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RNA-seq analysis of glycosylation related gene expression in STZ-induced diabetic rat kidney inner medulla.

Qian X, Li X, Ilori TO, Klein JD, Hughey RP, Li CJ, Alli AA, Guo Z, Yu P, Song X, Chen G - Front Physiol (2015)

Bottom Line: In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions.In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes.Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Emory University School of Medicine Atlanta, GA, USA ; Department of Cardiology, The Fourth Affiliated Hospital of Harbin Medical University Harbin, China.

ABSTRACT

Unlabelled: The UT-A1 urea transporter is crucial to the kidney's ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, β-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)-induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes.

Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the protein's glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.

No MeSH data available.


Related in: MedlinePlus

GST-galectin pulldown assays. (A) Equal amount of cell membrane fractions from rat IM tip were incubated with freshly prepared GST-galectin proteins for 1 h at 4°C. After washing with buffer A containing 14 mM β-mercaptoethanol and buffer containing 0.1 M sucrose, the galectin specific binding proteins were eluted by 0.1 M lactose (specific-binding) and subsequently analyzed by Western blot with UT-A1 antibody (Top). The same membrane was stained by Coomassie brilliant blue to verify GST fusion galectin proteins (Bottom). (B) Densitometry analysis of UT-A1 protein bands from three separate experiments (n = 3). Each GST-galectin precipitated UT-A1 was normalized with UT-A1 from input (means ± SD, *P < 0.05, **P < 0.01, NS, no significance).
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Figure 2: GST-galectin pulldown assays. (A) Equal amount of cell membrane fractions from rat IM tip were incubated with freshly prepared GST-galectin proteins for 1 h at 4°C. After washing with buffer A containing 14 mM β-mercaptoethanol and buffer containing 0.1 M sucrose, the galectin specific binding proteins were eluted by 0.1 M lactose (specific-binding) and subsequently analyzed by Western blot with UT-A1 antibody (Top). The same membrane was stained by Coomassie brilliant blue to verify GST fusion galectin proteins (Bottom). (B) Densitometry analysis of UT-A1 protein bands from three separate experiments (n = 3). Each GST-galectin precipitated UT-A1 was normalized with UT-A1 from input (means ± SD, *P < 0.05, **P < 0.01, NS, no significance).

Mentions: Galectins are a group of small lectin-like proteins (14–30 kDa) that bind β-galactose-containing glycoconjugates. Each galectin has unique binding specificities (Poland et al., 2011). To investigate whether the change of the UT-A1 glycan structure under diabetic conditions would result in alteration of UT-A1 binding to galectin proteins, we performed the GST-galectin pulldown assay with control and STZ rat IM samples as described in Materials and Methods. GST-galectin proteins pre-bound to glutathione beads were incubated with equal amounts of lipid raft membrane fractions from kidney IM and eluted with lactose. The binding of UT-A1 was examined by Western blot analysis of the eluted material. Galectin proteins are predicted to bind only the high glycosylation form of 117 kDa, as the 97 kDa form exhibits only immature high mannose Gal-deficient N-glycans (Chen et al., 2011 and Figure 1). As shown in Figure 2A, we found that the 117 kDa form of UT-A1 from control rat IM binds to primarily Gal-3 and Gal-7 with a small amount binding to Gal-8 and Gal-9C. However, we observed increased binding of the 117 kDa UT-A1 to Gal-3, -7, -8, and -9, particularly the enhanced binding to Gal-8 and -9 indicating that the N-glycans are changed on UT-A1 in diabetic rat kidney. The increased UT-A1 bound to Gal-3, -7, -8, and -9 is not because of protein overloading since UT-A1 bound to Gal-1 and -4 is not increased. Figure 2B shows the signal quantification and statistical analysis from three independent studies. Galectin-9 has two carbohydrate recognition domains (CRD). Since GST-Gal-9 is aggregated in the bacteria, we prepared N-terminal and C-terminal CRDs separately as GST-Gal-9N (residues 1–148) and GST-Gal-9C (residues 225–355) (Poland et al., 2011). Only C-terminal, but not N-terminal, CRD in galectin-9 interacted with UT-A1. Additionally, we observed that the 117 kDa form of UT-A1 from diabetic tissue migrates further upon electrophoresis, reflecting different glycosylation modifications occurred in the diabetic animal.


