A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

Christoffer K. Gotha, Adnan Halima, Sumeet A. Khetarpalb,c, Daniel J. Raderb,c, Henrik Clausena, and Katrine T.-B. G. Schjoldagera,1

aCopenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; bDepartment of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and cDepartment of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

Edited by Monty Krieger, Massachusetts Institute of Technology, Cambridge, MA, and approved October 9, 2015 (received for review June 8, 2015) Regulated shedding of the ectodomain of membrane cleavage sites and the specific ADAM involved have been by proteases is a common process that releases the extracellular identified (5). Interestingly, cleavage sites in the extracellular juxta- domain from the cell and activates cell signaling. Ectodomain membrane region are often found in close proximity to O-glycosyl- shedding occurs in the immediate extracellular juxtamembrane ation sites, hence glycosylation may affect susceptibility for proteolytic region, which is also where O-glycosylation is often found and processing of proteins (6). examples of crosstalk between shedding and O-glycosylation have GalNAc-type O-glycosylation (hereafter simply O-glycosylation) been reported. Here, we systematically investigated the potential is found on the majority of proteins passing through the secretory of site-specific O-glycosylation mediated by distinct polypeptide pathway and often in linker and stem regions of proteins (7). This GalNAc- (GalNAc-T) isoforms to coregulate ectodomain type of glycosylation is unique in being differentially shedding mediated by the A And regulated by a large family of up to 20 distinct polypeptide GalNAc- (ADAM) subfamily of proteases and in particular ADAM17. We (GalNAc-Ts), which initiate O-glycosylation by analyzed 25 membrane proteins that are known to undergo catalyzing addition of the first GalNAc residue to Ser and Thr ADAM17 shedding and where the processing sites included Ser/ residues (and possibly Tyr residues) (8). These GalNAc-T isoforms ± Thr residues within 4 residues that could represent O-glycosites. have different yet partly overlapping acceptor substrate CELL BIOLOGY We used in vitro GalNAc-T and ADAM cleavage assays to specificities, and have different expression patterns in cells and demonstrate that shedding of at least 12 of these proteins are α tissues, which opens for a high degree of differential and possibly potentially coregulated by O-glycosylation. Using TNF- as an dynamic regulation of the O-glycoproteome and individual example, we confirmed that shedding mediated by ADAM17 is O-glycosites in proteins (8). Previously it has been shown that coregulated by O-glycosylation controlled by the GalNAc-T2 iso- site-specific O-glycosylation specifically inhibits Furin proprotein form both ex vivo in isogenic cell models and in vivo in mouse processing ± 3 residues of cleavage sites (9), and in general Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding. O-glycosylation is known to confer structure and stability to stem regions of proteins and preventing . Classic studies ACE | IL-6RA | ErbB4 | ADAM10 | TACE demonstrated the importance of O-glycosylation in the stem region of the low-density-lipoprotein (LDL) using the CHO ldlD cell system that enables analysis of the general effect of O-glyco- ctodomain shedding, i.e., the proteolytic release of the ex- sylation (10). A number of other examples have confirmed this, tracellular domain of membrane proteins is a very common E and extended it to related receptors (11–13). Recently, it was and highly regulated process, in which specific proteases selectively shown that mutational loss of O-glycosylation in the stem region cleave substrate proteins in juxtamembrane regions. Ectodomain of the Interleukin-2 receptor subunit alpha (IL2Rα)resultin shedding is usually regulated in response to appropriate stimuli and increased shedding of the ectodomain (14). However, it is currently affects a large number of different classes of proteins including cell membrane receptors, growth factors, , and molecules (1). The shedding event may induce different effects in- Significance cluding activation, inactivation, or modulation of the functional properties of proteins (2) and are important in regulating a number Ectodomain shedding is a central event in a range of biological of biological processes such as signal transduction, adhesion, pro- processes