Targeting branched N-glycans and fucosylation sensitizes ovarian tumors to immune checkpoint blockade.

Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, it's unclear whether homologous recombination (HR) status-dependent glycosylation can be therapeutically explored. Here, we show that the inhibition of branched N-glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe an enrichment of branched N-glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N-glycans. The branched N-glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8+ T cells. In orthotopic, syngeneic EOC models in female mice, inhibiting branched N-glycans using 2-Deoxy-D-glucose sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N-glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion.

Impaired glycosylation of gastric mucins drives gastric tumorigenesis and serves as a novel therapeutic target.

BACKGROUND: Gastric cancer is often accompanied by a loss of MUC6, but its pathogenic role in gastric carcinogenesis remains unclear. METHOD: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, A4gnt-/- mice were also used. Histology, DNAs and RNAs, proteins, and sugar chains were analyzed by whole exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and LC-MS analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULT: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on MAPK activation, mediated by Golgi stress-induced upregulation of GOLPH3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with Banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. MAPK activation, Golgi stress responses, aberrant mannose expression are found in a separate Cosmc- and A4gnt-deficient mouse models which lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSION: We propose that Golgi stress responses and aberrant glycans are important drivers of, and promising new therapeutic targets for gastric cancer.

Aberrant Glycosylation in Pancreatic Ductal Adenocarcinoma 3D Organoids Is Mediated by KRAS Mutations.

Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells, to gasp the comprehensive cancer cell surface glycan expression profile using lectin microarray and transcriptomic analyses. Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. We successfully established 16 3D organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS was mutated in 13 (7 G12V, 5 G12D, 1 Q61L) and wild in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Lectin reactivity of AAL (Aleuria aurantia) and AOL (Aspergillus oryzae) with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. Meanwhile, mannose-binding lectin (rRSL [Ralstonia solanacearum] and rBC2LA [Burkholderia cenocepacia]) signals were higher while those of galactose-binding lectins (rGal3C and rCGL2) were lower in the KRAS mutants. We demonstrated here that PDAC 3D-cultured organoids with KRAS mutations were dominantly covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers.

Droplet-Based Glycan and RNA Sequencing for Profiling the Distinct Cellular Glyco-States in Single Cells.

Plate-based single-cell glycan and RNA sequencing (scGR-seq) is previously developed to realize the integrated analysis of glycome and transcriptome in single cells. However, the sample size is limited to only a few hundred cells. Here, a droplet-based scGR-seq is developed to address this issue by adopting a 10x Chromium platform to simultaneously profile ten thousand cells' glycome and transcriptome in single cells. To establish droplet-based scGR-seq, a comparative analysis of two distinct cell lines is performed: pancreatic ductal adenocarcinoma cells and normal pancreatic duct cells. Droplet-based scGR-seq revealed distinct glycan profiles between the two cell lines that showed a strong correlation with the results obtained by flow cytometry. Next, droplet-based scGR-seq is applied to a more complex sample: peripheral blood mononuclear cells (PBMC) containing various immune cells. The method can systematically map the glycan signature for each immune cell in PBMC as well as glycan alterations by cell lineage. Prediction of the association between the glycan expression and the gene expression using regression analysis ultimately leads to the identification of a glycan epitope that impacts cellular functions. In conclusion, the droplet-based scGR-seq realizes the high-throughput profiling of the distinct cellular glyco-states in single cells.

Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions.

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.

Single-Cell Glycan and RNA Sequencing (scGR-seq).

Single-cell multi-omics analysis has emerged as a crucial tool in modern biology due to its capacity to uncover the intricate molecular heterogeneity within individual cells. Glycan structural diversity on the cell surface creates a "cell signature" that varies by cell type and state. However, single-cell glycan analysis remains a challenge. This protocol presents a series of techniques and procedures that enable the simultaneous measurements of glycan and RNA at the single-cell level via DNA-barcoded lectin-based sequencing (scGR-seq). The techniques include (1) the preparation of DNA-barcoded lectins, (2) a step-by-step protocol for single-cell glycan/RNA sequencing, and (3) a data analysis approach for the integration of glycan and RNA data. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of DNA-barcoded lectins Basic Protocol 2: Single-cell glycan/RNA sequencing.

Lectin Blotting of Cell Lysates.

