The Interaction between Collagen 1 and High Mannose Type CD133 Up-Regulates Glutamine Transporter SLC1A5 to Promote the Tumorigenesis of Glioblastoma Stem Cells.

Targeting the niche components surrounding glioblastoma stem cells (GSCs) helps to develop more effective glioblastoma treatments. However, the mechanisms underlying the crosstalk between GSCs and microenvironment remain largely unknown. Clarifying the extracellular molecules binding to GSCs marker CD133 helps to elucidate the mechanism of the communication between GSCs and the microenvironment. Here, it is found that the extracellular domain of high mannose type CD133 physically interacts with Collagen 1 (COL1) in GSCs. COL1, mainly secreted by cancer-associated fibroblasts, is a niche component for GSCs. COL1 enhances the interaction between CD133 and p85 and activates Akt phosphorylation. Activation of Akt pathway increases transcription factor ATF4 protein level, subsequently enhances SLC1A5-dependent glutamine uptake and glutathione synthesis. The inhibition of CD133-COL1 interaction or down-regulation of SLC1A5 reduces COL1-accelerated GSCs self-renewal and tumorigenesis. Analysis of glioma samples reveals that the level of COL1 is correlated with histopathological grade of glioma and the expression of SLC1A5. Collectively, COL1, a niche component for GSCs, enhances the tumorigenesis of GSCs partially through CD133-Akt-SLC1A5 signaling axis, providing a new mechanism underlying the cross-talk between GSCs and extracellular matrix (ECM) microenvironment.

Loss of α-1,2-mannosidase MAN1C1 promotes tumorigenesis of intrahepatic cholangiocarcinoma through enhancing CD133-FIP200 interaction.

CD133 is widely used as a marker to isolate tumor-initiating cells in many types of cancers. The structure of N-glycan on CD133 is altered during the differentiation of tumor-initiating cells. However, the relationship between CD133 N-glycosylation and stem cell characteristics remains elusive. Here, we found that the level of α-1,2-mannosylated CD133 was associated with the level of stemness genes in intrahepatic cholangiocarcinoma (iCCA) tissues. α-1,2-mannosylated CD133+ cells possessed the characteristics of tumor-initiating cells. The loss of the Golgi α-mannosidase I coding gene MAN1C1 resulted in the formation of α-1,2-mannosylated CD133 in iCCA-initiating cells. Mechanistically, α-1,2-mannosylation promoted the cytoplasmic distribution of CD133 and enhanced the interaction between CD133 and the autophagy gene FIP200, subsequently promoting the tumorigenesis of α-1,2-mannosylated CD133+ cells. Analysis of iCCA samples showed that the level of cytoplasmic CD133 was associated with poor iCCA prognosis. Collectively, α-1,2-mannosylated CD133 is a functional marker of iCCA-initiating cells.

Siglec15 promotes the migration of thyroid carcinoma cells by enhancing the EGFR protein stability.

PURPOSE: Sialic acid-bound immunoglobulin-like lectin 15 (Siglec15) has emerged as a novel therapeutic target in tumor immunotherapy. This study is designed to investigate the function and mechanism of Siglec15 in thyroid carcinoma (THCA). MATERIALS AND METHODS: The information on patients with THCA from TGCA and GEO database were used to analyze the expression of Siglec15 in THCA. THCA cells were treated with Siglec15-mFc, a recombinant fusion protein consisting of the extracellular domain of human Siglec15 and murine IgG Fc. THP-1 cells expressing human Siglec15 and its mutant were co-cultured with THCA cells to mimic the contact between Siglec15-expressing tumor-associated macrophages and THCA cells. Wound-healing assay and transwell migration assay were used to examine the migration abilities of BCPAP and C643 cells. Pull-down assay was performed to examine the interaction between Siglec15 and epidermal growth factor receptor (EGFR) on the cancer cells. Cycloheximide (CHX) assay was used to evaluate the stability of the protein. RESULTS: The expression of Siglec15 in thyroid carcinoma tissues is higher than in normal tissues. Siglec15 promotes the migration of THCA cells by binding to EGFR in a sialic acid-dependent manner and increases EGFR protein expression. Inhibition of the EGFR pathway blocks the effect of Siglec15 on the migration of THCA cells. CONCLUSIONS: Our findings reveals that Siglec15 promotes the migration of thyroid carcinoma cells by enhancing the EGFR protein stability.

The Interaction between DNMT1 and High-Mannose CD133 Maintains the Slow-Cycling State and Tumorigenic Potential of Glioma Stem Cell.

