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1.
J Biol Chem ; 295(4): 1009-1020, 2020 01 24.
Article in English | MEDLINE | ID: mdl-31831622

ABSTRACT

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5-2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.


Subject(s)
Antibodies, Neoplasm/immunology , N-Acetylneuraminic Acid/metabolism , Neoplasms/immunology , Stage-Specific Embryonic Antigens/metabolism , Antibodies, Neoplasm/chemistry , Antibody Specificity/immunology , Carbohydrate Conformation , Humans , Immunoglobulin Fab Fragments/metabolism , Ligands , Molecular Dynamics Simulation , Polysaccharides/chemistry , Polysaccharides/metabolism , Stage-Specific Embryonic Antigens/chemistry
2.
J Vis Exp ; (151)2019 09 06.
Article in English | MEDLINE | ID: mdl-31545316

ABSTRACT

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and >50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.


Subject(s)
Oligosaccharides/chemical synthesis , Stage-Specific Embryonic Antigens/chemistry , Glycosylation , Humans , Software
3.
Glycoconj J ; 34(6): 713-723, 2017 12.
Article in English | MEDLINE | ID: mdl-27325407

ABSTRACT

The application of human stem cell technology offers theoretically a great potential to treat various human diseases. However, to achieve this goal a large number of scientific issues remain to be solved. Cell surface carbohydrate antigens are involved in a number of biomedical phenomena that are important in clinical applications of stem cells, such as cell differentiation and immune reactivity. Due to their cell surface localization, carbohydrate epitopes are ideally suited for characterization of human pluripotent stem cells. Amongst the most commonly used markers to identify human pluripotent stem cells are the globo-series glycosphingolipids SSEA-3 and SSEA-4. However, our knowledge regarding human pluripotent stem cell glycosphingolipid expression was until recently mainly based on immunological assays of intact cells due to the very limited amounts of cell material available. In recent years the knowledge regarding glycosphingolipids in human embryonic stem cells has been extended by biochemical studies, which is the focus of this review. In addition, the distribution of the human pluripotent stem cell glycosphingolipids in human tissues, and glycosphingolipid changes during human stem cell differentiation, are discussed.


Subject(s)
Embryonic Stem Cells/metabolism , Glycosphingolipids/metabolism , Blood Group Antigens/chemistry , Blood Group Antigens/metabolism , Glycosphingolipids/chemistry , Humans , Stage-Specific Embryonic Antigens/chemistry , Stage-Specific Embryonic Antigens/metabolism
4.
Anal Chim Acta ; 881: 124-30, 2015 Jun 30.
Article in English | MEDLINE | ID: mdl-26041528

ABSTRACT

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells.


Subject(s)
Biosensing Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pluripotent Stem Cells/cytology , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antigens, Tumor-Associated, Carbohydrate/chemistry , Antigens, Tumor-Associated, Carbohydrate/immunology , Biosensing Techniques/instrumentation , Cell Culture Techniques , DNA, Single-Stranded/analysis , Equipment Design , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Nucleic Acid Amplification Techniques/instrumentation , Pluripotent Stem Cells/immunology , Sensitivity and Specificity , Stage-Specific Embryonic Antigens/chemistry , Stage-Specific Embryonic Antigens/immunology
5.
Glycobiology ; 25(8): 902-17, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25978997

ABSTRACT

Stage-specific embryonic antigen-4 (SSEA-4) is a glycosphingolipid, which is overexpressed in some cancers and has been linked to disease progression. However, little is known about the functions of SSEA-4 and the characteristics of SSEA-4 expressing tumor cells. Our studies identified SSEA-4 expression on a subpopulation of cells in many solid tumor cell lines but not in leukemic cell lines. Fluorescence-activated cell sorting-sorted SSEA-4(+) prostate cancer cells formed fibroblast-like colonies with limited cell-cell contacts, whereas SSEA-4(-) cells formed cobblestone-like epithelial colonies. Only colonies derived from SSEA-4(+) cells were enriched for pluripotent embryonic stem cell markers. Moreover, major epithelial cell-associated markers Claudin-7, E-cadherin, ESRP1 and GRHL2 were down-regulated in the SSEA-4(+) fraction of DU145 and HCT-116 cells. Similar to cell lines, SSEA-4(+) primary prostate tumor cells also showed down-regulation of epithelial cell-associated markers. In addition, they showed up-regulation of epithelial-to-mesenchymal transition as well as mesenchymal markers. Furthermore, SSEA-4(+) cells escape from adhesive colonies spontaneously and form invadopodia-like migratory structures, in which SSEA-4, cortactin as well as active pPI3K, pAkt and pSrc are enriched and colocalized. Finally, SSEA-4(+) cells displayed strong tumorigenic ability and stable knockdown of SSEA-4 synthesis resulted in decreased cellular adhesion to different extracellular matrices. In conclusion, we introduce SSEA-4 as a novel marker to identify heterogeneous, invasive subpopulations of tumor cells. Moreover, increased cell-surface SSEA-4 expression is associated with the loss of cell-cell interactions and the gain of a migratory phenotype, suggesting an important role of SSEA-4 in cancer invasion by influencing cellular adhesion to the extracellular matrix.


