. To our knowledge the potential for GalNAc-Ts functioning with peptide substrates for other types of O-glycosylation have not been explored, but it seems likely that there are functional reasons

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for the segregation of GalNAc-Ts in Golgi and most other initiating enzymes controlling O-glycosylation in the ER. Apart from the competition between different pathways of glycosylation, the location of the initiation step in ER also offers access to protein substrates early on in the folding and dimerization events. These examples point to a more dynamic regulation of glycosyltransferases than previously thought, and we anticipate that many more enzymes, and in particular the GalNAc-Ts, will turn out to be regulated not only at the gene expression level but also at the protein level. Thus, the GalNAc-T family provides great potential capacity for dynamic and differential co-regulation of limited proteolytic processing events such as PC and ADAM protein processing. The examples discussed here were identified by chance. Thus methods for a more systematic discovery of co-regulation of processing by site-specific O-glycosylation are needed. In the following section we discuss promising strategies to achieve this goal. 5.1. Protein sequence motifs predictive of O-glycosylation — global and GalNAc-T isoform specific approaches Considerable efforts have been devoted to identifying sequence motifs predictive of GalNAc-type O-glycosylation, however clear motifs similar to those identified for N-glycosylation and several other types of O-glycosylation have not been found yet [130]. Several decades ago, using bovine submaxillary gland GalNAc-transferase extracts and synthetic peptide substrates, it was demonstrated that prolines surrounding the acceptor hydroxyamino acids were important for enzyme activity [131], and later compilation of known acceptor sites revealed that prolines in position − 1 and + 3 to the site of glycosylation were found with a higher frequency while charged amino acids were found with a lower frequency [132–134]. These observations were later supported by in vitro and in vivo assays [135,136]. Now we realize that the GalNAc-transferase activity is shared among 20 enzyme isoforms and characterization of the individual GalNAc-transferases have revealed overlapping yet distinct substrate specificities for these [20,22,23,36,137–145]. Using random peptide and glycopeptide substrate libraries Gerken et al. [25,35,146] have systematically analyzed the individual in vitro specificities of GalNAc-T1, -T2, -T3, -T5, -T10 and -T12 revealing specific N-terminal amino acid requirements and overall substrate charge preferences. For instance GalNAc-T2 has a preference for proline in position − 1 whereas GalNAc-T1 and -T3 prefer valine and GalNAc-T3 and -T5 prefer overall basic peptide substrates, whereas GalNAc-T1 and -T2 prefer more acetic peptide substrates. Concurrent strategies to decipher GalNAc-transferase preferences through prediction algorithms have been very fruitful. The earliest was based on matrix statistic methods, and later artificial neural network training has led to e.g. NetOGlyc prediction server [132,147–149]. The latest version of NetOGlyc (v3.1) was trained on 421 experimentally verified sites of O-glycosylation many of which were found in mucins, and as shown in Steentoft et al. [7] the prediction has a bias towards clustered O-glycosylation sites as it fails to predict a substantial fraction of the single or more isolated sites identified more recently. Thus, further insight into the O-glycoproteome is necessary to further refine such algorithms. Hopefully implementing the large amount of data generated by recent high through-put strategies [6,7,29,34,50] into predictors as NetOGlyc will massively improve the overall prediction accuracy. 6. GWAS points to more functions of site-specific o-glycosylation in health and disease A serendipitous discovery that led to the first clear example of an essential interplay between site-specific O-glycosylation and PC processing in FGF23 was reached using a classical strategy for gene association with disease [38]. Defects in the human glycogenome that