The quiescent/slow-cycling state preserves the self-renewal capacity of cancer stem cells (CSCs) and leads to the therapy resistance of CSCs. The mechanisms maintaining CSCs quiescence remain largely unknown. Here, it is demonstrated that lower expression of MAN1A1 in glioma stem cell (GSC) resulted in the formation of high-mannose type N-glycan on CD133. Furthermore, the high-mannose type N-glycan of CD133 is necessary for its interaction with DNMT1. Activation of p21 and p27 by the CD133-DNMT1 interaction maintains the slow-cycling state of GSC, and promotes chemotherapy resistance and tumorigenesis of GSCs. Elimination of the CD133-DNMT1 interaction by a cell-penetrating peptide or MAN1A1 overexpression inhibits the tumorigenesis of GSCs and increases the sensitivity of GSCs to temozolomide. Analysis of glioma samples reveals that the levels of high-mannose type N-glycan are correlated with glioma recurrence. Collectively, the high mannose CD133-DNMT1 interaction maintains the slow-cycling state and tumorigenic potential of GSC, providing a potential strategy to eliminate quiescent GSCs.

Siglec-15 promotes the migration of liver cancer cells by repressing lysosomal degradation of CD44.

Sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15) has been identified as a novel potential target for cancer immunotherapy. Here, we explored the role of Siglec-15 in human hepatoma cells. In this study, we found that the expression of Siglec-15 is substantially upregulated in liver cancer tissues in comparison with the nontumor tissues. Functionally, in vitro experiments show that Siglec-15 promotes the migration of hepatoma cells. Furthermore, the data demonstrated an interaction between Siglec-15 and CD44, a transmembrane glycoprotein that mediates tumor progression and metastasis. In addition, we show that CD44 is modified by α2,6-linked sialic acids on N-glycans in hepatoma cells and that CD44 sialylation affects its interaction with Siglec-15. Removal of the sialic acid residues from CD44 resulted in suppressed interaction between Siglec-15 and CD44. We further demonstrate that Siglec-15 interacts and promotes the stability of CD44 by preventing its lysosomal-mediated degradation. Taken together, our findings demonstrate that Siglec-15 promotes the migration of hepatoma cells by regulating the CD44 protein stability.

The binding of LDN193189 to CD133 C-terminus suppresses the tumorigenesis and immune escape of liver tumor-initiating cells.

The tumor-initiating cell (TIC) marker CD133 promotes TIC self-renewal and tumorigenesis through the tyrosine phosphorylation of its c-terminal domain. Therefore, finding compounds that target the phosphorylation of CD133 will provide an effective method for inhibiting TICs characteristics. Here, through small molecule microarray screening, compound LDN193189 was found to bind to the c-terminus of CD133 and influenced its tyrosine phosphorylation. LDN193189 inhibited the interaction between CD133 and p85, accompanied by a reduction in the self-renewal and tumorigenicity of liver TIC. In addition, LDN193189 inhibited the expression and transcription of Galectin-3 by reducing the tyrosine phosphorylation of CD133. Galectin-3 secreted by liver TICs inhibited the proliferation of activated CD8+ T cells by binding to PD-1. LDN193189 suppressed the immune escape ability of liver TICs by downregulating Galectin-3. Taken together, LDN193189 suppressed the tumorigenesis and immune escape of liver CSCs by targeting the CD133-Galectin-3 axis.

N-acetylglucosaminyltransferase I promotes glioma cell proliferation and migration through increasing the stability of the glucose transporter GLUT1.

Abnormal alteration of N-glycosylation structure contributes to glioma progression. N-acetylglucosaminyltransferase I (MGAT1) plays an essential role in the conversion of processed high-mannose cores into complex or hybrid N-linked oligosaccharide structures. The function of MGAT1 in glioma development remains largely unknown. Here, we found that the expression of MGAT1 is higher in glioblastoma compared to normal brain tissues. Inhibition of EGFR signalling pathway or serum starvation reduces MGAT1 expression. Knockdown of MGAT1 inhibits glioma cell proliferation and migration. Furthermore, MGAT1 promotes complex N-glycosylation of glucose transporter 1 (Glut1) and increases Glut1 protein levels. In summary, our findings indicate that MGAT1 is highly expressed in glioblastoma and promotes glioma cells at least partly through upregulation of Glut1 protein.

IL-17A secreted from lymphatic endothelial cells promotes tumorigenesis by upregulation of PD-L1 in hepatoma stem cells.