and pathways, however the underlying mechanisms liferation and differentiation (1, 3). Ectodomain shedding is pre- are still not fully understood. Here we present evidence that dominantly mediated by Zinc-dependent , and site-specific O-glycosylation regulated by individual GalNAc- one of the key classes of proteases involved is the ADAM subfamily. Transferase isoforms, serve to coregulate ectodomain shed- Members of the ADAM family are type 1 transmembrane proteins ding, predominantly through blocking of cleavage. Our general that share conserved domains, including a prodomain that serves as finding is exemplified by the specific role of a single GalNAc-T isoform (GalNAc-T2) in coregulating TNF-alpha release in vitro, an inhibitor of the proteolytic activity; a metalloprotease domain Galnt2 that may or may not contain an with the conserved motif ex vivo in isogenic cell models, and in vivo in mouse (HEXGHXXGXXHD) common for Zinc-binding proteases; a knockouts. Adding the large family of GalNAc-T isoforms to regulation of ectodomain shedding substantially increase the disintegrin domain and a cysteine-rich region, which are both ability to fine-tune this important process on a substrate level. believed to be involved in substrate recognition; an EGF-like

domain; a transmembrane domain; and a cytoplasmic domain Author contributions: C.K.G., S.A.K., D.J.R., H.C., and K.T.-B.G.S. designed research; C.K.G., important for intracellular signaling. Dysregulation of ectodo- A.H., and S.A.K. performed research; C.K.G., A.H., S.A.K., and K.T.-B.G.S. analyzed data; main shedding is involved in a variety of diseases including in- and C.K.G., H.C., and K.T.-B.G.S. wrote the paper. flammation, cardiovascular diseases, diabetes, Alzheimer disease, The authors declare no conflict of interest. and (1, 4). However, the underlying mechanisms for dys- This article is a PNAS Direct Submission. regulation are poorly understood. Defined consensus motifs for 1To whom correspondence should be addressed. Email: [email protected]. ADAM cleavage sites have not been identified, but for a number of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. membrane proteins undergoing regulated ectodomain shedding the 1073/pnas.1511175112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1511175112 PNAS | November 24, 2015 | vol. 112 | no. 47 | 14623–14628 Downloaded by guest on September 26, 2021 unclear to what extent O-glycosylation in the stem region serves a protective role for proteolytic processing in general or more spe- cific and regulated roles in ectodomain shedding by, e.g., ADAMs. O-glycosylation has been proposed to specifically affect ADAM-mediated ectodomain shedding (15, 16). Tumor ne- crosis factor alpha (TNF-α), a type 2 transmembrane precursor protein that undergoes ectodomain shedding to produce a soluble truncated form, was shown to be O-glycosylated near a known ADAM cleavage site in lymphoblastic leukemia B cells (16), and Minond and colleagues showed that glycosylation of synthetic pep- tides covering the TNF-α cleavage site affected in vitro ADAM processing (15). Furthermore, it was recently found that also ADAM mediated cleavage of the extracellular N-terminal tail of the GPCR β1-adrenergic receptor may be affected by O-glycosylation (17). We hypothesized that important roles of site-specific O-glycosylation would have to be carried out by individual GalNAc-T isoforms to enable tuneable coregulation. This hypothesis leaves the substrate specificities of GalNAc-T isoforms and their unique functions as a central element in deciphering the potential for O-glycosylation to Fig. 1. In vitro O-glycosylation analysis of known ADAM substrates. (A)Peptidesof20–28 residues covering published ADAM cleavage coregulate ectodomain shedding. This function was recently ex- sites. O-glycosylated residues are indicated by GalNAc (square). (B) In vitro emplified with the GalNAc-T11 isoform identified as a candidate O-glycosylation with GalNAc-T1, -T2, -T3, -T4, -T5, -T11, -T12, -T13, -T14, disease in new-born heart heterotaxy (18). Thus, in vitro and -T16. The number of incorporated sites is shown in parenthesis. studies suggested that the GalNAc-T11 enzyme exhibited selec- tive substrate specificity for the juxtamembrane region of Notch1 and coregulated ADAM17 processing of Notch1 (19). Among the 12 candidates for combined cleavage and glyco- In the present study, we have systematically investigated the sylation