Section : Erythrocytic proteins are O-GlcNAcylated
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We initially tested the extent of GlcNAcylation in erythrocytic proteins by using a pan-specific O-GlcNAc antibody (CTD 110.6)
Immunoblot data showed that multiple erythrocytic proteins are O-GlcNAc modified (Fig. 1)
Control immunoblotting with O-GlcNAc antibody but in the presence of excess free GlcNAc yielded little to no signal, suggesting the specificity of the blotting (data not shown)
We next wanted to assess whether the GlcNAcylation cycles in erythrocytes
Both OGT and O-GlcNAcase , the enzymes responsible for O-GlcNAc cycling, are detected in erythrocytes (Fig. 1)
The donor substrate for OGT , UDP-GlcNAc , and O-GlcNAcase activity are also present in the erythrocytes (K.P., C.S., G.H., unpublished data)
These data suggest that O-GlcNAc cycling may exist in human erythrocytes
Section : Selective enrichment and identification of putative GlcNAcylated proteins
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Enrichment is key for mass spectrometric identification of GlcNAcylated proteins because of low stoichiometry and ion suppression by unmodified peptide ions in the mass spectrometer (21)
Immunoisolation by pan-specific antibodies suffers from low efficiency due to relatively low binding affinity of the antibodies
Using lectins to enrich O-GlcNAc proteins suffers from low specificity because lectins may bind strongly to proteins with other forms of glycosylation (rev. in 21)
In this study, a highly selective tagging method was used (18)
This method takes advantage of the mutant UDP-galactose transferase (Y289L GalT1) (19), which has an enlarged donor-substrate binding pocket and can accommodate UDP-galactose analogs, in this case, UDP-Gal-ketone
GalT1 was used to enzymatically tag GlcNAc modifications on erythrocytic proteins with Gal-ketone
PNGase F was used to remove N-glycans
After enzymatic labeling, the ketone group was chemically tagged with an aminooxy biotin , which allowed capturing of O- GlcNAcylated proteins with strept avidin beads
Enriched proteins were then eluted, separated by SDS-PAGE, and identified by an ion trap mass spectrometer after in-gel digestion (Fig. 2)
By using this method, 25 erythrocyte proteins were identified as putatively GlcNAcylated (Table 1)
A mock experiment with no UDP-Gal-ketone added yielded no signal when blotted with horseradish peroxidase–conjugated avidin , indicating the specificity of the approach (Fig. 2)
We further confirmed some of the putative GlcNAc proteins by first immunoprecipitating the proteins and then Western blotting with O-GlcNAc antibody (Fig. 2)
O-GlcNAc antibody competition with excess free GlcNAc was routinely performed and eliminated the signals in immunoblotting, documenting the antibody specificity (data not shown)
Section : Mapping O-GlcNAcsites and site-specific quantitation
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We first attempted to evaluate the overall dynamics of O-GlcNAc on whole protein levels by immunoprecipitation and imunoblotting, but no conclusive result was observed
This may not be surprising because the extent of GlcNAcylation is site-specifically regulated and because O-GlcNAc antibody may not be sensitive enough to recognize significant change of O-GlcNAc on one specific site when the protein is modified on multiple sites
Furthermore, one of the eventual goals of this study is to develop site specific O-GlcNAc antibodies
Thus, we wanted to map O-GlcNAcsites and quantitate O-GlcNAcsite specifically
A recently developed chemoenzymatic tagging method was used to enrich O-GlcNAc at peptide levels (17,20)
Y289L GalT1 was similarly used to label the GlcNAc moieties as described previously but on trypsin-digested peptides instead of whole proteins
With increased accessibility of the enzyme and donor substrate, the labeling with UDP-GalNAz (an analog of UDP-galactose with azide function group) is nearly 100% after overnight incubation at 4°C (21)
Biotinylation was performed with the highly efficient copper-catalyzed cycloaddition reaction or “click” chemistry under mild conditions (22) (Fig. 3)
For this chemoenzymatic approach using UDP-GalNAz and biotin- PEG-alkyne, the tags added to the O-GlcNAcpeptide exceed 772 Da in mass
Although the biotin- PEG-alkyne tags allow for highly selective enrichment of GlcNAcylated peptides , they are problematic; not only do they negatively affect the ionization efficiency, but the heavy tags also impose other challenges for mass spectrometric analysis
For example, fragmentation of the biotin moiety and the PEG linker arm on CAD makes the MS/MS spectra noisy and difficult to interpret
In addition, the tagging does not change the extremely labile nature of the β-O-linkage, which undergoes neutral loss before peptide backbone fragmentation in CAD (Fig. 3)
To resolve these issues, we modified and combined a previously developed chemical derivatization method called BEMAD (β-elimination followed by Michael addition with DTT) (23) with the chemoenzymatic enrichment method
The BEMAD chemical derivatization was performed directly on the solid phase after the tagged peptides were captured by avidin beads (Fig. 3, inset)
The derivatized peptides were released from the solid phase with the O-GlcNAc , and tags were replaced by a DTT via Michael addition
The resulting DTT modification is stable and can be easily identified by mass spectrometry
This approach also circumvents the need to break the strong biotin-avidin interaction with harsh conditions
Mass spectrometric quantitation of O-GlcNAcpeptides is also readily enabled by isotopic labeling with deuterated DTT (DTT-d6), which introduces a 6-Da mass difference between the peptide pairs (e.g., normal vs. diabetic)
The overall approach is shown as a flow chart in Fig. 3 and described in detail in research design and methods
Of course, it is possible that the apparent changes in GlcNAcylation may arise from different dynamics of protein expression or turnover
To address this factor, we labeled the flow-through of avidin chromatography, containing mostly unmodified peptides , with iTRAQ reagents and used it to quantitate relative changes of protein expression levels
