glyc1

Biochimica et Biophysica Acta 1762 (2006) 94 – 102 http://www.elsevier.com/locate/bba

Early glycation products of endothelial plasma membrane proteins in experimental diabetes Sarah Nguyen, Mirela Pascariu, Lucian Ghitescu * Department of Pathology and Cell Biology, Universite´ de Montre´al, CP 6128 Succ. Centre-Ville, Montre´al, Que´bec, Canada H3C 3J7 Received 3 June 2005; received in revised form 9 August 2005; accepted 11 August 2005 Available online 24 August 2005

Abstract The participation of glucose and two intermediates of glucose metabolism: glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P) to the formation of early glycation products was comparatively evaluated in the endothelial plasma membrane of streptozotocin-induced diabetic rats. Antibodies risen to a carrier protein reductively glycated by each of the sugars mentioned above were used to probe by immunoblotting the proteins of the lung microvascular endothelium plasmalemma purified from normal and diabetic rats. The amount of glycated endothelial plasma membrane proteins was below the limit of detection in normoglycemic animals but increased dramatically in diabetic animals for glucose and G6P. In contrast, no signal was found in diabetic rats for Gald3P, indicating that either the contribution of this phosphotriose to the glycation of intracellular proteins is negligible in vivo, or the Schiff base generated by this sugar transforms very rapidly into products of advanced glycation. Globally, the endothelial plasma membrane proteins bound on average 300 times more glucose than G6P proving that, in spite of its low in vitro potency as glycating agent, glucose represents the main contributor to the intracellular formation of early glycation products. The most abundant glycated proteins of the lung endothelial plasma membrane were separated by two dimensional electrophoresis and identified by mass spectrometry.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Endothelium; Plasma membrane; Glycation; Protein; Diabetes

1. Introduction Reducing sugars such as glucose are able to bind covalently to the free amino residues of proteins—a process called glycation. Protein glycation is a reaction proceeding in steps, in the absence of any enzyme intervention. The first product is a labile Schiff base that is rearranged over a period of days into the more stable, Amadori products. These sugar-peptide adducts are known as ‘‘early glycation products’’. Through oxidation and fragmentation, the Amadori products evolve gradually towards a heterogeneous and still incompletely characterized population of macromolecules, labeled ‘‘advanced glycation end products’’ or AGEs [1]. These altered proteins carry highly reactive free radicals and may contain extensive intra and intermolecular cross-links. As a nonenzymatic process, the extent of the protein glycation depends

* Corresponding author.

E-mail address: ghitescd@patho.umontreal.ca (L. Ghitescu).

0925-4439/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2005.08.004

essentially on the concentration of sugar; it is therefore minimal in physiological, normoglycemic conditions, but increases dramatically in both types of diabetes, where hyperglycemia greatly accelerates the reaction [2].

Both early and advanced glycation products are seen as important players in the mechanism of diabetic vascular complications. Numerous studies have been focused on the detrimental role of glycation on the physiology of endothelial cells; either glycated extracellular matrix components [3] or AGE-modified circulating proteins [4] have been implicated.

Comparatively, very little is known about the glycation of intracellular endothelial proteins, although the general view is that this process might alter their functions. Identification of the intracellular glycated proteins of endothelium appears therefore as an important step for understanding the complexity of the diabetes-induced endothelial dysfunction.

Studies performed in vitro have shown that glucose is a relatively weak glycation agent while other reducing sugars, such as the intermediates of the glucose metabolism: glucose6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P),

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are significantly more potent. Consequently, it was inferred that these sugars would dominate the process of intracellular proteins glycation [5– 8].

The goal of the present investigation was to evaluate comparatively the contribution of glucose, G6P and Gald3P to the glycation of the endothelial plasma membrane proteins and to identify the targeted proteins. We choose to study this cellular compartment because it plays a crucial role in the majority of the endothelial/vascular functions: control of the vascular tone and of the coagulation, diapedesis of leucocytes, selection and transmural transport of plasma macromolecules.

