Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo

The advanced stage of the glycation process (also called the "Maillard reaction") that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. AGEs elicit a wide range of cell-mediated responses that might contribute to diabetic complications, vascular disease, renal disease, and Alzheimer's disease. Recently, it has been proposed that AGE are not only created from glucose per se, but also from dicarbonyl compounds derived from glycation, sugar autoxidation, and sugar metabolism. However, this advanced stage of glycation is still only partially characterized and the structures of the different AGEs that are generated in vivo have not been completely determined. Because of their heterogeneity and the complexity of the chemical reactions involved, only some AGEs have been characterized in vivo, including N-carboxymethyllysine (CML), pentosidine, pyrraline, and crosslines. In this article, we provide a brief overview of the pathways of AGE formation and of the immunochemical methods for detection of AGEs, and we also provide direct immunological evidence for the existence of five distinct AGE classes (designated as AGE-1 to -5) within the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis. We also propose pathways for the in vivo formation of various AGEs by glycation, sugar autoxidation, and sugar metabolism.     

Oxygen is not required for the browning and crosslinking of protein by pentoses: relevance to Maillard reactions in vivo

The chemical modification and crosslinking of proteins by the Maillard or browning reaction contributes to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications. Metal-catalyzed autoxidation reactions catalyze the browning of proteins by glucose, a process known as autoxidative glycosylation, but the effects of oxidative conditions on browning of proteins by smaller sugars has not been reported. In this work we studied the browning and crosslinking of the model protein, RNase A, by pentoses. Although antioxidative conditions inhibited the formation of glyoxal and the advanced glycation end-product, N epsilon-(carboxymethyl)lysine from arabinose, browning and crosslinking, and formation of the fluorescent crosslink pentosidine proceeded at comparable rates under oxidative and antioxidative conditions. These studies and other work on smaller dicarbonyl compounds indicate that Maillard reactions of simpler carbohydrates proceed efficiently in the absence of oxygen and suggest that antioxidant therapy for treatment of diabetic complications may have limited clinical efficacy.     

Covalent coupling of reduced glutathione with ribose: loss of cosubstrate ability to glutathione peroxidase

Glycation (nonenzymatic glycosylation of proteins) is known to be increased as a result of hyperglycaemia in diabetes. Moreover, cell glutathione concentration has been found to be lower in diabetics and such depletion may impair the cell defence against toxic radical species. Ribose being a potent reducing sugar expected to be increased in cells of diabetics where the pentose phosphate pathway is enhanced, its putative condensation with glutathione was investigated. Reduced glutathione (GSH) was incubated with ribose and the structure of the resultant product was assessed by mass spectrometry, as well as the measurement of its remaining thiol group. A covalent reaction clearly occurred between the reducing sugar and GSH, to give an adduct named N-ribosyl-1-glutathione. This adduct appears to be the Amadori product resulting from the condensation of the primary amine group of GSH with the aldehyde group of ribose. Interestingly, the adduct could not be used as a proper substrate by glutathione peroxidase although it keeps its thiol group. We conclude that the coupling of GSH with a monosaccharide such as ribose might contribute to the decreased cell GSH and glutathione peroxidase activity observed in diabetics.     

Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo

BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.     

Conformational changes induced in lens alpha- and gamma-crystallins by modification with glucose 6-phosphate. Implications for cataract.

There is good evidence that the non-enzymic chemical modification of proteins plays a role in the aetiology of cataract and diabetic sequelae. This paper presents new evidence that glycosylation of two major lens structural crystallins, alpha- and gamma-crystallins, by glucose 6-phosphate (G6P) induces conformational changes in the proteins. In addition the surface charge on the molecules is altered. These changes would affect protein-protein and protein-water interactions within the lens and could lead to disruption of the short-range order of the lens proteins which is essential for lens transparency. Conformational changes to lens proteins are known to occur in human cataractous lenses but their cause in vivo is not established. Cumulative chemical modification of proteins, over a period of decades, is a strong candidate as a causal agent.     

