Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth
2023; Elsevier BV; Volume: 299; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2023.105128
ISSN1083-351X
AutoresKenichiro Nagai, Y. Muto, Saori Miura, Kazuto Takahashi, Yu Naruse, Ryo Hiruta, Yuko Hashimoto, Miwa Uzuki, Yoshimi Haga, Risa Fujii, Koji Ueda, Yasushi Kawaguchi, Masazumi Fujii, Shinobu Kitazume,
Tópico(s)Galectins and Cancer Biology
ResumoGliomas 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. 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. Gliomas are the most common primary tumors in the central nervous system (CNS) (1Louis D.N. Perry A. Reifenberger G. von Deimling A. Figarella-Branger D. Cavenee W.K. et al.The 2016 World Health Organization Classification of tumors of the central nervous system: a summary.Acta Neuropathol. 2016; 131: 803-820Crossref PubMed Google Scholar, 2Brain tumor registry of Japan (2005-2008).Neurol. Med. Chir. (Tokyo). 2017; 57: 9-102Crossref PubMed Scopus (99) Google Scholar). High-grade gliomas—such as oligodendroglioma, isocitrate dehydrogenase (IDH) mutant, and 1p/19q-codeleted, World Health Organization (WHO) grade 3; astrocytoma, IDH mutant, WHO grade 3/4; and glioblastoma, IDH wildtype, WHO grade 4—exhibit highly invasive and proliferative phenotypes. Despite advances in surgery, radiation, and chemotherapy treatments, the median survival of patients with glioblastoma, the highest-grade glioma, is only 15 months (3Stupp R. Mason W.P. van den Bent M.J. Weller M. Fisher B. Taphoorn M.J. et al.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.N. Engl. J. Med. 2005; 352: 987-996Crossref PubMed Scopus (15528) Google Scholar). Protein tyrosine phosphatase receptor type Z (PTPRZ) is a membrane protein that is abundantly expressed in CNS glial cells (4Maeda N. Hamanaka H. Shintani T. Nishiwaki T. Noda M. Multiple receptor-like protein tyrosine phosphatases in the form of chondroitin sulfate proteoglycan.FEBS Lett. 1994; 354: 67-70Crossref PubMed Scopus (97) Google Scholar), including oligodendrocyte precursor cells, astrocytes, and oligodendrocytes (5Zhang Y. Chen K. Sloan S.A. Bennett M.L. Scholze A.R. O'Keeffe S. et al.An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.J. Neurosci. 2014; 34: 11929-11947Crossref PubMed Scopus (3129) Google Scholar). Although the multiregulatory roles of PTPRZ—such as in the control of oligodendrocyte precursor cell development (6Lamprianou S. Chatzopoulou E. Thomas J.L. Bouyain S. Harroch S. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17498-17503Crossref PubMed Scopus (66) Google Scholar), regulation of the remyelination process (7Kuboyama K. Fujikawa A. Masumura M. Suzuki R. Matsumoto M. Noda M. Protein tyrosine phosphatase receptor type z negatively regulates oligodendrocyte differentiation and myelination.PLoS One. 2012; 7e48797Crossref PubMed Scopus (48) Google Scholar, 8Kuboyama K. Fujikawa A. Suzuki R. Noda M. Inactivation of protein tyrosine phosphatase receptor type Z by pleiotrophin promotes remyelination through activation of differentiation of oligodendrocyte precursor cells.J. Neurosci. 2015; 35: 12162-12171Crossref PubMed Scopus (43) Google Scholar), and the formation of perineuronal nets (9Eill G.J. Sinha A. Morawski M. Viapiano M.S. Matthews R.T. The protein tyrosine phosphatase RPTPzeta/phosphacan is critical for perineuronal net structure.J. Biol. Chem. 2020; 295: 955-968Abstract Full Text Full Text PDF PubMed Google Scholar)—are well discussed, PTPRZ-deficient mice exhibit no obvious abnormalities (10Harroch S. Palmeri M. Rosenbluth J. Custer A. Okigaki M. Shrager P. et al.No obvious abnormality in mice deficient in receptor protein tyrosine phosphatase beta.Mol. Cell. Biol. 2000; 20: 7706-7715Crossref PubMed Scopus (99) Google Scholar). PTPRZ is abundant in gliomas (11Julien S.G. Dube N. Hardy S. Tremblay M.L. Inside the human cancer tyrosine phosphatome.Nat. Rev. Cancer. 