Quantitative evaluation of glycan-binding specificity of recombinant concanavalin A produced in lettuce (Lactuca sativa).

Concanavalin A (ConA), a mannose (Man)-specific leguminous lectin isolated from the jack bean (Canavalia ensiformis) seed extracts, was discovered over a century ago. Although ConA has been extensively applied in various life science research, recombinant mature ConA expression has not been fully established. Here, we aimed to produce recombinant ConA (rConA) in lettuce (Lactuca sativa) using an Agrobacterium tumefaciens-mediated transient expression system. rConA could be produced as a fully active form from soluble fractions of lettuce leaves and purified by affinity chromatography. From 12 g wet weight of lettuce leaves, 0.9 mg rConA could be purified. The glycan-binding properties of rConA were then compared with that of the native ConA isolated from jack bean using glycoconjugate microarray and frontal affinity chromatography. rConA demonstrated a glycan-binding specificity similar to nConA. Both molecules bound to N-glycans containing a terminal Man residue. Consistent with previous reports, terminal Manα1-6Man was found to be an essential unit for the high-affinity binding of rConA and nConA, while bisecting GlcNAc diminished the binding of rConA and nConA to Manα1-6Man-terminated N-glycans. These results demonstrate that the fully active rConA could be produced using the A. tumefaciens-mediated transient expression system and used as a recombinant substitute for nConA.

Carcinoembryonic antigen as a specific glycoprotein ligand of rBC2LCN lectin on pancreatic ductal adenocarcinoma cells.

The rBC2LCN lectin, known as a stem cell marker probe that binds to an H type 3 fucosylated trisaccharide motif, was recently revealed to also bind to pancreatic ductal adenocarcinoma (PDAC) cells. A lectin-drug conjugate was generated by fusing rBC2LCN with a cytocidal toxin, and it showed a strong anticancer effect in in vitro and in vivo PDAC models. However, it is unclear which molecules are carrier proteins of rBC2LCN on PDAC cells. In this study, we identified a rBC2LCN-positive glycoprotein expressed in PDAC. Tumor lysates of PDAC patient-derived xenografts (PDXs) were coprecipitated with rBC2LCN lectin and analyzed by liquid chromatography-mass spectrometry. A total of 343 proteins were initially identified. We used a web-based database to select five glycoproteins and independently evaluated their expression in PDAC by immunohistochemistry (IHC). Among them, we focused on carcinoembryonic antigen 5 (CEA) as the most cancer-specific carrier protein in PDAC, as it showed the most prominent difference in expression rate between PDAC cells (74%) and normal pancreatic duct cells (0%, P > .0001). rBC2LCN lectin and CEA colocalization in PDAC samples was confirmed by double-staining analysis. Furthermore, rBC2LCN-precipitated fractions were blotted with an anti-CEA polyclonal antibody (pAb), and CEA pAb-precipitated fractions were blotted with rBC2LCN lectin. The results demonstrate that CEA is in fact a ligand of rBC2LCN lectin.

Monoclonal antibodies specific for podocalyxin expressed on human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are useful starting materials for the generation of cell therapy products, due to their pluripotency and ability to self-renew. Quality control of hiPSCs is extremely important in creating a stable supply of hPSC-derived products. Previously we identified an hiPSC-specific lectin probe, rBC2LCN, which binds specifically to α1,2-fucosylated glycan and recognizes podocalyxin (PODXL) as a glycoprotein ligand. In this study, we produced monoclonal antibodies (mAbs) specific for α1,2-fucosylated PODXL expressed on hiPSCs. PODXL was recombinantly expressed in fucosyltransferase 1 (FUT1)-transfected HEK293, followed by immunization into mice. Monoclonal antibodies, which bind to PODXL/FUT1-transfected cells, but not to cells transfected with only one of PODXL or FUT1, were screened by flow cytometry. The two mAbs generated (179-6B8C9 and 179-7E12E10), termed α1,2-fucosylated PODXL-specific mAbs (FpMabs), showed binding specificity to PODXL/FUT1-transfected cells. The FpMabs bound to hiPSCs but never to human adipose-derived mesenchymal stem cells, human dermal fibroblasts, or hiPSC-derived mesoderm. Altogether, FpMabs are highly specific probes for hiPSCs, which might be a powerful tool for the characterization of hiPSCs used in regenerative medicine.

SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells.

Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1+ glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1+ deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1+ FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1+ deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine.

Oriented immobilization of rBC2LCN lectin for highly sensitive detection of human pluripotent stem cells using cell culture supernatants.