Superoxide-dependence of the short chain sugars-induced mutagenesis

Short chain sugars such as glycolaldehyde are produced at the initial stages of nonenzymatic glycosylation. Because their carbonyl groups cannot be blocked by cyclization, such compounds tautomerize to enediols, which are prone to autoxidation. Superoxide radical serves as an initiator and a propagator of this autoxidation. The biological importance of the involvement of superoxide in sugar autoxidation in vivo was examined using superoxide dismutase (SOD)-deficient and SOD-replete strains of Escherichia coli. Glycolaldehyde, glyceraldehyde, and dihydroxyacetone greatly enhanced the mutation rates in SOD-deficient E. coli. The effect was oxygen-dependent and was suppressed by SOD or by a SOD mimetic. The mutagenic effect of glycolaldehyde coincided with intracellular accumulation of glyoxal, a product of glycolaldehyde autoxidation.     

O-GlcNAc-specific antibody CTD110.6 cross-reacts with N-GlcNAc2-modified proteins induced under glucose deprivation

Modification of serine and threonine residues in proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a feature of many cellular responses to the nutritional state and to stress. O-GlcNAc modification is reversibly regulated by O-linked β-N-acetylglucosamine transferase (OGT) and β-D-N-acetylglucosaminase (O-GlcNAcase). O-GlcNAc modification of proteins is dependent on the concentration of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), which is a substrate of OGT and is synthesized via the hexosamine biosynthetic pathway. Immunoblot analysis using the O-GlcNAc-specific antibody CTD110.6 has indicated that glucose deprivation increases protein O-GlcNAcylation in some cancer cells. The mechanism of this paradoxical phenomenon has remained unclear. Here we show that the increased glycosylation induced by glucose deprivation and detected by CTD110.6 antibodies is actually modification by N-GlcNAc(2), rather than by O-GlcNAc. We found that this induced glycosylation was not regulated by OGT and O-GlcNAcase, unlike typical O-GlcNAcylation, and it was inhibited by treatment with tunicamycin, an N-glycosylation inhibitor. Proteomics analysis showed that proteins modified by this induced glycosylation were N-GlcNAc(2)-modified glycoproteins. Furthermore, CTD110.6 antibodies reacted with N-GlcNAc(2)-modified glycoproteins produced by a yeast strain with a ts-mutant of ALG1 that could not add a mannose residue to dolichol-PP-GlcNAc(2). Our results demonstrated that N-GlcNAc(2)-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6. We therefore propose that the glycosylation status of proteins previously classified as O-GlcNAc-modified proteins according to their reactivity with CTD110.6 antibodies must be re-examined. We also suggest that the repression of mature N-linked glycoproteins due to increased levels of N-GlcNAc(2)-modified proteins is a newly recognized pathway for effective use of sugar under stress and deprivation conditions. Further research is needed to clarify the physiological and pathological roles of N-GlcNAc(2)-modified proteins.     

Carbonyl compounds cross-link cellular proteins and activate protein-tyrosine kinase p60c-Src

Glyoxal, a dicarbonyl compound, is produced under oxidative stress by the autoxidation of glucose and reacts with the protein amino group to form Schiff base. In vitro treatment of murine thymocytes and fibroblasts with glyoxal induced extensive tyrosine phosphorylation of multiple proteins, which was drastically inhibited by the addition of OPB-9195, an inhibitor of the carbonyl reaction with proteins. Glyoxal induced cross-linking of a number of cellular proteins, including glycosylphosphatidylinositol (GPI)-anchored cell surface Thy-1. We then demonstrated that treatment of cells with glyoxal promptly induced activation of non-receptor protein-tyrosine kinase c-Src, which was partially inhibited by OPB-9195. It is suggested from these results that carbonyl amine reaction quickly activates c-Src, possibly through cross-linkage of GPI-anchored proteins or putative specific receptors.     

Controlling glycation of recombinant antibody in fed-batch cell cultures

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.     

