The Non-Enzymatic Glycation of Ubiquitin: A Structural and Functional Study
Mark Esposito and Roger Sandwick
Department of Chemistry and Biochemistry
Middlebury College, Middlebury, Vt. 05753
Monitoring the Ubiquitination/ Proteolysis of Yeast cytochrome c:
Abstract:
The glycation of ubiquitin with ribose 5-phosphate (R5P) and glucose was studied to determine the
effect of post-translationally modified ubiquitin on intracellular proteolysis. Intracellular proteolysis is
initiated by the conjugation of ubiquitin to protein substrates via an ensemble of enzymes to 1) form
a thiol ester bond to the C-terminus of ubiquitin, 2) transport conjugated ubiquitin to target protein
substrates and 3) ligate ubiquitin to surface ε-amino groups of a target protein substrate.
Polyubiquitination then can occur in a processive manner through ε-amino groups on ubiquitin. Our
investigations focused on identifying the location of glycation sites on the ubiquitin protein and on
developing a method for assessing the effect that glycation has on ubiquitin activity. R5P-glycated
and glucose-glycated ubiquitin isolated by boronate and anion exchange chromatography showed as
many as three glycation events within a 72 h period. Trypsin digestion studies suggest glycation
occurs preferentially at certain ε-amino groups. A novel method which employs yeast cytochrome c
as a ubiquitination target substrate and LC-MS for subsequent analysis is under development for use
in assessing the functionality of modified ubiquitin. Preliminary results suggest that this is a robust
method for the detection of ubiquitination. Further refinement of this method is necessary before
the effects of glycation on ubiquitination can be analyzed.
Intens.
x10 6
4
A
2
0
x10 6
6
4
2
7_27CT 02.D: EIC 616.3 +All MS
B
0
x10 6
7_27CT 03.D: EIC 616.3 +All MS
6
4
2
C
0
5
10
15
20
25
30
T i me [mi n]
Figure 2. Extracted ion chromatogram for heme group of urea-arrested proteolysis of
cytochrome c with wild-type ubiquitin. A) 0 minute arrest, integration yields 0 MIU. B) 2.5
minute arrest, integration yields 24.2 MIU. C) 5 minute arrest, integration yields 84.6 MIU.
Intens.
x10 6
6
4
7_27CT21.D: EIC 616.3 +All MS
1
A
2
0
x10 6
6
Introduction:
The intracellular proteolytic degradation of short-lived and abnormal proteins is achieved
through the conjugation and ligation of ubiquitin to protein substrates. Ubiquitin is a 8.6 kDa
protein highly conserved throughout the entire phylogenic tree. The biochemical pathway
resulting in covalent ligation is illustrated below. (Ciechanover and Hersko, Annu. Rev. Biochem. 1998)
7_27CT 01.D: EIC 616.3 +All MS
4
1
7_27CT22.D: EIC 616.3 +All MS
B
2
2
0
x10 6
6
C
7_27CT23.D: EIC 616.3 +All MS
1
4
2
0
Results:
Purification of Glycated Ubiquitin:
5
10
15
20
25
30
Time [min]
Figure 3. Extracted ion chromatogram for heme group of urea-arrested proteolysis of
cytochrome c with R5P-glycated ubiquitin A) 0 minute arrest, integration yields 49.9 MIU. B)
2.5 minute arrest, integration yields 68.7 MIU. C) 5 minute arrest, integration yields 54.2 MIU
Intens.
x109
A
7_27CT01.D: TIC +All MS
B
7_27CT02.D: TIC +All MS
C
7_27CT03.D: TIC +All MS
1
0
x109
4
The crucial importance of lysine residues in both initial ubiquitin ligation and subsequent
polyubiquitination events makes non-enzymatic glycation of proteins a pertinent field of
study. It has been previously established that glycation of protein substrates inhibits
proteolysis, however the effects of glycation on the ubiquitin pathway have not yet been
explored. Generally, the glycation of ε-amino groups is depicted below. (Watkins et. al., J. Bio. Chem. 1985 )
2
0
x109
2
0
Intens.
x10 9
0.75
5
10
15
20
25
30
D
Time [min]
7_27CT21.D: TIC +All MS
0.50
0.25
0.009
x10
1.00
0.75
7_27CT22.D: TIC +All MS
E
0.50
0.25
0.009
x10
1.00
Objectives:
1) In order to hypothesize the effects of glycation, the preferential sites of glycation must be
established.