Glycans are composed of branched structures consisting of monosaccharides, such as glucose and galactose linked by glycosidic bonds. Glycans are often bound to proteins and lipids and are localized at the cell surface. They are deeply involved in a wide range of multicellular systems inside and outside the cells, such as the quality control of glycoproteins, cell-cell communication, and various diseases. While western blotting uses antibodies to detect proteins, lectin blotting uses lectins, which are glycan-binding proteins, to detect glycans on glycoconjugates, such as glycoproteins. Lectin blotting was first reported in the early 1980s and has been widely used in life science for several decades. However, it is not straightforward to obtain consistent data using lectin blotting, which tends to show high backgrounds and lab-to-lab variation. Here, we describe the protocol used in our laboratory for lectin blotting following protein separation by SDS-PAGE to detect glycoproteins extracted from cell membrane fractions. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Extraction and quantification of proteins from cell lysate Basic Protocol 2: Lectin peroxidase labeling and lectin blotting.

Novel positron emission tomography imaging targeting cell surface glycans for pancreatic cancer: 18 F-labeled rBC2LCN lectin.

Advancement in early detection modalities will greatly improve the overall prognoses of pancreatic ductal adenocarcinoma (PDAC). For this purpose, we report a novel class of tumor-specific probes for positron emission tomography (PET) based on targeting cell surface glycans. The PDAC-targeting ability of rBC2LCN lectin, combined with fluorine-18 (18 F) ([18 F]FB-rBC2LCN), resulted in reproducible, high-contrast PET imaging of tumors in a PDAC xenograft mouse model. [18 F]N-succinimidyl-4-fluorobenzoate ([18 F]SFB) was conjugated to rBC2LCN, and [18 F]FB-rBC2LCN was successfully prepared with a radiochemical purity >95%. Cell binding and uptake revealed that [18 F]FB-rBC2LCN binds to H-type-3-positive Capan-1 pancreatic cancer cells. As early as 60 min after [18 F]FB-rBC2LCN (0.34 ± 0.15 MBq) injection into the tail vein of nude mice subcutaneously bearing Capan-1 tumors, tumor uptake was high (6.6 ± 1.8 %ID/g), and the uptake increased over time (8.8 ± 1.9 %ID/g and 11 ± 3.2 %ID/g at 150 and 240 min after injection, respectively). Tumor-to-muscle ratios increased over time, up to 19 ± 1.8 at 360 min. High-contrast PET imaging of tumors relative to background muscle was also achieved as early as 60 min after injection of [18 F]FB-rBC2LCN (0.66 ± 0.12 MBq) and continued to increase up to 240 min. Our 18 F-labeled rBC2LCN lectin warrants further clinical development to improve the accuracy and sensitivity of early-stage pancreatic cancer detection.

rBC2LCN-reactive SERPINA3 is a glycobiomarker candidate for pancreatic ductal adenocarcinoma.

Early detection is urgently needed to improve the patient's pancreatic ductal adenocarcinoma (PDAC) survival. Previously, we identified a novel tumor-associated glycan, H-type3, which is expressed on PDAC cells and is detected by rBC2LCN (recombinant N-terminal domain of BC2L-C identified from Burkholderia cenocepacia) lectin. Here, we identified that SERPINA3 is an rBC2LCN-reactive glycoprotein (BC2-S3) secreted from PDAC cells into the blood in patients with PDAC by liquid chromatography-tandem mass spectrometry analysis and lectin blotting. In immune staining, BC2-S3 was detected specifically in the tumor but not in normal tissues of PDAC. Lectin-ELISA was then developed to measure the serum level of BC2-S3 in healthy control (HC, n = 99) and patients with PDAC (n = 88). BC2-S3 exhibited higher in patients with PDAC than in those with HC. BC2-S3 showed similar diagnostic performance in all stages of PDAC (stages IA-IV, true positive rate = 76.1%, true negative rate = 81.8%) to CA19-9 (72.7%, 75.8%). Remarkably, BC2-S3 showed a significantly higher detection rate (89.7%) for early stage PDAC (IA-IIA) than CA19-9 (62.1%, P = 0.029). The combination of BC2-S3 and CA19-9 further improved the diagnostic ability for all stages of PDAC (81.8%, 87.9%). In conclusion, BC2-S3 is a glycobiomarker candidate for PDAC.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Core fucose-specific Pholiota squarrosa lectin (PhoSL) as a potent broad-spectrum inhibitor of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.

Hyporesponsiveness against donor's ABO antigens of renal grafts after ABO-incompatible kidney transplantation.