This study was performed on lung microvascular endothelium. The richness of the pulmonary vascular bed yields enough endothelial plasma membrane to allow the handling of each animal in experience as an independent sample. The data in the literature on endothelial glucose transporters indicate that in diabetes all types of endothelial cells are loaded with glucose [9– 11]. Therefore, it was plausible to assume that a nonenzymatic process such as protein glycation progresses similarly in all endothelia; in this perspective, the lung endothelium represents a convenient model of investigating this process.

2. Materials and methods 2.1. Preparation of antibodies recognizing early glycation products of proteins Keyhole limpet hemocyanin (KLH) was glycated in sterile conditions with glucose, G6P or Gald3P (Sigma Aldrich, St. Louis MO). Sodium cyanoborohydride (150 mM) present throughout the incubation transformed all the labile early glycation products into stable epitopes (mostly glucitollysine, glucitol-6phosphate lysine and glyceraldehyde-3-phosphate lysine) that allow the subsequent immunodetection of the proteins glycated by each of the three sugars [12]. The glycated KLH was used as an immunogen to generate rabbit polyclonal antibodies against the adducts of lysine with each of the three sugars listed above. The anti KLH-glucose, KLH-G6P and KLH-Gald3P immunoglobulins were purified on Protein A-Sepharose (Amersham-Pharmacia Biotechnol., Piscataway, NJ) and cleared of the anti-carrier (KLH) antibodies on an immobilized KLH-Sepharose column (Sigma Aldrich).

The ability of these antibodies to recognize selectively the corresponding sugar haptens on proteins different from the immunogen was tested on glycated bovine serum albumin (BSA). BSA fraction V (Sigma Aldrich) was glycated in reductive conditions with glucose, fructose, G6P and Gald3P, respectively.

BSA reductively modified by carboxymethyllysine (CML) and carboxyethyllysine (CEL), two AGEs-characteristic epitopes, was prepared according to Nagai et al. [13]. The unbound sugar was removed by extensive dialysis against PBS, the concentration of glycated BSA was evaluated spectrophotometrically (A 280 nm = 0,7 for 1 mg/ml BSA) and the extent of glycation was determined in each sample by the fluorescamine method [14]. The number of sugar residues introduced by glycation per BSA molecule was calculated by taking into account that each native BSA molecule has 59 available lysine ( NH2 groups.

An average sugar/BSA molar ratio of 57, 14.7, 46.6 and 8.3 was found for BSA-glucose, BSA-fructose, BSA-G6P and BSA-Gald3P respectively. The degree of substitution of amino residues for BSA-CML and BSA-CEL was of 84% and 98%, respectively.

To assess the specificity and the sensitivity of the antibodies to particular sugar—lysine adducts, native, unglycated BSA was mixed with each of the glycated BSA variants in proportions keeping the total protein concentration constant (1 mg/ml) but serially decreasing the amount of bound sugar from 128 to 1 nmol/ml. Parallel rows of dots were made by absorbing 1 Al of the above serial samples on nitrocellulose membranes that were subsequently blocked for 1 h in 1% non-fat dry milk and probed separately with each of the antibodies described. The primary antibody was followed by anti-rabbit Ig-HRP (Jackson

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ImmunoResearch; West Grove, PA) and the signal was detected by enhanced chemiluminescence (ECL kit, Roche Diagnostics BM).

2.2. Animal model Six male Sprague – Dawley rats, 125 g body weight (Charles River; StLaurent, QC) were rendered diabetic by a single i.p. injection of streptozotocin (STZ) (70 mg/kg of body weight) and maintained for a period of 2 months.

Their glycemia was tested with a GlycoTest II kit (Roche Diagnostics, Laval, QC). An equal number of age-matched rats were used as normoglycemic controls. All animals were permitted free access to a standard diet and water.

The following parameters were measured at the time of sacrifice for validating the diabetic state of the animals in the experimental lot : body weight (289 T 40 g for diabetic and 392 T 41 g for control rats), glycemia (>29 mM vs. 7.1 T 0.8 mM), and the percentage of glycated plasma proteins measured by the aminophenylboronate affinity chromatography [15] (2.6% T 0.3 in the diabetic vs. 1.1% T 0.1 in the normal lot). The experiments were approved by the institutional Committee of Deontology for the Experimentation on Animals.