BACKGROUND & AIMS: The microenvironment regulates hepatoma stem cell behavior. However, the contributions of lymphatic endothelial cells to the hepatoma stem cell niche remain largely unknown; we aimed to analyze this contribution and elucidate the mechanisms behind it. METHODS: Associations between lymphatic endothelial cells and CD133+ hepatoma stem cells were analyzed by immunofluorescence and adhesion assays; with the effects of their association on IL-17A expression examined using western blot, quantitative reverse transcription PCR and luciferase reporter assay. The effects of IL-17A on the self-renewal and tumorigenesis of hepatoma stem cells were examined using sphere and tumor formation assays. The role of IL-17A in immune escape by hepatoma stem cells was examined using flow cytometry. The expression of IL-17A in hepatoma tissues was examined using immunohistochemistry. RESULTS: CD133+ hepatoma stem cells preferentially interact with lymphatic endothelial cells. The interaction between the mannose receptor and high-mannose type N-glycans mediates the interaction between CD133+ hepatoma stem cells and lymphatic endothelial cells. This interaction activates cytokine IL-17A expression in lymphatic endothelial cells. IL-17A promotes the self-renewal of hepatoma stem cells. It also promotes their immune escape, partly through upregulation of PD-L1. CONCLUSION: Interactions between lymphatic endothelial cells and hepatoma stem cells promote the self-renewal and immune escape of hepatoma stem cells, by activating IL-17A signaling. Thus, inhibiting IL-17A signaling may be a promising approach for hepatoma treatment. LAY SUMMARY: The microenvironment is crucial for the self-renewal and development of hepatoma stem cells, which lead to the development of liver cancer. Lymphatic endothelial cells are an important component of this niche microenvironment, helping hepatoma stem cells to self-renew and escape immune attack, by upregulating IL-17A signaling. Thus, targeting IL-17A signaling is a potential strategy for the treatment of hepatoma.

Complex N-glycan promotes CD133 mono-ubiquitination and secretion.

CD133 is a widely used cell surface marker of cancer stem cells that plays an important role in tumor initiation and metastasis. Increasing evidence shows that CD133 is secreted to the extracellular space. However, the underlying mechanisms of CD133 secretion remain largely unknown. In this study, we report that secreted CD133 has a complex-type N-glycosylation and is modified by beta1,6GlcNAc N-glycan. We found that inhibition of CD133 complex-type N-glycosylation by swainsonine does not affect the membrane localization of CD133, but significantly reduces CD133 secretion and promotes its accumulation in early endosomes. Moreover, swainsonine reduces CD133 secretion by reducing its mono-ubiquitination and inhibiting the interaction between CD133 and Tsg101. These findings reveal a new mechanism of glycosylation-dependent secretion of CD133.

O-GlcNAc transferase activates stem-like cell potential in hepatocarcinoma through O-GlcNAcylation of eukaryotic initiation factor 4E.

O-GlcNAcylation catalysed by O-GlcNAc transferase (OGT) is a reversible post-translational modification. O-GlcNAcylation participates in transcription, epigenetic regulation, and intracellular signalling. Dysregulation of O-GlcNAcylation in response to high glucose or OGT expression has been implicated in metabolic diseases and cancer. However, the underlying mechanisms by which OGT regulates hepatoma development remain largely unknown. Here, we employed the lentiviral shRNA-based system to knockdown OGT to analyse the contribution of OGT in hepatoma cell proliferation and stem-like cell potential. The sphere-forming assay and western blot analysis of stem-related gene expression were used to evaluate stem-like cell potential of hepatoma cell. We found that the level of total O-GlcNAcylation or OGT protein was increased in hepatocellular carcinoma. OGT activated stem-like cell potential in hepatoma through eukaryotic initiation factor 4E (eIF4E) which bound to stem-related gene Sox2 5'-untranslated region. O-GlcNAcylation of eIF4E at threonine 168 and threonine 177 protected it from degradation through proteasome pathway. Expression of eIF4E in hepatoma was determined by immunostaining in 232 HCC patients, and Kaplan-Meier survival analysis was used to determine the correlation of eIF4E expression with prognosis. High glucose promoted stem-like cell potential of hepatoma cell through OGT-eIF4E axis. Collectively, our findings indicate that OGT promotes the stem-like cell potential of hepatoma cell through O-GlcNAcylation of eIF4E. These results provide a mechanism of HCC development and a cue between the pathogenesis of HCC and high glucose condition.