BACKGROUND: ABO antigens expressed on the red blood cells (RBCs) are not identical to those expressed on the renal endothelial cells. The isohemagglutinin assay employing the RBCs is the gold standard for evaluating anti-ABO antibody (Ab) levels. However, it remains unclear whether the anti-ABO Abs detected by the isohemagglutinin assay after ABO-incompatible (ABOi) kidney transplantations (KTx) that are not associated with antibody-mediated rejection can bind to renal graft endothelial cells. METHODS: Ninety plasma samples were collected from patients with stable graft function after ABO-compatible (ABOc) or ABOi KTx. Anti-ABO Ab titers were examined by both the isohemagglutinin assay and the CD31-ABO microarray, which was developed as a mimic of the ABO antigens expressed on the renal endothelial cells. RESULTS: The antibody titers detected by the isohemagglutinin assay and the CD31-ABO microarray after the ABOc KTx relatively correlated with each other. However, the CD31-ABO microarray results showed low antibody levels against donor blood group antigens after ABOi KTx and did not correlate with the isohemagglutinin assay. In contrast, the antibody levels against non-donor blood group antigens after ABOi KTx were comparable to those after the ABOc KTx. Fourteen patients received graft biopsies, and no antibody-mediated rejection was observed in ABOi KTx recipients, except for two patients who had anti-donor-HLA Abs. CONCLUSION: The present study suggested that the anti-ABO Abs detected by the isohemagglutinin assay after ABOi KTx with stable graft function were hyporeactive to the ABO antigen of graft renal endothelial cells.

Lectin-based phototherapy targeting cell surface glycans for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to current treatments but lectin-based therapy targeting cell surface glycans could be a promising new horizon. Here, we report a novel lectin-based phototherapy (Lec-PT) that combines the PDAC targeting ability of rBC2LCN lectin to a photoabsorber, IRDye700DX (rBC2-IR700), resulting in a novel and highly specific near-infrared, light-activated, anti-PDAC therapy. Lec-PT cytotoxicity was first verified in vitro with a human PDAC cell line, Capan-1, indicating that rBC2-IR700 is only cytotoxic upon cellular binding and exposure to near-infrared light. The therapeutic efficacy of Lec-PT was subsequently verified in vivo using cell lines and patient-derived, subcutaneous xenografting into nude mice. Significant accumulation of rBC2-IR700 occurs as early as 2 hours postintravenous administration while cytotoxicity is only achieved upon exposure to near-infrared light. Repeated treatments further slowed tumor growth. Lec-PT was also assessed for off-target toxicity in the orthotopic xenograft model. Shielding of intraperitoneal organs from near-infrared light minimized off-target toxicity. Using readily available components, Lec-PT specifically targeted pancreatic cancer with high reproducibility and on-target, inducible toxicity. Rapid clinical development of this method is promising as a new modality for treatment of pancreatic cancer.

Bioorthogonal Photoreactive Surfaces for Single-Cell Analysis of Intercellular Communications.

Methods to construct single-cell pairs of heterogeneous cells attract attention because of their potential in cell biological and medical applications for analyzing individual intercellular communications such as immune and nerve synaptic interactions. Photoactivatable substrate surfaces for cell anchoring are promising tools to achieve single-cell pairing. However, conventional surfaces that photoactivate a single type of cell anchoring moiety restrict the combination of cell pair types and their applications. We developed a photoresponsive material comprising a bioorthogonal photoreactive moiety and non-cell adhesive hydrophilic polymer. The material-coated surface allows conjugation with various cell anchoring molecules in response to light at specific timings and consequently achieves light-induced anchoring of a variety of cells at defined regions. Using the platform surface, an array of cancer cell and natural-killer (NK) cell pairs was constructed on a flat substrate surface and the dynamic morphological changes of the cancer cells were monitored by cytotoxic interaction with NK cells at a single-cell level. The photoreactive surface is a useful tool for image-based investigation of the communications between a variety of cell types.

Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A).

N-Glycosylation is a common post-translational modification, and the number of GlcNAc branches in N-glycans impacts glycoprotein functions. N-Acetylglucosaminyltransferase-IVa (GnT-IVa, also designated as MGAT4A) forms a ß1-4 GlcNAc branch on the α1-3 mannose arm in N-glycans. Downregulation or loss of GnT-IVa causes diabetic phenotypes by dysregulating glucose transporter-2 in pancreatic ß-cells. Despite the physiological importance of GnT-IVa, its structure and catalytic mechanism are poorly understood. Here, we identify the lectin domain in mouse GnT-IVa's C-terminal region. The crystal structure of the lectin domain shows structural similarity to a bacterial GlcNAc-binding lectin. Comprehensive glycan binding assay using 157 glycans and solution NMR reveal that the GnT-IVa lectin domain selectively interacts with the product N-glycans having a ß1-4 GlcNAc branch. Point mutation of the residue critical to sugar recognition impairs the enzymatic activity, suggesting that the lectin domain is a regulatory subunit for efficient catalytic reaction. Our findings provide insights into how branching structures of N-glycans are biosynthesized.

Glycan Profiling by Sequencing to Uncover Multicellular Communication: Launching Glycobiology in Single Cells and Microbiomes.