2011; 11: 35-49Crossref PubMed Scopus (391) Google Scholar, 12Müller S. Kunkel P. Lamszus K. Ulbricht U. Lorente G.A. Nelson A.M. et al.A role for receptor tyrosine phosphatase zeta in glioma cell migration.Oncogene. 2003; 22: 6661-6668Crossref PubMed Google Scholar, 13Ulbricht U. Brockmann M.A. Aigner A. Eckerich C. Müller S. Fillbrandt R. et al.Expression and function of the receptor protein tyrosine phosphatase zeta and its ligand pleiotrophin in human astrocytomas.J. Neuropathol. Exp. Neurol. 2003; 62: 1265-1275Crossref PubMed Google Scholar), and its soluble cleaved form (sPTPRZ) is detected at high concentrations in the cerebrospinal fluid (CSF) of glioma patients, indicating that CSF sPTPRZ might be a diagnostic marker for glioma (14Yamanoi Y. Fujii M. Murakami Y. Nagai K. Hoshi K. Hashimoto Y. et al.Soluble protein tyrosine phosphatase receptor type Z (PTPRZ) in cerebrospinal fluid is a potential diagnostic marker for glioma.Neurooncol. Adv. 2020; 2vdaa055PubMed Google Scholar). Furthermore, several reports have suggested that PTPRZ-dependent signaling via its ligand pleiotrophin, which is abundantly secreted from tumor-associated macrophages (15Shi Y. Ping Y.F. Zhou W. He Z.C. Chen C. Bian B.S. et al.Tumour-associated macrophages secrete pleiotrophin to promote PTPRZ1 signalling in glioblastoma stem cells for tumour growth.Nat. Commun. 2017; 815080Crossref Scopus (178) Google Scholar), neural precursor cells (16Qin E.Y. Cooper D.D. Abbott K.L. Lennon J. Nagaraja S. Mackay A. et al.Neural precursor-derived pleiotrophin mediates subventricular zone invasion by glioma.Cell. 2017; 170: 845-859.e19Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and glioma cells (17Lu K.V. Jong K.A. Kim G.Y. Singh J. Dia E.Q. Yoshimoto K. et al.Differential induction of glioblastoma migration and growth by two forms of pleiotrophin.J. Biol. Chem. 2005; 280: 26953-26964Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), supports glioma growth and invasion and the maintenance of glioma stem cells (15Shi Y. Ping Y.F. Zhou W. He Z.C. Chen C. Bian B.S. et al.Tumour-associated macrophages secrete pleiotrophin to promote PTPRZ1 signalling in glioblastoma stem cells for tumour growth.Nat. Commun. 2017; 815080Crossref Scopus (178) Google Scholar). These findings indicate that targeting PTPRZ may be a promising glioma therapy. Indeed, both small interfering RNA targeting PTPRZ and small-molecule PTPRZ inhibitors significantly reduce tumor growth in vivo (18Ulbricht U. Eckerich C. Fillbrandt R. Westphal M. Lamszus K. RNA interference targeting protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta suppresses glioblastoma growth in vitro and in vivo.J. Neurochem. 2006; 98: 1497-1506Crossref PubMed Scopus (67) Google Scholar, 19Fujikawa A. Nagahira A. Sugawara H. Ishii K. Imajo S. Matsumoto M. et al.Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.Sci. Rep. 2016; 620473Crossref Scopus (42) Google Scholar, 20Fujikawa A. Sugawara H. Tanaka T. Matsumoto M. Kuboyama K. Suzuki R. et al.Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells.Sci. Rep. 2017; 7: 5609Crossref PubMed Scopus (36) Google Scholar). However, the relatively large catalytic pocket of PTPRZ (19Fujikawa A. Nagahira A. Sugawara H. Ishii K. Imajo S. Matsumoto M. et al.Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.Sci. Rep. 2016; 620473Crossref Scopus (42) Google Scholar) has hampered the development of small-molecule inhibitors that can cross the blood–brain barrier. Brain PTPRZ undergoes several types of glycosylation (21Nagai K. Fujii M. Kitazume S. Protein tyrosine phosphatase receptor type Z in central nervous system disease.Int. J. Mol. Sci. 2022; 23: 4414Crossref PubMed Scopus (7) Google Scholar, 22Dwyer C.A. Katoh T. Tiemeyer M. Matthews R.T. Neurons and glia modify receptor protein-tyrosine phosphatase zeta (RPTPzeta)/phosphacan with cell-specific O-mannosyl glycans in the developing brain.J. Biol. Chem. 2015; 290: 10256-10273Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), such as by chondroitin sulfate (23Kuboyama K. Fujikawa A. Suzuki R. Tanga N. Noda M. Role of chondroitin sulfate (CS) modification in the regulation of protein-tyrosine phosphatase receptor type Z (PTPRZ) activity: pleiotrophin-ptprz-a signaling is involved in oligodendrocyte differentiation.J. Biol. Chem. 2016; 291: 18117-18128Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), keratan sulfate (24Narentuya Takeda-Uchimura Y. Foyez T. Zhang Z. Akama T.O. Yagi H. et al.GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain.Sci. Rep. 2019; 9: 4387Crossref PubMed Scopus (16) Google Scholar), N-glycans, GalNAc-type O-glycans, and O-mannosyl (O-Man) glycans. Notably, several types of PTPRZ glycans are brain specific. The brain-specific human natural killer-1 (HNK-1) epitope is attached to the nonreducing ends of both N-glycans and O-Man core M1 and M2 glycans of PTPRZ (22Dwyer C.A. Katoh T. Tiemeyer M. Matthews R.T. Neurons and glia modify receptor protein-tyrosine phosphatase zeta (RPTPzeta)/phosphacan with cell-specific O-mannosyl glycans in the developing brain.J. Biol. Chem. 2015; 290: 10256-10273Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 25Yaji S. Manya H. Nakagawa N. Takematsu H. Endo T. Kannagi R. et al.Major glycan structure underlying expression of the Lewis X epitope in the developing brain is O-mannose-linked glycans on phosphacan/RPTPbeta.Glycobiology. 2015; 25: 376-385Crossref PubMed Scopus (20) Google Scholar, 26Kizuka Y. Oka S. Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate Cell.Mol. Life Sci. 2012; 69: 4135-4147Crossref PubMed Scopus (44) Google Scholar, 27Morise J. Takematsu H. Oka S. The role of human natural killer-1 (HNK-1) carbohydrate in neuronal plasticity and disease.Biochim. Biophys. Acta Gen. Subj. 2017; 1861: 2455-2461Crossref PubMed Scopus (28) Google Scholar). Formation of the core M2 branch structure is initiated by the brain-specific N-acetylglucosaminyltransferase IX (GnT-IX) (28Abbott K.L. Matthews R.T. Pierce M. Receptor tyrosine phosphatase beta (RPTPbeta) activity and signaling are attenuated by glycosylation and subsequent cell surface galectin-1 binding.J. Biol. Chem. 2008; 283: 33026-33035Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 29Kanekiyo K. Inamori K. Kitazume S. Sato K. Maeda J. Higuchi M. et al.Loss of branched O-mannosyl glycans in astrocytes accelerates remyelination.J. Neurosci. 2013; 33: 10037-10047Crossref PubMed Scopus (56) Google Scholar). To date, HNK-1-capped O-Man core M2 glycans have only been identified in PTPRZ. Here, we used CRISPR genome-editing technology and demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, suggesting that PTPRZ may be a potential therapeutic target for glioma. Because an important role of protein glycosylation is to protect proteins (30Varki A. Biological roles of glycans.Glycobiology. 2017; 27: 3-49Crossref PubMed Scopus (1354) Google Scholar) and a previous study has reported that GnT-IX-deficient mice have reduced PTPRZ in the brain (29Kanekiyo K. Inamori K. Kitazume S. Sato K. Maeda J. Higuchi M. et al.Loss of branched O-mannosyl glycans in astrocytes accelerates remyelination.J. Neurosci. 