Cancer Malignancy Is Correlated with Upregulation of PCYT2-Mediated Glycerol Phosphate Modification of α-Dystroglycan.

The dystrophin-glycoprotein complex connects the cytoskeleton with base membrane components such as laminin through unique O-glycans displayed on α-dystroglycan (α-DG). Genetic impairment of elongation of these glycans causes congenital muscular dystrophies. We previously identified that glycerol phosphate (GroP) can cap the core part of the α-DG O-glycans and terminate their further elongation. This study examined the possible roles of the GroP modification in cancer malignancy, focusing on colorectal cancer. We found that the GroP modification critically depends on PCYT2, which serves as cytidine 5'-diphosphate-glycerol (CDP-Gro) synthase. Furthermore, we identified a significant positive correlation between cancer progression and GroP modification, which also correlated positively with PCYT2 expression. Moreover, we demonstrate that GroP modification promotes the migration of cancer cells. Based on these findings, we propose that the GroP modification by PCYT2 disrupts the glycan-mediated cell adhesion to the extracellular matrix and thereby enhances cancer metastasis. Thus, the present study suggests the possibility of novel approaches for cancer treatment by targeting the PCYT2-mediated GroP modification.

Carbohydrate Sulfation As a Mechanism for Fine-Tuning Siglec Ligands.

The immunomodulatory family of Siglecs recognizes sialic acid-containing glycans as "self", which is exploited in cancer for immune evasion. The biochemical nature of Siglec ligands remains incompletely understood, with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs─CHST1, CHST2, or CHST4─significantly enhance the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, a pharmacological blockade of N- and O-glycan maturation reveals a cell-type-specific pattern of importance for either class of glycan. Production of a highly homogeneous Siglec-3 (CD33) fragment enabled a mass-spectrometry-based binding assay to determine ≥8-fold and ≥2-fold enhanced affinity for Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc and Neu5Acα2-3Galß1-4(6-O-sulfo)GlcNAc, respectively, over Neu5Acα2-3Galß1-4GlcNAc. CD33 shows significant additivity in affinity (≥28-fold) for the disulfated ligand, Neu5Acα2-3(6-O-sulfo)Galß1-4(6-O-sulfo)GlcNAc. Moreover, joint overexpression of CHST1 with CHST2 in cells greatly enhanced the binding of CD33 and several other Siglecs. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a general mechanism for tuning Siglec ligands on cells, including in cancer.

Establishment of a novel monoclonal antibody against truncated glycoforms of α-dystroglycan lacking matriglycans.

α-Dystroglycan (α-DG) is a glycoprotein specifically modified with O-mannosyl glycans bearing long polysaccharides, termed matriglycans, which comprise repeating units of glucuronic acid and xylose. The matriglycan is linked to the O-mannosyl glycan core through two ribitol phosphate units that can be replaced with glycerol phosphate (GroP) units synthesized by fukutin and fukutin-related protein that transfer GroP from CDP-Gro. Here, we found that forced expression of the bacterial CDP-Gro synthase, TagD, from Bacillus subtilis could result in the overproduction of CDP-Gro in human colon carcinoma HCT116 cells. Western blot and liquid chromatography-tandem mass spectrometry analyses indicated that α-DG prepared from the TagD-expressing HCT116 cells contained abundant GroP and lacked matriglycans. Using the GroP-containing recombinant α-DG-Fc, we developed a novel monoclonal antibody, termed DG2, that reacts with several truncated glycoforms of α-DG, including GroP-terminated glycoforms lacking matriglycans; we verified the reactivity of DG2 against various types of knockout cells deficient in the biosynthesis of matriglycans. Accordingly, forced expression of TagD in HCT116 cells resulted in the reduction of matriglycans and an increase in DG2 reactivity. Collectively, our results indicate that DG2 could serve as a useful tool to determine tissue distribution and function of α-DG lacking matriglycans under physiological and pathophysiological conditions.

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/ß-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through ß-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma.

