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Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

ABSTRACT: Gliomas are the most prevalent primary tumor of the central nervous system. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor type Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 capped O-mannosyl core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology, we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of human natural killer-1-capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

SUBMITTER: Nagai K

PROVIDER: S-EPMC10480537 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

Nagai Kenichiro K Muto Yui Y Miura Saori S Takahashi Kazuto K Naruse Yu Y Hiruta Ryo R Hashimoto Yuko Y Uzuki Miwa M Haga Yoshimi Y Fujii Risa R Ueda Koji K Kawaguchi Yasushi Y Fujii Masazumi M Kitazume Shinobu S

The Journal of biological chemistry 20230804 9

Gliomas are the most prevalent primary tumor of the central nervous system. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor type Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 capped O-mannosyl cor ...[more]

PMID: 37543361

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Project description:N-Acetylglucosaminlyltransferase-V (GnT-V) synthesizes GlcNAcbeta1,6Man branched N-glycans both in vitro and in vivo. A paralog, GnT-Vb (or GnT-IX), has also been shown to synthesize both GlcNAcbeta1,6Man branched N- and O-glycans. GnT-V is expressed in most human and rodent tissues while GnT-Vb expression is limited mainly to neural tissue and testes. It is of interest, therefore, to compare the catalytic properties and reaction kinetics of these sister enzymes. The results demonstrate that while GnT-V was fully active without exogenous cation and in the presence of EDTA, the activity of GnT-Vb was stimulated over 4-fold in the presence of 10 mM Mn(++). The pH optimum for GnT-V was in the range of 6.5-7.0, while that of GnT-Vb was 8.0. common for glycosyltransferases active in brain. Both enzymes transferred GlcNAcbeta1,6 to the Man residue of the GlcNAcbeta1,2Man moiety of glycan substrates, and both enzymes acted effectively on a synthetic GlcNAcbeta1,2Manalpha1,2Glc-O-octyl trisaccharide acceptor. Moreover, although both enzymes utilized an N-linked asialo-agalacto-biantennary glycan as an acceptor, GnT-Vb displayed an almost 2.5-fold higher apparent K(m) value compared to GnT-V. Conversely, GnT-Vb very efficiently glycosylated a synthetic glycopeptide, Ac-H(2)N-Val-Glu-Pro-(GlcNAcbeta1,2-Man-O-)Thr-Ala-Val-CO-Ac, while GnT-V showed relatively poor activity toward this O-Man-linked glycopeptide acceptor, with a K(m) value of 20-fold higher than that of GnT-Vb. When the N-linked asialo-agalacto-biantennary glycan acceptor was utilized with GnT-Vb, the expected triantennary beta1,6-branched product was observed up to 8 h incubation. An additional product with two beta1,6-linked GlcNAc resides, however, was observed after prolonged (>8 h) incubation, consistent with an earlier report. This unusual tetraantennary product was observed with GnT-Vb only after substantial accumulation of the first triantennary product and not during the early stages of incubation.

Project description:BackgroundVascular endothelial growth factor (VEGF) signaling pathway plays an important role in the progression of diabetic retinopathy (DR). The glycosylation modification process of many key functional proteins in DR patients is abnormal. However, the potential involvement of abnormal N-glycoproteins in DR progression remains unclear.MethodsGlycoproteomic profiling of the vitreous humor was performed. The level of protein and N-glycoprotein was confirmed by Western blot and Lectin blot, respectively. The cell viability and migration efficiency were detected by CCK-8 and Transwell assay. Flow cytometry was conducted to analyze the level of cell apoptosis and reactive oxygen specie. Malondialdehyde, superoxide dismutase activity and VEGF content were detected by Enzyme linked immunosorbent assays. The interaction of metalloproteinase 1 (TIMP-1) with N-acetylglucosamine transferase V (GnT-V) was detected by GST pull-down. Hematoxylin and eosin staining and choroidal and retinal flat mount stained with fluorescein isothiocyanate-Dextran assay were used for functional research in vivo.ResultsWe found that N-glycosylation was up-regulated in DR rats and high glucose (HG)-induced human retinal pigment epithelium cell line ARPE-19. HG-induced inhibited the viability of ARPE-19 cells and promoted cell apoptosis and oxidative stress (OS), but these effects were reversed with kifunensine treatment, GnT-V knockdown and TIMP-1 mutation. Additionally, GnT-V binds to TIMP-1 to promote N-glycosylation of TIMP-1. Over-expression of GnT-V inhibited the viability of ARPE-19 cells and promoted cell apoptosis, OS and VEGF release, which these effects were reversed with TIMP-1 mutation. Interestingly, over-expression of GnT-V promoted retinal microvascular endothelial cells (RMECs) angiogenesis but was revered with TIMP-1 mutation, which was terminally boosted by VEGF-A treatment. Finally, knockdown of GnT-V relieved DR progression.ConclusionThe findings indicate that GnT-V can promote RMECs angiogenesis and ARPE-19 cells injury through activation VEGF signaling pathway by increasing TIMP-1 N-glycosylation level, which provides a new theoretical basis for the prevention of DR.

Dataset Information

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

Publications

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

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