With relative abundance of both O-GlcNAcpeptides and corresponding protein levels, RORs of O-GlcNAc could then been calculated using a simple equation (see research design and methods)
Erythrocyte lysates from normal and diabetic blood donors (10 each; Table 2) were pooled separately and used as the starting materials after partial depletion of abundant hemoglobins
Three independent experiments were performed according to the flow chart shown in Fig. 3
Using the standard of at least one unique peptide with a >99% confidence level, 206 erythrocyte proteins were identified and quantified (supplemental data, available in an online appendix at http ://dx.doi.org/10.2337/db08-0994)
Although most proteins were equally abundant, changes were observed for a few proteins between normal and diabetic samples (Fig. 3)
Thirty-five O-GlcNAcsites originating from 17 proteins were identified
The relative occupancy rates of O-GlcNAc at these sites between diabetic and normal states were calculated (Table 3)
A negative control sample was first treated with hexosaminidase (an enzyme that removes GlcNAc) before enrichment and yielded no identification of a GlcNAcylated protein (Fig. 3), indicating the specificity of the overall approach
Differentially regulated GlcNAcylation was observed on multiple sites originating from several proteins (Table 3; Fig. 4)
This regulation is clearly site specific, as observed in the cases of ankyrin-1 , hemoglobin α, and catalase (Table 3)
Section : Erythrocytes are probably among the simplest of human cells
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For a long time, erythrocytes had been regarded as a cytoplasm surrounded by a simplified membrane and consisting mainly of hemoglobins
A recent in-depth analysis of the erythrocyte proteome indicated that there are likely far more complex cellular processes inside erythrocytes than previously known (24)
Results presented here suggest that O-GlcNAc actively cycles on erythrocyte proteins
Some of the earliest known GlcNAcylated proteins were detected in human erythrocytes (25)
The challenges of studying O-GlcNAc by mass spectrometry come from its low stoichiometry, suppressed ionization efficiency in presence of unmodified peptides , and intrinsic lability in gas phase (21)
In this study, highly efficient enrichment methods based on chemoenzymatic tagging addressed the first two challenges
Solid-phase chemical derivatization successfully circumvented the lability issue
As the exploratory phase of a project aimed at using O-GlcNAc as a potential biomarker for diagnostic of diabetes, we identified 25 O-GlcNAc modified proteins , mapped 35 O-GlcNAcsites , and compared the O-GlcNAc RORs between erythrocyte lysates obtained from normal and diabetic individuals
By using a rigorous mass spectrometric standard, we also identified 206 erythrocytic proteins and compared their abundance between normal and diabetic samples
A few proteins , such as carbonic anhydrase 1 (diabetic:normal 0.51), glutathione transferase ω 1 (0.79), GLUT1 (0.88), superoxide dismutase (1 .21), and isocitrate dehydrogenase (1.29), were observed as differentially regulated in normal and diabetic samples
Although these protein level dynamics might not be conclusive because of relatively small sample size and inherent variation among individuals, these observations may reflect hyperglycemia and increased oxidative stress in diabetic patients
Clinical diagnosis of diabetes has been evolving since the diagnostic criteria were first initiated in 1979 by the National Diabetes Data Group report (26)
The glycemic criteria have been based on levels of glucose that associate with microvascular, specifically retinopathic, changes characteristic of diabetes
There are major limitations in the current criteria used for the diagnosis of diabetes
Fasting plasma glucose reflects only one aspect of glucose metabolism, which may be stated as the postabsorptive balance of hepatic glucose production and peripheral glucose uptake
It does not reflect the free-living, daily glycemic patterns, the prolonged fasted state, or the even postprandial state
The oral glucose tolerance test, in addition to being clinically cumbersome, is also nonphysiological (assuming most meal ingestion does not include 75 g concentrated sucrose)
Assessing glucose tolerance with the single measure of plasma glucose 2 h after the oral glucose is therefore of limited usefulness
Another commonly used test is to assay for A1C
A1C values reflect an average glycemic status over several months’ time (27)
A1C assay has been recently proposed as a diagnostic criterion (28)
Perhaps the most apparent functional aspect of O-GlcNAc is its role in regulation of insulin signaling and as a mediator of glucose toxicity (2–15,29)
Increasing global GlcNAcylation in adipocytes or muscle blocks insulin signaling at several points (12,29,30)
Moderately increased UDP-GlcNAc levels in muscle induced insulin resistance (31), whereas overexpression of OGT in muscle or adipose causes insulin resistance and hyperleptinemia in transgenic mice (11)
Diabetes is an extremely complicated syndrome
Although some controversies still exist about the roles of O-GlcNAc in diabetes (32), the results presented in this report along with the rapid cycling nature of GlcNAcylation and its sensitivity toward changes in glucose metabolism give site-specific GlcNAcylation on erythrocyte proteins great potential as biomarker(s) for detecting the early stages of diabetes
Given the exploratory nature of the current study, quantitative measurements were based on relatively small sample sizes
Completion of the discovery phase will be followed by a validation phase, for which targeted high-throughput mass spectrometry will be adopted to determine the prevalence of O-GlcNAc dynamics on preselected sites among a large amount of samples
Polyclonal and monoclonal antibodies against O-GlcNAc on specific sites will be developed and used to screen a large number of samples from normal, pre-diabetic, and diabetic patients to further evaluate the feasibility of this approach
In any case, this study not only has identified important GlcNAcylated proteins and sites of modification in human erythrocytes but also suggests that O-GlcNAc cycling plays a role in erythrocyte biology