Review Fine-Tuning Limited Proteolysis: A Major Role for Regulated Site-Specific O-Glycosylation Christoffer K. Goth, 1, * Sergey Y. Vakhrushev, 1 Hiren J. Joshi, 1 Henrik Clausen, 1 and Katrine T. Schjoldager 1, * Limited proteolytic processing is an essential and ubiquitous post-translational modification (PTM) affecting secreted proteins; failure to regulate the process is often associated with disease. Glycosylation is also a ubiquitous protein PTM and site-specific O-glycosylation in close proximity to sites of proteolysis can regulate and direct the activity of proprotein convertases, a disintegrin and metalloproteinases (ADAMs), and metalloproteinases affecting the activation or inactivation of many classes of proteins, including G-protein-coupled recep- tors (GPCRs). Here, we summarize the emerging data that suggest O-glyco- sylation to be a key regulator of limited proteolysis, and highlight the potential for crosstalk between multiple PTMs. Modulation of Protease Substrate Sites Is Key Limited proteolysis by proteases is one of the most prevalent PTMs regulating numerous cellular and physiological processes [1,2]. The functional consequences of limited proteolysis are diverse and can lead to the activation, inactivation, and change of protein function. A large number of proteases involved in regulating protein functions through limited proteolysis are targets for drug development [3], and understanding underlying molecular mechanisms is essential in health and disease. Distinct families of proteases have evolved to serve limited proteolytic events at different locations and for different protein classes [4]. In total, the human genome encodes over 560 different proteases, some of which are known to have broad specificities, whileothers have distinct private functions. Importantly, the number of known and potential protein substrates vastly outnumbers the number of proteases, and additional mechanisms for differential regulation of the function of individual proteins appear to be needed. A plethora of different mechanisms regulate activities of proteases involved in limited proteolysis, including expression, localization, and transport. Furthermore, some proteases are produced as inactive zymogens that need the proteolytic removal of their inactivating prodomains before becoming catalytically active. Endog- enous inhibitors constitute another important level of regulation and several hundred different inhibitors have been identified with important co-regulatory functions of many different proteases [5]. Here, we propose that the true magnitude in differential regulation and specificity is further derived from the crossroads of multiple PTMs at specific protease substrate sites, with site- specific O-glycosylation being particularly suited for this interplay. Co-regulation at the substrate site by PTM crosstalk offers an appealing mechanism to provide selectivity and fine-tuning for individual proteins. This has been demonstrated for several PTMs, where ubiquitination [6], oxidation, nitrosylation, citrullination, and isomerization of aspartates [7] regulate proteolysis. One of the more well described PTMs affecting cleavage is intracellular Highlights Site-specific O-glycosylation has emerged as a general co-regulator of proprotein processing and ectodo- main shedding. N-terminal cleavage of GPCRs is modulated by site-specific O-glycosy- lation. Evidence points to a general phenomenon. Proximal (or adjacent) site-specific O- glycosylation may reduce, enhance, or displace proteolytic cleavage sites as well as direct the proteases involved. Interplay between site-specific O-gly- cosylation and extracellular phosphor- ylation may be emerging as a fine- tuning process for regulated proteolysis. 1 Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark *Correspondence: goth@sund.ku.dk (C.K. Goth) and schjoldager@sund.ku.dk (K.T. Schjoldager). TIBS 1432 No. of Pages 16 Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.tibs.2018.02.005 1 © 2018 Elsevier Ltd. All rights reserved.