Promotion by phosphate of Fe(III)- and Cu(II)-catalyzed autoxidation of fructose

Although the oxidative destruction of glucose and fructose has been studied by several investigators over the past century, the mechanism by which phosphate promotes these oxidation reactions is not known. A wide range of oxidation products have been used to monitor the oxidation of sugars and free radicals have been shown to be involved. The influence of phosphate concentration on the rate of production of free radicals and several sugar oxidation products has been studied. It was found that fructose is much more susceptible to autoxidation than glucose, galactose, or sucrose. The promotion of sugar oxidation by phosphate was found to be iron dependent. Addition of the iron chelators, diethylenetriaminepentaacetic acid (DTPA) and desferrioxamine completely suppressed the oxidation reactions, even at high concentrations of phosphate. Formaldehyde was positively identified as a product of fructose oxidation by HPLC analysis of its acetylacetone adduct. A mechanism is proposed in which phosphate cleaves the oxo bridges of the iron(III)-fructose complex, based on UV spectral analysis and magnetic susceptibility measurements, and thereby catalyzes the autoxidation of fructose.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination

The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of ¹⁴C-labeled glucosamine, it appeared that not all of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins.     

Dolichol-phosphate mannose synthase: structure, function and regulation

Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and dolichol-phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from dolichol-phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of dolichol-phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.     

Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells

Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.     

Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.     

Elevated cAMP level attenuates 2-deoxy-d-ribose-induced oxidative damage in pancreatic beta-cells

Glucose toxicity to pancreatic beta-cells is defined as irreversible beta-cell damage, including apoptosis, caused by chronic exposure to high glucose levels in type 2 diabetes. Oxidative stress is an important mechanism for glucose toxicity to pancreatic beta-cells. Reducing sugars produce reactive oxygen species through autoxidation and protein glycosylation. 2-Deoxy-d-ribose (dRib) is a reducing sugar with high reactivity. We investigated whether cAMP-stimulating agents could protect beta-cells from dRib-induced oxidative damage. HIT-T15 cells were cultured with various concentrations of dRib for 24 h. We measured cell survival, intracellular cAMP and H2O2 levels, and apoptosis. dRib decreased cell survival in a dose- and time-dependent manner and markedly increased intracellular H2O2 levels and apoptosis. N-Acetyl-l-cysteine decreased dRib-induced rises in intracellular H2O2 and apoptosis to control levels. Forskolin, IBMX, and dbcAMP markedly elevated intracellular cAMP levels and significantly attenuated dRib-induced cytotoxicity and apoptosis, but had no influence on the dRib-induced rise in intracellular H2O2 levels. These results demonstrate that dRib produced oxidative stress and apoptosis in pancreatic beta-cells and that elevated intracellular cAMP levels reduced dRib-induced damage, independent of reactive oxygen species metabolism.     

Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins

Tom20 and Tom34 are mammalian liver proteins previously identified by others to be components of the mitochondrial import translocation apparatus. It has been shown that Tom20 interacts with the leader sequence of nuclear coded matrix space precursor proteins. Here we show with recombinantly expressed Tom proteins that Tom34 binds the mature portion of the precursor and not the leader. Both these Tom proteins inhibited the import of newly translated precursor of aldehyde dehydrogenase in an in vitro assay. Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase. Antibodies against Tom20 coprecipitated both the precursor of aldehyde dehydrogenase (pALDH) and of dihydrofolate reductase (pA-DHFR). Antibodies against Tom34 interacted only when the mature portion of aldehyde dehydrogenase was present. Similar import inhibition patterns were found when other precursor and chimeric constructs we investigated. When Tom34-green fluorescence protein was transfected to HeLa cells it was observed that Tom34 was found through out the cell. It is concluded from our observation that Tom34 is a cytosolic protein, whose role appeared to be to interact with mature portion of some preproteins and may keep them in an unfolded, import compatible state.     

Glucosylation of beta-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect protein thermodynamics

Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded beta-lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of epsilon-amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by approximately 3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5 degrees C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications.     

Sugar binding residue affects apparent Na+ affinity and transport stoichiometry in mouse sodium/glucose cotransporter type 3B

SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.