Subject(s)
Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Prostate/metabolism , Stage-Specific Embryonic Antigens/genetics , Cadherins/genetics , Cadherins/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Movement , Claudins/genetics , Claudins/metabolism , Cortactin/genetics , Cortactin/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epithelial Cells/pathology , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Humans , Male , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Prostate/pathology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Signal Transduction , Stage-Specific Embryonic Antigens/chemistry , Stage-Specific Embryonic Antigens/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolism
6.
Transfusion ; 55(1): 115-28, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25039255

ABSTRACT

BACKGROUND: "Weak P" is a rare red blood cell (RBC) phenotype, characterized by a global decrease in P(k) and P antigens. We now describe a second weak P individual who also typed LKE-negative (LKE-N) and possessed a clinically significant anti-LKE. STUDY DESIGN AND METHODS: Patient RBCs and plasma were examined by standard serology and flow cytometry. Glycosphingolipids (GSLs) from patient, P(k) , and LKE-strong (LKE-S) RBCs were isolated and analyzed by high-performance thin-layer chromatography (HPTLC). To confirm antibody specificity, patient serum and 30 human polyclonal controls, including alloanti-P and anti-PP1 P(k) , were tested against a panel of GSLs by HPTLC immunostaining. RESULTS: The patient typed P1 +, P+, and LKE-N and possessed a "P-like" panagglutinin. In a two-stage indirect antiglobulin test, the patient's plasma caused hemolysis of LKE-S cells but not p, P(k) , or LKE-N cells. Clinically, transfusion of P+ RBCs compatible by a prewarmed technique had shortened RBC survival with laboratory evidence of hemolysis. Analysis of the patient's isolated RBC GSLs showed a 30% relative decrease in Gb3 (P(k) ) and Gb4 (P) and a 90% decrease in monosialogalactosylgloboside (MSGG, LKE), accompanied by increased lactosylceramide (CDH), paragloboside, and GM3. On HPTLC immunostaining, the patient's plasma strongly bound MSSG with weak binding to galactosylgloboside (Gb5). Binding to MSGG, Gb5, and Gb4 was also observed with some examples of alloanti-P from P(k) individuals, but not anti-PP1 P(k) , autoanti-P, or normal controls. CONCLUSIONS: We describe the first example of a clinically significant anti-LKE in the setting of a rare weak P background. Human alloanti-LKE and some alloanti-P recognized Gb5 and MSGG.


Subject(s)
Anemia, Hemolytic, Autoimmune/blood , Globosides/immunology , Glycosphingolipids/immunology , Isoantibodies/immunology , P Blood-Group System/immunology , Stage-Specific Embryonic Antigens/immunology , Anemia, Hemolytic, Autoimmune/diagnosis , Anemia, Hemolytic, Autoimmune/genetics , Anemia, Hemolytic, Autoimmune/immunology , Antibody Specificity , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Blood Grouping and Crossmatching/methods , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Coombs Test , Erythrocyte Transfusion , Fatal Outcome , Humans , Lymphoma, Large B-Cell, Diffuse/complications , Lymphoma, Large B-Cell, Diffuse/diagnosis , Lymphoma, Large B-Cell, Diffuse/drug therapy , Male , Middle Aged , Phenotype , Stage-Specific Embryonic Antigens/chemistry , Transfusion Reaction/etiology
7.
Carbohydr Res ; 397: 1-6, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-25163606