2.3. Immunochemical detection of the glycated proteins of the endothelial plasma membrane The apical aspect (blood front) of the endothelial plasma membrane was isolated from the rat lung microvasculature by the cationic colloidal silica method [16,17]. Protein concentration was measured in quadruplicates by the Amidoblack method [18] in samples previously solubilized in 1% SDS. The glycation of the endothelial membrane (operationally labeled P2 fraction) proteins by glucose, G6P and Gald3P was first explored using onedimensional electrophoresis and immunoblotting with the corresponding antibodies. For each animal, normal and diabetic, 30 Ag P2 were resolved by SDS-PAGE. The proteins were electrotransferred to nitrocellulose and the membranes were incubated in 50 mM sodium borohydride (Sigma Aldrich) for 15 min to irreversibly transform the early glycation products into the stable forms: glucitollysine, G6P-lysine and Gald3P-lysine, detectable by our antibodies. The membranes were blocked in 1% defatted milk and probed with the polyclonal anti-glucitollysine (1:2000 dilution), anti-G6P-lysine (1:200) and anti-Gald3P-lysine (1:500) antibodies overnight, followed by 1h incubation with a 1:10.000 dilution of anti-rabbit Ig-HRP and development

by ECL. To comparatively evaluate the extent of glycation by each of the sugars taken into consideration, a semi-quantitative immunochemical assay was employed. As the previously described immunoblotting experiment did not detect any early glycation products made by Gald3P, the quantification assay was performed for glucose and G6P only. Five to twenty micrograms of total lung endothelial membrane proteins purified from diabetic rats (P2 fraction), solubilized in 1% SDS, were absorbed on nitrocellulose as dots along with standards made of serial mixtures of BSA and reductively glycated BSA, as described in the first paragraph. The glucitollysine and G6P-lysine epitopes were revealed by immunoblotting with the corresponding antibodies and the intensity of the signal was measured on film by densitometry for each spot using the Scion Image program (Scion Corp, Frederick, MA). A standard curve was created for each hapten, and the extent of glycation of the P2 proteins was evaluated by interpolation.

2.4. Two dimension electrophoretic separation and mass spectrometry identification of the glycated proteins Eight hundred micrograms of lung endothelial plasma membrane proteins from a control, normoglycemic animal were solubilized for isoelectric focussing in 20 mM Tris buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 40 mM dithioerythriol and 2% ampholytes mixture for immobilized 4 – 7 pH gradient [19]. After removal of the silica nanoparticles by microcentrifugation, the proteins were loaded onto an Immobiline Dry strip pH 4 – 7, 18 cm (Amersham Biosciences, Piscataway, NJ). The first dimension run was followed by SDS-PAGE in a 10% acrylamide, 20-cm gel in the second dimension. The gel was stained by Sypro Ruby (Molecular Probes, Eugene, OR) and scanned (BioRad Molecular Imager ProPlusFX) to obtain the global

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pattern of endothelial membrane proteins. Two other samples (200 Ag each), this time from a diabetic rat, were resolved by 2D electrophoresis in identical conditions, electrotransferred to nitrocellulose, reduced by NaBH4, and stained with Sypro Ruby Protein Blot Stain (BioRad Laboratories Canada, Mississauga, ON) before being separately submitted to immunoblotting with antiglucitollysine and anti-G6P-lysine antibodies, as described above. The comparisons between the three patterns (immunoblot, Sypro Ruby on nitrocellulose and Sypro Ruby in gel) allowed for an unambiguous identification of the position, in the gel, of each glycated polypeptide, within the spectrum of all endothelial membrane proteins. This operation was facilitated by the lack of qualitative differences in the 2D electrophoretic pattern of endothelial membranes of normo- and hyperglycaemic rats. To check for reproducibility, this experiment was repeated three times using different, independently purified endothelial plasma membrane from normal and diabetic animals.

The major spots corresponding to the glycated proteins were manually cut out of the Sypro Ruby stained gel using a biopsy needle and stored in 1% acetic acid. The proteins contained were digested with trypsin (6 ng/Al) for 5 h on a MassPrep robotic workstation (Micromass). Peptides were extracted in a final volume of 45 Al of 0.5% formic acid (v/v) and 9% acetonitrile (v/v).

