Cell Surface and Functional Features of Cortical Bone Stem Cells

The newly established mouse cortical-bone-derived stem cells (mCBSCs) are unique stem cells compared to mouse mesenchymal stem cells (mMSCs). The mCBSC-treated hearts after myocardial infarction have been reported to have greater improvement in myocardial structure and functions. In this study, we examined the stemness features, cell surface glycan profiles, and paracrine functions of mCBSCs compared with mMSCs. The stemness analysis revealed that the self-renewing capacity of mCBSCs was greater than mMSCs; however, the differentiation capacity of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles by lectin array analysis revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. In contrast, the lactosamine (Galβ1-4GlcNAc) structure, poly lactosamine- or poly N-acetylglucosamine structure, and α2-3sialic acid on both N- and O-glycans were more highly expressed in mCBSCs. Moreover, we found that mCBSCs secrete a greater amount of TGF-β1 compared to mMSCs, and that the TGF-β1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Together, these results suggest that unique characteristics in mCBSCs compared to mMSCs may lead to advanced utility of mCBSCs for cardiac and noncardiac repair.

Dectin-2 mediates phagocytosis of Lactobacillus paracasei KW3110 and IL-10 production by macrophages

Lactic acid bacteria (LAB) are most generally used as probiotics and some strains of LAB are known to have anti-inflammatory effects. A specific strain of lactic acid bacteria, Lactobacillus paracasei KW3110 (KW3110), activates macrophages to produce interleukin-10 (IL-10), an anti-inflammatory cytokine; however, the biological mechanism remains unclear. In this study, we showed that the amount of incorporated KW3110 into a macrophage cell line, RAW 264.7, was higher than other genetically related strains using fluorescence microscopy. RNA-seq analysis indicated that treatment of macrophages with KW3110 induced Dectin-2 gene expression, which is a pattern recognition receptor, recognizing α-mannose. In addition, antibody treatment and knock down of Dectin-2, or factors downstream in the signaling pathway, decreased the amount of incorporated KW3110 and IL-10 production. Substantial lectin array analysis also revealed that KW3110 had higher binding affinities to lectins, which recognize the carbohydrate chains comprised of α-mannose, than two other LAB. In conclusion, KW3110 is readily incorporated into macrophages, leading to IL-10 production. Dectin-2 mediated the phagocytosis of KW3110 into macrophages and this may be involved with the characteristic carbohydrate chains of KW3110.

Brugia malayi Glycoproteins Detected by the Filariasis Test Strip Antibody AD12.1

BackgroundRapid and accurate prevalence mapping of lymphatic filariasis (LF) is necessary to eliminate this disfiguring and disabling neglected tropical disease. Unfortunately, rapid tests such as the filariasis test strip (FTS) for Wuchereria bancrofti, the causative agent of LF in Africa, can cross-react with antigens circulating in some persons infected by the African eye worm, Loa loa, rendering the test unreliable in eleven co-endemic nations. The intended target of the FTS is a heavily glycosylated W. bancrofti circulating filarial antigen (Wb-CFA). Previously, we determined that the FTS monoclonal antibody, AD12.1, which detects a carbohydrate epitope on Wb-CFA, also detects multiple L. loa proteins in cross-reactive sera from persons with loiasis. Since the carbohydrate epitope recognized by AD12.1 is present on glycoproteins of other parasitic nematodes, including Brugia species, it is unclear why reactive glycoproteins are not detected in infections with other filarial parasites.MethodsTo gain a better understanding of the proteins recognized by the FTS diagnostic antibody, we used proteomics and lectin array technology to characterize filarial glycoproteins that are bound by the AD12.1 antibody using Brugia malayi as a model.ResultsDistinct but overlapping sets of AD12 glycoproteins were identified from somatic and excretory/secretory worm products. One of the identified proteins, Bm18019 was confirmed as a secreted AD12-reactive glycoprotein by in-gel proteomics and immunoassays. Based on lectin binding patterns, Brugia AD12-reactive glycoproteins express glycans including core fucose, galactose, N-acetylglucosamine and galactose(β1-3)N-acetylgalactosamine in addition to the epitope recognized by AD12.1. None of the lectins that bound B. malayi AD12 glycoproteins had affinity for the Wb-CFA, highlighting a key difference between it and other AD12 glycoproteins.ConclusionsB. malayi somatic and excretory/secretory proteins are similar to L. loa antigens found in FTS-positive human sera, bolstering the hypothesis that circulating L. loa AD12 antigens result from worm tissue damage or death. The difference in glycan and protein composition between the Wb-CFA and other AD12 glycoproteins can be used to differentiate LF from cross-reactive loiasis.

