\chapter{Conclusion}
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\begin{quote}
``Isn't it funny how day by day nothing changes,\\
but when you look back, everything is different?''\newline\\
-- C.S. Lewis (Prince Caspian)
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%Summary, conclusions, future directions.
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The works presented in Chapters~\ref{ch:hcfiscleaved}, \ref{ch:mechanism} and~\ref{ch:aspartate} illuminate details of the function of OGT \emph{beyond} Ser/Thr glycosylation. Chapter~\ref{ch:hcfiscleaved} showed us that HCF-1, a transcriptional co-regulator of human cell-cycle progression, is subject to cleavage \emph{directly} by OGT. Further, OGT effects this cleavage using the chemistry employed for Ser/Thr glycosylation; that is, the same active site, and the same co-substrate, UDP-GlcNAc - a common cellular metabolite, which links OGT to cellular metabolism - creating the potential to link cell-cycle regulation with cellular metabolic status. While tempting, this remains to be shown definitively. Furthermore, little is known about local (e.g. within sub-cellular compartments) concentration dynamics for UDP-GlcNAc within a cell, with measurements to date being limited to whole-cells~\cite{Bosch:2004ha}. Notably, the concentration of UDP-GlcNAc required for HCF-1 cleavage \textit{in vitro} is higher than that for Ser/Thr glycosylation, a feature that may be the result of an uncoupling between UDP-GlcNAc consumption and amide bond scission events. Even if cellular UDP-GlcNAc levels vary over a narrow range of concentrations, such an uncoupling would amplify these differences.\par
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From our mechanistic studies into HCF-1 cleavage, we wonder whether there is a biological significance to the pyroglutamate product formed in the cleavage process. Mutation of HCF-1 to replace the proteolytic repeats recognized by OGT with an ectopic protease recognition site (Taspase1) failed to rescue the phenotype of blocking HCF-1 proteolysis \cite{Capotosti:2011kd}. Pyroglutamate residues are found at the N-terminus of many antibodies and secreted proteins/peptides \cite{Burstein:1976ud,Liu:2011jk,Prasad:1975vh,Shih:2014dy}; pyroglutamate formation can occur spontaneously, and while it more commonly occurs at glutamine residues, it can also occur for glutamate \cite{Twardzik:1972wg}. An enzyme, glutaminyl cyclase, has been shown to accelerate this process \textit{in vivo} \cite{Liu:2011jk,Schilling:2008ks,Shih:2014dy}. Whether the pyroglutamate residue formed during cleavage persists \textit{in vivo}, is unknown, and given the misassignment of the cleavage site initially suggests that at least a portion of HCF-1 isolated from cells no longer bears the pyroglutamate residue. Efforts to prepare an antibody that selectively recognizes the N-terminal pyroglutamate of an HCF cleavage product have so far been unsuccessful. However, obtaining HCF-1 from cells using general HCF-1 antibodies, followed by MS analysis may reveal the relative abundances of HCF-1 polypeptides with and without this modification; cellular HCF-1 has already been examined by MS \cite{Myers:2013ix}, but whether peptides were observed where the pyroglutamate had been lost has not been described.\par
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With regard to the slow rate for HCF-1 cleavage, we hypothesized that amide bond hydrolysis occurs after dissociation from the enzyme, and suggested that this was the rate-determining step of cleavage. However, another possibility is that dissociation from OGT is the rate-determining step in this process. This notion may be supported by the fact that in Chapter \ref{ch:aspartate}, when we compared the relative rates of starting material consumption for cleavage, serine glycosylation and aspartate isomerization, at least \textit{in vitro}, the glutamate-containing acceptor (E10-32) was consumed far slower than S10-35 or D10-32. Curiously, it appeared as if wild-type OGT was not able to undergo multiple turnovers of HCF-1 cleavage, even when the substrate was supplied in excess. This could be the result of strong product inhibition, where the C-terminal cleavage product may remain bound in the TPRs of OGT and prevent the binding of the next substrate molecule, or an extremely inefficient coupling between glutamate GlcNAcylation and UDP-GlcNAc turnover that results in complete consumption of the co-substrate. Whatever the reason for this, it raises questions about whether OGT effects HCF-1 cleavage in a catalytic or stoichiometric fashion inside cells. It also raises questions about the number of repeats within the full-length HCF protein: is the origin of multiple repeats the result of increasing the probability that cleavage will occur? Are multiple cleavage events needed for HCF-1 to become functional? Or are multiple repeats required to prevent product inhibition?\par
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Having directly observed and isolated a glutamate-linked glycopeptide we cemented our mechanistic picture for the steps involved in HCF-1 cleavage~\cite{Janetzko:2016er}. We were prompted from these studies to examine whether aspartate was a suitable acceptor~\cite{Janetzko:2017ab}, yet the fact that it reacted was less a surprise than that upon reaction, and forming a glycosyl aspartate, this led to isomerization of the acceptor residue to isoaspartate. In hindsight, the behavior of these glycosyl-glutamate and glycosyl-aspartate species should not have been a surprise at all. Amino acid side chain activation has long been established: numerous examples exist wherein glutamic acids have been shown to be susceptible to activation by peptide coupling reagents, and this activation leads to the formation of pyroglutamate residues or succinimides \cite{JR9560002076,JR9610000524,Bodanszky:1975br,Chulin:2005ks,Feinberg:1975eu,leQuesne:1952il,Nalbone:2016fw,Ondetti:1968ew,Tofteng:2009gp,Yang:1976vg}. In the case of internal pyroglutatamtes, this activation has been shown to predispose the peptide bond to spontaneous hydrolysis at that site \cite{JR9560002076,JR9610000524,Chulin:2005ks,Feinberg:1975eu,leQuesne:1952il,Nalbone:2016fw,Tofteng:2009gp}. Similar hydrolytic instability has been seen for \textit{N}-acylurea-containing peptides, generated by oxidation of asparagine residues \cite{Tanabe:2014ko}. Furthermore, when a glycosidase is trapped with a reactive sugar fluoride, it, too, became susceptible to backbone amide bond cleavage, presumably through the intermediacy of the same species described in Chapter \ref{ch:mechanism} \cite{Kotzler:2016br}.\par
