Glycation Of Membrane Proteins Research Articles

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.

Erythrocyte membrane glycation and NA +–K + levels in NIDDM

Although there are many investigations on protein glycation in diabetic patients, many detailed studies are needed on this subject. In this study, the correlation between red cell membrane and serum protein glycation was investigated in NIDDM. The relation of membrane glycation to intracellular Na + and K + levels was also considered. Forty patients with NIDDM and 22 healthy subjects were included in the study. The membrane proteins were isolated, and total protein (TP m) and fructosamine (FA m) levels were determined. Serum glucose, fructosamine (FA s) and total protein (TP s) levels were also measured. HbA 1C, red blood cell (RBC) and reticulocyte (RET) counts in whole blood were made in all samples. NA + and K + levels of both serum and RBC were determined. The patient group had lower levels of K + RBC ( P<.001) and Na + s ( P<.05) and RBC count ( P<.05), and higher levels of FA m ( P<.001), Na + RBC ( P<.01), K + s ( P<.01), glucose ( P<.001) and HbA 1C ( P<.001) than those of controls. The ratios of FA s/TP s ( P<.001) and FA m/TP m ( P<.001) were higher in patients than in control. As a result, HbA 1C levels and the ratio of FA m/TP m were high in NIDDM patients ( P<.001) and these patients have slight negative correlations in FA m/TP m and FA s/TP s ( P<.05). On the other hand, that there is no correlation between RBC membrane protein glycation and RBC Na + and K + levels may be caused by the fact that the membrane protein glycation is lower than that of other soluble proteins and that the membrane proteins are functional with respect to Na +–K + transport.

Determination of membrane protein glycation in diabetic tissue.

Diabetes-associated hyperglycemia causes glycation of proteins at reactive amino groups, which can adversely affect protein function. Although the effects of glycation on soluble proteins are well characterized, there is no information regarding membrane-associated proteins, mainly because of the lack of reproducible methods to determine protein glycation in vivo. The current study was conducted to establish such a method and to compare the glycation levels of membrane-associated proteins derived from normal and diabetic tissue. We present a detailed sample preparation protocol based on the borohydride-periodate assay, modified to allow manipulation of animal tissue. Assay noise associated with extraction protocols and nonproteinaceous buffer components was eliminated by the using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) as a membrane detergent, applying desalting columns, and including a protein precipitation step. The glycation level of membrane proteins from diabetic rats is elevated to 4.89 nmol/mg protein (standard deviation [SD] 0.48) compared with normoglycemic control tissue (2.23 nmol/mg protein, SD 0.64). This result is consistent with and correlated to the total glycated hemoglobin levels in diabetic and normoglycemic rats. Using <100 microg protein, the described methods allow further study of protein glycation effects on the function of individual transporter proteins and the role of these modifications in diabetes.

Diabetes Mellitus Alters the Effect of Peptide and Protein Ligands on Membrane Fluidity of Blood Platelets

The increased nonenzymatic glycosylation of platelet membrane proteins has been suggested to underlie platelet hypersensitivity in diabetes and the relationship of this to the reduced membrane lipid fluidity has been reported. As the modulation in membrane fluidity may determine the degree of accessibility of membrane receptors, the consequent alterations in membrane lipid-protein interactions in diabetes mellitus may also underlie the differentiated effects of various thrombotic and fibrinolytic agents on platelet membrane lipid bilayer. In the present study we employed electron paramagnetic resonance and fluorescence spectroscopy to explore the ligand-induced platelet membrane fluidity changes in diabetic state, i.e. under conditions when the membrane architecture is considerably altered. The yield of the excimer formation of pyrenemaleimide (PM), which depends directly upon the collisional rate and distances between molecules, was elevated in diabetic platelet membranes, thus pointing to the occurrence of some constraints in the structure/conformation of platelet membrane proteins in diabetes mellitus. Such an immobilization of PM was accompanied by the significant elevation in membrane protein glycation in diabetic platelets. The effects of various interacting ligands on platelet membrane fluidity were significantly lower in diabetic platelets, and the differences were much more distinct at the lower depths of a lipid bilayer. Nevertheless, the alterations in membrane lipid fluidity observed upon the interaction of a given ligand occurred with an approximately equal frequency in control and diabetic platelets. Moreover, the probability that these alterations were less profound in diabetic platelets was the same for all types of ligands studied. In diabetic patients the interaction of RGDS and tissue-type plasminogen activator with platelet membranes resulted in much smaller reductions of the h+/h0 parameters in 5-DOXYL-Ste acid-labelled platelets, thus indicating a lesser rigidization of membrane lipid bilayer in diabetes. Likewise, the fluidizing effect of both fibrinogen itself and fibrinogen-derived peptides containing gamma-chain carboxy-terminal sequence H-12-V was less pronounced in diabetic platelet membranes.

Decreased Platelet Membrane Fluidity Due to Glycation or Acetylation of Membrane Proteins

Platelets from diabetic subjects and animals are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function and previously we observed reduced membrane fluidity in platelets from diabetic patients associated with hypersensitivity to thrombin. We previously reported that decreased fluidity of isolated platelet membranes from diabetic patients is associated with increased glycation of platelet membrane proteins, but not with any change in the cholesterol to phospholipid molar ratio. We have now examined in vitro whether incubation of platelet membranes in a high glucose medium causes sufficient glycation to reduce membrane fluidity. Incubation of platelet membranes from control subjects in a high glucose (16.1 mM) medium for 10 days at 37 degrees C led to an increase in the extent of glycation of membrane proteins and a decrease in membrane fluidity (indicated by an increase in steady state fluorescence polarization); most of the changes occurred within the first 3 days of incubation. Incubation of platelet membranes with 5.4 mM glucose had less effect. In contrast, incubation of platelet membranes with the same concentrations of 1-0-methylglucose did not cause a change in either the extent of glycation of proteins or membrane fluidity. We also determined if acetylation by aspirin or acetyl chloride of the sites available for glycation on platelet membrane proteins leads to a similar reduction in membrane fluidity. Pretreatment of platelet membranes with aspirin or acetyl chloride diminished the extent of glycation that occurred when platelet membranes were subsequently incubated with glucose, but membrane fluidity was reduced even in the absence of glucose; subsequent incubation with glucose caused no further reduction in membrane fluidity.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycation of lens membrane intrinsic proteins.