The glycome of one of the largest and most exposed human organs, the skin, as well as glycan changes associated with non-melanoma skin cancers have not been studied in detail to date. Skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are among the most frequent types of cancers with rising incidence rates in the aging population. We investigated the healthy human skin N- and O-glycome and its changes associated with BCC and SCC. Matched patient samples were obtained from frozen biopsy and formalin-fixed paraffin-embedded tissue samples for glycomics analyses using two complementary glycomics approaches: porous graphitized carbon nano-liquid chromatography electro spray ionization tandem mass spectrometry and capillary gel electrophoresis with laser induced fluorescence detection. The human skin N-glycome is dominated by complex type N-glycans that exhibit almost similar levels of α2-3 and α2-6 sialylation. Fucose is attached exclusively to the N-glycan core. Core 1 and core 2 type O-glycans carried up to three sialic acid residues. An increase of oligomannose type N-glycans and core 2 type O-glycans was observed in BCC and SCC, while α2-3 sialylation levels were decreased in SCC but not in BCC. Furthermore, glycopeptide analyses provided insights into the glycoprotein candidates possibly associated with the observed N-glycan changes, with glycoproteins associated with binding events being the most frequently identified class.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring ß-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

Direct Mapping of Additional Modifications on Phosphorylated O-glycans of α-Dystroglycan by Mass Spectrometry Analysis in Conjunction with Knocking Out of Causative Genes for Dystroglycanopathy.

Dystroglycanopathy is a major class of congenital muscular dystrophy caused by a deficiency of functional glycans on α-dystroglycan (αDG) with laminin-binding activity. Recent advances have led to identification of several causative gene products of dystroglycanopathy and characterization of their in vitro enzymatic activities. However, the in vivo functional roles remain equivocal for enzymes such as ISPD, FKTN, FKRP, and TMEM5 that are supposed to be involved in post-phosphoryl modifications linking the GalNAc-ß3-GlcNAc-ß4-Man-6-phosphate core and the outer laminin-binding glycans. Herein, by direct nano-LC-MS2/MS3 analysis of tryptic glycopeptides derived from a truncated recombinant αDG expressed in the wild-type and a panel of mutated cells deficient in one of these enzymes, we sought to define the full extent of variable modifications on this phosphorylated core O-glycan at the functional Thr317/Thr319 sites. We showed that the most abundant glycoforms carried a phosphorylated core at each of the two sites, with and without a single ribitol phosphate (RboP) extending from terminal HexNAc. At much lower signal intensity, a novel substituent tentatively assigned as glycerol phosphate (GroP) was additionally detected. As expected, tandem RboP extended with a GlcA-Xyl unit was only identified in wild type, whereas knocking out of either ISPD or FKTN prevented formation of RboP. In the absence of FKRP, glycoforms with single but not tandem RboP accumulated, consistent with the suggested role of this enzyme in transferring the second RboP. Intriguingly, the single GroP modification also required functional FKTN whereas absence of TMEM5 significantly hindered only the addition of RboP. Our findings thus revealed additional levels of complexity associated with the core structures, suggesting functional interplay among these enzymes through their interactions. The simplified analytical workflow developed here should facilitate rapid mapping across a wider range of cell types to gain better insights into its physiological relevance.

Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity.

Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3ß (GSK3ß) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by ß-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3ß binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3ß and ß-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3ß inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.

Facile removal of high mannose structures prior to extracting complex type N-glycans from de-N-glycosylated peptides retained by C18 solid phase to allow more efficient glycomic mapping.

The relative amount of high mannose structures within an N-glycomic pool differs from one source to another, but quite often it predominates over the larger size complex type structures carrying biologically important glyco-epitopes. An efficient method to separate these two classes of N-glycans would significantly aid in detecting the lower abundant components by MS. Capitalizing on an initial observation that only high mannose type structures were recovered in the flow-through fraction when peptide-N-glycosidase F digested peptides were passed through a C18 cartridge in 0.1% formic acid, we demonstrated here that native complex type N-glycans can be retained by C18 cartridge and to be efficiently separated from both the smaller high mannose type structures, as well as de-N-glycosylated peptides by stepwise elution with increasing ACN concentration. The weak retention of the largely hydrophilic N-glycans on C18 resin is dependent not only on size but also increased by the presence of α6-fucosylation. This was shown by comparing the resulting N-glycomic profiles of the washed and low-ACN eluted fractions derived from both a human cancer cell line and an insect cell line.

Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

Polysaccharides purified from the submerged culture of Ganoderma formosanum stimulate macrophage activation and protect mice against Listeria monocytogenes infection.

