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- Materials and Methods
- Supporting Information
Protein O-glycosylation is important in numerous processes including the regulation of proteolytic processing sites by O-glycan masking in select newly synthesized proteins. To investigate O-glycan-mediated masking using an assay amenable to large-scale screens, we generated a fluorescent biosensor with an O-glycosylation site situated to mask a furin cleavage site. The sensor is activated when O-glycosylation fails to occur because furin cleavage releases a blocking domain allowing dye binding to a fluorogen activating protein. Thus, by design, glycosylation should block furin from activating the sensor only if it occurs first, which is predicted by the conventional view of Golgi organization. Indeed, and in contrast to the recently proposed rapid partitioning model, the sensor was non-fluorescent under normal conditions but became fluorescent when the Golgi complex was decompartmentalized. To test the utility of the sensor as a screening tool, cells expressing the sensor were exposed to a known inhibitor of O-glycosylation extension or siRNAs targeting factors known to alter glycosylation efficiency. These conditions activated the sensor substantiating its potential in identifying new inhibitors and cellular factors related to protein O-glycosylation. In summary, these findings confirm sequential processing in the Golgi, establish a new tool for studying the regulation of proteolytic processing by O-glycosylation, and demonstrate the sensor's potential usefulness for future screening projects.
Protein O-glycosylation is an important post-translational modification occurring in the secretory pathway. Currently there are 25 well-defined medical syndromes linked to defects in O-glycosylation . Additionally, aberrations in glycosylation may also relate to certain forms of heart disease [2, 3] as well as tumor formation and metastasis . O-linked glycosylation begins in early Golgi cisternae  where a family of at least 24  highly conserved polypeptide N-acetylgalactosaminyltransferases (ppGalNTases) add N-acetylgalactosamine (GalNAc) to secretory cargo on serine or threonine residues that are typically adjacent to proline residues . The subsequent additions of sugar moieties to the initial carbohydrate group are thought to proceed in an orderly fashion as cargo moves through each successive Golgi cisterna, each containing a unique mix of glycosylation extending enzymes [5, 8, 9].
The ability of an added O-glycan moiety to regulate cleavage of an adjacent proteolytic processing site in newly synthesized cargo is an intriguing and medically relevant aspect of protein O-glycosylation . Glycan addition next to a protease recognition site can sterically block access of the protease to this site, and aberrations in this interplay between glycosylation and proteolysis can lead to disease. Familial Tumoral Calcinosis is thought to arise when mutations block glycosylation of the bone growth factor FGF23 thereby allowing a proprotein convertase to access and cleave the growth factor leading to its inactivation [11, 12]. Similarly, hypoglycosylation of apolipoprotein(a) leads to its proteolytic digestion, creating fragments in the blood stream that compete for binding to the extracellular matrix in atherosclerotic lesions . Conversely, the glycosylation of angiopoietin-like 3 blocks it's processing and activation causing altered triglyceride homeostasis [10, 13, 14]. Likewise, glycosylation inhibits cleavage-mediated activation of natriuretic peptide B, which regulates sodium excretion during heart failure [3, 15].
The mechanism of regulation of protease sites by glycan masking is incompletely understood. A scalable cell-based screen for defects in this process offers great promise toward identification of the full complement of the involved cellular factors. Similarly, a screen to identify small-molecule inhibitors of O-glycosylation would potentially lead to novel therapeutic approaches given that the known inhibitors are few, toxic  and offer little specificity toward individual members of the large ppGalNTase family.
Surprisingly, even the question of whether O-glycan addition necessarily occurs upstream of protein processing has been, at least indirectly, challenged. The conventional view of secretory traffic is that cargo moves in an orderly fashion from early Golgi cisternae where the ppGalNTases reside to the trans cisternae and trans Golgi network where the processing proteases are localized. Compartmentalization of these processing steps and the sequential flow of cargo ensure that O-glycosylation precedes proteolytic processing. This allows protease sites adjacent to O-glycosylation sites to be regulated by changes in expression or activity of ppGalNTases. Recently, however, in their rapid partitioning model, Patterson et al.  have challenged this basic premise of Golgi functional organization. While still maintaining that lipids and enzymes are distributed in a polarized fashion, they argue that incoming cargo rapidly exchanges among all cisternae, mixing with earlier arriving cargo before it is non-preferentially exported from partitioned domains present in all cisternae. This model predicts that cargo molecules could exit the Golgi stacks before complete processing and that later enzymes, namely proteases, could also have access to cargo before glycosylation protection, making glycan masking ineffective at best.
As a means toward identifying the cellular factors regulating O-glycan-mediated masking of proteolytic sites as well as novel inhibitors of O-glycosylation, we developed a fluorescent biosensor with the potential to be used in large-scale screens. Herein, we report the design and ‘proof of principle’ tests of such a sensor. Additionally, sensor behavior is used to examine predictions made by conventional versus rapid partitioning models of cargo traffic through the Golgi complex.
