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Actin and Annexins I and II Are Among the Main
Endothelial Plasmalemma-Associated Proteins Forming
Early Glucose Adducts in Experimental Diabetes
Lucian D. Ghitescu,
1
Alejandro Gugliucci,
1
and France Dumas
2
An immunochemical and biochemical study was per-
formed to reveal which of the endothelial plasma mem-
brane proteins become glycated during the early phases
of diabetes. The blood front of the lung microvascular
endothelial plasmalemma was purified by the cationic
colloidal silica method from normal and diabetic (strep-
tozotocin-induced) rats and comparatively analyzed by
two-dimensional electrophoresis. No major qualitative
differences in the general spectrum of endothelial plas-
malemmal proteins were recorded between normoglyce-
mic and hyperglycemic animals. By probing with anti-
glucitollysine antibodies, we found that at 1 month after
the onset of diabetes, several endothelial membrane
polypeptides contained glucose covalently linked to their
lysyl residues. Ten days of insulin treatment restored
euglycemia in the diabetic animals and completely abol-
ished the membrane nonenzymatic glycosylation. All the
glycated polypeptides of the endothelial plasma mem-
brane belong to the peripheral type and are associated
with its cytoplasmic face (cell cortex). They were solu-
bilized by buffers of high pH and were not detected in
the lung cytosolic fraction (100,000 g). By microse-
quencing, the major proteins labeled by the anti-gluci-
tollysine have been identified as being actin, annexin I,
annexin II, the p34 subunit of the Arp2/3 complex, and
the Ras suppressor protein-1. Conversely, the intrinsic
endothelial membrane proteins do not seem to be af-
fected by hyperglycemia. This defines the internal face
of the endothelial plasma membrane, particularly the
cortical cytoskeleton, as a preferential target for
nonenzymatic glycosylation in diabetes, with possible
consequences on the fluidity of the endothelial plasma-
lemma and impairment of the endothelial mechano-
transducing ability. Diabetes 50:1666 –1674, 2001
The leading cause in the morbidity and mortality
of diabetic patients is related to vascular com-
plications such as atheroma, hypertension, and
microangiopathy. Abnormalities induced by dia-
betes are found at the level of circulating plasma proteins
and blood cells, but they equally affect the components of
the vessel walls (1). The most obvious of these injuries are
encountered in the microcirculation of the retina, skin,
nerves, and kidney, although it is widely accepted that
none of the segments of the vasculature are spared (2,3). It
is therefore plausible to consider that on a general back-
ground of alterations affecting all blood vessels, local
factors exacerbate the disease, conferring clinical and
morphologic particularities to the vasculopathies encoun-
tered in certain tissues.
Most of the endothelial biological responses are trig-
gered by events taking place at the interface between the
blood and the vessel wall, namely at the luminal aspect of
the endothelial plasma membrane. On the premise that
pathological conditions induce modifications at this level,
we have undertaken a study aiming to detect putative
alterations in the endothelial plasmalemmal proteins dur-
ing the early phase of diabetes.
The hallmark of diabetes is hyperglycemia. Depending
on the type of glucose transporters in each tissue, this can
be accompanied by elevated intracellular concentrations
of glucose and glycolytic intermediates. The mechanisms
by which hyperglycemia exerts its detrimental effects have
not yet been elucidated, and several hypotheses are cur-
rently considered (4). Among these, a possible explanation
of the injuries induced to cells and extracellular material
by hyperglycemic conditions stems from the ability of
glucose, as a reducing sugar, to bind to the amino groups
in the proteins. These glucose adducts spontaneously
evolve toward a heterogeneous population of molecules,
generically termed “advanced glycation end products”
(AGEs), mostly characterized by extensive intra- and
intermolecular cross-linking, acquired fluorescence, and
chromogenicity. It is clearly documented that both early
glycated proteins, as well as AGEs, exert deleterious
effects (5,6). A large number of studies have been dedi-
cated to the consequences of the nonenzymatic glyco-
sylation of circulating proteins such as albumin,
immunoglobulins, and lipoproteins (7). Similarly, the
structural and functional changes of the connective tissue
and extracellular matrix have been extensively analyzed,
From the
1
De´partement de Pathologie et Biologie Cellulaire, Universite´de
Montre´al; and the
2
Institut de Recherche en Biotechnologie de Montre´al,
Montre´al, Canada.
A.G. is currently affiliated with Touro University, College of Osteopathic
Medicine, Vallejo, California.