were six membrane receptors: TGF-beta receptor type-1 potential of site-specific O-glycosylation mediated by distinct (TGFR-1), Receptor tyrosine-protein kinase erbB-4 (ErbB4), GalNAc-T isoforms to coregulate known ADAM processing. We Interleukin-6 receptor subunit alpha (IL6-RA), Tumor necrosis chose ADAM17 (also known as TACE) as a representative ex- factor receptor superfamily member 1B (TNF-R2), IFN alpha/beta ample identified 25 membrane proteins with known ADAM17 receptor 2 (IFN-R-2) and Growth hormone receptor (GHR); two processing sites in juxtamembrane regions and putative proximal proteases: ADAM10 and Angiotensin-converting enzyme (ACE); O-glycosylation sites defined as Ser/Thr residues within ± 4 two growth factors: Betacellulin (BTC) and Proheparin-binding residues of the processing site. Synthetic covering such EGF-like growth factor (HB-EGF); as well as the TNF-α, regions from approximately half of the analyzed proteins were and Myelin basic protein (MBP). Interestingly, the peptide sub- O-glycosylated by one or more GalNAc-T isoforms and in most strates designed for TNF-α, TGFR-1, ErbB4, and HB-EGF cases glycosylation modulated in vitro ADAM cleavage. We used were only glycosylated at one or two sites by a single GalNAc-T TNF-α as an example to demonstrate that shedding by ADAM17 isoform (Fig. 1). Several peptide substrates were glycosylated by is coregulated by site-specific O-glycosylation controlled by the two GalNAc-T isoforms (IFN-R2, p75, and ACE), whereas the GalNAc-T2 isoform ex vivo and in vivo. Our study provides evi- peptides for BTC, MBP, ADAM10, IL6-RA, GHR, and ACE dence for a wider role of site-specific O-glycosylation in coregulation were glycosylated at one or more sites by multiple GalNAc-Ts of ectodomain shedding. (Fig. 1). Thus, the first group of proteins has potential for being differentially O-glycosylated by a single GalNAc-T isoform, but Results the second group may also be regulated in cells where subsets of Screening for Site-Specific O-Glycosylation Adjacent to Known ADAM GalNAc-T isoforms are expressed. Cleavage Sites. ADAM17 is the best-characterized member of the ADAM family with respect to ectodomain Site-Specific O-Glycosylation Modulates in Vitro ADAM Processing. cleavage sites, so we chose to focus on ADAM17 to investigate To evaluate the effect of GalNAc O-glycosylation on ADAM the potential role of site-specific O-glycosylation. We data mined processing of the 12 candidate peptides we analyzed in vitro the literature for experimentally validated ADAM17 cleavage ADAM cleavage of peptides and produced by the sites with Ser/Thr residues ± 4 residues of the relevant GalNAc-Ts. The O-glycosites of these glycopeptides cleavage sites, and identified 25 cleavage sites from 23 different were characterized by ESI-LIT-FT-MS, and five recombinant membrane proteins (Fig. 1 and Fig. S1). We then designed short human ADAM proteases (ADAM8, -9, -10, -12, and -17) were (20mer) peptides covering the cleavage and putative adjacent tested in in vitro enzyme assays, where cleavage product for- glycosylation sites, and used in vitro enzyme time-course analyses mation was monitored by time-course MALDI-TOF MS as de- monitored by MALDI-TOF mass spectrometry to evaluate poten- scribed (21). This assay provides a relative estimate of the tial for O-glycosylation with a panel of recombinant GalNAc-Ts. efficiencies of ADAMs in cleaving the (GalNAc)-peptides in vitro, We tested 10 human recombinant GalNAc-Ts covering the major and more detailed analysis of kinetic properties of the ADAMs isoforms expressed in most cells and found that 12 of the 25 with these substrates is beyond this study. Ten out of 12 peptides designed peptide substrates served as substrates for O-glycosylation were cleaved in vitro by one or more ADAM isoforms and, among by one or more of the (Fig. 1 and Fig. S1). Consider- the corresponding glycopeptides, 8 of 10 cleavages by one or more able data indicate that in vitro GalNAc-T enzyme analysis of ADAMs were affected by glycosylation (Fig. 2). In most cases, short peptide substrates correlates well with in vivo glycosylation glycosylation inhibited cleavage (TNF-α, ADAM10, GHR, IL-6R, in cells (7, 20), and in a large scale analysis of peptides designed to ErbB4, HB-EGF, and ACE), and in one case glycosylation en- cover known O-glycosites about 40% were not glycosylated (20). hanced cleavage (ErbB4). For TNF-α glycosylation of S80 resulted This finding suggests, if anything, that in vitro analysis appears in partial protection from ADAM17, -9, and -10 processing at to underestimate O-glycosylation capacity (20), and hence that 76A↓V77, whereas ADAM12 processing at 79S↓S80 was completely additional candidates among the 25 identified in fact may be blocked(Figs.2and3).ForADAM10T625, glycosylation blocked glycosylated in vivo. cleavage at 621G↓R622 (Fig. 2). Glycosylation of T250 and S255 in