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affect pathways in glycosylation of proteins and lipids, are collectively referred to as congenital disorders of glycosylation (CDG). CDGs identified to date are often associated with serious and multi-systemic clinical manifestations of which a large proportion involves neurological presentations [150]. Deficiencies in genes involved in N-linked glycosylation comprise the largest CDG group. The most common CDG, PMM2-CDG, affects the GDP-mannose pathway and is found in more than 700 cases worldwide [150]. Defects in genes involved in O-linked glycosylation have so far primarily been identified in the O-mannose and O-xylose glycosylation pathways, and patients suffering from these defects present with muscular dystrophies (O-Man) [151] and bone/cartilage abnormalities (O-Xyl) [152], respectively (Fig. 5). Disease discovery in the GalNAc-T family has in contrast been slow, and until now only two diseases/syndromes caused by genetic defects in mucin-type O-glycosylation has been described: the Tn-syndrome caused by somatic mutations in COSMC [153] and FTC and HHS diseases caused by mutations in GALNT3 [32,38]. The apparent lack of congenital diseases associated with deficiencies in GalNAc O-glycosylation is likely to stem from the great level of functional redundancy provided by the many GalNAc-Ts, which probably signifies that genetic deficiencies will not result in complex and multi-systemic diseases, but rather present as more subtle phenotypes or diseases similar to what was found with defects in GALNT3 (Fig. 4). This view is partly supported by studies of gene deficiencies in animal models. Results of knock-out studies of GalNAc-T genes in mice have ranged from viable and fertile without any obvious phenotypes (galnt4, galnt5 and galnt8) [154,155] to viable and non-fertile with distinctive but subtle phenotypes (galnt1, galnt3 and galnt13). Thus, galnt1 −/− mice suffered from a bleeding disorder and B-lymphocyte homing deficiency probably caused by decreased L-selectin ligand on B-cell surface glycoproteins [156]. Galnt13 null mice were also viable but displayed decreased Tn antigen in the cerebellum [145]. Galnt3 deficient mice showed biochemical signs of FTC as high serum phosphate levels and low active FGF23 levels but no ectopic calcification. Furthermore male galnt3 −/− mice showed growth retardation and infertility [157]. GalNAc-T3 is probably the only GalNAc-T expressed in human testicular germ cells and sperm cells pointing to other specific functions of the enzyme, e.g. in sperm cell maturation [158]. A second report found that another galnt3 null mouse exhibited ectopic calcification as well as increased phosphate level in good agreement with the phenotype of FTC as well as male infertility [159,160]. Interestingly, oligoazoospermia has also been reported in one FTC patient [57], but reports of offspring from other FTC patients have been postulated [56,161]. In contrast to these subtle phenotypes found in mice, several GalNAc-Ts in Drosophila melanogaster have been shown to be essential. Mutagenesis studies in Drosophila showed that pgant35, ortholog of human GALNT11, was essential for Drosophila development [162,163] and recent studies show that ubiquitous RNAi knock-down of pgant4, pgant5, pgant7 and a putative GalNAc-T CG30463 all resulted in a complete loss of viability [164], pointing to specific non-redundant functions of several of the individual GalNAc-Ts in the fly. Surprisingly, it was not possible to rescue the pgant35 (l(2)35Aa) phenotype by substituting the catalytic domain with that of the human ortholog GALNT11 despite the finding that the two enzymes was shown to have similar peptide substrate specificities [165]. Furthermore, genome-wide RNAi screens in Drosophila have also identified specific GalNAc-Ts important for subcellular organization and hence site-specific O-glycosylation as having other functions in the secretory process [166,167]. While the functions of individual GalNAc-Ts in Drosophila largely remain obscure at present, RNAi mediated knock-down of the 9 genes encoding 13 GalNAc-Ts in Caenorhabditis elegans did not affect cytokinesis perhaps suggesting functional redundancy in this model organism [168]. It has proven a major challenge to decipher the specific biological functions of individual GalNAc-Ts using the conventional reverse genetics approach as well as animal models. Consequently, our

