Aspartate Glycosylation Triggers Isomerization to Isoaspartate

Abstract

O-Linked β-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor 1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. We report here that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells but was previously thought to be exclusively non-enzymatic. Our findings suggest it may also be enzyme-catalyzed. In addition to OGT, enzymes that may catalyze aspartate to isoaspartate isomerization include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.

Figure 1

An aspartate-containing substrate is modified by OGT. (a) Sequences of peptides used in this study. The ‘^*’ denotes that S10-35 bears 3 N-terminal lysine residues not found in the other peptides. (b) EIC traces for [M+GlcNAc] for both D10-32 (top) and A10-32 (bottom) after 5 min incubation with OGT. (c) EIC traces of 841.6279 [M] (left panel) and 837.1253 [M-H₂O] (right panel) for D10-32 show conversion of starting material (S.M.) over time to a species isobaric with starting material (S.M. isomer) and to an anhydropeptide.

Figure 2

The anhydropeptide formed from glycosyl aspartate breakdown is a succinimide. (a) Two plausible pathways for the breakdown of a glycosyl aspartate intermediate. (b) EIC traces for the anhydropeptide (left), [M-H₂O] = 850.3983 (4+), and starting material (right), [M] = 854.9009 (4+), for the R9D10-32 peptide after treatment with OGT for 60 min.

Figure 3

¹⁸O is incorporated into D10-32 by OGT. (a) Succinimide hydrolysis can occur at either C1 or C2 to produce isoaspartate and aspartate, respectively. (b) Isotope envelope (z = 4+) for D10-32 prior to (top) and 45 minutes after treatment with OGT and ¹⁸O-water (bottom). At 45 minutes, two peaks were present in the EIC; the isotope envelope shown is averaged over both peaks. Isotopomers are labeled A+0 through A+5; vertical axes are normalized. (c) A plot of A+4/A+0 over time for D10-32 treated with wild-type OGT and ¹⁸O water (■), with wild-type OGT and ¹⁶O water (●), and with OGT K842M and ¹⁸O water (▲). The red and blue dotted lines denote the expected ratio for incorporation of zero and one ¹⁸O, respectively. All points are mean ± s.e.m. (n=2).

Figure 4

OGT converts aspartic acid to isoaspartate. (a) PIMT catalyzes isoaspartate methylation, producing a succinimide that hydrolyzes to a mixture of aspartate and isoaspartate. (b) EIC traces for 841.6279 (4+), corresponding to D10-32 starting material and isomer. Traces are after treatment with OGT alone (top) or with OGT followed by PIMT (bottom). (c) Normalized succinimide abundance for OGT and PIMT treated D10-32 (left), pure isoD10-32 (middle) or pure D10-32 (right); abundances were measured by integrating the respective EIC traces for succinimide, 837.1253 (4+).