Fig 1: Melibiose directly binds to GLO1 and enhances its enzymatic activity. (A) GSEA of the AGE‐RAGE signaling pathway in diabetic complications based on HGEC transcriptomic data. (B) Schematic diagram showing the workflow for identifying melibiose‐binding proteins using the DARTS approach. (C) Representative immunoblots and quantification from the DARTS assay showing the binding between melibiose and GLO1 (n = 3). (D) Representative immunoblots and quantification from the CETSA assay assessing GLO1 stability at different temperatures with or without melibiose treatment (n = 3). (E) Microscale thermophoresis (MST) assay showing the direct binding affinity between melibiose and GLO1. (F) GLO1 enzyme activity in HGECs (n = 3). (G) Methylglyoxal levels in HGECs (n = 3). (H,I) Methylglyoxal levels in mouse kidney (n = 5–6). (J–O) Representative immunoblotting (J) and quantitative analysis of GLO1 (K), ET‐1 (L), OCLN (M), ICAM‐1 (N), and ZO‐1 (O) proteins in HGECs following GLO1 overexpression (n = 3). P–U) Representative immunoblotting (P) and quantitative analysis of GLO1 (Q), ET‐1 (R), OCLN (S), ICAM‐1 (T), and ZO‐1 (U) proteins in HGECs following GLO1 knockdown (n = 3). V) GLO1 enzyme activity in HGECs following GLO1 knockdown (n = 3). W) Molecular docking between melibiose and GLO1. Overview of the binding pose and close‐up views of interactions with residues R38, N104, E111, K157, and M158. X) Enzyme activity of GLO1 wild‐type and point mutants re‐expressed in GLO1‐knockdown HGECs (n = 3). Data are presented as mean ± SD (C, F–I, K–O, Q–V, X). Statistical analysis was performed using the one‐way ANOVA (C, F–I, K–O, Q–U) and the two‐way ANOVA (V, X). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05.
Fig 2: The protective effect of melibiose on the glomerular endothelium is mediated by GLO1. (A) Experimental scheme of the endothelial cell‐specific GLO1 knockout mice. Male GLO1fl/fl, cre− (GLO1fl/fl) and GLO1fl/fl, cre+ (GLO1−/−) mice were fed either a chow diet (CON) or a high‐fat diet combined with STZ to induce diabetes. Diabetic mice received melibiose (1.0 g kg−1, MEL) treatment or PBS (DM). (B) Schematic diagram showing Cre‐mediated recombination between loxP sites flanking exon 2 of the GLO1 allele. Recombination results in the excision of exon 2, generating a GLO1 knockout allele. (C) Genotyping of endothelial cell‐specific GLO1 knockout mice. Representative agarose gel electrophoresis image of PCR products showing six genotypes: Cre− and Cre+ combined with GLO1+/+, GLO1fl/+, and GLO1fl/fl alleles, using genomic DNA extracted from tail tissue of 4‐week‐old mice. (D,E) Fasting blood glucose (D) and body weight (E) levels in mice (n = 6). (F–H) UACR (F), Cr (G), and BUN (H) levels in mice (n = 6). (I) Representative images of H&E, Masson's trichrome, and PAS staining showing DKD‐associated glomerular pathology. Scale bar, 20 µm. (J) Representative TEM and SEM images showing glomerular endothelial fenestration structures. Scale bar, 500 nm. (K) Morphometric quantification of the endothelial cell fenestration density per unit length, performed on TEM images (n = 3). (L) Morphometric quantification of the endothelial cell fenestration area density, performed on SEM images (n = 3). (M–R) Representative immunoblotting (M) and quantitative analysis of ET‐1 (N), SDC1 (O), OCLN (P), ICAM‐1 (Q), and ZO‐1 (R) proteins in the kidney (n = 3). (S–U) Plasma TNF‐α (S), plasma IL‐6 (T), and renal methylglyoxal (U) levels in mice (n = 6). Data are presented as mean ± SD (D–H, K, L, N–U). Statistical analysis was performed using the one‐way ANOVA (F–H, K, L, N–U) and the two‐way ANOVA (D, E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05.
Supplier Page from Abcam for Recombinant human GLO1 protein (His tag C-Terminus)