Analysis of extracted tryptic peptides by LC-QTOF mass spectrometry (MS) was performed by applying the samples to a reverse phase column (10 cm by 75 Am PicoFrit column filled with BioBasic C18). The column bound material was eluted in-line at 200 nl/min, over a 30-min interval, with a linear gradient of 10 – 95% acetonitrile containing 0.1% formic acid throughout. A 2000-V charge was applied to the PicoFrit column such that the eluted peptides were electrosprayed into a capillary liquid chromatography quadrupole time-offlight MS (LC-Micro Q-TOF) (Waters). Double or triple charged detected ions were selected for passage into a collision cell where fragmentation was induced by collision with argon gas. Fragmentation data were collected in 1 s. scans for up to 5 s. The MS/MS data were peaklisted with Masslynx (MicroMass) and submitted to Mascot (MatrixScience) software for database search analysis against the NCBI non-redundant database. The MALDI MS survey scans were used to generate peak lists for peptide map searches using the peptide mass fingerprint search option in Mascot.

Fig. 1. The sensitivity and specificity of the antibodies against glucitol-lysine (A), glucitol-6-phosphate lysine (B), and glyceraldehyde-3-phosphate lysine (C) evaluated by immunoblotting of serum albumin glycated by various sugars.

Each dot contains 15 pmol (1Ag) BSA and varying amounts (1 – 128 pmol) of bound sugars.

3. Results The specificity and sensitivity of the antibodies obtained from rabbits immunized with glycated KLH was evaluated by a dot blot assay, against BSA reductively glycated with glucose, fructose, G6P, Gald3P, CML and CEL (Fig. 1). The test demonstrated that the antibodies showed a high degree of specificity towards their corresponding epitopes. No reaction existed between the antibodies and native, un-glycated BSA. Also, there were no reactions between our anti-glucitollysine antibody with G6P-BSA, the anti-G6P-lysine antibody with glucose-BSA, and our anti Gald3P-lysine antibody with BSA glycated by either glucose or G6P. Both anti-glucitollysine and anti-G6P-lysine show a slight cross-reaction with Gald3P-BSA but, as it will be seen further on, this does not alter the validity of the results and conclusions. No reaction was recorded between any of the three antibodies and BSA glycated with fructose or carrying AGE epitopes (CML and CEL). Identical results were obtained when the test was repeated with glycated hemoglobin (not shown), stressing the fact that our antibodies recognized the early-glycation related epitopes irrespective of their carrier protein.

It has been previously documented by others [16] and by us [17] that the cationic colloidal silica method yields very highly purified preparations of lung endothelial plasma membranes (luminal or blood front). The proteins of these membranes, separately purified from each animal, were

resolved by SDS-PAGE and comparatively probed by Western blotting with each of the previously described antibodies. Within each of the two experimental groups (diabetic and control), the results were the same, so Fig. 2 illustrates a representative pair from these lots only. The silver-stained SDS-PAGE gel shows a virtually identical profile for endothelial plasma membrane proteins from normal and hyperglycemic rats (Fig. 2A). However, significant differences between these two categories are detected at the level of protein glycation. Effectively, no signal for glycation was detected in the membranes obtained from normal rats and incubated with anti-glucitollysine antibodies (Fig. 2B), and only two bands of relatively low intensity were revealed by both the anti-G6P-lysine and anti-Gald3P-lysine (Fig. 2C and D). In contrast, a high level of glycation was exposed by the anti-glucitollysine and anti-G6P-lysine antibodies in endothelial membranes purified from hyperglycemic animals. The pattern of bands produced by these two antibodies in samples from diabetic rats is practically the same, with a significantly more intense reaction for anti-glucitollysine (Fig. 2B and C).

Surprisingly, there was a complete absence of signal when the P2 fraction from diabetic rats was probed with the antiGald3P-lysine antibody (Fig. 2D).