Address correspondence and reprint requests to Lucian Ghitescu, De´parte-
ment de Pathologie et Biologie Cellulaire, Universite´ de Montre´ al, P.O. Box
6128, Succursale Centre-ville, Montre´al (Que´bec) H3C 3J7, Canada. E-mail:
ghitescd@patho.umontreal.ca.
Received for publication 3 April 2000 and accepted in revised form 20 March
2001.
2D, two-dimensional; BSA, bovine serum albumin; ECL, enhanced chemi-
luminescence; IPG, immobilized pH gradients; rsp-1, Ras suppressor protein-1;
PVDF, polyvinylidine fluoride; TFA, trifluoroacetic acid.
1666 DIABETES, VOL. 50, JULY 2001
particularly in the renal glomerulus and lens crystallins
(5,6).
Considerable effort is currently being made to define the
alterations induced by diabetes at the level of cell proteins.
A reduction in the efficacy but not in the number of the
glucose transporters was reported to occur in diabetic rat
brain microvessels (8); conversely, in skeletal muscle
sarcolemma, it was the number of transporters that de-
creased by two-thirds (9). In the rat liver plasma mem-
brane, a one-half reduction in serine proteinase activity
and in epidermal growth factor receptor number was
recorded in experimental diabetes (10,11). This reduction
was reversed by insulin treatment. Intra-endothelial glyca-
tion of the basic fibroblast growth factor was accompanied
by the loss of its mitogenic activity (12). All these results
indicate that as a consequence of hyperglycemia and/or
hypoinsulinemia, cell proteins (or at least some of them)
do change.
Nonenzymatic glycosylation of erythrocyte and platelet
membrane proteins in diabetes has been detected by
chemical assays (13), but so far only a single attempt has
been made to identify these proteins (14) using tritiated
borohydride to reduce and label their early glycosylation
adducts. The ability of the agarose-immobilized aminophe-
nylboronic acid to bind carbohydrates containing a pair of
adjacent cis-configurated hydroxyl groups has been tenta-
tively used to assess the existence of glycated polypep-
tides in the liver and kidney membranes (15) and in the
liver cytosol (16) of diabetic animals. However, this
method is not free of interference from the naturally
occurring glycoconjugates, which are particularly abun-
dant in plasma membrane preparations.
Consequently, we have decided to reveal the endothelial
membrane proteins modified by early glycation by an
alternative method using a monoclonal antibody that
specifically recognizes the glucitollysine residue, irrespec-
tive of the carrier protein (17).
RESEARCH DESIGN AND METHODS
Experimental animal model. Twenty-seven Sprague Dawley male rats, 125 g
body weight (Charles River, St. Constant, QB, Canada), were used throughout
the experiment. One-third was kept as a control, and the rest was rendered
diabetic by a single intraperitoneally injected 9-mg dose of streptozotocin. All
injected rats became diabetic, as revealed by their glycemia, glycosuria, and
ketonuria (Miles, Rexdale, ON, Canada). The animals were fed ad libitum with
standard laboratory diet. After 1 month, three rats from the diabetic lot were
intraperitoneally injected once a day with Humulin U (ultraslow) insulin (Eli
Lilly). The amount of injected insulin (between 5 and 15 U/dose) was adjusted
for each animal so that its glycemia, taken 2 h after each injection, fell to
between 2.8 and 5.2 mmol/l (close to the normal range). This treatment was
continued for 10 days. At 40 days after the initial injection of streptozotocin,
all animals were killed, and the lungs were harvested according to the
procedure described below. Glycemic control and response to the treatment
was further monitored by measuring the extent of plasma protein glycation in
blood samples drawn just before death (using the GlycoTest II kit from Pierce
Chemical). The experiments were approved by the Committee of Deontology
for Experimentations on Animals, Universite´ de Montre´al.
Purification and electrophoretic analysis of the endothelial plasma
membranes. The blood front (luminal domain) of the lung microvascular
endothelial plasma membrane was isolated by the cationic colloidal silica
method as described in detail previously (18). Basically, this method involves
the perfusion of the lung blood vessels with positively charged 20- to 40-nm
colloidal silica particles, adhering by electrostatic interactions to the anionic
sites present in high density on the luminal front of the endothelial cells. The
cationic colloidal silica was prepared according to the method of Channey and
Jacobson (19). The continuous layer of silica attached to the endothelial
surface was hindered from other interactions by a second coat, obtained by
infusing anionic sodium polyacrylate through the vasculature. The endothelial
membranes, rendered very heavy by the attached silica, were separated from
any other cellular organelles by two centrifugation steps of the lung homog-
enate through dense layers of Nycodenz (Life Technologies). The purity of the
isolated membrane fractions, labeled P
2
, was assessed by electron micros-
copy. Throughout the purification protocol and subsequent electrophoretic
procedures, a cocktail of protease inhibitors (1 mmol/l phenylmethylsulfonyl
fluoride, 5 mmol/l benzamidine, and Complete [Boehringer]) was present.