Glycans are essential building blocks of life that are located at the outermost surface of all cells from mammals to bacteria and even viruses. Cell surface glycans mediate multicellular communication in diverse biological processes and are useful as "surface markers" to identify cells. Various single-cell sequencing technologies have already emerged that enable the high-throughput analysis of omics information, such as transcriptome and genome profiling on a cell-by-cell basis, which has advanced our understanding of complex multicellular interactions. However, there has been no robust technology to analyze the glycome in single cells, mainly because glycans with branched and heterogeneous structures cannot be readily amplified by polymerase chain reactions like nucleic acids. We hypothesized that the generation of lectins conjugated with DNA barcodes (DNA-barcoded lectins) would enable the conversion of glycan information to gene information, which may be amplified and measured using DNA sequencers. This technology will enable the simultaneous analysis of glycan and RNA in single cells. Based on this concept, we developed a technology to analyze glycans and RNA in single cells, which was referred to as scGR-seq. Using scGR-seq, we acquired glycan and gene expression profiles of individual cells constituting heterogeneous cell populations, such as tissues. We further extended Glycan-seq to the profiling of the surface glycans of bacteria and even gut microbiota. Glycan-seq and scGR-seq are new technologies that enable us to elucidate the function of glycans in cell-cell and cell-microorganism communication, which extends glycobiology to the level of single cells and microbiomes.

Glycan expression profile of signet ring cell gastric cancer cells and potential applicability of rBC2LCN-targeted lectin drug conjugate therapy.

BACKGROUND: Signet ring cell carcinoma (SRC) is a distinct subtype of gastric cancer (GC); however, the specific characteristics of cancer cell surface glycans and glycosylation remain unclear. In this study, we investigated SRC-specific glycans using lectin microarray and evaluated the potential applicability of a glycan-targeting therapy. METHODS: SRC cell lines (NUGC-4 and KATO-III) and non-SRC (NSRC) cell lines (NCI-N87, SNU-1, and MKN-45) were subjected to lectin microarray analysis to identify the SRC-specific glycans. Additionally, we performed immunohistochemical lectin staining and evaluated the anti-tumor effects of lectin drug conjugates (LDCs) using high-affinity lectins for SRC. RESULTS: Among the 96 lectins tested, 11 high-affinity and 8 low-affinity lectins were identified for SRC. Glycan-binding motifs varied in the high-affinity lectins, but 5 (62.5%) low-affinity lectins bound the same glycan structure, α2-6-linked sialic acids. The ratio of signal intensity in SRC to NSRC (SRC/NSRC) was highest in the rBC2LCN lectin (1.930-fold), followed by the BPL lectin (1.786-fold). rBC2LCN lectin showed high affinity for both SRC cell lines and one of the three NSRC cell lines (NCI-N87). The therapeutic effects of the LDC, rBC2LCN-PE38 (rBC2LCN, and Pseudomonas exotoxin A), showed cytocidal effects in vitro and tumor regression in in vivo mouse xenograft models. CONCLUSION: We reported specific glycan profiles in SRC cells, showing reduced α2-6-linked sialic acids. Additionally, we found a targeted therapy using rBC2LCN lectin might be applicable as an alternative treatment option for patients with SRC.

A Novel Method of CD31-Combined ABO Carbohydrate Antigen Microarray Predicts Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation.

Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO antigens expressed on RBCs are not identical to those of kidney and antibody titers do not always correlate with clinical outcome. We previously reported that CD31 was the main protein linked to ABO antigens on kidney endothelial cells (KECs), which was different from those on RBCs. We developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO antigens (CD31-ABO microarray). Mass spectrometry analysis suggested that rCD31 and native CD31 purified from human kidney had similar ABO glycan. To confirm clinical use of CD31-ABO microarray, a total of 252 plasma samples including volunteers, hemodialysis patients, and transplant recipients were examined. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value of AABMR, compared to isohemagglutinin assays. Use of a CD31-ABO microarray to determine antibody titer specifically against ABO antigens expressed on KECs will contribute to precisely predicting AABMR or preventing over immunosuppression following ABOi KTx.

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.

The therapeutic capacity of bone marrow MSC-derived extracellular vesicles in Achilles tendon healing is passage-dependent and indicated by specific glycans.

The therapeutic potential of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) for various diseases and tissue repair is attracting attention. Here, EVs from conditioned medium of human bone marrow MSCs at passage 5 (P5) and passage 12 (P12) were analysed using mouse Achilles tendon rupture model and lectin microarray. P5 MSC-EVs accelerated Achilles tendon healing compared with P12 MSC-EVs. Fucose-specific lectin TJA-II was indicated as a glycan marker for therapeutic MSC-EVs. The present study demonstrated that early passaged MSC-EVs promote Achilles tendon healing compared with senescent MSC-EVs. Glycans on MSC-EVs might provide useful tools to establish a quality control and isolation system for therapeutic MSC-EVs in regenerative medicine.