In order to assess comparatively the level of early glycation products generated among endothelial plasma membrane proteins by each of the three sugars under study, a semi-

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Fig. 2. Representative silver-stained electrophoretic pattern of the endothelial plasma membrane fractions purified from a normal (N) and a diabetic (D) rat (A). Western blotting of the same samples with anti-glucitollysine (B), antiG6P-lysine (C) and anti-Gald3P-lysine antibodies (D).

quantitative assay was employed. This assay compares the intensity of the western blotting signal recorded for each of the corresponding antibodies with that of a series of standards made from BSA carrying various amounts of sugar-lysyl residues (Fig. 3, insets). We found a linear relationship between the intensity of the immunochemical signal and the amount of sugar in the domain encompassing the signal revealed for the P2 proteins (Fig. 3A and B). By interpolation, we have calculated that at 2 months of diabetes the rat lung endothelial plasma membrane proteins carry simultaneously, on average, 1900 pmol of glucose and only 6.5 pmol of G6P per mg of protein (Fig. 3C). As previously mentioned, virtually no products of early glycation by Gald3P were detected among the endothelial plasmalemmal proteins.

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In order to identify which are the lung endothelial plasma membrane proteins forming early glycation products with glucose and G6P, the P2 fraction was resolved by 2D electrophoresis and probed with the corresponding antibodies.

The vast majority of the glycated proteins of this fraction are distributed in the acidic part of the pI spectrum [20], so the isoelectric focusing was performed, for a greater resolution, in the narrower pH range of 4 –7. Gel staining with either silver nitrate or Sypro Ruby Red (Fig. 4A) revealed, in this domain, more than five hundred individual spots forming a reproducible 2D electrophoretic pattern of rat lung endothelial plasma membrane proteins. This spectrum of spots was present in all samples, independently purified from various individual animals (n = 6), and the relative position of the spots was unchanged when the endothelial plasma membrane fraction from normal rats was compared to that from diabetic rats, or when the two were co-migrated in the same gel (not shown).

The glycated proteins revealed by western blotting with either anti-glucitollysine or anti-G6P-lysine antibodies represent only a fraction of the global spectrum of lung endothelial plasmalemma proteins. Less than one hundred individual spots or spot clusters were found to be positive for glucitollysine (Fig. 4B) and only about fifty responded to the anti-G6P-lysine antibody (Fig. 4C). Except the spots labeled 7a –c in Fig. 4C, all polypeptides glycated by G6P contain glucose adducts too.

In spite of the higher sensitivity of the anti-G6P-lysine antibody (Fig. 1), the intensity of the immunochemical signal was significantly higher for glucitollysine, indicating to us that glucose was the major intracellular glycating reagent.

The 2D electrophoretic pattern of endothelial plasma membrane proteins from normal and diabetic rats were consistently identical. No variations in the position of the

Fig. 3. Semi-quantitative evaluation of the extent of protein glycation in the lung endothelial plasma membrane preparations from diabetic rats. Total proteins of lung endothelial plasma membrane were adsorbed as dots on a nitrocellulose membrane along a series of standards (glycated BSA containing various amounts of bound glucose or glucose-6-phosphate). The membranes were incubated with anti-glucitollysine (A) or anti-glucitol-6-phosphate lysine (B) and developed by ECL (insets).

The intensity of the signal was measured by densitometry for each spot, used to construct a standard curve for each hapten (A) and (B), and calculate by interpolation the level of global protein glycation (C). Filled circles: the optical density (arbitrary units) of the standards; open squares: the photometric readings for the membrane proteins.

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Fig. 4. The spectrum of rat lung endothelial plasma membrane proteins comprised between pI 4.0 and 7.0. In gel staining with Sypro Ruby Red (A). Identification of the glycated endothelial plasma membrane proteins by immunoblotting with anti-glycitol-lysine (B) and anti-glucitol-6-phosphate-lysine antibodies (C).

spots between the samples containing glycated or nonglycated proteins were ever recorded, and the images of the two could be perfectly overlapped. This, in conjunction with the relative simplicity of the electrophoretic pattern, allowed us to select and excise the spots corresponding to 29 major endothelial proteins found to be glycated in diabetes. We had to ignore several other spots and spot clusters that, in spite of exhibiting a strong signal for glucitollysine (Fig. 4B, upper left quadrant), were present in amounts too small for their identification in the Sypro Ruby Red stained gels. Mass spectrometry analysis of the selected polypeptides (Fig. 4A, encircled spots) has produced a list of rat lung endothelial plasma membrane proteins susceptible to glycation in a hyperglycemic condition (Table 1). Three of these – dipeptidyl peptidase IV, integrin alpha 3 and chloride intracellular channel 5 – were integral membrane proteins. The majority of the glycated proteins were found to be cytoskeletal, belonging to the cell cortex, and known or putative peripheral plasma membrane proteins. Only three polypeptides could be bona fide considered as contami-

nants, either from the circulation (albumin) or extracellular matrix origin (laminin and collagen type VI).