The protein concentration was measured by the bicinchoninic method
(Sigma Chemical). The membranes were solubilized in 1% SDS, sonicated,
boiled, and microfuged, and the supernatants, free of interfering colloidal
silica, were used for the protein assay. As a control for the reproducibility of
the results, endothelial plasma membranes, as well as the cytosol, were
separately purified for each animal and run as individual distinct samples
throughout the subsequent analysis.
The global polypeptide composition of the membranes purified from
normal diabetic and insulin-treated diabetic animals was compared by two-
dimensional (2D) electrophoresis. Immobilized linear pH gradient (IPG)
18-cm pH 3-10L strips (Amersham Pharmacia Biotechnology) were used for
the first dimension, followed by SDS-PAGE in 10% acrylamide (20-cm gel)
reducing conditions (20). For the isoelectric focusing step, the samples were
solubilized in IPG buffers containing 4% CHAPS, 7 mol/l urea, and 2 mol/l
thiourea (21) and loaded concomitantly to the rehydration of the IPG strips
(22). The proteins were revealed by silver staining (23).
For further fractionation, 100 g of each P
2
membrane preparation was
sequentially incubated for 1 h at 4°C with 500 l of 50 mmol/l Na carbonate,
pH 11.0, and 500 l of 1.0 mol/l NaCl in 25 mmol/l HEPES, pH 7.2, and
microfuged to recover the supernatants. Each of these steps solubilized a
distinct set of cytoplasmically attached peripheral membrane proteins (la-
beled S
carb
and S
NaCl
, respectively), leaving as a pellet a membrane fraction
still attached to the silica, named P
3
and highly enriched in integral plasmale-
mmal proteins (24). In preparation for electrophoresis, the soluble fractions
were precipitated with TCA and washed with ethanol/ether; the silica-
attached membranes were concentrated by microcentrifugation. The cytosol
was obtained by spinning 1.5 ml of each lung homogenate at 100,000gfor1h
in a Beckman SW60 rotor (Beckman).
Immunochemical detection of nonenzymatically glycated proteins.
First, a comprehensive immunochemical screening of all the animals used
throughout the experiment was performed to detect whether a consistent
pattern of nonenzymatic glycosylation of endothelial membrane proteins
could be defined for each experimental condition. For each animal, 20 g lung
homogenate, cytosol, and endothelial plasma membrane fractions was re-
solved by SDS-PAGE in reducing conditions (20) using a Minigel system
(Bio-Rad) and electrotransferred to nitrocellulose membranes. The mem-
branes were incubated in 100 mmol/l Na borohydride for1htoirreversibly
transform the glucose-derived early glycation products (ketoamines and
aldimines) into a stable glucitollysine. Alternatively, the borohydride treat-
ment was performed before preparing the samples for electrophoresis to
avoid a possible breakdown of the labile lysyl-glucose adducts during the
electrophoretic run. No differences were recorded between the results ob-
tained with these two variations of the protocol. After quenching with 1%
nonfat dry milk in Tris-buffered saline, the nitrocellulose membranes were
probed with a mouse monoclonal anti-glucitollysine antibody (clone G8C11;
supplied by Drs. L. Curtis and J. Witztum, Scripps Research Institute and
University of California at San Diego, respectively), followed by a horseradish
peroxidase–conjugated anti-mouse immunoglobulin (Ig) (Amersham, Ontario,
Canada), and developed by enhanced chemiluminescence (ECL).
The specificity of the antibody, although previously demonstrated (25), was
further tested by performing immunoblotting on samples of native serum
albumin and glycated bovine serum albumin (BSA)—the latter prepared
according to a published protocol (26). The glycated BSA contained an
average of 2.5 mol glucose residues per 1 mol BSA, as measured by a
thiobarbituric assay.