14624 | www.pnas.org/cgi/doi/10.1073/pnas.1511175112 Goth et al. Downloaded by guest on September 26, 2021 252 253 GHR blocked ADAM9 and -17 cleavages at P↓Q . Glycosyl- A B ation of T352,S359,andS360 in IL6-RA blocked ADAM9 cleavage at 359P↓V360. IFN-R2 glycosylation at T228 blocked ADAM9 cleav- age at 228T↓L229. Cleavage of ACE by ADAM17 at 1232R↓S1233 was blocked by glycosylation at S1230 and S12333, and glycosylation of HB-EGF at T75 and T85 blocked ADAM17 processing at 72L↓R72 C D and 72R↓V73; no difference in efficiency of the two cleavage sites was observed. In ErbB4, glycosylation at T643 and T650 ledtoin- creased cleavage by ADAM10 at 637P↓W638, and the ADAM17 cleavage sites shifted from 641H↓S642 and from 645P↓Q646 to 637P↓W638, no difference in cleavage efficiency of 645P↓Q646 and 637P↓W638 was observed. In addition, the ADAM9 cleavage at 641H↓S642 was blocked and ADAM8 cleavage at 645P↓Q646 was unaffected (Fig. 2). We did not observe any effect of glycosylation on cleavage of p75 (ADAM17 and -9 at 261P↓V262)orMBP(ADAM17at202H↓Y203,and ADAM8, -10, and -12 at 207P↓Q208)(Fig.2). Fig. 3. TNF-α is selectively O-glycosylated by GalNAc-T2, and this modulates Ectodomain Shedding of TNF-α Is Coregulated by GalNAc-T2. We in vitro ADAM cleavage. (A) Schematic drawing of TNF-ectodomain shed- selected TNF-α as a model for ex vivo validation in cells because ding zooming in on the juxtamembrane part that undergoes cleavage with O-glycosylation has been speculated to coregulate the shedding indicated cleavage and glycosylation sites. (B) In vitro glycosylation of the juxtamembrane part of TNF-α with GalNAc-T2. Horizontal arrow indicates of TNF-α and because it served as an example of GalNAc-T α B mass shift due to GalNAc glycosylation. (C) In vitro cleavage analysis of TNF- isoform-specific (GalNAc-T2) glycosylation (Fig. 3 and Fig. S1 ). peptide and by ADAM17 monitored by MALDI-TOF. Products TNF-α is produced as a type II transmembrane precursor (26 kDa) formed after 30 min, 1 h, and 4 h are shown. ADAM17 cleaved at 76A↓V. that undergoes regulated ectodomain shedding of a 17-kDa Cleavage of the glycopeptide was less efficient. (D) ADAM12 cleaved at 79S↓S. C-terminal form that assembles into functional homotrimers and Cleavage of the glycopeptide was almost completely abolished. ADAM9 and enters the bloodstream (22). To probe the role of glycosylation for -10 had similar cleavage patterns as ADAM17 (Fig. 2). ADAM8 was not ob- TNF-α shedding ex vivo we expressed an alkaline phosphatase- served to cleave the peptide. Sequence, cleavage site (scissors), and site of glycosylation (square) are shown above each spectra. CELL BIOLOGY tagged juxtamembrane TNF-α reporter construct (Fig. 4A)ina panel of isogenic cell lines with and without specific GalNAc-Ts. Δ Human liver HepG2 isogenic cells without GalNAc-T1 (HepG2 T1) analyzed by Western blotting (Fig. 4B). Comparing HepG2ΔT2 to Δ or GalNAc-T2 (HepG2 T2), as well as the latter with GalNAc-T2 WT and ΔT1 isogenic cells, we observed specific increased shed- Δ + reintroduced (HepG2 T2 T2) were transiently transfected for 48 h ding in T2 KO cells, which were abrogated when HepG2ΔT2 α using the TNF- reporter construct, and total cell lysates were clones were rescued by site-directed AAVS1 insertion of GalNAc- T2 (Fig. 4B). The increased shedding was verified in four different HepG2ΔT2 clones and in two GalNAc-T2 rescue clones in three independent experiments (Fig. 4B and Fig. S2). To further confirm this finding and exclude cell-specific factors, we also tested the TNF-α reporter construct in a panel of HEK293 isogenic cell lines (HEK293 WT, HEK293ΔT2, and HEK293ΔT3), which express a different repertoire of GalNAc-Ts with the major difference being GalNAc-T3. Similar to HepG2 cells we found se- lective increased shedding of TNF-α (∼40%) in the HEK293ΔT2 clones, confirming that GalNAc-T2 may play a critical role for processing and shedding of the TNF-α reporter construct in agreement with the in vitro analysis (Fig. 4 C and D).