2013; 33: 10037-10047Crossref PubMed Scopus (56) Google Scholar), we also explored whether disruption of the GnT-IX gene results in reduced glioma growth. Indeed, GnT-IX knockdown in glioma cells led to reduced cellular PTPRZ and a marked decrease in xenograft tumor growth. Our data demonstrate that, similar to PTPRZ, GnT-IX is a promising target for glioma therapies. Using antibodies, small-molecule inhibitors, or small hairpin RNA in glioma tumors in mouse xenograft models, several groups have reported that PTPRZ is a promising therapeutic target for glioma (19Fujikawa A. Nagahira A. Sugawara H. Ishii K. Imajo S. Matsumoto M. et al.Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.Sci. Rep. 2016; 620473Crossref Scopus (42) Google Scholar, 20Fujikawa A. Sugawara H. Tanaka T. Matsumoto M. Kuboyama K. Suzuki R. et al.Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells.Sci. Rep. 2017; 7: 5609Crossref PubMed Scopus (36) Google Scholar, 31Foehr E.D. Lorente G. Kuo J. Ram R. Nikolich K. Urfer R. Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model.Cancer Res. 2006; 66: 2271-2278Crossref PubMed Scopus (70) Google Scholar). To confirm this, we used CRISPR genome-editing technology and disrupted the PTPRZ gene in LN-229Luc cells (human glioma LN-229 cells that stably express luciferase and GFP). As a result of alternative mRNA splicing, PTPRZ has multiple mRNA isoforms; in humans, there are two main groups: PTPRZ-long and PTPRZ-short (Fig. 1A) (32Hillier L.W. Fulton R.S. Fulton L.A. Graves T.A. Pepin K.H. Wagner-McPherson C. et al.The DNA sequence of human chromosome 7.Nature. 2003; 424: 157-164Crossref PubMed Scopus (214) Google Scholar). Quantitative PCR analysis revealed that LN-229 expressed both PTPRZ isoforms, whereas levels of both types of PTPRZ expression were negligible in the PTPRZ knockdown clone Δ-PTPRZ-LN-229Luc (Fig. 1, B and C). Western blot analysis revealed that PTPRZ-long and PTPRZ-short levels were decreased in ΔPTPRZ-LN-229Luc compared with LN-229Luc (Fig. 1D). Furthermore, in vitro cell proliferation assays demonstrated that cell growth rates were significantly lower in ΔPTPRZ-LN-229Luc than in LN-229-Luc from day 2 of incubation (Fig. 1E). We also investigated whether PTPRZ knockdown suppressed tumor growth in a xenograft glioma model. LN-229Luc or ΔPTPRZ-LN-229Luc were transplanted into the brains of SCID-Beige mice, which are defective in T- and B-cell development and natural killer cell activity; tumor growth was monitored every week using an in vivo imaging system. Although LN-229Luc-transplanted mice showed tumor growth over time, tumor growth was significantly suppressed with ΔPTPRZ-LN-229Luc transplantation; marked differences were observed from week 4 after transplantation (Fig. 1F). Similar to previous reports (19Fujikawa A. Nagahira A. Sugawara H. Ishii K. Imajo S. Matsumoto M. et al.Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.Sci. Rep. 2016; 620473Crossref Scopus (42) Google Scholar, 20Fujikawa A. Sugawara H. Tanaka T. Matsumoto M. Kuboyama K. Suzuki R. et al.Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells.Sci. Rep. 2017; 7: 5609Crossref PubMed Scopus (36) Google Scholar, 31Foehr E.D. Lorente G. Kuo J. Ram R. Nikolich K. Urfer R. Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model.Cancer Res. 2006; 66: 2271-2278Crossref PubMed Scopus (70) Google Scholar), our results indicate the important role of PTPRZ in glioma growth. The extracellular region of PTPRZ is heavily glycosylated, cleaved, and shed (33Chow J.P. Fujikawa A. Shimizu H. Suzuki R. Noda M. Metalloproteinase- and gamma-secretase-mediated cleavage of protein-tyrosine phosphatase receptor type Z.J. Biol. Chem. 2008; 283: 30879-30889Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and the resulting soluble form is known as sPTPRZ or phosphacan (Fig. 2A). We have previously reported that sPTPRZ is detectable in CSF; its level is ten times higher in patients with glioma than in those with other brain diseases such as multiple sclerosis (14Yamanoi