N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to ϳ1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of 125 I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the upregulation of the endocytosis of EGFR.
The glycation reaction (Maillard reaction) plays a major role in diabetic complications, since some reaction intermediates are responsible for the modification and cross-linking of long-lived proteins, resulting, in turn, in a deterioration of normal cell function. The reaction intermediates include methylglyoxal (MG) and 3-deoxyglucosone (3-DG), both of which are cytotoxic dicarbonyl compounds and are elevated during hyperglycemia. Aldehyde reductase (ALR) catalyzes the reduction of both compounds. To examine the intracellular role of ALR in the diabetic complications of neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells, which normally express a low level of ALR. Western blot analysis showed that ALR protein in the ALR gene-transfected cells was more than twice as much as in the control cells. In the parental cells, cytotoxicity, including apoptotic cell death, which was determined by fluorescent microscopy using the fluorescent DNA binding dye Hoechst 33258, was observed at 100 microM MG. In the ALR gene-transfected cells, the cytotoxicity of both MG and 3-DG and apoptotic cell death were decreased. This suggests that intracellular ALR protects neural cells from the cytotoxicity of 3-DG or MG, and that neural cells, which normally express a low level of ALR, might be susceptible to diabetic complications caused by intermediate products of the Maillard reaction, such as 3-DG and MG.
An antibody has been raised against fructated lysine in proteins by immunizing fructated lysine-conjugated ovalbumin in rabbits. The affinity-purified antibody specifically recognized proteins incubated with fructose but not with other reducing sugars such as glucose, galactose or ribose, as judged by immunoblotting and ELISA techniques. Competitive binding to this antibody was observed specifically by fructated lysine but not by glucated lysine, glucose, fructose or lysine. The antibody binds specifically to fructated lysine residues in the protein but not to borohydride-reduced material or advanced glycation end products, indicating that the antibody recognizes only the reducing, carbonyl-containing forms produced in the early stage of the fructation reaction. When BSA was incubated with various concentrations of fructose, the reactivity of the antibody increased in a dose- and time-dependent manner. When soluble proteins prepared from either normal or streptozotocin-induced diabetic rat eyes were analysed by ELISA with this antibody, an increase in the reactive components was observed as a function of aging as well as under diabetic conditions. Western blotting analysis showed that lens crystallin reacted highly with this antibody. Because fructose is biosynthesized largely through the polyol pathway, which is enhanced under diabetic conditions, and lens is known to have a high activity of enzymes in this pathway, this antibody is capable of recognizing fructated proteins in vivo. Thus it is a potentially useful tool for investigating two major issues that seem to be involved in diabetic complications, namely the glycation reaction and the polyol pathway.
2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.
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