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 cell membrane proteins cleavage sites and the specific ADAM proteases 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-transferase (GalNAc-T) isoforms to coregulate ectodomain type of protein glycosylation is unique in being differentially shedding mediated by the A Disintegrin And Metalloproteinase regulated by a large family of up to 20 distinct polypeptide GalNAc- (ADAM) subfamily of proteases and in particular ADAM17. We transferases (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 peptide acceptor substrate CELL BIOLOGY We used in vitro GalNAc-T enzyme 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 proteolysis. Classic studies ACE | IL-6RA | ErbB4 | ADAM10 | TACE demonstrated the importance of O-glycosylation in the stem region of the low-density-lipoprotein (LDL) receptor 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, cytokines, and cell adhesion 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 metalloproteinases, 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 active site 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 cancer (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 gene 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