Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration

Abstract

Glioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative growth of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts revealed a significantly higher expression of Glycosyltransferase 8 domain containing 1 (GLT8D1) compared to normal brain tissue and could be associated with impaired patient survival. Increased in vitro expression of GLT8D1 significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in the predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration.

Keywords: Biochemistry; Cancer; Cell biology; Glycobiology.

Graphical abstract

Figure 1

GLT8D1 gene expression and protein expression in clinical GBM samples (A) GLT8D1 mRNA expression in non-tumor vs. GBM samples (GlioVis platform, The Cancer Genome Atlas (TCGA_GBM) dataset, HG-U133A platform, total 538 entries). n = 10, median 8.24, mean 8.27, SD 0.15 (non-tumor), n = 528, median 9.12, mean 9.08, SD 0.43 (GBM), Tukey's Honest Significant Difference (HSD), ∗∗∗p < 0.001. (B) Kaplan-Meier survival analysis of the patients with the GBM (both primary and secondary tumors) showing low vs. high expression of GLT8D1 (GlioVis platform, TCGA_GBM dataset, HG-U133A platform, total 538 entries). Median cutoff; n = 266 for GLT8D1 high, n = 259 for GLT8D1 low; Log rank ∗∗p = 0.0015; Wilcoxon ∗∗p = 0.0026. (C) Kaplan-Meier survival analysis of the patients with the GBM (primary tumors only) showing low vs. high expression of GLT8D1 (GlioVis platform, TCGA_GBM dataset, HG-U133A platform, total 538 entries). Median cutoff; n = 247 for GLT8D1 high, n = 250 for GLT8D1 low; Log rank ∗p = 0.0395; Wilcoxon p = 0.0524. (D) GLT8D1 expression in the cryo-preserved specimens of normal appearing brain (GM – gray matter, WM – white matter) vs. GBM WHO grade IV (qPCR). Mean of three technical replicates per sample + SEM is shown. (E and F) Semiquantitative analysis of GLT8D1 protein expression determined on immunohistochemistry staining of FFPE sections from patient-derived normal appearing brain (NAB; n = 4) and GBM WHO grade IV specimens (GBM; n = 9). Analyzed was the average DAB intensity per nucleus. Data are represented as mean + SEM (F) Representative immunohistochemical DAB staining of GLT8D1 in the FFPE sections of the normal appearing brain (NAB) and of the GBM WHO grade IV specimen in two magnifications (scale bar 100μm).

Figure 2

Predicted UDP-hexosyltransferase active site in GLT8D1 (A) Predicted active site in the homology-based 3D-model of GLT8D1 (residues 62-354). (B) Location of the plausible substrate groove in the model based on UPG and substrate placeholder molecules. (C) Approximate orientation of the predicted active site residues in a close-up, DxD centered view with UPG and metal ion (M²⁺) locations (colored sticks/sphere). Label colors reflect positive (blue), negative (red), neutral (gray) charge at physiological pH, metal-coordinating residues are underlined. See also Figure S1 and Table S4.

Figure 3

Impact of GLT8D1 expression on GBM cell migration (A) GLT8D1 expression in NCH644 and NCH601 cells (qPCR). Unpaired t-test, mean + SEM, n = 3 (∗∗p = 0.0090). (B) Representative immunoblot of GLT8D1 staining in the cell extracts of NCH644 and NCH601 cells and densitometric analysis. Unpaired t-test, mean + SEM, n = 3 (∗p = 0.0434). (C) Immunofluorescence images of GLT8D1 localization in the NCH601 cells as detected by proximity ligation assay. The red immunosignal indicates the interaction of GLT8D1 with the organelle-specific proteins. ER – endoplasmic reticulum (PDI), GA – Golgi apparatus (Golgin97), LS – lysosomes (LAMP1), MT – mitochondria (MTC02), PX – peroxisomes (Catalase). NC – negative control. Scale bar 10μm. (D) GLT8D1 expression in the GLT8D1-overexpressing NCH644 cells (qPCR). ANOVA, mean + SEM, n = 3 (∗p = 0.0204 for GLT8D1-HA #1; ∗p = 0.0192 for GLT8D1-HA #2). (E) Quantitative analysis of cell migration in the GLT8D1-overexpressing NCH644 cells after 48h (Boyden chamber assay). ANOVA, mean + SEM, n = 6 (∗p = 0.0204 for GLT8D1-HA #1; ∗∗∗p = 0.0004 for GLT8D1-HA #2). (F) Representative images of the migrated GLT8D1-overexpressing NCH644 cells after 48h (Boyden chamber assay). Scale bar 100μm. (G) GLT8D1 expression in the GLT8D1-knockdown NCH601 cells (qPCR). ANOVA, mean + SEM, n = 4 (∗p = 0.0265 for sh-GLT8D1 #1; ∗p = 0.0127 for sh-GLT8D1 #2). (H) Quantitative analysis of the cell migration in the GLT8D1-knockdown NCH601 cells after 48h (Boyden chamber assay). ANOVA, mean + SEM, n = 6 (∗∗∗p < 0.0001 for sh-GLT8D1 #1; ∗∗∗p = 0.0004 for sh-GLT8D1 #2). (I) Representative images of the migrated GLT8D1-knockdown NCH601 cells after 48h (Boyden chamber assay). Scale bar 100μm. (J) GLT8D1 expression in the GLT8D1-knockdown NCH601 cells followed by rescue with the wild type GLT8D1 (qPCR). ANOVA, mean + SEM, n = 4 (∗p = 0.0489 for sh-GLT8D1 + Neo; ∗p = 0.0418 for sh-GLT8D1 + rescue). (K) Quantitative analysis of the cell migration in the GLT8D1-knockdown NCH601 cells followed by rescue with the wild type GLT8D1 after 48h (Boyden chamber assay). ANOVA, mean + SEM, n = 7 (∗∗∗p = 0.0006 for sh-GLT8D1 + Neo; ∗p = 0.0172 for sh-GLT8D1 + rescue). (L) Representative images of the migrated GLT8D1-knockdown NCH601 cells or rescue NCH601 cells after 48h (Boyden chamber assay). Scale bar 100μm. See also Figures S2 and S3.

Figure 4

Glycosyltransferase activity of GLT8D1 and its impact on GBM cell migration (A) Domain organization and mutations in GLT8D1 proteins illustrated using DOG 2.0 software (Ren et al., 2009). (B) Graph representing glycosyltransferase reaction rate of wild type GLT8D1 (black), mAS1 (orange) and mAS2 (red) using UDP-Galactose as donor substrate (expressed as mean of n = 3). (C) Graph representing glycosyltransferase reaction rate of wild type GLT8D1, mAS1, and mAS2 (expressed as mean of n = 3) using UDP-Glucose as donor substrate. (D) Representative images of the migrated GLT8D1-knockdown NCH601 cells followed by rescue with GLT8D1, carrying mutations in the active site (mAS1 or mAS2) after 48h (Boyden chamber assay). (E) Quantitative analysis of the cell migration in the GLT8D1-knockdown NCH601 cells followed by rescue with GLT8D1, carrying mutations in the active site (mAS1 or mAS2) after 48h (Boyden chamber assay). ANOVA, mean + SEM, n = 5 (∗∗∗p < 0.0001 for sh-GLT8D1 + Neo; ∗∗p = 0.0065 for sh-GLT8D1 + mAS2). See also Figure S4.