Changes occurring at the membrane are believed to be the decisive factors in the initiation of diabetic cataract. During diabetic hyperglycemia lens crystallins were shown to undergo glycation. Several studies indicated that glycation brings about protein conformational changes thus implicated in cataractogenesis. Since the membrane proteins are the first targets for glycation, in this study we measured the glycation of alkali washed urea-insoluble membrane proteins from control and diabetic rats by two different methods, phenyl-boronate affinity chromatography and [3H]NaBH4 reduction, and confirmed by amino acid analysis. There was a significant increase in the glycation of membrane proteins in diabetic cataract lenses when compared to controls. It appears that lysine is the major site of glycation. Concomitant to early glycation, there was an increase in non-tryptophan fluorescence (Ex: 350 nm/Em: 440 nm) in the diabetic lens membrane proteins suggesting the presence of advanced glycation mediated protein cross-links. In order to identify whether the major membrane intrinsic protein, MIP26, undergoes glycation, we isolated MIP26 along with its degradatory product MIP22 as one peak on molecular sieve HPLC. HPLC isolated MIP26/MIP22 was further separated on SDS-PAGE followed by slicing and counting. This analysis revealed that MIP26 and MIP22 were more or less equally glycated in controls, however, in diabetic rats glycation of MIP22 was glycated slightly higher than MIP26. Moreover, the proportion of MIP22 increased by about 2-fold in diabetic lenses compared to controls. Thus it appears that major glycation sites are still retained in MIP22 in diabetic rat lenses. In vitro glycation studies with bovine lens membranes were also done using 14C glucose, followed by SDS-PAGE and autoradiography. The major protein glycated in vitro also seems to be MIP26. Interestingly, MIP22 was less glycated than MIP26 in vitro.

The relationship of chemical modification of membrane proteins and plasma lipoproteins to reduced membrane fluidity of erythrocytes from diabetic subjects.

The significance of the two most common hallmarks of the diabetic state, hyperglycaemia and hyperlipidaemia, was investigated in terms of disorders of cell membrane dynamics. In order to examine whether the alterations in cell membrane lipid bilayer dynamics are somehow related to protein chemical modifications in plasma low-(LDL) and high-density lipoproteins (HDL) and blood cell membranes, we compared 19 poorly controlled diabetic subjects with 19 age- and sex-matched controls. The extent of (non-enzymatic) glycation, lipid peroxidation and the cholesterol/phospholipid ratio were increased in plasma low density lipoproteins and high density lipoproteins from diabetic patients. The mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5-hexatriene-labelled isolated erythrocyte membranes from diabetic subjects were significantly greater than from control subjects (0.186 +/- 0.008 vs 0.173 +/- 0.006, p < 0.001); the fluorescence polarization values in erythrocyte membranes from diabetic and control subjects positively correlated with the extent of membrane protein glycation, lipid peroxidation and the cholesterol content. The cholesterol to phospholipid molar ratios in low density lipoproteins and high-density lipoproteins from diabetic and control subjects correlated significantly with the fluorescence polarization values in erythrocyte membranes from these subjects. Furthermore, the extent of glycation of low density lipoproteins appears to be strongly correlated with the extent of lipoprotein lipid peroxidation (r = 0.789, p < 0.001). The atherosclerotic potential of plasma lipoproteins in diabetes mellitus was discussed in terms of membrane and plasma protein chemical modifications.

Association between the glycation of erythrocyte membrane proteins and membrane fluidity

Erythrocyte membrane ghosts prepared from normal individuals were incubated in vitro at different glucose concentrations for 72 h. Incubation with increasing concentrations of glucose resulted in increased glycation (nonenzymatic glycosylation) of membrane proteins and a decrease of erythrocyte membrane fluidity measured by the use of the fluorescent label pyrene. It would therefore seem likely that the changes in erythrocyte membrane fluidity previously reported in diabetic subjects (1-3) are related to in vivo glycation of membrane proteins as well as to changes in lipid composition, as was previously suggested (2).

Glucose transport in erythrocytes of diabetic and healthy children as related to hemoglobin A1c.

We studied glucose transport under physiological conditions (38 degrees C, pH 7.2, 5 mmol of glucose per liter) in erythrocytes of nine diabetic children with hemoglobin A1c values ranging from 6.6 to 13.8%, and in erythrocytes from six healthy children. Glucose transport was determined to be 2.38 (SD 0.16) X 10(-10) mol/cm2 X s (n = 18), and 2.47 (SD 0.18) X 10(-10) mol/cm2 X s (n = 12) in erythrocytes from diabetics and controls, respectively. The corresponding values for hemoglobin A1c were 11.0% (SD 2.3%) for the diabetics and 5.6% (SD 0.3%) for the controls. Thus the concentration of hemoglobin A1c, which reflects the degree of glycation of membrane proteins, differs significantly (p less than 0.001) between the two groups, whereas there was no significant variation (p greater than 0.1) in D-glucose transport. We conclude that glycation of the integral membrane protein that mediates glucose transport has no effect on transport function under physiological conditions.