Monoubiquitination of Cancer Stem Cell Marker CD133 at Lysine 848 Regulates Its Secretion and Promotes Cell Migration.

CD133, a widely known marker of cancer stem cells, was recently found in extracellular vesicles. However, the mechanisms underlying CD133 translocation to the extracellular space remain largely unknown. Here we report that CD133 is monoubiquitinated. Ubiquitination occurs primarily on complex glycosylated CD133. The lysine 848 residue at the intracellular carboxyl terminus is one of the sites for CD133 ubiquitination. The K848R mutation does not affect CD133 degradation by the lysosomal pathway but significantly reduces CD133 secretion by inhibiting the interaction between CD133 and tumor susceptibility gene 101 (Tsg101). Furthermore, knockdown of the E3 ubiquitin protein ligase Nedd4 largely impairs CD133 ubiquitination and vesicle secretion. Importantly, CD133-containing vesicles are taken up by recipient cells, consequently promoting cell migration. The K848R mutation reduces cell migration induced by CD133. Taken together, our findings show that monoubiquitination contributes to CD133 vesicle secretion and promotes recipient cell migration. These findings provide a clue to the mechanisms of CD133 secretion and cancer stem cell microenvironment interactional effects.

ß1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells.

Malignant glioblastoma multiforme is one of the most aggressive human cancers, with very low survival rates. Recent studies have reported that glioma stem-like cells transdifferentiate into endothelial cells, indicating a new mechanism for tumor angiogenesis and potentially providing new therapeutic options for glioblastoma treatment. Glioma malignancy is strongly associated with altered expression of N-linked oligosaccharide structures on the cell surface. We have previously reported that ß1,4-galactosyltransferase V (ß1,4GalTV), which galactosylates the GlcNAcß1-6Man arm of the branched N-glycans, is highly expressed in glioma and promotes glioma cell growth in vitro and in vivo However, the mechanism by which ß1,4GalTV stimulates glioma growth is unknown. Here we demonstrate that short hairpin RNA-mediated ß1,4GalTV knockdown inhibits the tumorigenesis of glioma stem-like cells and reduces their transdifferentiation into endothelial cells. We also found that ß1,4GalTV overexpression increased glioma stem-like cell transdifferentiation into endothelial cells and that this effect required ß1,4GalTV galactosylation activity. Moreover, ß1,4GalTV promoted ß1,4-galactosylation of Notch1 and increased Notch1 protein levels. Of note, ectopic expression of activated Notch1 rescued the inhibitory effect of ß1,4GalTV depletion on glioma stem-like cell transdifferentiation. In summary, our findings indicate that ß1,4GalTV stimulates transdifferentiation of glioma stem-like cells into endothelial cells by activating Notch1 signaling. These detailed insights shed important light on the mechanisms regulating glioma angiogenesis.

The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration.

CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells.

ER stress inducer tunicamycin suppresses the self-renewal of glioma-initiating cell partly through inhibiting Sox2 translation.

Glioma-initiating cells possess tumor-initiating potential and are relatively resistant to conventional chemotherapy and irradiation. Therefore, their elimination is an essential factor for the development of efficient therapy. Here, we report that endoplasmic reticulum (ER) stress inducer tunicamycin inhibits glioma-initiating cell self-renewal as determined by neurosphere formation assay. Moreover, tunicamycin decreases the efficiency of glioma-initiating cell to initiate tumor formation. Although tunicamycin induces glioma-initiating cell apoptosis, apoptosis inhibitor z-VAD-fmk only partly abrogates the reduction in glioma-initiating cell self-renewal induced by tunicamycin. Indeed, tunicamycin reduces the expression of self-renewal regulator Sox2 at translation level. Overexpression of Sox2 obviously abrogates the reduction in glioma-initiating cell self-renewal induced by tunicamycin. Taken together, tunicamycin suppresses the self-renewal and tumorigenic potential of glioma-initiating cell partly through reducing Sox2 translation. This finding provides a cue to potential effective treatment of glioblastoma through controlling stem cells.

Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth.

The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with ß-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and ß-catenin and inhibited the activation of ß-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies.

Activation of PI3K/Akt pathway by CD133-p85 interaction promotes tumorigenic capacity of glioma stem cells.