knowledge of functions of site-specific O-glycosylation orchestrated by the large GalNAc-T enzyme family is highly limited. However, the last decade has provided an explosion of GWAS, and several of these have linked GALNT genes to certain traits or syndromes making it possible for the community to take on a forward genetics approach to decipher the biological functions of the GalNAc-Ts. As mentioned earlier, the first GWAS linking a GalNAc-T to disease traits described a polymorphism near GALNT2 that was related to serum HDL-C and TG levels. Already now two functional pathways in which GalNAc-T2 could exert its role in lipid metabolism have been suggested [26,169]. The regulation of PC processing and activation of ANGPTL-3 as described above and more recently a study described a heterozygous missense mutation (D314A) in GALNT2 identified in two individuals with increased postprandrial triglyceride clearance and reduced GalNAc-T2 activity. Interestingly, the authors of this study found a minor fraction of aberrantly glycosylated ApoC-III in affected individuals and carriers, and they concluded that deficient O-glycosylation of ApoC-III affected its lipase inhibition and thereby resulted in increased triglyceride clearance [169]. While this is clearly possible, it is noteworthy that the original study demonstrating direct function of GalNAc-T2 on HDL-C using transgenic manipulation of GalNAc-T2 levels in the liver was performed in mice [78], and the mouse and rat ApoC-III amino acid sequences around the O-glycosylation site differ substantially. Thus, the rat sequence has multiple O-glycosylation sites and the glycosylation of these are not GalNAc-T2 isoform specific [79]. In a GWAS designed to explore the genetic background for osteoporosis in post-menopausal women, several loci were identified in or near GALNT3 associated with bone mineral density giving rise to the intriguing hypothesis that dysfunctional GALNT3 might be involved in the pathway leading to a very common disease [160]. Such involvement seems appropriate considering the role GalNAc-T3 plays in FTC. In 2008 a single nucleotide polymorphism (SNP) in GALNT1 was associated with a reduced risk of ovarian cancer, however, recently in a larger study the same authors have not been able to show a significant association of this SNP with ovarian cancer [170,171]. Another GWAS identified an SNP near GALNT10 associated with obesity and high body mass index (BMI) in African Americans and the authors propose GALNT10 as a new potential candidate for the regulation of adiposity [172]. Other genetic association studies have also linked GalNAc-Ts to disease, e.g. GALNT11 was associated with the severe congenital heart disease heterotaxy and Xenopus knock-down of galnt11 showed a disrupted morphological left-right development [173]. In addition GALNT12 expression is down-regulated in colon cancer cells and human colorectal cancer [174], and inactivating mutations have been identified in individuals suffering from colorectal cancer [175,176]. Moreover, GALNT4 was identified as a risk gene of coronary artery diseases (CAD) [177] and GalNAc-T14 is involved in O-glycosylation of death receptor 5 and apoptopic signaling [178]. In line with the genetic associations of GalNAc-Ts with cancer, aberrant expression and activity of several GalNAc-Ts in humans have been associated with a number of malignant states. Especially GalNAc-T6 has been shown as a biomarker for breast [179], gastric [180], and pancreatic cancers [181], and recently isoform and site-specific glycosylation of fibronectin by GalNAc-T6, and its close homolog T3 in a breast cancer cell line was shown to stabilize fibronectin and inhibit endogenous degradation [182]. In addition it was shown that GalNAc-T3 and T6 knock-down suppressed the production of oncofetal fibronectin (O-glycosylated fibronectin) and epithelial-mesenchymal transition process [183]. Noticeable, induction of oncofetal fibronectin expression did not alter the expression levels of GalNAc-T3 or -T6 [183]. There is thus substantial evidence for highly specific and nonredundant biological functions of many of the GalNAc-Ts, but our

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Processing

Initiation

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Disease association

β

1 gene

OGT lethal [205]

1 gene

Not known

OGT β

EOGT

POMGnT1

2 genes

POMT1/2 β

β3Gal-T6 β4Gal-T7

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Rodent XYLT2 liver and kidney disease [201] B4GALT7, Ehlers Danlos Syndrome [202]

XylT-1 α

β4Gal−T1 β3Glc-T Fringe GT

POMT1/2 , Walker Warburg Syndrome [200,207] POMGnT1, Muscle Eye Brain disease [208]