2) Though very sensitive, currents methods for examining ubiquitin mediated degradation
through radioactive counting are impractical for rapidly examining the effects of proteolysis.
Thus a novel method for monitoring proteolysis will be developed.
3) To quantify the effects of glycation, a population of ubiquitin glycated by either R5P or
glucose must be purified. Methods will be developed for the purification of both species of
modified ubiquitin.
4) Purification and proteolysis methods will be synthesized to determine the effects of
glycation on ubiquitin mediated proteolysis.
Methods:
Ubiquitin Glycation and Purification: Ubiquitin was reacted with R5P or glucose for 24 - 72
hours. Glucose glycated samples were purified through boronate affinity chromatography.
R5P glycated samples were purified through anion exchange chromatography. Relative
concentration was established by the BCA assay.
Glycation Analysis: Purified samples were subjected to trypsin digest and analyzed by LC-MS.
Development of Novel Proteolysis Method: Bovine erythrocytes were obtained from Vermont
Livestock S & P. Cells were depleted of ATP and lysed with DTT. The ensemble of enzymes
necessary for proteolysis were purified through ion exchange chromatography. Reactions
buffered at low ionic strength containing ubiquitin, ATP, an ATP regeneration system , purified
erythrocyte fractions, and yeast cytochrome c were arrested with urea and monitored at
varying time scales with LC-MS. Heme growth and UV absorbance were quantified to
establish the extent of proteolysis.
Purified glycated ubiquitin was used in conjunction with proteolysis reagents to assess effects
of glycation.
0.75
7_27CT23.D: TIC +All MS
F
0.50
0.25
0.00
5
Figure 1. A) Sample incubated at 37° C for 72 hr at 1 M glucose and 1
mg/mL Ubiquitin. B) Glucose incubated ubiquitin purified through
boronate chromatography. C) Sample incubated at 37° C for 24 hr at 0.1
M R5P and 1 mg/mL Ubiquitin. D) R5P incubated sample purified
through anion exchange chromatography.
Trypsin Digest of Glycated Ubiquitin Enrichment:
Table 1. Trypsinated fragment sequence and LC-MS analysis of
trypsinated, R5P-glycated ubiquitin. Percent fragment present obtained
by comparison to control integrations and reference to fragment 10.
Fragment
1
2
3
4
5
6
7
8
9
10
11
12
Fragment Sequence
MQIFVK
TLTGK
TITLEVEPSDTIENVK
AK
IQDK
EGIPPDQQR
LIFAGK
QLEDGR
TLSDYNIQK
ESTLHLVLR
LR
GG
Percent Fragment Present
18.4
6.1
33.9
N/A
12.4
20.9
68.4
9.7
7.6
100.0
N/A
N/A
10
15
20
25
30
Time [min]
Figure 4. Total ion chromatogram of cytochrome c proteolysis. A, B, C) Proteolysis conducted
with wild-type ubiquitin at 0, 2.5, 5 minutes. D, E, F) Proteolysis conducted with R5P
glycated ubiquitin at 0, 2.5, 5 minutes.
Conclusions:
1.
2.
3.
4.
Methods used to purify glycated ubiquitin completely isolate wild-type ubiquitin.
Trypsin analysis indicates preferential glycation sites putatively exist, and exclusive
glycation sites do not exist unless located on Arg74.
Comparison of long time scale wild-type ubiquitin-mediated proteolysis to control
samples indicates that A) Conjugated ubiquitin putatively exists in purified
erythrocyte extracts. B) Cytochrome c is oxidized by experimental conditions. C)
Oxidation does not result in heme release. D) Both ubiquitin and cytochrome c are
rapidly modified when reacted with erythrocyte extracts and ATP. E) Heme release is a
direct result of either ubiquitination alone or ubiquitination followed by proteolysis.
Comparison of short time scale wild-type ubiquitin-mediated proteolysis to glycated
ubiquitin samples indicates that A) Glycation of ubiquitin likely inhibits the ubiquitinmediated proteolysis of cytochrome c.
Acknowledgements:
I would like to thank Professor Sandwick for his guidance throughout the entire project,
and Vermont Livestock for generously donating bovine blood. I would also like to thank
Middlebury College and Charles Bliss Allen ‘62 for financial support.