Y. Fujii M. Murakami Y. Nagai K. Hoshi K. Hashimoto Y. et al.Soluble protein tyrosine phosphatase receptor type Z (PTPRZ) in cerebrospinal fluid is a potential diagnostic marker for glioma.Neurooncol. Adv. 2020; 2vdaa055PubMed Google Scholar). To explore the glycosylation status of PTPRZ in glioma, we treated sPTPRZ in CSF from glioma patients with several kinds of glycosidases: chondroitinase ABC (ChABC), end-β-galactosidase, sialidase, and peptide-N-glycosidase (PNGase). We used three different antibodies to detect PTPRZ: Cat-315, anti-PTPRZ (Santa Cruz), and antiphosphacan. Antiphosphacan is raised against recombinant phosphacan (sPTPRZ) (34Morise J. Kizuka Y. Yabuno K. Tonoyama Y. Hashii N. Kawasaki N. et al.Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains.Glycobiology. 2014; 24: 314-324Crossref PubMed Scopus (32) Google Scholar), whereas Cat-315 detects the HNK-1-capped O-Man glycan plus PTPRZ peptide region (35Dino M.R. Harroch S. Hockfield S. Matthews R.T. Monoclonal antibody Cat-315 detects a glycoform of receptor protein tyrosine phosphatase beta/phosphacan early in CNS development that localizes to extrasynaptic sites prior to synapse formation.Neuroscience. 2006; 142: 1055-1069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 36Sakuda K. Kizuka Y. Yamaguchi Y. Tanaka K. Ogiwara K. Segawa T. et al.Reactivity of anti-HNK-1 antibodies to branched O-mannose glycans associated with demyelination.Biochem. Biophys. Res. Commun. 2017; 487: 450-456Crossref PubMed Scopus (10) Google Scholar). The epitope information of anti-PTPRZ (Santa Cruz) has not yet been investigated in detail, but both Cat-315 and anti-PTPRZ (Santa Cruz) antibodies react with sPTPRZ-long and sPTPRZ-short in CSF (14Yamanoi Y. Fujii M. Murakami Y. Nagai K. Hoshi K. Hashimoto Y. et al.Soluble protein tyrosine phosphatase receptor type Z (PTPRZ) in cerebrospinal fluid is a potential diagnostic marker for glioma.Neurooncol. Adv. 2020; 2vdaa055PubMed Google Scholar). After ChABC and end-β-galactosidase digestions to remove chondroitin sulfate and keratan sulfate, a sPTPRZ-long signal (300–500 kDa) was detected with antiphosphacan, Cat-315, and anti-PTPRZ (Santa Cruz), indicating that the domain specific to PTPRZ-long is modified with these glycosaminoglycan chains to mask epitope regions (lanes 1 and 2, Figs. 2B and S1), as reported previously (23Kuboyama K. Fujikawa A. Suzuki R. Tanga N. Noda M. Role of chondroitin sulfate (CS) modification in the regulation of protein-tyrosine phosphatase receptor type Z (PTPRZ) activity: pleiotrophin-ptprz-a signaling is involved in oligodendrocyte differentiation.J. Biol. Chem. 2016; 291: 18117-18128Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). These antibodies also detected sPTPRZ-short (∼200 kDa). Sialidase digestion reduced the molecular weights of sPTPRZ-long and sPTPRZ-short, indicating that both forms are sialylated (lanes 2 and 3). After PNGase digestion to remove N-glycans, the bands corresponding to sPTPRZ-long and sPTPRZ-short were shifted, indicating that both forms have N-glycans (lanes 2 and 4) (35Dino M.R. Harroch S. Hockfield S. Matthews R.T. Monoclonal antibody Cat-315 detects a glycoform of receptor protein tyrosine phosphatase beta/phosphacan early in CNS development that localizes to extrasynaptic sites prior to synapse formation.Neuroscience. 2006; 142: 1055-1069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 36Sakuda K. Kizuka Y. Yamaguchi Y. Tanaka K. Ogiwara K. Segawa T. et al.Reactivity of anti-HNK-1 antibodies to branched O-mannose glycans associated with demyelination.Biochem. Biophys. Res. Commun. 