Role Of Enzyme-catalyzed Glycosylation In Diabetic Retinopathy

Purpose To investigate (1).whether hyperglycemia affect the expression ofglycosyltransferases and Enzyme-catalyzed glycosylation in the retina of diabetic rat.(2).the role of enzyme-catalyzed glycosylation in diabetic retinopathy.Methods (1).Realtime-PCR was used to analyze the mRNA level of sixglycosyltransferases in the retina of steptozocin diabetic rats; Lectin blot assay withRCA-I or PHA-L was performed to investigate the level of Galβ1→4GlcNAc orN-glycans on total retinal glycoproteins. (2).Bovine Retinal Pericyte (BRP) wascultured in medium with increasing concentrations of glucose (5mM, 10mM, 20mMand 30mM) for 72 hours, 5mM glucose as control. Gene expression ofβ1,4Galactosyltransferase-1(GalT-1) in BRP was studied with Reverse TranscriptionPolymerase Chain Reaction (RT-PCR). Lectin blot assay with RCA-I was performedto explore the level of the groups of Galβ1→4GlcNAc on cell total proteins and thelevel of apoptosis of BRP was examined by flow cytometry analysis. (3).HumanUmbilical Vein Endothelial Cell (HUVEC) was cultured in medium with high-glucose,and Gene expression of theβ1,4Galactosyltransferase family in HUVEC was studiedwith RT-PCR. Lectin blot assay with RCA-I was performed to explore the level of thegroups of Galβ1→4GlcNAc on cell total proteins. Using RNA interference todecrease the expression of GaiT-1 in HUVEC, The level of apoptosis of HUVEC wasexamined by flow cytometry analysis and the expression of cleave Caspase-3 andspecific protein-1(Sp1) was studied with Western blot assay. After transfected withGalT-1 promoter construct p-1652-luc plasmid, HUVEC cultured with high-glucosefor 24 hours in the presence or absence of Mithramycin A and the luciferase activitieswere measured using a Dual Luciferase assay. (4).Gene and protein expression ofintercellular adhesion molecule-1(ICAM-1) in Bovine Retinal Endothelial Cell(BREC), which cultured with increasing concentrations of glucose in the presence orabsence of tunicamycin, were studied with RT-PCR and Western blot assay, and theexpress level of ICAM-1 on the surface of BREC was examined byimmunofluorescence analysis. Lectin blot assay with PHA-L was performed toexplore the level of N-glycans of immunoprecipitated ICAM-1 treated or untreatedwith high glucose. Results (1).The mRNA level ofβ1,4Gaiactosyltransferase-1 was up-regulated in theretina of streptocin diabetic rats. Consistent with this, the level of Galβ1→4GlcNAc of glycoproteins in streptocin diabetic rat's retina was strengthenedcompared with that in control rats. Additionally, the level of N-glycans ofglycoproteins in streptocin diabetic rat's retina was also up-regulation.(2).High-glucose could increase the expression of GAIT-1 at mRNA level and increasethe groups of Galβ1→4GlcNAc in BRP. Simultaneously, the apoptosis of BRP wasup-regulated by high-glucose: (5 mM glucose: 7.35±1.82%; 10mM glucose: 16.34±2.25%; 20mM glucose: 29.85±6.66%; 30mM glucose: 39.48±6.48%). (3).High-glucose caused up-regulation of GaiTⅠmRNA and a significant increase of thebinding of total glycoprotein with RCA-Ⅰwas observed in high glucose-treatedHUVEC. The treatment of HUVEC with Mithramycin A blocked the positive effectof high glucose on GaiTⅠpromoter. In addition, the expression of SP1 was increasedin response to high glucose treatment. Decreasing GaiT-1 expression by transfectedwith GalT-1 RNAi could decrease the level of high glucose-induced apoptosis andinhibit the cleavage of Caspase-3 increased by high glucose. (4).Both the mRNA andthe protein level of ICAM-1, as well as the level of ICAM-1 on BREC surface, weresignificantly upregulated by increasing the concentration of glucose in culturemedium. Consistent with this, a dramatic increase in the N-glycosylation of ICAM-1in BREC cultured with high-glucose was observed. The upregulation of ICAM-1 atBREC surface which induced by high-glucose could be partially attenuated by thetreatment with tunicamycin.Conclusions The results from this study showed that (1).Hyperglycemia couldup-regulate the expression of GalT-1 and strengthed the level of Galβ1→4GlcNAc ofglycoproteins in the retina of streptocin diabetic rats. (2). High-glucose couldup-regulate the expression of GalT-1 in BRP, and GalT-1 might participate in theinduction of apoptosis in BRP cultured with high-glucose. (3). High concentrations ofglucose increase the mRNA level of GalT-1 in a SP1-dependent manner in HUVEC.High glucose induces HUVEC apoptosis via up-regulation of GalT-1. (4).Highglucose-induced upregulation of ICAM-1 on the surface of BREC could be ascribedto the alterations in its N-glycosylation at least in part. Our initial results willcontribute to the research for the relation between the Enzyme-catalyzedGlycosylation and the etiopathogenesis of diabetic retinopathy.