Two major sulfoglycolipids, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol) exist in mammals. Sulfatide is abundant in the myelin sheath and seminolipid is unique to the ...spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is identical and synthesized by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). We have purified CST homogenously, cloned the CST gene and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. Analysis of CST-null mice has revealed that sulfatide is an essential component for the axo-glial junction at the paranode region and regulates terminal differentiation of oligodendrocytes, and that seminolipid is responsible for the formation of a functional lactate transporter assembly to take up the critical energy source for spermatocytes. We have developed a new analytical method termed EMARS to identify co-clustered molecules in the membrane microdomains in order to elucidate the functional molecules that collaborate with sulfoglycolipids.
Sulfation confers negative charge on glycolipids and the attached sulfate group presents a part of determinants for the molecular interactions. Mammalian sulfoglycolipids are comprised of two major ...members, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is biosynthesized via sequential reactions catalyzed by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). To elucidate the biological function of sulfoglycolipids, we have purified CST, cloned the CST gene, and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis. CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms underlying the biological events for which sulfoglycolipids are essential are ongoing. (Communicated by Kunihiko SUZUKI, M.J.A.)
Display omitted
For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid–poly(ethylene glycol)–chitosan oligosaccharide lactate (FA–PEG–COL) nanoparticles is ...vital for delivery to cancer site(s). siRNA/FA–PEG–COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA–PEG–COL conjugate was characterized by MALDI-TOF-MS, FT-IR and 1H NMR. The average size of siRNA/FA–PEG–COL nanoparticles was approximately 200nm, and the surface charge was +8.4mV compared to +30.5mV with siRNA/COL nanoparticles. FA–PEG–COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76–62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA–PEG–COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA–PEG–COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA–PEG–COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy.
Ganglioside GD2 is specifically expressed in small‐cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes ...malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme‐mediated activation of radical sources combined with mass spectrometry in GD2+ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid‐enriched microdomain/rafts in GD2+ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2+ SCLC cells were enhanced by glutamine uptake, and were suppressed by L‐γ‐glutamyl‐p‐nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid‐enriched microdomain/rafts in GD2+ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.
We defined ASCT2, a glutamine transporter as a ganglioside GD2‐associating molecule with novel methods. ASCT2 plays key roles in the enhancement of malignant properties of small cell lung cancer cells by collaborating with cancer‐associated antigen, GD2, and would be a target of cancer treatment.
Protein-protein interactions are essential in biological reactions and fundamental to cell-cell communication (e.g., the binding of secreted proteins, such as hormones, to cell membrane receptors) ...and the subsequent intracellular signal transduction cascade. Several studies have been extensively carried out on protein-protein interactions because they have the potential to resolve various problems in molecular biology. Biochemical methods, such as chemical cross-linking and immunoprecipitation, have long been used to analyze which proteins interact with each other. However, there are some problems, such as unphysiological states and non-specific binding, that require the development of more useful experimental methods. This chapter discusses the "proximity labeling (Proteomics)" analysis technique, which has been attracting attention in protein-protein interaction analysis in recent years and is used in many biological studies. "Membrane proximity labeling (proteomics)," which analyzes the interaction of cell membrane proteins, and "intracellular proximity labeling (proteomics)" will be explained in-depth.
Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for ...treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1
mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1
endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin β3 was disturbed in St6gal1
mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-β3, indicating that the reduction of cell surface integrin-β3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.
Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, ...identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains.
The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane ...organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.
Glioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the ...evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid-polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth.
To examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt-8 assay in mouse glioma cells. The efficacy of siRNA therapy-targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis.
In vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146-cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice.
CD146 is a potential therapeutic target, and folic acid-conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.
Cancer‐specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective ...cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane‐resident “cis‐bimolecular complex” as a possible cancer target (cis‐bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme‐mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co–expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.
Here, we examined the cancer cell membrane‐resident “cis‐bimolecular complex” as a possible cancer target (cis‐bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT has the potential to contribute to several molecular targeted strategies in future.
PDF