4. Discussion The ability of reducing sugars to bind covalently and nonenzymatically to proteins depends on their concentration and on the proportion at which these sugars are present in an acyclic form [6]. Studies performed in vitro have shown that the formation of Schiff bases between proteins and G6P proceeds about ten times faster than for glucose, and that Gald3P can glycate in the same time interval over 200 times more aminoacid residues as the equimolar amounts of glucose [5,21]. Consequently, glucose is considered as a weak glycating sugar although, in the hyperglycemic condition of diabetes, glucose is the glycation agent for plasma and extracellular matrix proteins.

Glucose transport across the lung microvascular endothelium plasma membrane is facilitated by the insulin-insensitive,

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Table 1 List of the glycated proteins on the rat lung endothelial plasma membrane Spot no.

MS-identified proteins

Accession number

No. of peptides

MW (Da) calculated

pI calculated

1 2 3a 3b 4a 4b 4c 4d 5a 5b 5c 5d 6a 6b 7a 7b 7c 8a 8b 9 10a 10b 11a 11b 11c 11d 11e 11f 11g 11h 11i 11j 12 13 14 15a 15b 16 17 18 19a 19b 20 21a 21b 22a 22b 23 24a 24b 25 26 27 28a 28b 29a 29b

Laminin gamma 1 chain precursor Procollagen type VI alpha 1 Procollagen type VI alpha 2

XP341134 NP034063 XP342116

172481 109562 122067

5.12 5.2 6.00

Dipeptidyl-peptidase IV membrane-bound form

P14740

89090

5.57

Similar to Integrin alpha 3

XP340885

120843

6.43

Gelsolin

NM001004080

86067

5.75

Ezrin

AAR91694

69390

5.83

Heat shock cognate protein 70

NP077327

70871

5.37

Annexin VI Albumin

P48037 P02770

75754 70670

5.39 6.09

Moesin/Radixin

AAB61666/AAR91693

69405

5.83

Chaperonin containing TCP-1 (5() Tubulin alpha 1 Vimentin Similar to Septin 8 protein

AAH79441 AAH62238 P31000 XP220423

59955 50135 53601 53003

5.81 4.94 5.06 5.82

ARP 3 homolog Tubulin, beta Actin, beta TPA: type I keratin KA19

XM341113 CAE84031 P60711 DAA04480

55287 50096 41736 44635

8.85 4.78 5.29 5.21

Septin 2 Eukaryotic translation initiation factor 2, Subunit 1 (alpha) Protein phosphatase 1 subunits h/ y/ g

NP476489 NP062229

41592 36108

6.15 5.02

37248

5.84

Annexin I Annexin II

P07150 NP063970

39147 38939

6.97 7.55

Annexin V Annexin IV Annexin III Chloride intracellular channel 5

2RAN NP077069 LURT3 Q9EPT8

35458 35848 36363 28298

4.97 5.31 5.96 5.64

Similar to Polymerase I transcript release factor

XP220982

3 12 3 3 7 9 7 7 8 7 6 10 2 8 12 8 13 6 12 20 1 1 10 3 3 38 10 15 12 12 17 16 6 8 14 4 2 8 9 10 13 8 3 3 4 4 13 14 10 12 22 4 4 11 4 4 2

43908

5.43

NP037197/1703464D/I76573

low affinity and high capacity GLUT2. As the expression of this transporter is not down regulated in streptozotocin-induced diabetic rats (our unpublished results), the glucose concentration is expected to be significantly higher than normal in the cytosol of the pulmonary endothelium of hyperglycemic

animals. No direct measurement of the sugar at this location was ever made, but the assertion above is eloquently supported by the high level of endothelial protein glycation by glucose in diabetic rats (Fig. 2B). It is plausible to infer that in diabetes, the increased availability of the substrate (glucose) accelerates

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the glycolysis and increases the level of glycolytic intermediates such as G6P and Gald3P. It was therefore of a particular interest to assess the involvement of all these sugars in the glycation of intracellular proteins.