Once the consistency of the endothelial plasma membrane glycation
pattern was demonstrated in one dimension, the affected polypeptides were
further pinpointed in the 2D electrophoretic spectrum. For this, 125 gofthe
Scarb fraction of endothelial membranes from diabetic rats was precipitated
with methanol/chloroform (27), solubilized in the IPG buffer, and loaded on
13-cm IPG 3-10L strips. After transfer on nitrocellulose, the proteins were
stained with a 15-nm colloidal gold suspension containing 0.1% Tween-20 (28)
and were submitted to the immunoblotting protocol as previously described.
At the end of the immunoblotting sequence, the pattern of proteins on
nitrocellulose was intensified by a silver amplification step (29) and aligned
with the image generated by immunoblotting and ECL.
Identification of the glycated proteins by microsequencing. The major
glycated proteins contained in the Western blotting image were unambigu-
ously identified in the 2D pattern of the Scarb proteins from normal animals.
L.D. GHITESCU, A. GUGLIUCCI, AND F. DUMAS
DIABETES, VOL. 50, JULY 2001 1667
This was possible because of the perfect reproducibility of the 2D electro-
phoretic spectra generated by the IPG technique and because no major
qualitative differences were found between the 2D patterns of endothelial
membranes from diabetic and normal rats.
For microsequencing, 1 mg Scarb proteins from the endothelial mem-
branes of normoglycemic animals was resolved by 2D electrophoresis and
transferred on ProBlott polyvinylidine fluoride (PVDF) membranes (Applied
Biosystems, Foster City, CA) using a 3-(cyclohexylamino)-1-propanesulfonic
acid buffer (30). Selected spots corresponding to major glycated proteins were
cut and submitted to the automated Edman degradation to obtain the
NH
2
-terminal amino acid sequences. This was performed on a 494-cLC-Procise
HS sequencer using the general protocol of Hewick et al. (31). For proteins
found to be NH
2
-terminally blocked, a protocol of digestion “in gel” and
peptide separation were applied. Protein spots were cut out of the Coomassie
Brilliant Blue R-250 lightly stained 2D gel, reduced with dithiothreitol, and
alkylated with iodoacetamide before trypsin digestion (32,33). The enzyme
used was the modified sequencing grade trypsin from Promega. The peptides
were extracted from the gel and separated on a Brownlee HPLC microbore
C18 column (OD-300, 7 m,1⫻50 mm) using an applied Biosystem 130A
Separation System. Peptides were eluted at 150 l/min with the following
gradient program: 0–40 min (0– 80% solvent B) and 40 –57 min (80 –100%
solvent B), with solvent A being 0.1% trifluoroacetic acid (TFA)/H
2
O and
solvent B being 0.08% TFA in 70% acetonitrile/H
2
O. The peptides were
detected by their absorbance at 220 nm. Fractions were adsorbed on a
TFA-treated glass fiber filter disk coated with 0.374 mg polybrene and 0.025
mg NaCl (Biobrene Plus; ABI) before analysis by Edman degradation as
described previously. The sequence fragments found were compared with the
primary structures of known proteins using the BLAST system (34).
Immunochemical confirmation of the microsequencing data. The iden-
tity of the main glycated species was further confirmed by performing
sequential immunoblotting with relevant antibodies on the same nitrocellu-
lose membrane carrying the spots of interest, resolved by 2D electrophoresis.
Besides anti-glucitollysine, the following antibodies were used according to
the previously described protocol: rabbit anti–-actin (Sigma Aldrich Canada,
Oakville, ON, Canada) and goat anti–annexins I and II (Santa Cruz Biotech-
nology, Santa Cruz, CA). The secondary antibodies conjugated to horseradish
peroxidase were from Amersham Pharmacia Biotechnology (anti-mouse and
anti-rabbit Ig) and Dako (Carpinteria, CA) (anti-goat Ig). The immunostaining
was revealed by ECL, and between the incubations with different primary
antibodies, the membranes were stripped in 0.1 mol/l HCl-glycine for 1 h. At
the end, the gold staining was silver-amplified to facilitate the alignment of the
immunoblots with the global spectrum of the proteins.
RESULTS
The level of plasma protein glycation is generally taken as
an assessment of the effect of the glycemic condition,
averaged for periods up to 2 weeks. In our experimental
setting, at the moment of death, glycated plasma proteins
amounted to 3.03 ⫾0.84% (n⫽3) for the diabetic group
vs. 0.96 ⫾0.23% in the control rats (P⬍0.01). In the third
group, comprised of diabetic insulin-treated animals, the
proportion of glycated plasma proteins fell to 1.54 ⫾0.41%
(P⬍0.05 vs. diabetic nontreated rats).