ABSTRACT

Globo-series glycans are human cell-surface carbohydrates that include stem-cell marker SSEA-4 and cancer-cell antigen Globo H. These two hexasaccharides differ only in their terminal saccharide: N-acetylneuraminic acid in SSEA-4 and L-fucose in Globo H. Herein, we evaluated the affinity of the monoclonal antibodies α-SSEA-4 and α-GH for the glycans SSEA-4 and Globo H. Using fluorescence polarization, we find that the two monoclonal antibodies have affinity for their cognate glycan in the low nanomolar range, and have negligible affinity for the non-cognate glycan. Using surface plasmon resonance, we find that each cognate affinity is ∼20-fold greater if the glycan is immobilized on a surface rather than free in solution. We conclude that the terminal saccharide plays a dominant role in the ability of monoclonal antibodies to recognize these Globo-series glycans and that the extraordinary specificity of these antibodies supports their use for identifying and sorting stem-cells (α-SSEA-4) and as an agent in cancer immunotherapy (α-GH).


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Tumor-Associated, Carbohydrate/immunology , Stage-Specific Embryonic Antigens/immunology , Antibody Specificity , Antigens, Tumor-Associated, Carbohydrate/chemistry , Boron Compounds/chemistry , Fluorescence Polarization , Humans , Solutions , Stage-Specific Embryonic Antigens/chemistry , Surface Plasmon Resonance
8.
Anal Biochem ; 436(2): 160-4, 2013 May 15.
Article in English | MEDLINE | ID: mdl-23416186

ABSTRACT

A lateral flow biosensor based on immunoassay has been developed for the detection of human stem cells for the first time. Antibody specific for a human stem cell surface antigen, SSEA-4, is coated onto gold nanoparticles, whereas antibody against another human pluripotent stem cell surface antigen, SSEA-3, is immobilized on the test zone of the NC membrane. Target cells bind to the antibody coated on the gold nanoparticles to form nanoparticles-stem cell complexes, and the complexes are then captured by another antibody immobilized on the test zone to form a red line for visual detection. This biosensor has been successfully applied to human embryonic stem cells and induced pluripotent stem cells. It is capable of detecting a minimum of 10,000 human embryonic stem cells by the naked eye and 7000 cells with a portable strip reader within 20 min. This approach has also shown excellent specificity to distinguish other types of cells. The biosensor shows great promise for specific and handy detection of human pluripotent stem cells.


Subject(s)
Biosensing Techniques/methods , Pluripotent Stem Cells , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antigens, Tumor-Associated, Carbohydrate/chemistry , Antigens, Tumor-Associated, Carbohydrate/immunology , Biosensing Techniques/instrumentation , Embryonic Stem Cells/cytology , Equipment Design , Gold , Humans , Immunoglobulin G/chemistry , Nanoparticles , Sensitivity and Specificity , Stage-Specific Embryonic Antigens/chemistry , Stage-Specific Embryonic Antigens/immunology
9.
Biotechnol Lett ; 35(1): 55-65, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23007446

ABSTRACT

Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95 % confidence level of the isotype control; however, this study found that adjusting the control gate to a 99 % confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99 % can be implemented on a more routine basis.


Subject(s)
Embryonic Stem Cells/chemistry , Embryonic Stem Cells/cytology , Flow Cytometry/methods , Antigens, Surface/analysis , Antigens, Surface/chemistry , Biomarkers/analysis , Biomarkers/chemistry , Humans , Lewis X Antigen/analysis , Lewis X Antigen/chemistry , Proteoglycans/analysis , Proteoglycans/chemistry , Stage-Specific Embryonic Antigens/analysis , Stage-Specific Embryonic Antigens/chemistry
10.
Stem Cell Rev Rep ; 7(4): 987-96, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21373881

ABSTRACT

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small, physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode, with a stimulation threshold of <30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary, whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF, while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition, a method to aid expansion and survival of stem cells, significantly increased the motility, but reduced directionality of iPS cells in an EF by 70-80%. Thus, our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells.


Subject(s)
Cell Movement , Electrophysiological Phenomena , Induced Pluripotent Stem Cells/physiology , Amides/pharmacology , Biomarkers/analysis , Biomarkers/chemistry , Cell Line , Electric Stimulation , Electrodes , Humans , Induced Pluripotent Stem Cells/chemistry , Induced Pluripotent Stem Cells/cytology , Pyridines/pharmacology , Signal Transduction , Stage-Specific Embryonic Antigens/chemistry , Time-Lapse Imaging , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/chemistry
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