Despite the evidence that G6P and Gald3P are capable of glycating proteins to a far greater extent than glucose in vitro, our results point to glucose as the most important intracellular glycating sugar. In the diabetic animals, more endothelial proteins were glycated, and in far more extent, by glucose than

by G6P; virtually no protein modification by Gald3P was recorded. A possible explanation for the discrepancy in levels of glycation by the three sugars might reside in their intracellular concentration. Our results point to a moderate increase in G6P leading to the glycation of endothelial proteins by this hexosephosphate (Fig. 2C), although at a level approximately 300 times lower than that made by glucose (Fig. 3C). The similarity of the electrophoretic patterns of proteins carrying glucitollysine and G6P-lysine (Fig. 2B and C and Fig. 4B and C) indicates that the two sugars compete for the same polypeptides.

The surprising lack of detection, in the diabetic rats, of lung endothelial plasma membrane proteins glycated by Gald3P cannot be explained by a lack of reactivity of these proteins with that particular phosphotriose. When endothelial plasma membrane fractions isolated from normoglycemic animals were incubated in vitro, in non reducing conditions, with Gald3P a substantial increase in the level of glycation by Gald3P was recorded, well correlated with the time of incubation (not shown). Also, the ability to detect early glycation products of Gald3P in vitro makes less plausible the hypothesis that Gald3P-protein adducts progress so rapidly towards AGEs [22] that they do not accumulate above the detection limit of the method we used. In our view, the only plausible explanation for the lack of detectable early Gald3P glycated products in diabetic rats is the fact that, in spite of a rise in intracellular level of glucose and G6P, the concentration of Gald3P in the endothelial cells remains low. The competition between these sugars might well account for the disappearance of the two faint Gald3P positive bands recorded among the endothelial membrane proteins of normoglycemic rats (Fig. 2D). To our knowledge, very few systematic measurements of the phosphorylated intermediates of the glycolysis have been made in nucleated cells, and none concerns endothelial cells. In rat erythrocytes, the concentration of Gald3P is hundred of times smaller than that of glucose [23]. Interestingly, the red blood cells of diabetic patients contain double the amount of hexosephosphates (G6P, fructose-6-phosphate and fructose1,6 –bis-phosphate) found in the erythrocytes of normoglycemic subject, but the level of the triosephosphates remains unaltered [24]. Therefore, in spite of its well-documented, high glycation potency in vitro, Gald3P does not appear to participate significantly to the in vivo formation of early glycation products with intracellular proteins.

We have previously shown that the majority of the endothelial plasma membrane proteins targeted by glycation are peripheral, plasmalemma-associated polypeptides [20].

Seventeen of the 29 proteins identified in this study as being

glycated belong indeed to this category. Six forms of annexins (I to VI) each defined by several unique peptides were found among the glycated polypeptides. Annexins are known to be involved in a large variety of cell structure and physiology aspects (reviewed in [25]). They seem to act as organizers/ stabilizers of the plasma membrane lipid microdomains or rafts (annexins II and VI), as a link between the membrane and the cortical cytoskeleton (annexin II); they also appear to be involved in exo- and endocytosis (annexins I, II, V and VI) by modulating vesicle docking and fusion, and presumably, able to form transmembrane ion channels [26]. Directly concerning the vascular endothelial functions is the anti-inflammatory contribution of annexin I as a downregulator of the leukocyte extravasation [27], as well as the status of a pro-fibrinolytic, simultaneous receptor for plasminogen and tissue plasminogen activator for annexin II [28]. Finding that these proteins are all modified by glucose and G6P in hyperglycaemic conditions opens to future investigations the question whether there is any relationship between the glycation of annexins and diabetesassociated endothelial dysfunction.