The positively charged colloidal silica particles that
were perfused through the vasculature attached them-
selves to the luminal front of the endothelial cells only
(Fig. 1A, black dots); they did not cross the endothelial
barrier. Consequently, the plasma membrane fractions
obtained by this technique were virtually free of contam-
ination, purely endothelial, and lacked detectable endo-
membranes, as confirmed in the present work by the
electron microscope examination (Fig. 1B) and, in previ-
ous articles, by enzyme and immunochemical assays
(18,35).
This purity translates into a remarkably reproducible 2D
electrophoretic pattern of the membranes (fraction P
2
)
isolated from individual animals. A systematic comparison
between normal and diabetic animals revealed that no
major alterations were induced by diabetes in the global
2D electrophoretic pattern of lung endothelial plasma
membrane proteins. An example of this is presented in Fig.
2. By performing Western blotting with anti-glucitollysine
antibodies, we have searched for glycated proteins in the
purified endothelial membranes of the diabetic animals.
The specificity of these antibodies for the reduced form of
glucose conjugated to the epsilon amino group of lysine
was demonstrated by an immunoblotting test in which
only the glycated albumin, but not its native nonglycated
variant, is labeled (Fig. 3). A clear signal was elicited from
only 0.37 fmol glucose bound to albumin and loaded on gel
(glucose/BSA molar ratio ⫽2.5). This gives a measure of
the sensitivity with which glucitollysine was detected
throughout this study.
In all diabetic animals examined, several P
2
polypep-
tides (220, 74, 57, 42, 36, 34, and 22 kDa of apparent MW)
were found to react with the antibody (Fig. 4, lanes D
1–3
),
indicating that these membrane proteins are the target for
the glucose attachment during the early phases of hyper-
glycemia. Insulin treatment in the diabetic animals com-
pletely abolished the glycation of the membrane proteins
(Fig. 4, lanes T
1–3
) that was also absent in the membranes
isolated from normoglycemic animals (Fig. 4, lanes C
1
and
C
2
).
The glycated proteins found in the endothelial mem-
brane of diabetic animals belong to the cell cortex—
namely the polypeptides electrostatically attached to the
FIG. 1. A: Rat lung perfused with cationic colloidal silica and Na
polyacrylate. Note that silica particles decorate the luminal front of
the endothelial cells only. B: Purified lung endothelial plasma mem-
branes (fraction P
2
). Note the lack of contaminants from other cellular
compartments. A, alveolar space; CL, capillary lumen; Si, silica parti-
cles. Bar equals 2.5 m.
ENDOTHELIAL PLASMA MEMBRANE GLYCATION IN DIABETES
1668 DIABETES, VOL. 50, JULY 2001
cytoplasmic face of the plasma membrane. They were
virtually completely solubilized by the high pH conditions
known to detach peripheral membrane proteins (Fig. 5).
As previously shown (24), none of these carbonate-soluble
proteins are labeled in situ by membrane impermeable
tracers (sulfo-N-hydroxysuccinimidyl ester-biotin [sulfo-
NHS-biotin]), a fact that demonstrates their localization on
the internal cytoplasmic face of the plasmalemma. Surpris-
ingly, no specific glycated species were observed among
the intrinsic endothelial membrane polypeptides, except
for the faint residues left by carbonate solubilization (Fig.
5, lane P
3
).
When the distribution of glycated proteins in diabetic
rats was comparatively assessed in the whole lung homog-
enate, in the cytosolic fraction, and in the purified endo-
thelial membranes (fraction P
2
), it was observed that the
glucose-modified species in the cytosol and in the plasma
membrane are distinct and complementary to each other
in respect to the whole homogenate (Fig. 6).
Spreading the endothelial membrane proteins by 2D
electrophoresis before the immunoblotting step revealed
that some of the major anti-glucitollysine–reactive bands
previously recorded contain a relatively complex distribu-
tion of proteins of similar apparent molecular weight (Fig.
7Aand B). To facilitate the comparison of the traditional
immunoblotting pattern with that generated by the 2D
technique, samples of total Scarb proteins, either directly
solubilized in Laemmli’s buffer (Fig. 7A) or previously
resolved by isoelectric focusing in IPG strips (Fig. 7B),
were simultaneously run in the same 2D gel, transferred,
FIG. 2. Comparative 2D electrophoretic profiles of the endothelial P
2
membrane proteins (100 g/sample) purified from normal (A) and
diabetic (B) rats. Molecular weight (MW) and pI standards (Bio-Rad
kit) have been incorporated in the sample (*, **, ***) for alignment,
comparison of the patterns, and MW/pI scale drawing.
