Characterization of the specificity of CTD110.6, an O-GlcNAc reactive antibody, under conditions of cellular starvation and stress

Under normal cellular conditions, the modification of nuclear,
cytoplasmic and mitochondrial proteins by O-linked β-
N-acetyl-D-Glucosamine (O-GlcNAc) has been shown to dynamically
modify and regulate over 1500 proteins. O-GlcNAc is analogous
to protein phosphorylation and has been implicated in
regulating signal transduction, protein-protein interactions, transcription,
the cell cycle, nutritional signaling and the cellular stress
response. Detecting changes in the levels of O-GlcNAc can be
assayed using a wide variety of techniques. One commonly used
antibody that recognizes O-GlcNAc, CTD110.6, has recently had
its specificity called into question. It has been reported that under
conditions of glucose starvation, N-glycan biosynthesis is attenuated
resulting in an increase in N-linked chitobiose, which crossreacts
with CTD110.6. These data have suggested that
stress-induced changes in O-GlcNAc may be due to changes in
N-linked chitobiose rather than O-GlcNAcylation. To investigate
this possibility, we have examined CTD110.6 reactivity in response
to a variety of forms of cellular stress in OGT wild-type
and null cells. Our findings confirm that the increased signal we
observe using CTD110.6 during stress treatments are in fact due to
increases in O-GlcNAc and not N-linked chitobiose. As cells
become glucose starved during injuries such as
ischemia-reperfusion injury, we have investigated changes in
O-GlcNAc and chitobiose during times of glucose starvation. Our
studies have confirmed that glucose starvation results in increases
in O-GlcNAc, but that severe nutrient deprivation (glucose/FBS)
results in changes in N-linked chitobiose, which react with
CTD110.6. To delineate between O-GlcNAc and N-linked chitobiose,
we have developed a western blot approach that can be used
to confirm the specificity of a wide variety of O-GlcNAc-specific
antibodies and lectins. This protocol uses a combination of
PNGaseF treatments and on-blot mild β-elimination. Currently, we
are determining which other O-GlcNAc-specific antibodies crossreact
with the N-linked chitobiose modification and are focusing
on identifying the factor(s) in FBS which suppresses the induction
of chitobiose. This project is funded in part by a Program of
Excellence in Glycosciences (P01HL107153).