2017; 487: 450-456Crossref PubMed Scopus (10) Google Scholar). Sialic acid was also present on O-glycan because sialidase digestion reduced the molecular weight of sPTPRZ-long without N-glycans (lanes 4 and 5). Compared with Cat-315 and anti-PTPRZ (SantaCruz), antiphosphacan reacted weakly with sPTPRZ even after the removal of glycosaminoglycan chains; however, additional glycosidase treatment enhanced the sPTPRZ signals, indicating that glycosylation hinders the epitope of the antiphosphacan antibody. Because both Cat-315 and anti-PTPRZ (Santa Cruz) clearly detected CSF sPTPRZ and their Western blot patterns of glycosidase-treated samples were similar, we speculated that both antibodies recognize brain-specific HNK-1 epitopes on PTPRZ, and that this epitope is crucial for detecting CSF sPTPRZ. To test this idea, we expressed human PTPRZ in human embryonic kidney 293T (HEK293T) cells with or without two types of glycosyltransferases, GlcAT-P (pIRES-glucuronyltransferase) and HNK-1 sulfotransferase (HNK-1ST) (GlycoT X2), which are key enzymes for HNK-1 epitope synthesis. We first expressed full-length PTPRZ-long; however, sPTPRZ-long showed high instability and was almost undetectable in culture media, as has been previously reported (37Kido M. Asano M. Iwakura Y. Ichinose M. Miki K. Furukawa K. Presence of polysialic acid and HNK-1 carbohydrate on brain glycoproteins from beta-1,4-galactosyltransferase-knockout mice.Biochem. Biophys. Res. Commun. 1998; 245: 860-864Crossref PubMed Scopus (32) Google Scholar). We then expressed a shorter sPTPRZ, His-tagged-sPTPRZ754 (sPTPRZ754-His), which lacks transmembrane and cytoplasmic regions and has a His tag at the C terminus. Without GlycoT X2 expression, sPTPRZ754-His was detected using antiphosphacan but not Cat-315 or anti-PTPRZ (Santa Cruz) (lane 2, Fig. 2C). In contrast, with GlycoT X2 expression, sPTPRZ754-His was clearly detected not only with antiphosphacan but also with Cat-315 and anti-PTPRZ (Santa Cruz) (lane 3). These findings indicate that both Cat-315 and anti-PTPRZ (Santa Cruz) antibodies recognize the HNK-1 epitope on sPTPRZ, and that CSF sPTPRZ has this epitope. We then expected that PTPRZ in glioma cells would have the HNK-1 epitope. Unexpectedly, however, PTPRZ was detected in LN-229 with antiphosphacan but not with Cat-315 or anti-PTPRZ (Santa Cruz) (Fig. 2D), indicating the absence of the HNK-1 epitope. Emerging reports indicate that extrinsic signals—such as growth factors and neuronal activity, and resulting transcriptional and chromatin remodeling—are necessary for astrocyte maturation; in vitro cultured astrocytes lack the expression of many mature astrocyte-specific genes (38Hasel P. Dando O. Jiwaji Z. Baxter P. Todd A.C. Heron S. et al.Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism.Nat. Commun. 2017; 815132Crossref Scopus (163) Google Scholar, 39Lattke M. Goldstone R. Ellis J.K. Boeing S. Jurado-Arjona J. Marichal N. et al.Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture.Nat. Commun. 2021; 12: 4335Crossref PubMed Scopus (32) Google Scholar). Such extrinsic signals may also be critical to upregulate glycosylation enzyme genes for the addition of the HNK-1 epitope to PTPRZ in glioma. We therefore investigated whether PTPRZ was modified with the HNK-1 epitope in LN-229Luc transplanted into mouse brains. Unfortunately, Western blot analysis of fluorescence-activated cell sorting (FACS)-sorted LN-229Luc cells was unsuccessful because of severe proteolytic degradation. We therefore decided to analyze xenograft mouse brains without glioma separation. At 6 weeks after glioma injection, lysates from the contralateral cortex had endogenous PTPRZ signal detected using antiphosphacan and anti-PTPRZ (Santa Cruz), whereas lysates from the ipsilateral cortex had strong signal using anti-PTPRZ (Santa Cruz) and Cat-315; these findings indicate that the HNK-1 epitope was expressed in transplanted LN-229 cells. In