Cell surface carbohydrates of symbiotic dinoflagellates and their role in the establishment of cnidarian–dinoflagellate symbiosis

Symbiodiniaceae algae are often photosymbionts of reef-building corals. The establishment of their symbiosis resembles a microbial infection where eukaryotic pattern recognition receptors (e.g. lectins) are thought to recognize a specific range of taxon-specific microbial-associated molecular patterns (e.g. glycans). The present study used the sea anemone, Exaiptasia diaphana and three species of Symbiodiniaceae (the homologous Breviolum minutum, the heterologous-compatible Cladocopium goreaui and the heterologous-incompatible Fugacium kawagutii) to compare the surface glycomes of three symbionts and explore the role of glycan–lectin interactions in host–symbiont recognition and establishment of symbiosis. We identified the nucleotide sugars of the algal cells, then examined glycans on the cell wall of the three symbiont species with monosaccharide analysis, lectin array technology and fluorescence microscopy of the algal cell decorated with fluorescently tagged lectins. Armed with this inventory of possible glycan moieties, we then assayed the ability of the three Symbiodiniaceae to colonize aposymbiotic E. diaphana after modifying the surface of one of the two partners. The Symbiodiniaceae cell-surface glycome varies among algal species. Trypsin treatment of the alga changed the rate of B. minutum and C. goreaui uptake, suggesting that a protein-based moiety is an essential part of compatible symbiont recognition. Our data strongly support the importance of D-galactose (in particular β-D-galactose) residues in the establishment of the cnidarian–dinoflagellate symbiosis, and we propose a potential involvement of L-fucose, D-xylose and D-galacturonic acid in the early steps of this mutualism.

Cell Surface and Functional Features of Cortical Bone Stem Cells

Background: The newly established mouse cortical bone–derived stem cells (mCBSCs) are unique stem cells compared with mouse mesenchymal stem cells (mMSCs), and can improve cardiac function after myocardial infarction. However, the mCBSCs’ characterizations including their stem cell features, non-cardiac therapeutic potential, and cell surface features have not been fully understood. In this study, we examined stem cell features, cell surface glycan profiles, and cell functional features in mCBSCs compared to the bone marrow-derived mMSCs. Methods: The stem cell features were compared between mCBSCs and mMSCs by immunoblotting of stem cell markers, self-renewal assay, and multilineage differentiation. The cell surface glycan profiles were examined by lectin array analysis and fluorescence-activated cell sorting analysis using lectins. The production of transforming growth factor (TGF)-β1 from mCBSCs were examined by ELISA. The effects of TGF-β1 released from mCBSCs on self-migration and on activation of fibroblast were examined by migration assay and immunocytostaining, respectively. Results: The stem cell feature, including the self-renewing ability in mCBSCs was higher than that in mMSCs. In contrast, the differentiation ability of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. Additionally, the lactosamine (Galβ1-4GlcNAc)-structure, poly lactosamine- or poly N-acetylglucosamine-structure, and α2-3sialic acid on both N- and O-glycans are more highly expressed in mCBSCs compared with mMSCs. Furthermore, these highly expressed glycans were increased with cellular aging of mCBSCs. We found that TGF-β1 was released from mCBSCs and the released TGF-β1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Conclusions: These results reveal the differences between mCBSCs and mMSCs, and it is proposed that there is the potential use of mCBSCs for infarct healing and wound healing.