Human pluripotent stem cells (hPSCs) are considered ideal cell sources for regenerative medicine, but their clinical and industrial applications are hindered by their tumorigenic potential. We previously identified an hPSC-specific lectin, rBC2LCN, that recognizes the podocalyxin glycoprotein secreted by undifferentiated hPSCs into the culture media. Using biotinylated rBC2LCN and a peroxidase-labeled R-10G antibody, we developed a sandwich assay for the detection of tumorigenic hPSCs. In this assay, the lectin is randomly immobilized on streptavidin-coated microplates to capture hPSC-derived podocalyxin. In the present study, rBC2LCN was genetically fused with polystyrene-binding peptides (PS-tags) for direct, site-specific, and oriented immobilization on polystyrene microplates. rBC2LCN lectins fused with PS-tags at the C-terminus were successfully overexpressed as a soluble form in Escherichia coli and then purified by affinity chromatography. We optimized the various parameters (protein and NaCl concentration, buffer pH, and blocking agents) of the sandwich assay by using PS-tagged rBC2LCN and the R-10G antibody. Finally, the lower limit of detection (LLOD) of the sandwich assay for hPSCs was examined. The LLOD was 2.2-fold lower than that achieved with the previous method. Considering that the developed method does not require the precoating of polystyrene microplates with streptavidin, it provides a cost-effective approach for the highly sensitive detection of hPSCs residing in hPSC-derived cell therapeutics.

Photoactivable Elimination of Tumorigenic Human Induced Pluripotent Stem Cells by Using a Lectin-Doxorubicin Prodrug Conjugate.

Human pluripotent stem cells (hPSCs) are attractive resources for regenerative medicine, but medical applications are hindered by their tumorigenic potential. Previously, a hPSC-specific lectin probe, rBC2LCN, was identified through comprehensive glycome analysis by using high-density lectin microarrays. Herein, a lectin-doxorubicin (DOX) prodrug conjugate, with controllable photolysis activation for the elimination of tumorigenic human induced pluripotent stem cells, has been developed. rBC2LCN was fused with a biotin-binding protein, tamavidin (BC2Tama), and the fusion protein was expressed in Escherichia coli and purified by means of affinity chromatography. BC2Tama was then conjugated with doxorubicin-photocleavable biotin (DOXPCB). The BC2Tama-DOXPCB conjugates were observed to bind to hPSCs followed by internalization. Upon exposure to ultraviolet light, DOX was released inside the cells, which allowed specific killing of the hPSCs. Thus, BC2Tama-DOXPCB should be useful for the targeted elimination of hPSCs contained in hPSC-derived cell therapy products. This is the first report of the generation of lectin-prodrug conjugates. BC2Tama should be applicable for the targeted delivery of various types of biotinylated compounds into hPSCs.

Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray.

Glycans are one of the major building blocks of extracellular vesicles (EVs). However, their roles and applications have not been completely explored. Here, we analyzed the glycome of EVs derived from human induced pluripotent stem cells (hiPSCs) using high-density lectin microarray. The glycan profiles of hiPSC-derived EVs were different from those of non-hiPSC-derived EVs. Moreover, rBC2LCN that shows specific binding to hiPSCs, showed strong specificity for hiPSC-derived EVs but not non-hiPSCs-derived EVs. Further, other hiPSC-specific probes, such as anti-TRA-1-60, anti-SSEA4, and anti-R-10G, exhibited specific, but weaker binding to hiPSC-derived EVs than rBC2LCN. We then developed a sandwich assay using rBC2LCN and a phosphatidylserine receptor, Tim4, to specifically detect hiPSC-derived EVs. The Tim4-rBC2LCN sandwich assay allowed for specific detection of hiPSC-derived EVs but not non-hiPSC-derived EVs, indicating that rBC2LCN could also be used for the specific detection of hiPSC-derived EVs. Together, our findings demonstrate that the characteristic glycan signature of hiPSCs are retained by EVs derived from them. The EV glycome could be novel targets for the identification and characterization of stem cells for use in regenerative medicine.

α2-6 sialylation is a marker of the differentiation potential of human mesenchymal stem cells.

Human somatic stem cells such as human mesenchymal stem cells (hMSCs) are considered attractive cell sources for stem cell-based therapy. However, quality control issues have been raised concerning their safety and efficacy. Here we used lectin microarray technology to identify cell surface glycans as markers of the differentiation potential of stem cells. We found that α2-6Sia-specific lectins show stronger binding to early passage adipose-derived hMSCs (with differentiation ability) than late passage cells (without the ability to differentiate). Flow cytometry analysis using α2-6Sia-specific lectins supported the results obtained by lectin microarray. Similar results were obtained for bone marrow-derived hMSCs and cartilage tissue-derived chondrocytes. Little or no binding of α2-6Sia-specific lectins was observed for human dermal fibroblasts, which are unable to differentiate, suggesting that the binding of α2-6Sia-specific lectins is associated with the differentiation ability of cells, but not to their capacity to proliferate. Quantitative analysis of the linkage mode of Sia using anion-exchange chromatography showed that the percentage of α2-6Sia linkage type was higher in early passage adipose-derived hMSCs than late passage cells. Integrinα5 was found to be a carrier protein of α2-6Sia. Sialidase treatment significantly reduced the differentiation efficiency of bone marrow-derived hMSCs. Based on these findings, we propose that α2-6sialylation is a marker of differentiation potential in stem cells such as adipose-derived hMSCs, bone marrow-derived hMSCs, and cartilage tissue-derived chondrocytes.