2 genes

Rodent pofut1 [206] lethal B3GALTL Peters Plus syndrome [204]

O-FucT-1

α

2 genes

Drosophila poglut (Rumi) lethal [203]

2 genes

Not known

Initiation controlled by 1-2 genes

α

HCLP46

GXYLT1/2

β

Core2 structures

α

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C1GalT1 Core4 structures

GalNAc-T1-20 β3GnT6

GalNAc Gal GlcNAc

Glc

Man

Xyl

T3 FTC T2 HDL/TG T1 B-cell homing T4 CAD T10 BMI T11 Heterotaxy T12 Colon cancer T14 Death receptor Cosmc Tn syndrome

Initiation controlled by 20 genes

GLT25D1/2

Fuc

Fig. 4. Initiation and processing steps of mammalian O-glycosylation. An overview of the biosynthetic initiation and processing steps of mammalian O-linked glycosylation, and the diseases or syndromes associated with defects in genes involved in these two steps. Remarkably enzymes encoded by one or two genes initiate O-GlcNAc, O-Man, O-Xyl, O-Fuc, O-Glc and O-Gal glycosylation, whereas enzymes encoded by 20 different genes initiate O-GalNAc glycosylation. Diseases or syndromes listed in italics are not confirmed to be caused by defects in the corresponding genes, but rather associations have been made through genetic screens. O-linked GlcNAc stands out in that it is initiated in the cytosol and nucleus and is not further processed. Disease association references OGT [205], POMT1/2 [200,207], POMGnT1 [208], XYLT2 [201], B4GALT7 [202], pofut1 [206], B3GALTL [204], poglut (Rumi) [203].

insight into the molecular aspects of these are quite limited. This is at least partly due to technical difficulties in predicting and identifying sites of O-glycosylation in proteins. As more studies reveal

phenotypic associations with GalNAc-Ts, we are in need of tools to further clarify the substrate specificities and specific functions of each GalNAc-T and we predict that many of these functions will