LPS-Induced Plasma Levels of TNF-α Is Elevated in T2 KO Mice Compared with WT. We next tested the in vivo effect of com- plete GalNAc-T2 deficiency on levels of circulating TNF-α after lipopolysaccharide (LPS) stimulation. We compared TNF-α re- −/− lease in WT and Galnt2 KO mice. were injected using 0.5 mg/kg body weight of LPS by IP injection and plasma levels were measured by ELISA at 0, 1-h, 3-h, and 6-h time −/− points. Galnt2 mice showed a statistically significant increase in TNF-α plasma levels at all measured time points (1 h, 3 h, and 6 h) (Fig. 4E), confirming that GalNAc-T2 plays a role in TNF-α release and regulation. Furthermore, we observed no difference in the plasma levels of IL6-RA before or after LPS induction in Fig. 2. Summary of in vitro ADAM cleavage of naked peptides and glyco- these animals (Fig. S2). Finally, expression analysis showed that peptides. Peptide sequences covering known ADAM processing sites (arrows TNF-α expression levels were not changed between WT and KO and numbers indicate cleavage sites and ADAM isoforms, respectively). animals (Fig. S2). ADAM8, -9, -10, -12, and -17 were tested on all peptides that were in vitro O-glycosylated. Numbers in red indicate that we observed protection from Congenital Gene Variants with Potential Dysregulated Ectodomain cleavage after glycosylation, numbers in black indicate no effect, and num- bers in green indicate that cleavage was increased. No cleavage was observed Shedding due to Loss of O-Glycosylation. Several diseases or syn- of BTC and TGFR1. *The ADAM17 cleavage of ERBb4 shifted from H642↓STLP↓Q dromes have been linked to deficiencies in GalNAc-T isoforms of the naked peptide to 637P↓W of the glycopeptide, and ERBb4 glycosylation by various genetic association studies. The molecular mecha- increased cleavage by ADAM10. nisms behind these associations are in most cases unresolved, but

Goth et al. PNAS | November 24, 2015 | vol. 112 | no. 47 | 14625 Downloaded by guest on September 26, 2021 AB was much more efficiently cleaved, which is likely to account for the increased serum levels in people carrying this genotype. Nevertheless, the glycosylation was still found to inhibit ADAM mediated cleavage of the IL6-RA peptide. Our results highlight the importance of considering the glycosylation status of the jux- tamembrane part of a protein when investigating shedding and ac- tivation of membrane receptors. Finally, we tested the artificially produced ErbB4 mutant Q646C, which has been reported to induce a constitutively active receptor CED predicted to undergo constitutive dimerization (27). ErbB4 is also known to undergo Regulated Intramembrane Proteolysis (RIP) leading to intracellular signaling (28), and this mechanism could also play a role in the increased receptor activity. We tested the mutant Q646C peptide by in vitro assays and observed a dramatic reductionintheglycosylationcomparedwiththeWTpeptide (Fig. S3B). Discussion

Fig. 4. Ectodomain shedding of TNF-α is coregulated by GalNAc-T2 in Cell O-glycosylation in the juxtamembrane region of cell membrane line models and in vivo. (A)DepictionoftheTNF-α reporter construct proteins have for long been known to confer general stability and containing GalNAc-T2 specific glycosylation and ADAM cleavage sites. extended structure to the stem region that may be important for (B) Western blot visualizing cleavage patterns in HepG2 WT, ΔT2, ΔT1, and protrusion of the receptor domains (6). However, the extent to ΔT2+T2 cells transiently transfected (48 h) with TNF-α reporter construct. which isolated distinct sites of O-glycosylation play specific roles (C) Visualization of AP activity in SDS-polyacrylamide gel in cell lysates and for regulated proteolytic processing events such as ectodomain supernatants from HEK293 wild type and ΔT2 of full-length reporter and cleaved shedding has remained unclear. Moreover, provided distinct Δ Δ fragment. (D) Relative levels of shedding in HEK293 wild type, T2, and T3. O-glycosites serve such functions it seems conceivable that the Alkaline activity was measured in media and lysate. (E) Plasma levels (pg/mL) of glycosylation of these sites would be directed by distinct mem- TNF-α after LPS induction in WT and GalNAc-T2 KO mice. Statistical analyses were performed using the Student’s t test. Error bars show SDs. bers of the large GalNAc-T family to enable reasonable regu- lation. Here, through systematic analysis of membrane proteins with known ADAM17 processing sites, we demonstrate that deficiencies in distinct GalNAc-T isoform functions have been proximal site-specific O-glycosylation is likely to serve a common shown to cause diseases due to lack of coregulation of proteolytic coregulatory role in ectodomain shedding. We further demon- processing by site-specific O-glycosylation (19, 23, 24). In strate that most of the identified O-glycosites were controlled by line with these observations, we considered whether any of only one or a few GalNAc-T isoforms, which opens the possi- the O-glycosites