FIG. 3. Test for the specificity of anti-glucitollysine antibodies toward
the glucose (gluc)-modified proteins. BSA (3 g) and decreasing
amounts of nonenzymatically glycated BSA (glucose/protein molar
ratio of 2.5) were submitted to SDS-PAGE (A), transferred to nitro-
cellulose, and probed with the anti-glucitollysine antibodies (B). Only
the protein containing glucitollysine was labeled.
FIG. 4. Nonenzymatically glycated proteins of the endothelial mem-
branes (P
2
fraction) purified from diabetic rats (lanes D
1–3
) revealed
by Western blotting with the anti-glucitollysine antibody: P
2
protein
(20 g) per lane. Note the absence of glycated species in diabetic
animals treated with insulin (lanes T
1–3
) as well as in the control
euglycemic rats (lanes C
1
and C
2
).
L.D. GHITESCU, A. GUGLIUCCI, AND F. DUMAS
DIABETES, VOL. 50, JULY 2001 1669
and probed together with the anti-glucitollysine antibody.
The picture obtained demonstrates that the spectrum of
glycated proteins revealed by SDS-PAGE/immunoblotting
is reproduced without losses and further enriched in
resolution by the 2D electrophoretic approach.
Several major glycated polypeptides, marked from 1 to 7
in Fig. 7B, were pinpointed in the Coomassie-stained
PVDF transfers or in gel, cut out, and identified by
microsequencing. The internal or NH
2
-terminal sequence
fragments found for these proteins presented a high ho-
mology or were identical (Fig. 8) to those of the following
known polypeptides: rat actin (protein 1) (36), rat annexin
I (protein 2) (37), rat annexin II (protein 3) (38), annexin
fragments (proteins 4 and 5) (37), the subunit p34 of the
human actin-related protein (Arp2/3) complex (protein 6)
(39), and human Ras suppressor protein (protein 7) (40).
To further confirm the identity of the glycated species, two
segments of a nitrocellulose membrane carrying the whole
2D spectrum of endothelial Scarb proteins of a diabetic
animal were cut as outlined in Fig. 7 and stained with
colloidal gold. The two membrane fragments were incu-
bated sequentially with anti-glucitollysine antibodies and
anti–-actin (Fig. 7C) or anti-glucitollysine, anti–annexin
II, and anti–annexin I (Fig. 7D), with membrane stripping
in between. The perfect alignment of the spots positive for
these antibodies validated the identities of the glycated
proteins established by microsequencing. Moreover, it
allowed us to conclude that three more spots, positive for
glucitollysine, located on the acidic side of proteins 2 and
3 and previously unchecked by their amino acid sequence,
were also fragments of annexins I and II, respectively
(Figs. 7Band D).
DISCUSSION
The first step of this study addresses the question of
whether diabetes induces major alterations in the spec-
trum of endothelial membrane proteins. Because the nu-
clear factor B, a factor that plays a pivotal role in early
gene responses, was shown to be activated by hypergly-
cemia in cultured endothelial cells (41), we expect that the
endothelial cells from diabetic animals might express new
proteins at their surface. As illustrated in Fig. 2, at least
during the early phases of streptozotocin-induced diabe-
tes, the spectrum of the proteins making the endothelial
surface does not change significantly. However, it must be
stressed that the simple comparison of silver-stained gels,
although entirely adequate for revealing qualitative differ-
ences, has limitations in assessing quantitative variations,
particularly for the low-abundance proteins. In fact, the
literature contains several reports regarding quantitative
variations in the expression of certain endothelial mem-
FIG. 5. The glycated proteins of the endothelial membranes are
peripheral proteins solubilized by the membrane treatment with a high
pH buffer. Forty micrograms of the endothelial membrane P
2
fraction,
of the proteins solubilized by Na carbonate (S
C
) and by 1 mol/l NaCl
(S
NaCl
), and of the endothelial membrane stripped of the peripheral
proteins (fraction P
3
) were resolved by SDS-PAGE. Coomassie blue
staining (A) and Western blotting with anti-glucitollysine (B) are
shown.
FIG. 6. Forty micrograms of protein/lane of homogenate (Hom), cy-
tosol (Cyt), and endothelial plasmalemma (P
2
) of diabetic untreated
animals were separated by SDS-PAGE. Coomassie blue staining (A)
and Western blotting with glucitollysine (B) are shown. Note that in
respect to the homogenate, the cytosolic and plasma membrane gly-
cated proteins exhibit complementary but clearly distinct patterns.