Elements of the plasma membrane-associated cytoskeleton also represent an important target for nonenzymatic glycosylation. The proteins of the microfilaments (actin h, and actin related protein 3) and their associated proteins (gelsolin and septins 2 and 8) and well as those of the microtubules (tubulin a and h) and intermediate filaments (vimentin and cytokeratin K19) were found to be all glycated by glucose and G6P. Ezrin, moesin and radixin are members of a family of proteins involved in the functional association between the cell surface and the cytoskeleton [29]. The large polymorphism of these three proteins on the isoelectric focussing scale (Fig. 4) is not surprising; up to 12 consensus sites for serine/threonine phosphorylation are present in the sequence of moesin. Finding that in diabetes many proteins of the endothelial cell cortex are targeted by glycation allows us to hypothesize that this process might be at the origin of the documented hyperglycemiaassociated alterations of the plasma membrane fluidity [30,31].

A group of five other glycated polypeptides – heat shock cognate protein 70 (Hsc70), T-complex-containing protein 1 (TCP-1), eukaryotic translation initiation factor 2, protein phosphatase 1 and polymerase I transcript release factor (PTRF) – could be seen as elements of the cell internal compartments contaminating the plasma membrane preparation. However, recurrent evidence suggests that some of them are physically and functionally associated with the plasma membrane too. Although localized in the cytosol and nucleus (under stress), Hsc70 was detected at the plasma membrane and putative roles in the immune response and signal transduction have been attributed to his protein [32]; the plasma membrane lipid rafts of adipocytes and HeLa cells concentrate most of the PTRF, otherwise, a nuclear factor required for the termination of the ribosomal RNA synthesis [33]. TCP-1 is a chaperonin involved in the correct folding of actin and tubulin [34] and its association with the cortical cytoskeleton appears therefore natural.

Only three integral plasmalemmal proteins were recognized among the endothelial glycated polypeptides (Table 1). The

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first is a cell surface enzyme—dipeptidyl peptidase IV. Its activity was showed to decrease by 25% in the heart vasculature of the STZ-induced diabetic rats [35]. In the perspective of our results this loss of function might be the consequence of this protein glycation. Although several other integrins have been detected by a comprehensive proteomic study at the luminal front of the rat lung endothelial plasma membrane ([36], and our unpublished results), only the integrin alpha 3 was consistently found to be glycated. The third glycated integral membrane protein was identified as the chloride intracellular channel 5 or CLIC5, which is a member of a recently described family of proteins believed to be involved in the control of the cellular water and electrolyte balance. Although initially reported as residents of intracellular compartments (hence the name), the members of the CLIC family and particularly CLIC-5 have been recently localized by biochemical fractionation and immunofluorescence at the apical plasma membrane of polarized epithelia [37], in close association with the cortical actin cytoskeleton [38].

An important question related to glycation is how many sugar molecules are bound by each targeted protein in the real conditions of diabetes. It is intriguing that the 2D electrophoretic patterns of the glycated (from diabetic), and nonglycated (from normoglycemic rats) endothelial plasmalemma proteins are virtually identical and that they overlap perfectly. Glycation was expected to produce a shift in the electric charge of the protein, particularly evidenced by isoelectric focussing in a narrow pI domain. We did not observe any alteration of this sort for any of the spots corresponding to glycated proteins. To us, the only plausible explanation is that in each protein targeted by glycation the number of aminoacid residues affected is too small to change the electric balance of the macromolecule. This is in accord with the few data in the literature concerning the extent of glycation of intracellular proteins in diabetes. Only 1% of the total (-amino groups of the hemoglobin were found to be glycosylated in the erythrocytes of the diabetic patients [39], while in the skeletal muscle myofibrils of the diabetic rats this figure is approximately 0.05% [40]. In contrast, in long – lived proteins of extracellular matrix like the collagen, the extent of glycation is significantly higher: 2.5% of the total lysyl residues [41].

Several studies performed in vitro have emphasized that on proteins such as albumin, hemoglobin, LDL and RNase, sugars condense only with a few, selected aminoacid residues [9,41 –44]. These studies have also shown that, in spite of a very limited number of available sites for glycation, the reaction can have a pronounced effect on the protein ligand binding capacity, recognition by its receptor or enzymatic activity.

What is the impact of glycation, at levels existing in vivo, on the functions of endothelial cell proteins identified throughout this study is a question deserving further attention.

Acknowledgements The authors warmly thank Mrs. Line Roy (Montreal Proteomic Centre) for her help with the mass spectrometry

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