potentially involved in coregulation of ectodomain bility for selective regulation through tuning of the expression of shedding identified in this study were found to be mutated in specific GalNAc-T isoforms. We selectively studied ADAM17 disease. We therefore searched for known missense SNVs as- processing sites, but it is likely that these findings can be ex- sociated with phenotypes or functional change of the variant trapolated to other proteases involved in ectodomain shedding, protein that were located in the juxtamembrane regions in close stressing the need to include analysis of potential O-glycosylation proximity to our newly identified glycosylation sites presented adjacent to processing sites in future studies of ectodomain here (±5 amino acids). shedding. We also demonstrate the importance of considering We identified two missense SNVs in 2 (ACE and IL6-RA) of O-glycosylation when analyzing the effects SNVs may exert. the 10 proteins with putative O-glycosites affecting ADAM GalNAc-type O-glycosylation is highly abundant with perhaps processing within a distance of ±5 residues of the processing site. over 85% of all proteins trafficking the secretory pathway being O-glycosylated, and interestingly a majority of the identified Interestingly, these two variants of ACE and IL6-RA (P1228L O- only have very few O-glycosites in isolated positions and D358A, respectively) have been found to be associated with (7). Although our knowledge of the O-glycoproteome has greatly increased serum levels of their shed ectodomains (25, 26). To expanded in recent years with the introduction of different test whether these variations in amino acid sequence affected O-glycoproteomics strategies (29, 30), there are still technical dif- in vitro O-glycosylation and/or ADAM processing, we tested B ficulties in identifying, for example, O-glycosites close to the jux- peptides with the substitutions. As shown in Fig. S3 , the gly- tamembrane region due to lack of efficient proteolytic cleavage cosylation efficiency by GalNAc-T3 of the ACE P1228L variant sites for bottom-up mass spectrometry sequencing strategies (31). peptide was dramatically decreased. However, the IL6-RA Although prediction algorithms for O-glycosylation are available D358A variant was a better substrate for GalNAc-T1, GalNAc- (7), these may not have sufficient examples from juxtamembrane T2, and GalNAc-T3, i.e., more peptide were glycosylated of the regions to serve as good predictors for O-glycosites in this region. variant compared with the WT when comparing the glycosylation Another obstacle is our limited knowledge on the substrate B reactions at different time points (1 h, 2 h, 4 h) (Fig. S3 ). We specificities of individual GalNAc-T isoforms and in particular the also observed an additional fourth site added by GalNAc-T3, specific O-glycosites they control nonredundantly in cells. Only which was not detected before (Fig. S3B). The amino acid substi- with such information will it be possible to define the regulatable tution did not seem to affect the substrate efficiency for GalNAc- part of the O-glycoproteome. Progress has been made with dif- T11 and GalNAc-T13. To test if the amino acid substitutions alone ferential O-glycoproteomes using isogenic SimpleCells with and affected ADAM activity, we analyzed in vitro cleavage of the non- without individual GalNAc-T isoforms (32) and systematic in vitro glycosylated variant peptides. We found that the ACE variant pep- analysis of O-glycosylation (20), but we are still far from being able tide was cleaved with comparable efficiency as the WT peptide to identify O-glycosites that are being regulated by individual without glycosylation (Fig. S3C), indicating that it was not the GalNAc-Ts. We therefore used in vitro GalNAc-T enzyme assays amino acid substitution itself, but rather the loss of O-glycosylation, to identify putative O-glycosylation sites, which revealed potential that is responsible for increased shed ACE levels in individuals with O-glycosites within ±4 residues of almost 50% of the analyzed this gene variant. Conversely, the naked IL6-RA variant peptide ADAM17 cleavage sites and glycosylation of most of these

14626 | www.pnas.org/cgi/doi/10.1073/pnas.1511175112 Goth et al. Downloaded by guest on September 26, 2021 affected in vitro ADAM cleavage (Fig. 2). Interestingly, in most A number of diseases are associated with altered levels of shed cases glycosylation affected cleavage negatively, but in one case cell membrane receptor ectodomains in serum, and in some cleavage was positively affected, similar to what we have pre- cases mutations in the juxtamembrane region of these receptors viously reported (19). It is important to note that we only tested have been identified. More recently, somatic mutations in the the simplest O-glycoform with just a GalNAc residue attached, juxtamembrane region of the colony-stimulating factor 3 re- whereas most cells will produce larger structures mainly of core1 ceptor (CSF3R) that appear to