MW, molecular weight.
ENDOTHELIAL PLASMA MEMBRANE GLYCATION IN DIABETES
1670 DIABETES, VOL. 50, JULY 2001
brane proteins. Elevated expression of the cell adhesion
molecules intracellular adhesion molecule-1 and P-selectin
(42) and the endothelin A receptor (43) was recorded in
endothelia from diabetic animals. It was also suggested
that in diabetes, the activity of the endothelial Ca
2⫹
-
dependent protein kinase C is increased (44)—a fact
believed to imply the enzyme translocation from the
cytosol to the plasma membrane.
A second aspect explored in this study regards the
possibility that several of the endothelial membrane
polypeptides might be covalently modified by glucose
during an early phase of hyperglycemia and that these
modifications alter the endothelial membrane functions.
The glycated proteins were detected by immunoblotting
with anti-glucitollysine antibodies. No significant glycation
of total lung or endothelial plasmalemmal proteins was
noticed in normoglycemic animals. On the contrary, in
diabetic rats, clear and reproducible patterns of protein
glucose adducts were revealed (Fig. 4). Some of these
were traced in the purified endothelial plasma membrane
fraction (P
2
) only but not in the cytosol, recommending
them as specific plasmalemmal polypeptides (Fig. 6).
Decreasing glucose concentration in the blood by insulin
treatment was very efficient in canceling nonenzymatic
glycosylation (Fig. 4)—a fact that could be explained by
the turnover of these proteins (⬍10 days, the duration of
insulin treatment).
All the glycated proteins detected in the P
2
fractions
from diabetic animals were solubilized by buffers of high
pH (Fig. 5). This places them in the category of peripheral
membrane proteins, which accommodates factors of the
membrane signal transduction machinery as well as the
elements of the cortical actin cytoskeleton. The identifica-
tion by microsequencing of seven of the major glycated
polypeptides (Fig. 8) has shown that all of these are or are
believed to be related to the organization of the cortical
cytoskeleton. Actin constitutes the very building blocks of
this peri-plasmalemmal network. Annexin I is a calcium-
dependent phospholipid binding protein known to interact
with actin and profilin (45) and is considered an active
player in the regulation of membrane-cytoskeleton inter-
actions. We do not yet know whether the two fragments of
annexin I found among the glycated species (proteins 4
and 5) and beginning at residues 24 and 28, respectively,
are functionally significant or just artifacts produced de-
spite the continuous presence of protease inhibitors
FIG. 7. A total of 200 gofS
carb
proteins from a diabetic rat, previously resolved by isoelectric focusing (B), were loaded on the same 2D
SDS-PAGE gel (B) with another 50-gS
carb
solubilized directly in Laemmli’s buffer (A). After electrophoresis, the whole gel was transferred on
nitrocellulose (NC) stained with colloidal gold and submitted to immunoblotting with anti-glucitollysine antibody. The numbers label the
glucitollysine-positive spots subsequently submitted to identification by microsequencing. From a similar NC membrane, areas of interest were
cut, stained with colloidal gold, and sequentially probed with antibodies in the following order: anti-glucitollysine and anti–-actin (C), and
anti-glucitollysine, anti–annexin II, and anti–annexin I (D), respectively. Note the identity of the position of the proteins identified by
microsequencing and by immunoblotting with the corresponding antibodies. MW, molecular weight.
L.D. GHITESCU, A. GUGLIUCCI, AND F. DUMAS
DIABETES, VOL. 50, JULY 2001 1671
throughout the experimental procedures. Annexin II, a
third major glycated polypeptide, belongs to the same
family of Ca
2⫹
-, phospholipid-, and actin-binding proteins
and has been shown to localize to detergent-resistant
cholesterol-rich membrane microdomains, currently
equated to the caveolae (46). It is considered to be
implicated in the regulation of the vesicular traffic (47).
Along with these major glycated species in the lung
endothelial plasma membrane, two other proteins, which
are significantly less abundant, have been identified as
targets for nonenzymatic glycosylation. The actin-related
protein (Arp2/3) complex is recognized as a multifunc-
tional actin organizer. It caps the pointed end of actin
filaments and provides nucleation sites for actin polymer-
ization (48). The Arp2/3 complex contains seven subunits,
and one of these, p34-Arc, was found to be glycated
(protein 5). Finally, a relatively novel peptide, Ras sup-
pressor protein-1 (rsp-1), was also identified as carrying
glucitollysine residues (protein 7). Although relatively
little is known about the functional relationships of this
molecule within the cell, rsp-1 is highly homologous to a
Drosophila fli-1 gene product possessing a gelsolin-like
domain (48). It has been suggested that rsp-1 modulates
Ras signal transduction (49), and this can be, at least
speculatively, related to the fact that Ras proteins are
implicated in the regulation of the actin cytoskeleton.