RNA-seq analysis of glycosylation related gene expression in STZ-induced diabetic rat kidney inner medulla.

Qian X, Li X, Ilori TO, Klein JD, Hughey RP, Li CJ, Alli AA, Guo Z, Yu P, Song X, Chen G - Front Physiol (2015)

GST-galectin pulldown assays. (A) Equal amount of cell membrane fractions from rat IM tip were incubated with freshly prepared GST-galectin proteins for 1 h at 4°C. After washing with buffer A containing 14 mM β-mercaptoethanol and buffer containing 0.1 M sucrose, the galectin specific binding proteins were eluted by 0.1 M lactose (specific-binding) and subsequently analyzed by Western blot with UT-A1 antibody (Top). The same membrane was stained by Coomassie brilliant blue to verify GST fusion galectin proteins (Bottom). (B) Densitometry analysis of UT-A1 protein bands from three separate experiments (n = 3). Each GST-galectin precipitated UT-A1 was normalized with UT-A1 from input (means ± SD, *P < 0.05, **P < 0.01, NS, no significance).
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Figure 2: GST-galectin pulldown assays. (A) Equal amount of cell membrane fractions from rat IM tip were incubated with freshly prepared GST-galectin proteins for 1 h at 4°C. After washing with buffer A containing 14 mM β-mercaptoethanol and buffer containing 0.1 M sucrose, the galectin specific binding proteins were eluted by 0.1 M lactose (specific-binding) and subsequently analyzed by Western blot with UT-A1 antibody (Top). The same membrane was stained by Coomassie brilliant blue to verify GST fusion galectin proteins (Bottom). (B) Densitometry analysis of UT-A1 protein bands from three separate experiments (n = 3). Each GST-galectin precipitated UT-A1 was normalized with UT-A1 from input (means ± SD, *P < 0.05, **P < 0.01, NS, no significance).
Mentions: Galectins are a group of small lectin-like proteins (14–30 kDa) that bind β-galactose-containing glycoconjugates. Each galectin has unique binding specificities (Poland et al., 2011). To investigate whether the change of the UT-A1 glycan structure under diabetic conditions would result in alteration of UT-A1 binding to galectin proteins, we performed the GST-galectin pulldown assay with control and STZ rat IM samples as described in Materials and Methods. GST-galectin proteins pre-bound to glutathione beads were incubated with equal amounts of lipid raft membrane fractions from kidney IM and eluted with lactose. The binding of UT-A1 was examined by Western blot analysis of the eluted material. Galectin proteins are predicted to bind only the high glycosylation form of 117 kDa, as the 97 kDa form exhibits only immature high mannose Gal-deficient N-glycans (Chen et al., 2011 and Figure 1). As shown in Figure 2A, we found that the 117 kDa form of UT-A1 from control rat IM binds to primarily Gal-3 and Gal-7 with a small amount binding to Gal-8 and Gal-9C. However, we observed increased binding of the 117 kDa UT-A1 to Gal-3, -7, -8, and -9, particularly the enhanced binding to Gal-8 and -9 indicating that the N-glycans are changed on UT-A1 in diabetic rat kidney. The increased UT-A1 bound to Gal-3, -7, -8, and -9 is not because of protein overloading since UT-A1 bound to Gal-1 and -4 is not increased. Figure 2B shows the signal quantification and statistical analysis from three independent studies. Galectin-9 has two carbohydrate recognition domains (CRD). Since GST-Gal-9 is aggregated in the bacteria, we prepared N-terminal and C-terminal CRDs separately as GST-Gal-9N (residues 1–148) and GST-Gal-9C (residues 225–355) (Poland et al., 2011). Only C-terminal, but not N-terminal, CRD in galectin-9 interacted with UT-A1. Additionally, we observed that the 117 kDa form of UT-A1 from diabetic tissue migrates further upon electrophoresis, reflecting different glycosylation modifications occurred in the diabetic animal.

Bottom Line: In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions.In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes.Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Emory University School of Medicine Atlanta, GA, USA ; Department of Cardiology, The Fourth Affiliated Hospital of Harbin Medical University Harbin, China.

ABSTRACT

Unlabelled: The UT-A1 urea transporter is crucial to the kidney's ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, β-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)-induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes.

Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the protein's glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.

No MeSH data available.


Related in: MedlinePlus