eliminate O-glycosylation has structure (Galβ1–3GalNAcα1-O-Ser/Thr) with sialic acid cap- been identified as driver mutations in chronic neutrophilic leu- ping. We have, however, previously shown that the mere pres- kemia (41). In this case, the mechanism was proposed to involve ence of an O-glycan is sufficient to evaluate an effect on enhanced homodimerization of the receptor due to loss of proprotein processing (21), but also that the complete sialylated O-glycosylation and constitutive signaling. The data presented core1 structure may exert more pronounced effects (23). here provide an additional scenario where mutations may alter ef- In agreement with our starting hypothesis, we also found that the ficiency of site-specific O-glycosylation, which subsequently affect majority of the glycosites identified that modulated ADAM17 pro- the proteolytic cleavage event and ectodomain shedding. One cessing in vitro were glycosylated in a GalNAc-T isoform-specific illuminating example may be ACE in which a SNV (P1228L) is manner. The majority of the O-glycoproteome is covered by func- associated with 2.5-fold higher levels of shed circulating ACE tional redundancy among GalNAc-T isoforms, and the widely (25). Elevated plasma ACE activity have been suggested to be expressed isoforms, GalNAc-T1, T2, and T3, appear to cover the associated with increased risk of cardiovascular disease (42), majority of the O-glycosylation events in cells, whereas other although a later report has not been able to reproduce this effect GalNAc-T isoforms have more restricted and specialized functions (43). Similarly, an IL6-RA SNV (D358A) has also been linked to (8, 20). It is therefore striking that most of the O-glycosites pre- higher soluble levels of the receptor (44). We found that the D358A dicted to modulate ADAM17 ectodomain shedding events were mutation enhanced O-glycosylation and also enhanced ADAM17 controlled by nonredundant GalNAc-T isoform-specific functions, cleavage (Fig. S3C). Interestingly, site-specific O-glycosylation further supporting that they likely play coregulatory roles in ecto- appears to play a role in coregulating both variants and the domain shedding. In four cases (IFNAR2, ADAM10, GHR, and change in IL6-RA signalingcausedbytheD358ASNV BTC) the O-glycosites were potentially controlled by two or three has been suggested to have a pathogenic role in and is GalNAc-T isoforms with overlapping functions (Fig. 1), however, associated with more severe disease (45). Finally, an experi- because most cells express only a limited subset of GalNAc-T iso- mental mutation in ErbB4 in close proximity to the glycosylation forms these glycosites may still be regulated by individual isoforms. sites identified here has previously been shown to result in a CELL BIOLOGY In this respect, it is important to include the repertoire of GalNAc-Ts marked increase in signaling (27). Again, this mutation affected expressed in a given cell when considering redundancies of efficiency of site-specific O-glycosylation (Fig. S3B). It is possible GalNAc-T isoform activities (8, 32). HB-EGF was specifically gly- that the loss of O-glycosylation affects the signaling through cosylated by GalNAc-T3 at two residues, which blocked ADAM17 modulating the ectodomain shedding and/or leads to increase in cleavage (Figs. 1 and 2). Given that GalNAc-T3 is not expressed in dimerization and ligand independent signaling as recently de- normal liver (24), the hepatic form of HB-EGF is likely not gly- scribed for the CSF3R (41). cosylated in these sites, but GalNAc-T3 is widely expressed in other In summary, we present evidence that site-specific O-glycosyl- cellsandcanbestronglyup-regulatedindisease(33).HB-EGFhas ation regulated by distinct GalNAc-T isoforms may widely serve been shown to play a protective role in acute liver damage (34). to coregulate ADAM mediated ectodomain shedding. Although, Similarly GHR was glycosylated at one site by GalNAc-T1 and at the role of glycosylation predominantly appears to reduce shed- two sites by GalNAc-T3 and GalNAc-T13, suggesting that there ding, examples of enhancement of shedding are also found. Site- may be some degree of redundancy in control of O-glycosylation of specific O-glycosylation has potential for complex modulation of these sites. However, again, GalNAc-T3 is not expressed in liver and ectodomain shedding to regulate efficiency and direction of pro- GalNAc-T13 is predominantly expressed in the brain and a few cessing site and selectivity. Adding the many GalNAc-T other tissues (8), so it is plausible that GalNAc-T1 alone controls isoforms to regulation of ectodomain shedding substantially in- glycosylation of GHR in liver. These examples underline the com- crease the ability to fine-tune the selectivity of this important plexity in deciphering the roles site-specific O-glycosylation may process. Moreover, as demonstrated, disease-associated muta- exert on biological processes such as ectodomain shedding. tions in the juxtamembrane region of membrane