Glycans unique to the relapse-prone subset within triple-negative breast cancer as revealed by lectin array-based analysis of surgical specimens

Introduction Molecular and cellular characteristics of the relapse-prone subset within triple-negative breast cancer (TNBC) remain unclear. Aberrant glycosylation is involved in the malignant behavior of cancer cells. In the present study, we aimed to reveal glycan profiles unique to relapsed TNBC patients. Methods Thirty TNBC patients who did not undergo neoadjuvant chemotherapy but postoperative standard adjuvant therapy from 2009 through 2016 at Juntendo Hospital were investigated. TNBC cells were resected from primary breast cancer sections of formalin-fixed surgical specimens using laser-assisted microdissection. The binding intensities of the extracted glycoproteins to 45 lectins were quantified using lectin microarray and compared between relapsed and non-relapsed patients. Immunohistochemical staining with TJA-II lectin in specimen sections was performed. Results Five patients relapsed during the follow-up (range 37–123 months). Lectin microarray analysis revealed that 7 out of 45 lectins showed significant differences in binding intensity between the relapsed and the non-relapsed group. TJA-II, ACA, WFA, and BPL showed stronger binding in the relapsed group. PNGase F treatment of TNBC cell lysates suggested that TJA-II and ACA bind O-glycans. TJA-II staining of tissue sections revealed strong binding to cell surface membranes and to the cytoplasm of TNBC cells, but not to other types of cells. Significantly more TNBC cells were stained in tissue sections from relapsed than non-relapsed patients. Conclusions TNBC cells from relapsed patients showed a unique lectin reactivity, with higher levels of TJA-II (also WFA and BPL) binding than in non-relapsed patients. The results are potentially useful to develop new prognostic and therapeutic tools.

Novel urinary glycan profiling by lectin array serves as the biomarkers for predicting renal prognosis in patients with IgA nephropathy

In IgA nephropathy (IgAN), IgA1 molecules are characterized by galactose deficiency in O-glycans. Here, we investigated the association between urinary glycosylation profile measured by 45 lectins at baseline and renal prognosis in 142 patients with IgAN. The primary outcome was estimated glomerular filtration rate (eGFR) decline (> 4 mL/min/1.73 m2/year), or eGFR ≥ 30% decline from baseline, or initiation of renal replacement therapies within 3 years. During follow-up (3.4 years, median), 26 patients reached the renal outcome (Group P), while 116 patients were with good renal outcome (Group G). Multivariate logistic regression analyses revealed that lectin binding signals of Erythrina cristagalli lectin (ECA) (odds ratio [OR] 2.84, 95% confidence interval [CI] 1.11–7.28) and Narcissus pseudonarcissus lectin (NPA) (OR 2.32, 95% CI 1.11–4.85) adjusted by age, sex, eGFR, and urinary protein were significantly associated with the outcome, and they recognize Gal(β1-4)GlcNAc and high-mannose including Man(α1-6)Man, respectively. The addition of two lectin-binding glycan signals to the interstitial fibrosis/tubular atrophy score further improved the model fitness (Akaike’s information criterion) and incremental predictive abilities (c-index, net reclassification improvement, and integrated discrimination improvement). Urinary N-glycan profiling by lectin array is useful in the prediction of IgAN prognosis, since ECA and NPA recognize the intermediate glycans during N-glycosylation of various glycoproteins.

Microarray investigation of glycan remodeling during macrophage polarization reveals α2-6 sialic acid as an anti-inflammatory indicator

Glycosylation is a widely occurring posttranslational modification. Here, we applied a quick, convenient and high-throughput strategy (lectin array) to investigate the variation in glycans on different macrophage subtypes derived from...

Predictive modeling of complex ABO glycan phenotypes by lectin microarrays

Serological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.

Mammalian lectin arrays for screening host–microbe interactions

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen–receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.