contrast, these signals were absent in LN-229 lysates from cell culture (Fig. 3A). Moreover, LN-229 lysates had signal detected using anti-PTPRZ (Sigma), which recognizes a peptide portion specific to PTPRZ-long, whereas lysates from the ipsilateral cortex did not have positive signals. These results suggest that glioma PTPRZ in the xenograft model receives additional glycosylation, such as the HNK-1 epitope, which then hinders the epitopes for antiphosphacan and anti-PTPRZ (Sigma). The immunohistochemical analysis of tumor xenografts confirmed that Cat-315 signals were present in human glioma cells (detected with human-specific antigen TRA-1–85; Fig. 3B). We then expected that the expression of glycosylation enzymes for the synthesis of HNK-1 epitope would be suppressed in LN-229 cells cultured in vitro, whereas these expression levels would be upregulated in cells in the xenograft model, possibly by extrinsic signals in vivo. To explore this idea, we performed FACS on LN-229Luc cells from xenografted mouse brains and quantified the mRNA levels of a series of HNK-1-related glycosylation enzymes. Compared with the cells cultured in vitro, the mRNA levels of GnT-IX, β1,4-galactosyltransferase 2 (B4Gal-T2), and HNK-1ST were markedly lower in the in vivo sample (GnT-IX, 3.3%; B4Gal-T2, 25%; and HNK-1ST, 30%), whereas GlcAT-P mRNA levels in the cells in vivo were six times higher than those in vitro (Fig. 3C). These findings indicate that GlcAT-P expression in glioma cells is specifically upregulated by extrinsic signals in the brain. Indeed, ectopic GlcAT-P expression changed the glycan epitope of PTPRZ in LN-229 cells cultured in vitro; PTPRZ was detectable using anti-Cat-315 and -PTPRZ (Santa Cruz) antibodies, whereas reactivity with antiphosphacan disappeared (Fig. 3D). Notably, a higher molecular weight band—detected with anti-Cat-315 and -PTPRZ (Santa Cruz) antibodies—emerged with GlcAT-P expression, similar to that observed in brain lysates from xenograft glioma model mice (Fig. 3A). An important role of protein glycosylation is to protect carrier proteins (30Varki A. Biological roles of glycans.Glycobiology. 2017; 27: 3-49Crossref PubMed Scopus (1354) Google Scholar). Furthermore, it has previously been reported that GnT-IX deficiency results in reductions of both the Cat-315 epitope and PTPRZ in mouse brains (28Abbott K.L. Matthews R.T. Pierce M. Receptor tyrosine phosphatase beta (RPTPbeta) activity and signaling are attenuated by glycosylation and subsequent cell surface galectin-1 binding.J. Biol. Chem. 2008; 283: 33026-33035Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 29Kanekiyo K. Inamori K. Kitazume S. Sato K. Maeda J. Higuchi M. et al.Loss of branched O-mannosyl glycans in astrocytes accelerates remyelination.J. Neurosci. 2013; 33: 10037-10047Crossref PubMed Scopus (56) Google Scholar). We therefore considered whether O-Man core M2 glycan (synthesized by GnT-IX) plays a role in maintaining PTPRZ levels in glioma cells. Because GnT-IX expression was markedly downregulated in LN-229 in xenograft mouse brains compared with those in vitro (Fig. 3A), we first examined GnT-IX expression in human glioma samples. We immunohistochemically analyzed a series of gliomas (oligodendroglioma, IDH mutant and 1p/19q-codeleted, WHO grade 3; astrocytoma, IDH mutant, WHO grade 3; and glioblastoma, mot otherwise specified, WHO grade 4) to verify the expression of GnT-IX and PTPRZ with the HNK-1 epitope (Fig. 4A). All three glioma types were Cat-315 positive, and the plasma membranes were especially strongly stained. Moreover, all glioma types had GnT-IX-signals in the perinuclear region. These findings indicate that PTPRZ and GnT-IX are simultaneously expressed in glio
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