proteins may Precise gene editing tools offer unprecedented options to indirectly affect ectodomain shedding by affecting glycosylation dissect and validate nonredundant biological functions of gly- efficiency. Our study clearly underlines the importance of con- cosyltransferase isoenzymes (35). The knockout strategy used in sidering O-glycosylation and the GalNAc-T repertoire as co- this study overcome knockdown strategies issue with insufficient regulators of ectodomain shedding reduction in enzyme levels to produce substantial changes in Experimental Procedures glycosylation. We used a panel of ZFN edited isogenic cell lines to demonstrate that ectodomain shedding of TNF-α is specifically Glycosyltransferase Assays. Recombinant glycosyltransferases were expressed as soluble secreted truncated proteins in insect cells (45). In vitro activity coregulated by GalNAc-T2 site-specific glycosylation at Ser80 Galnt2−/− assays for GalNAc-T glycosylation of peptides (Schafer-N, NeoBioSci) were and a ZFN edited mouse model to validate this performed as described and monitored with MALDI-TOF (20). in vivo. TNF-α is a central mediator in several diseases including rheumatoid arthritis and Crohn’s disease (36), and blockage of Characterization of O-Glycosylation Sites by Electron Transfer Dissociation-MS2. TNF-α is a well established treatment regime for these and other Products of O-glycosylation reactions were characterized by electrospray ion- conditions (37). Several regulatory mechanisms of TNF-α release ization-linear ion trap-Fourier transform mass spectrometry (ESI-LIT-FT-MS) in and ectodomain shedding have been proposed (38–40), but the an LTQ-Orbitrap XL hybrid spectrometer (Thermo Scientific) equipped for 2 regulation and the importance of the different mechanisms still electron transfer dissociation for peptide sequence analysis by MS/MS (MS ) remain poorly understood. The coregulatory role of GalNAc-T2 with retention of glycan site-specific fragments (SI Experimental Procedures). offers new insight to the molecular mechanisms behind TNF-α ADAM Cleavage of Peptides and Glycopeptides. In vitro ADAM cleavage ac- release and may be important to consider in diseases with elevated – α GALNT2 tivity were assayed by adding 150 600 nM ADAM17, -10 (Enzo Life Sciences), secreted TNF- levels. play additional important roles -8, -9 (provided by Alexander Matthias Kotzsch, NNF Center for Protein in dyslipidaemia (24), and future studies need to address the ex- Research, Faculty of Health, University of Copenhagen, Copenhagen), or -12 tent to which these functions are regulated and fine-tuned. (a gift from the Kveiborg group, BRIC, Faculty of Health University of

Goth et al. PNAS | November 24, 2015 | vol. 112 | no. 47 | 14627 Downloaded by guest on September 26, 2021 Copenhagen), using 10 μg of peptide or glycopeptide substrate in a total The construct has been described in details by Zheng and colleagues (46) (SI volume of 25 μL(SI Experimental Procedures). Experimental Procedures).

Precise Gene Edited Cell Models. To confirm the in vitro assay findings, we T2 KO Mouse and Measurement of TNF-α Plasma Levels After LPS Induction. T2 developed cell line models capable of probing both the effect on cleavage by KO male mice (n = 4) and WT male controls (n = 8) were administered LPS site-specific glycosylation and the GalNAc-T isoform involved. Because GalNAc-Ts reconstituted in PBS at a dose of 0.5 mg/kg body weight by IP injection in are differentially expressed we used two different cell lines, HepG2 and HEK293, total volume of 250 μL. Blood was collected via retroorbital bleed before LPS representing different GalNAc-T repertoires. We used Zinc finger nuclease (ZFN) administration and at 1, 3, and 6 h after LPS injection. Plasma TNF-α was gene editing to knockout and/or knock in GALNT1 and GALNT2 (Sigma-Aldrich) measured from all timepoints by a mouse TNF-α ELISA (Quantikine ELISA, in HepG2 as described (32) (SI Experimental Procedures). R&D Systems). All procedures were approved by the Institutional Care and Use Committee of the University of Pennsylvania. TNF-α Shedding Assay. HepG2 WT, ΔT1, ΔT2, and T2 rescue and HEK293 WT, ΔT1, and ΔT2 were plated in six-well plates and transiently transfected with ACKNOWLEDGMENTS. This work was supported by the Danish Research the juxtamembrane-TNF-α-AP expression construct (generously provided by α Councils (including Sapere Aude Research Talent Grant to K.T.-B.G.S.), Carl Blobel, Cornell University, Ithaca, NY). The TNF- reporter construct a program of excellence from the University of Copenhagen, The Novo consists of a N-terminal intracellular FLAG tag fused to the first 87 amino Nordisk Foundation, a program of excellence from the University of acids of TNF-α, followed by alkaline phosphatase and a MYC tag. The full- Copenhagen (CDO2016), and The Danish National Research Foundation length fusion protein has a size of ∼70.2 kDa, and the shed part is ∼60.4 kDa. (DNRF107).

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