Glucitollysine represents a marker for only the first
stage of a continuous process. Before being removed
through protein turnover, the intracellular Amadori prod-
ucts can evolve into AGEs—some of them intermediates
and others genuine end products. AGEs are known to
exert deleterious effects on proteins by altering their
charge and therefore their conformation, by promoting
free radical–mediated oxidation leading to molecular frag-
mentation or blocking of metal-associated enzymes, and
by forming irreversible intra- and intermolecular cross-
links. AGE content increases dramatically in endothelial
cells after only 1 week of growth in hyperglycemic condi-
tions (12). The rapidity of this process was attributed to
the involvement of glycolytic intermediates, which are
significantly more reactive than glucose. Although AGEs
could be formed through pathways that do not involve
Amadori products (50,51), it has been shown that the latter
are the most significant precursor of AGEs in vivo (52). It
is therefore plausible to infer that the glycated membrane
proteins detected with anti-glucitollysine antibodies dur-
ing the early stage of diabetes also contain intermediate
and late glycation products. At least at this stage, they do
not seem to contain extensive intermolecular cross-links,
as suggested by the lack of shifts toward the high molec-
ular weight of actin or annexin I– and annexin II–positive
bands. The disappearance of the immunocytochemical
signal for glucitollysine in diabetic rats treated for 10 days
with insulin might be interpreted as the result of the
protein turnover as well as the effect of the gradual
evolution of these toward AGEs at the expense of early
glycation products.
The picture emerging from our data is that the proteins
involved in the organization of the cortical actin cytoskel-
eton represent the preferential target for nonenzymatic
glycosylation in early diabetes. The link between this
finding and the reported decrease in the plasma membrane
fluidity of several cell types in diabetes (53,54) appears
logical, particularly in light of the fact that the diabetes-
induced rigidity of plasmalemma could not be explained
by meaningful modifications of its lipid composition (13).
Membrane fluidity is very important in the physiology of
endothelial cells, as it modulates the sensitivity of the
stretch-activated ion channels in mechanotransduction
and controls the mechanism of nitric oxide synthase
activation by the shear stress. An alternative hypothetical
mechanism might be drawn from the colocalization of
actin, annexin II, and nitric oxide synthase at the caveolar
microdomains of the endothelial plasma membrane. Ama-
dori products (in this case from actin and annexin) can
release superoxide anion (55), which in turn can generate
other highly reactive free radicals that are able to induce
protein fragmentation and lipid peroxidation in the prox-
imity of their site of genesis. Moreover, superoxide anion
as well as actin- and annexin II–based AGEs could quench
nitric oxide directly. We therefore advance the idea that in
hyperglycemic conditions, the glycation of the actin cy-
toskeleton associated with the endothelial plasma mem-
brane might be responsible for the diabetes-induced
impairment of the endothelial cell’s ability to regulate
vascular tone.
FIG. 8. Matching of the sequence fragments found
for the glycated proteins with primary structures
of known proteins found in databases. Rat annex-
in II and the p34 subunit of the Arp2/complex
were identified by NH
2
-terminal microsequencing.
The others, which were NH
2
-terminally blocked,
were digested with trypsin, except protein 1, which
was fragmented by cyanogen bromide (*). All se-
quences beginning after K, R (trypsin cutting site),
or M (CNBr cut) are internal sequences (§). The
others represent NH
2
-terminal sequences (**).
ENDOTHELIAL PLASMA MEMBRANE GLYCATION IN DIABETES
1672 DIABETES, VOL. 50, JULY 2001
ACKNOWLEDGMENTS
This work was supported by the Natural Science and
Engineering Research Council of Canada and Association
Diabe`te Que´ bec grants to L.D.G.
The excellent technical help of Rozica Bolovan, Diane
Gingras, and Jean Leveille´ is gratefully acknowledged. The
anti-glucitollysine antibody was supplied by Drs. Linda
Curtis (Scripps Research Institute, La Jolla, CA) and
Joseph Witztum (University of California at San Diego).
We also thank Dr. Moise Bendayan (Universite´ de Mon-
tre´al) for his encouragement and for critically reading the
manuscript.
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