The biological significance of a known normal and cancer stem cell marker CD133 remains elusive. We now demonstrate that the phosphorylation of tyrosine-828 residue in CD133 C-terminal cytoplasmic domain mediates direct interaction between CD133 and phosphoinositide 3-kinase (PI3K) 85 kDa regulatory subunit (p85), resulting in preferential activation of PI3K/protein kinase B (Akt) pathway in glioma stem cell (GSC) relative to matched nonstem cell. CD133 knockdown potently inhibits the activity of PI3K/Akt pathway with an accompanying reduction in the self-renewal and tumorigenicity of GSC. The inhibitory effects of CD133 knockdown could be completely rescued by expression of WT CD133, but not its p85-binding deficient Y828F mutant. Analysis of glioma samples reveals that CD133 Y828 phosphorylation level is correlated with histopathological grade and overlaps with Akt activation. Our results identify the CD133/PI3K/Akt signaling axis, exploring the fundamental role of CD133 in glioma stem cell behavior.

Hepatitis B virus X protein blunts senescence-like growth arrest of human hepatocellular carcinoma by reducing Notch1 cleavage.

UNLABELLED: One of the serious sequelae of chronic hepatitis B virus (HBV) infection is hepatocellular carcinoma (HCC). Among all the proteins encoded by the HBV genome, hepatitis B virus X protein (HBx) is highly associated with the development of HCC. Although Notch1 signaling has been found to exert a tumor-suppressive function during HCC development, the mechanism of interaction between HBx expression and Notch1 signaling needs to be explored. In this study, we report that HBx expression in hepatic and hepatoma cells resulted in decreased endogenous protein levels of Notch1 intracellular domain (ICN1) and messenger RNA levels of its downstream target genes. These effects were due to a reduction of Notch1 cleavage by HBx through the suppression of presenilin1 (Psen1) transcription rather than inhibition of Notch1 transcription or its ligands' expression. Through transient HBx expression, decreased ICN1 resulted in enhanced cell proliferation, induced G1-S cell cycle progression, and blunted cellular senescence in vitro. Furthermore, the effect of blunted senescence-like growth arrest by stable HBx expression through suppression of ICN1 was shown in a nude mouse xenograft transplantation model. The correlation of inhibited Psen1-dependent Notch1 signaling and blunted senescence-like growth arrest was also observed in HBV-associated HCC patient tumor samples. CONCLUSION: Our results reveal a novel function of HBx in blunting senescence-like growth arrest by decreasing Notch1 signaling, which could be a putative molecular mechanism mediating HBV-associated hepatocarcinogenesis.

Regulation of the beta1,4-Galactosyltransferase I promoter by E2F1.

Cell surface carbohydrate chains are widely known to contribute to cell migration, recognition and proliferation. beta1,4-Galactosyltransferase I (beta1,4GalT I) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycan, and contributes to cell proliferation, differentiation and migration. Here, we identified beta1,4GalT I as a novel target gene of cell cycle regulator E2F1. E2F1 proteins interact with the promoter of the beta1,4GalT I gene in vivo, and E2F1 over-expression stimulates the activity of beta1,4GalT I promoter and the mRNA and protein expression of beta1,4GalT I, and augments the level of beta1, 4-galactosyltion. Site-specific mutagenesis revealed that this region which contains two E2F1 binding site (nt -215 to -207 and +1 to +6) is necessary for beta1,4GalT I activation by E2F1. Furthermore, down-regulation of beta1,4GalT I expression attenuates E2F1-induced DNA synthesis and cell cycle progression as well as the expression of cell-cycle regulator Cyclin D1. Thus, beta1,4GalT I is an important E2F1 target gene that is required for cell cycle progression in mammalian cells, which elicits a new mechanism of cell growth and a new mechanism of beta1,4GalT I transcription.

Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells.

Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

Alpha2,3-Sialylation regulates the stability of stem cell marker CD133.

CD133 is widely used as a marker for the isolation and characterization of normal and cancer stem cells. The dynamic alternation of CD133 glycosylation contributes to the isolation of normal and cancer stem cells, and is supposed to be associated with cell differentiation. Although CD133 has been identified as a N-glycosylated protein, the specific glycosylation status of CD133 remain unclear. Here, we found that CD133 could be sialylated in neural stem cells and glioma-initiating cells, and the sialyl residues attach to CD133 N-glycan terminal via alpha2,3-linkage. Furthermore, desialylation of CD133 by neuraminidase specifically accelerates its degradation in lysosomes-dependent pathway. Taken together, our results characterized CD133 as an alpha2,3-sialylated glycoprotein and revealed that the sialylation modification contributes to the stability of CD133 protein, providing clues to understanding the function of CD133 molecular and to understanding the utility of glycosylated CD133 epitopes in defining neural stem cells and tumour-initiating cells.