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turn out to be similar modulations of proteolytic processing sites. To this end, we have recently developed a new strategy that allows for a high-through-put proteome-wide identification of site-specific O-glycosylation events controlled by individual GalNAc-Ts ex vivo in human isogenic cell line models engineered by the zinc finger nuclease (ZFN) gene targeting technology. 7. A novel approach for discovery of functions of site-specific O-glycosylation using ZFN targeted gene knock-out The heterogenic, complex and dynamic natures of O-glycoproteins have hampered their identification and characterization and our knowledge of the O-glycoproteome is mainly derived from the analysis of individually isolated proteins. New technologies for high through-put glycoproteomics have appeared and enrichment strategies for sialylated glycoproteins followed by mass spectrometry have resulted in the discovery of a number of new glycoproteins from cerebrospinal fluid and urine [34,50,184]. Specific improvements for GalNAc-type O-glycoproteomics have incorporated lectin weak affinity chromatography (LWAC) enrichment, enzymatic simplification of O-glycans, and insights into fragmentation and data processing of electron transfer dissociation (ETD) spectra to facilitate site-specific characterization of O-glycopeptides and identification of O-glycoproteins [185–188]. A more comprehensive proteome-wide discovery strategy may be available with so-called SimpleCells [7] where ZFN engineering of cells have truncated and simplified O-glycosylation facilitating isolation of entire cell O-glycoproteomes and identification of glycosylation sites by mass spectrometry (Fig. 5A). The SimpleCell strategy is based on a genetic interference with an extension of O-glycan structures beyond the first GalNAc. O-glycosylation is initiated by an addition of GalNAc by the GalNAc-Ts to the protein backbone and followed by elongating core structures catalyzed by the Core1 β1,3-galactosyltransferase (C1GalT) or Core3 β1,3-GlcNAc-transferase (β3GlcNAc-T6). The most common and the only primary Core extension in most cell lines is Core1 that requires a chaperone Cosmc to be active [189,190]. It is well known that cells deficient in Cosmc produce only truncated GalNAcα1-O-Ser/Thr (Tn) O-glycans with or without sialylation (NeuAcα2-6GalNAcα1-O-Ser/Thr) [191], a feature that greatly simplifies identification of O-glycosylation sites by mass spectrometry. We previously used ZFN targeting of the X-linked COSMC to engineer 3 different human cell lines to produce homogenous and simple O-glycans. Proteins from these cell lines were then separated by LWAC and sequenced by nLC-MS/MS producing cell line O-glycoproteomes entailing hundreds of O-glycosylation sites [7]. In order to identify the contribution of individual GalNAc-T isoforms to the O-glycoproteome and functions of a non-redundant site-specific O-glycosylation, we have extended the SimpleCell strategy for glycoproteomics to include isogenic SimpleCell systems with deficiency in single GalNAc-T genes (Fig. 5). As a first approach we have applied this strategy to further explore the association of GalNAc-T2 to HDL and triglyceride metabolism and used a human liver cell line HepG2 as an example. By sequential knock-out of COSMC and GALNT2 followed by LWAC and comparative nLC–MS/ MS (Fig. 5A) we identified a number O-glycosylation sites of which approximately one third were not found in the COSMC/GALNT2 knock-out cells and thus considered GalNAc-T2 specific (Fig. 5C). Among these we found ApoC-III and validated Thr94 as a specific site for O-glycosylation by GalNAc-T2. As previously mentioned aberrant ApoC-III O-glycosylation is presumably not causal of the observed phenotype associated with GALNT2 polymorphisms, however, it may function as a biomarker for deficient GalNAc-T2 glycosylation. In fact ApoC-III has already been proposed as a biomarker for dysfunctional O-glycosylation [192], in the same way transferrin is a biomarker for deficient N-glycosylation, but what is being realized now is that the strict substrate specificities of the individual GalNAc-Ts requires that biomarkers for each isoform be discovered. More

importantly, the developed isogenic HepG2 SimpleCell pairs with and without GalNAc-T2 allowed us to confirm that site-specific O-glycosylation of ANGPTL3 as well as PC processing are directly controlled by this GalNAc-T isoform [79]. We demonstrated this in both wild type and SimpleCell HepG2 background and thus provide a new proteome-wide discovery strategy for the interplay between GalNAc-T isoform O-glycosylation and proteolytic processing events. We are currently expanding this strategy to include other GalNAc-T isoforms and monitoring a wide range of proteins undergoing processing in such isogenic cell models. We also envision combining these cell models with isogenic cell pairs with knock-out of individual PCs and other regulating proteases such as ADAMs to identify more proteins where proteolysis is modulated by GalNAc O-glycosylation. Furthermore expansion of isoform-specific O-glycoproteomes will add a great value to the on-going work exploring the existence of isoform-specific consensus motifs [35].

8. Conclusion and perspectives As we are beginning to appreciate the wide range of biological functions exerted by site-specific O-glycosylation orchestrated by the large GalNAc-T gene family, it is becoming clear that GalNAc-type protein O-glycosylation is an important novel player in modulating protein processing. O-linked glycans have long been known to have important biological functions [193], and here we have discussed how O-glycans at specific sites may have distinct functions and that these may be regulated in hitherto unappreciated dynamic manner through the different substrate specificities of the large GalNAc-T family. One major biological role of site-specific O-glycosylation is the co-regulatory role of PC processing, but we envision that many more roles will be uncovered with the application of new technologies and strategies such as the SimpleCells. One area where the functions of site-specific O-glycosylation remain virtually unexplored is cancer biology. Cancer cells almost always exhibit aberrant O-glycosylation typically with truncated O-glycans, but the GalNAc-T repertoire is also markedly altered in cancer cells. Only few studies have so far addressed the functional consequences of the changes in the GalNAc-T repertoire, but it is clear from the examples of functions exerted by site-specific O-glycosylation discussed here that such changes can have profound implications for the cancer cell phenotype. It is thus not unlikely that changes in the GalNAc-T repertoires are directly required in cancer cells for their growth, invasive and metastatic properties, as these properties at least partly are regulated by proteins undergoing regulated limited proteolysis. The major barrier to be overcome is to get insight into the O-glycoproteome as our knowledge of where O-glycosylation occurs in the human proteome is highly limited. The SimpleCell strategy holds great potential to provide this. Non-redundant contributions to the O-glycoproteomes of individual GalNAc-Ts can be identified by comparative analysis of isogenic SimpleCell lines with knock-outs of GalNAc-Ts. Systematic targeting of the entire GalNAc-T repertoire of a cell with this strategy should thus enable us to decipher the biological functions exerted by site-specific O-glycosylation.

