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. 2022 Sep 29:2022:5239006.
doi: 10.1155/2022/5239006. eCollection 2022.

N-Glycosylation on Asn50 of SND1 Is Required for Glioma U87 Cell Proliferation and Metastasis

Ying Zhou  1   2   3 , Qingyu Li  3 , Jianfeng Zheng  4 , Nengming Lin  1
Affiliations

N-Glycosylation on Asn50 of SND1 Is Required for Glioma U87 Cell Proliferation and Metastasis

Ying Zhou et al. J Immunol Res. .

Abstract

Staphylococcal nuclease domain-containing protein 1 (SND1) is an evolutionarily conserved multidomain protein, which has gained attention recently due to its positive regulation in several cancer progression and metastatic spread. However, the specific contribution of SND1 glycosylation in glioma remains uncertain. In the current study, we confirmed that SND1 was highly expressed in human glioma. Using site-directed mutagenesis, we created four predicted N-glycosylation site mutants for SND1 and provided the first evidence that SND1 undergoes N-glycosylation on its Asn50, Asn168, Asn283, and Asn416 residues in human glioma U87 cells. In addition, we found that removing the N-glycans on the Asn50 site destabilized SND1 and led to its endoplasmic reticulum-associated degradation. Furthermore, destabilized SND1 inhibits the glioma cell proliferation and metastasis. Collectively, our results reveal that N-glycosylation at Asn50 is essential for SND1 folding and trafficking, thus essential for the glioma process, providing new insights for SND1 as a potential disease biomarker for glioma.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Expression level of SND1 gene in different cancers and pathological stages. The expression status of the human SND1 gene in different cancers tissues (a), (b) GBM, or specific GBM subtypes (c) was analyzed through GEPIA 2 (http://gepia2.cancer-pku.cn/#analysis). (d) Immunohistochemical staining of SND1 in clinical glioma tissue samples and normal brain tissues were obtained from the Human Protein Atlas (http://www.proteinatlas.org). Normal tissue, access numbers: NOS (M-00100), patient ID: 1582; glioma, malignant: low grade (M-938031), Patient ID: 3120; glioma, malignant: high grade (M-938033), patient ID: 46. (e) The effect of SND1 on the overall survival and disease-free survival of glioblastoma patients was acquired from GEPIA 2.
Figure 2
Figure 2
Analysis of N-glycosylation in SND1. (a) Human glioma U87 cells were treated by tunicamycin (TM) and peptide -N-glycosidase F (PNGase F); the level of SND1 was determined by electrophoresis in 6% SDS-PAGE and then immunoblotted with anti-SND1 antibodies. (b) Three-dimensional structure for human SND1. Locations of four potential N-glycosylation sites (Asn50, Asn168, Asn283, and Asn416) were indicated in yellow. (c) The schematic illustrates structural domains of human SND1 and its potential N-glycosylation site mutagenesis (N50Q, N168Q, N283Q, and N416Q) SN: staphylococcal nuclease domains. Asn (N); Gln (Q). (d) Cell lysate from U87 cells expressing WT-SND1, or its single mutants were analyzed by 6% SDS-PAGE with an anti-GFP antibody. GAPDH was used as a loading control.
Figure 3
Figure 3
Subcellular localization of SND1 N-glycosylation mutants. (a) The localization of WT-SND1-EGFP and its four N-glycosylation mutants (green), CNX (red), and DAPI (blue) in U87 cells. (b) Immunostaining for WT-SND1-EGFP and its four N-glycosylation mutants (green), GM130 (red), and DAPI (blue) in U87 cells. Scale bar, 10 μm.
Figure 4
Figure 4
N-glycosylation of Asn50 induces ER-associated degradation of SND1. (a) U87 cells overexpressing wild-type SND1-EGFP were pretreated with 1 μg/mL TM for 24 h, followed by culture with 10 μM MG132 or 50 μM CQ for 0 h, 1.5 h, and 3 h. The expression level of exogenous SND1 was then determined by immunoblot. (b) U87 cells overexpressing wild-type SND1-EGFP and N50Q-EGFP were treated with MG132 for 3 h before collection of whole lysates. Exogenous expression was then determined by immunoblot. (c) Cells expressing EGFP, SND1-EGFP, and N50Q-EGFP were analyzed by western blot to determine the levels of GRP94, GRP78, CHOP, and CNX. GAPDH was used as a loading control. All these data are representative of at least three independent experiments.
Figure 5
Figure 5
Elimination of N-glycans at Asn50 inhibits cell proliferation and metastasis. (a) The proliferation ability of glioma cells was detected by CCK8 assay performed on both wild-type SND1-EGFP and N50Q-EGFP overexpressing cells with a duration of 4 days. (b) The cell invasion assay was detected by transwell assay administrated in the wild-type SND1-EGFP and N50Q-EGFP overexpressing cells, and invaded cells were fixed and stained with 0.5% crystal violet. Scale bar: 100 μm. (c) Representative images of the bottom surface were captured using a phase-contrast microscopy (upper). The numbers of migrated cells in nine random microscopic fields (×200) were counted and averaged. Data represent the mean ± SEM of three independent experiments. ∗p < 0.01; ∗∗p < 0.001.
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