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%Aspartic acid residues are known to undergo spontaneous cyclization to form succinimides, as discussed in Chapter~\ref{ch:aspartate}, but this process is slow. Thus, it stood to reason that catalyzing GlcNAc transfer to aspartic acid may lead to rapid succinimide formation. Indeed, this is not dissimilar from the reaction catalyzed by PIMT, which generates the methyl ester of isoaspartate, using SAM, to facilitate succinimide formation. I assure the reader, the irony that the enzyme I used to characterize the isoaspartate residue produced by OGT-catalyzed aspartate glycosylation employs highly similar chemistry has not been lost.\par
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One of the real surprises, given this work, is that at present there are no other examples of the use of UDP-GlcNAc as an electrophilic activator in biology. UDP-GlcNAc is one of the most abundant metabolites in cells, and other abundant metabolites like ATP~\cite{Funabashi:2010ev}, NAD\textsuperscript{+}~\cite{Feldman:2012ez}, and SAM~\cite{Funabashi:2010ev} have all been implicated as co-substrates for electrophilic activation. Does this imply that other glycosyltransferases, or enzymes otherwise annotated as glycosyltransferases, are able to modify glutamic acid or aspartic acid for the purpose of effecting the downstream chemistry that these species are inherently prone to? Is OGT able to glycosylate any other protein substrates on glutamic acid, and as a result promote amide bond cleavage in those substrates? Amide bond cleavage, as a result of glutamate glycosylation, would produce an N-terminal pyroglutamate on the C-terminal cleavage peptide. Unfortunately, at present, a robust method for pyroglutamate enrichment has not been worked out. However, if it were possible to enrich for pyroglutamate residues, one could examine the differences in pyroglutamate-containing peptides under conditions where OGT activity was perturbed (endogenous vs. chemically inhibited vs. OGT over-expression). With regard to aspartate glycosylation, proteomic studies into isoaspartate and succinimide formation have previously been described \cite{Alfaro:2008ku,Klaene:2014hx,Vigneswara:2006jj,Zhu:2006cg}. Treating protein samples with hydrazine derivatives will label many cellular electrophiles \cite{Matthews:2017eh}, however, using an aldehyde-derivatized resin, only carbonyl-based electrophiles would be retained (due to their ability to form a hydrazone) \cite{Alfaro:2008ku}. One can envision examining cellular succinimide levels under various conditions where OGT activity has been modulated, either chemically or genetically. It may be necessary to employ a PIMT+/-- background \cite{Qin:2015cs} to observe differences in cellular succinimide when OGT activity is perturbed. Alternatively, detection of isoaspartate may be better suited if the expected time-scale for OGT-mediated changes is long. Treating cell lysates first with PIMT (to generate succinimides) followed by hydrazinolysis should allow for enrichment of isoaspartate-containing peptides, indirectly.
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While it remains to be seen whether OGT is responsible for aspartate glycosylation \textit{in vivo}, efforts to examine this are underway.\par
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The chemical reactivity of glutamate and aspartate linked glycosyl esters is also interesting in the context of protein ADP-ribosylation. Many of the identified ADP-ribosylation sites have been mapped to Asp and Glu residues \cite{Daniels:2014kz,Hassa:2006iz,Martello:2016bq}; it remains to be examined whether any of these proteins are susceptible to backbone cleavage or isoaspartate formation. While the local peptide backbone conformations are likely to play a role in dictating whether a succinimide can form \cite{Clarke:1987bb}, this may simply mean that these chemistries are limited to relatively unstructured regions or regions prone to partial unfolding \cite{Johnson:1989wy}.\par
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Beyond its ability to modify Ser/Thr/Glu/Asp, as described here, it has been reported that Cys is also able to serve as a suitable glycosyl acceptor for OGT \cite{Maynard:2016hh}. It remains to be seen whether this of modification is of physiological relevance, these studies may suggest a revision of our picture of OGT and the diversity of substrates it may modify. There are other examples of glycosyltransferases which are able to tolerate multiple glycosyl acceptors~\cite{Lizak:1fw}; however, only PARPs or ADP-ribosyl transferases demonstrate the substrate breadth now seen with OGT \cite{Daniels:2014kz,Hassa:2006iz,Leidecker:2016ce,Martello:2016bq}. Furthermore, these studies raise many questions as to relative importance of each of these activities for OGT, particularly since the elements of OGT's function that are required for the survival of mammalian cells are still unknown \cite{Levine:2016du}. This is undoubtedly an exciting time to study OGT, and the answers to many of these questions are likely to offer a wealth of new biological insights.\par
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