Acknowledgements We thank Dr. Nabil Seidah and Steve Levery for their careful reading of the manuscript. This work was supported by Kirsten og Freddy Johansen Fonden, A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, The Carlsberg Foundation, The Novo Nordisk Foundation, The Danish Research Councils, a program of excellence from the University of Copenhagen and the Danish National Research Foundation.

K.T.-B. G. Schjoldager, H. Clausen / Biochimica et Biophysica Acta 1820 (2012) 2079–2094

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B GalNAc-T2 Tn (GalNAc)

A Core4 Core2

Core3

C1GalT1/ Cosmc

wild type

GalNAc-T Ser/Thr

Core1 (T)

Tn

GalNAc-T2-/-

Targeting COSMC

1

GALNT2

Targeting COSMC and GALNT2

. .

Cosmc-

Cosmc-/GalNAc-T2-/-

2

C SC/T2-/-

SC 3

73

116

30

ApoC-III

α-Tn α-T α - n

4

m/z

m/z

Fig. 5. Targeting GALNT2 and COSMC C1GalT1 chaperone in HepG2 cells. A. Biosynthetic pathways of mucin type O-glycosylation. GalNAc-transferases initiate O-glycan biosynthesis by adding GalNAc to Ser or Thr residues in protein backbones. This structure is then further elongated to produce Core3 or Core1 structures with possible further elongation to Core2 or Core4 structures. Most cell lines do not express Core3 [190] and the SimpleCell strategy is not intended for cells with Core3 elongated structures. Specific targeting of both the Core1 and Core3 synthases would expand the strategy to include such cell lines. In SimpleCells these elongation pathways are eliminated due to mutations in the C1GalT1 chaperone Cosmc and by further eliminating individual GalNAc-transferase isoforms we have developed a model to study isoform-specific glycosylation (top). Schematic drawing of the overall glycogene targeting strategy: (1) ZFN targeting of COSMC in HepG2 (SC) generates cell lines with homogenous and truncated O-glycosylation (GalNAc), and subsequent targeting of GALNT2 (SC/T2−/−) eliminates GalNAc-T2 non-redundant O-glycosylation allowing for a comparative analysis of O-glycoproteomes of isogenic cell lines with and without GALNT2; (2) proteins from total cell lysates or cell culture supernatants from HepG2-SC or SC/T2−/− are digested by trypsin; (3) GalNAc-glycopeptides are isolated and separated by lectin weak affinity chromatography; and (4) O-glycosylation sites are identified by nLC MS/MS and comparison between the two cell lines produces candidates for GalNAc-T2 specific contribution. B. Fluorescent images showing immunocytochemical stainings of HepG2 wild type, GALNT2−/−, COSMC−/− and GALNT2−/−/COSMC−/− cell lines stained with anti-Tn (5F5) or anti-GalNAc-T2 (4C4). C. Venn diagram illustrating the distribution of identified O-glycoproteins. ApoC-III was identified in HepG2-SC and SC/T2−/− secretomes and related to HDL and triglyceride metabolism and therefore selected for further analysis [79].

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