- Top of page
- Materials and Methods
- Supporting Information
Here we describe a biosensor of protein O-glycosylation based on O-glycan masking of a furin protease cleavage site. In the process, we demonstrate its prospective use in the development of novel O-glycosylation inhibitors. We also document its potential utility in broad based screens that study the factors contributing to and regulating the process of O-glycosylation in general and O-glycan masking of protease sites in particular.
The sensor takes advantage of the innate structure of the Golgi apparatus in which enzymes are organized into functionally distinct cisternae that are experienced in a temporally ordered manner by transiting cargo. While step-wise processing imposed by compartmentalization seems necessary for O-glycan masking, its existence was recently questioned. The rapid partitioning model states that the Golgi complex is effectively a single compartment. Because our sensor is setup such that activation depends on the order of processing, we were able to test this aspect of the model. That the sensor was largely silenced at steady state supports the conventional view that cisternal organization creates boundaries that order cargo movement. While there was some minor activation of the sensor, this is expected because of incomplete glycosylation of cargo under conditions of cargo over-expression such as in our cell line. Further, p115 knockdown to decompartmentalize the Golgi significantly increased the level of sensor activation showing that, when present in a single compartment, furin cleavage efficiently competes with the O-glycosyltransferases. Thus, Golgi structure plays a critical role in the process of O-glycan masking.
The influence of Golgi structure on processing emphasizes the point that O-glycosylation has many dependencies. One must consider more than just the sugar transporters and transferases. Indeed, it is possible that factors controlling Golgi organization may be manipulated to treat defects in glycosylation. Consistent with this point, both COG3 and GM130 are critical for Golgi organization and knockdown of either impairs glycosylation [24, 26]. COG3 is needed for recycling of a subset of Golgi resident proteins including some glycosylation enzymes  and GM130 links Golgi stacks to form a contiguous ribbon allowing for even enzyme distribution . The knockdowns we performed of these proteins indicate that the sensor can be used in conjunction with an siRNA library to screen for additional proteins involved in regulating O-glycosylation. One set of candidates is members of the Src signaling pathway that play a role in O-glycosylation . Generally, such a screen is expected to identify diverse factors acting in O-glycosylation enzymology, Golgi structure and signaling networks particularly as they relate to protease site masking.
Because O-glycosyltransferases are expressed in a tissue specific manner [6, 30, 31], the hits from screens using the sensor may vary depending on the cell types tested. That is, specific O-glycosyltransferases may have specific regulators. Currently, we do not know the identity of the ppGalNTase(s) that modify the sensor in HEK293 cells; however, we confirmed that GalNT-2 is expressed. Further, this enzyme remained Golgi-localized in the absence of GM130 and COG3 excluding mislocalization of this enzyme as the primary effect of these knockdowns (data not shown). In addition, we do not know the extent of extending and branching that takes place at the sensor O-glycosylation site. Masking requires O-glycosylation within 3 residues of the protease site  but how masking is affected by tree branching/elongation is not known. Interestingly, addition of a single GalNAc is sufficient for masking in vitro , whereas our observation in BAG-treated cells suggests that masking requires at least an additional galactose. Investigation of branching/elongation requirements could be accomplished using the sensor in cell lines that were generated to have limited types of extension on the initial GalNAc residue . At the very least, our sensor can be used as an easy test for a sequence that is suspected of being masked by an O-glycan.
It was somewhat surprising that fluorescence from the activated sensor was almost entirely at the cell surface with little or no fluorescence in the Golgi region. We attribute this to two factors. First, it is likely that the sensor rapidly exits the Golgi after cleavage. Indeed, there was a small detectable increase in Golgi region fluorescence in cells shifted to 20°C to delay exit (not shown). Second, the sensor takes time to develop fluorescence because after the blocking domain is removed it must dimerize to bind and activate the dye (Alan Waggoner, Carnegie Mellon University, personal communication).
As mentioned in the introduction, the misregulation of O-glycan masking leads to disease. In the case of hypoglycosylation of FGF23 and angiopoietin-like 3, the defective ppGalNTases are known (ppGalNT-3 and -2, respectively) [10, 12]. Therapeutically, it would be useful to individually target these ppGalNTases in order to alleviate symptoms; however, at present, specific inhibitors of O-glycosylation do not exist. In fact, current inhibitors act generally as competitors of sugar addition, are largely toxic and can have low cell permeability . One reason for their toxic effects could be that by inhibiting all O-glycosylation they affect many basic cellular functions. The sensor may allow identification of new, less toxic inhibitors that are specific to a particular ppGalNTase. Conceivably, such inhibitors may target the catalytic pocket, the lectin-binding domain [34-36] or factors specifically involved in the localization or expression of the enzyme. Hopefully targeted O-glycosylation treatments will be less disruptive and have fewer side effects.
O-glycan masking is not limited to the medically oriented examples mentioned. It also occurs in a large number of proteins including the cell surface receptors β1AR  and transferrin receptor [38, 39], the ion transporter copper transporter-1 [40, 41] and the metalloprotease MTI-MMP-1 [42, 43]. It is our hope that the biosensor described herein can be used to study the mechanisms regulating O-glycan masking in all cases where it occurs as well as in the development of therapies for disorders that arise from their misregulation.