The bioactive components of Ganoderma formosanum have not yet been characterized. We investigated the immunomodulatory activities of the extracellular polysaccharides produced from a submerged mycelial culture of G. formosanum. The polysaccharides were mainly composed of D-mannose, D-galactose and D-glucose. After gel filtration chromatography, three polysaccharide fractions (PS-F1, PS-F2 and PS-F3) were purified. PS-F2 stimulated mouse RAW264.7 macrophages to produce TNF-α and nitric oxide, and enhanced the phagocytic activity of macrophages. PS-F2 challenge in mice triggered an acute inflammatory response characterized by the recruitment of neutrophils and monocytes, which protected mice from subsequent infection of Listeria monocytogenes. The results indicate that the heteropolysaccharides produced by G. formosanum can activate the innate immune response on macrophages.

Glycomics and proteomics analyses of mouse uterine luminal fluid revealed a predominance of Lewis Y and X epitopes on specific protein carriers.

Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization. Using a concerted proteomics and glycomics approach with advanced mass spectrometry-based glycan sequencing capability, we show in this work that 24p3, an abundant mouse uterine luminal fluid (ULF) glycoprotein also called lipocalin 2 (Lcn2), is highly fucosylated in the context of carrying multiple Lewis X and Y epitopes on complex type N-glycans at its single glycosylation site. The predominance of Lewis X/Y along with Neu5Acalpha2-6 sialylation was found to be a salient feature of the ULF glycome, and several other protein carriers were additionally identified including the highly abundant lactotransferrin, which is N-glycosylated at two sites, both with a similar range of highly fucosylated N-glycans. A comparative glycomics analysis of the male genital tract fluids revealed that there is a gradient of glycomic complexity from the cauda to caput regions of the epididymis, varying from high mannose to sialylated complex type N-glycans but mostly devoid of fucosylation. The seminal vesicle fluid glycome, on the other hand, carries equally abundant multimeric Lewis X structures but is distinctively lacking in additional fucosylation of the terminal galactose to give the Lewis Y epitope typifying the glycome of female ULF. One-dimensional shotgun proteomics analysis identified over 40 proteins in the latter, many of which are reported for the first time, and a majority are notably involved in immune defense and antigen processing. Further sperm binding and motility assays suggest that the Lewis X/Y epitopes do contribute to the sperm motility-enhancing activity of 24p3, whereas lactotransferrin is largely inactive in this context despite being similarly glycosylated. These findings underline the importance of glycoproteomics in delineating both the specific glycan structures and their carriers in assigning glycobiological functions.

A transcriptomic and proteomic analysis of the effect of CpG-ODN on human THP-1 monocytic leukemia cells.

The CpG motif of bacterial DNA (CpG-DNA) is a potent immunostimulating agent whose mechanism of action is not yet clear. Here, we used both DNA microarray and proteomic approaches to investigate the effects of oligodeoxynucleotides containing the CpG motif (CpG-ODN) on gene transcription and protein expression profiles of CpG-ODN responsive THP-1 cells. Microarray analysis revealed that 2 h stimulation with CpG-ODN up-regulated 50 genes and down-regulated five genes. These genes were identified as being associated with inflammation, antimicrobial defense, transcriptional regulation, signal transduction, tumor progression, cell differentiation, proteolysis and metabolism. Longer stimulation (8 h) with CpG-ODN enhanced transcriptional expression of 58 genes. Among these 58 genes, none except one, namely WNTI inducible signaling pathway protein 2, was the same as those induced after 2 h stimulation. Proteomic analysis by two-dimensional gel electrophoresis, followed by mass spectrometry identified several proteins up-regulated by CpG-ODN. These proteins included heat shock proteins, modulators of inflammation, metabolic proteins and energy pathway proteins. Comparison of microarray and proteomic expression profiles showed poor correlation. Use of more reliable and sensitive analyses, such as reverse transcriptase polymerase chain reaction, Western blotting and functional assays, on several genes and proteins, nonetheless, confirmed that there is indeed good correlation between mRNA and protein expression after CpG-ODN treatment. This study also revealed that several anti-apoptotic and neuroprotective related proteins, not previously reported, are activated by CpG-DNA. These findings have extended our knowledge on the activation of cells by CpG-DNA and may contribute to further understanding of mechanisms that link innate immunity with acquired immune response(s).