RESUMEN
Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proliferación Celular/genética , Terapia de Inmunosupresión , Células Madre Mesenquimatosas/inmunología , 5'-Nucleotidasa/genética , Adenosina/biosíntesis , Adenosina Monofosfato/metabolismo , Animales , Antígenos CD/genética , Apirasa/genética , Vesículas Extracelulares/inmunología , Proteínas Ligadas a GPI/genética , Humanos , Tolerancia Inmunológica/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Stem cells have a unique ability to self-renew and differentiate into diverse cell types. Currently, stem cells from various sources are being explored as a promising new treatment for a variety of human diseases. A diverse set of functional and phenotypical markers are used in the characterization of specific therapeutic stem cell populations. The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. We have now discovered by mRNA analysis that a novel glycosyltranferase, epidermal growth factor (EGF) domain-specific O-linked GlcNAc transferase (EOGT), is highly expressed in stem cells. EOGT is responsible for adding O-linked N-acetylglucosamine (O-GlcNAc) to folded EGF domains on extracellular proteins, such as those on the Notch receptors. We were able to show by immunological assays that human umbilical cord blood-derived mesenchymal stromal cells display O-GlcNAc, the product of EOGT, and that O-GlcNAc is further elongated with galactose to form O-linked N-acetyllactosamine. We suggest that these novel glycans are involved in the fine tuning of Notch receptor signaling pathways in stem cells.
RESUMEN
We have recently developed an in vitro culture model enabling the large-scale expansion of switched-memory B lymphocytes, producing a polyclonal human IgG repertoire. Given the importance of glycosylation for the functions of immunoglobulins, we analyzed the N-glycosylation profiles of the immunoglobulin G (IgG) in this model. Switched-memory B cells were cultured for 38 days and, using liquid chromatography-mass spectrometry, we analyzed IgGs' glycosylation profiles which were then compared to the glycosylation patterns of commercial intravenous immunoglobulin (IVIG). We observed a reproducible proliferation rate, high viability through the cultures as well as a good maintenance of the switched-memory B cells repertoire. The glycosylation pattern analyses revealed a variety of the typical biantennary N-glycan structures with diverse terminal monosaccharides. While many similarities were detected in comparison to the glycosylation profile of IVIG, in vitro-produced polyclonal IgGs were bearing higher levels of bisecting GlcNAc known to affect the effector functions of therapeutic antibodies. This data highlights the need for monitoring of the glycoform distribution in antibodies produced in vitro.
Asunto(s)
Linfocitos B/metabolismo , Inmunoglobulina G/análisis , Linfocitos B/inmunología , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Glicosilación , Humanos , Memoria Inmunológica/inmunología , Espectrometría de MasasRESUMEN
Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen-positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology.
RESUMEN
There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the N-glycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri- and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.
Asunto(s)
Glicómica , Hexosaminas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Glicosilación , Humanos , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/metabolismo , Oligosacáridos/genética , Oligosacáridos/metabolismo , Antígeno Sialil Lewis XRESUMEN
Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and ß-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.
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Cromatografía de Fase Inversa/métodos , Espectrometría de Masas/métodos , Polisacáridos/química , Humanos , Inmunoglobulina G/química , Isomerismo , Metilación , Orosomucoide/química , Polisacáridos/aislamiento & purificaciónRESUMEN
Despite recent technical advances in glycan analysis, the rapidly growing field of glycomics still lacks methods that are high throughput and robust, and yet allow detailed and reliable identification of different glycans. LC-MS-MS(2) methods have a large potential for glycan analysis as they enable separation and identification of different glycans, including structural isomers. The major drawback is the complexity of the data with different charge states and adduct combinations. In practice, manual data analysis, still largely used for MALDI-TOF data, is no more achievable for LC-MS-MS(2) data. To solve the problem, we developed a glycan analysis software GlycanID for the analysis of LC-MS-MS(2) data to identify and profile glycan compositions in combination with existing proteomic software. IgG was used as an example of an individual glycoprotein and extracted cell surface proteins of human fibroblasts as a more complex sample to demonstrate the power of the novel data analysis approach. N-glycans were isolated from the samples and analyzed as permethylated sugar alditols by LC-MS-MS(2), permitting semiquantitative glycan profiling. The data analysis consisted of five steps: 1) extraction of LC-MS features and MS(2) spectra, 2) mapping potential glycans based on feature distribution, 3) matching the feature masses with a glycan composition database and de novo generated compositions, 4) scoring MS(2) spectra with theoretical glycan fragments, and 5) composing the glycan profile for the identified glycan compositions. The resulting N-glycan profile of IgG revealed 28 glycan compositions and was in good correlation with the published IgG profile. More than 50 glycan compositions were reliably identified from the cell surface N-glycan profile of human fibroblasts. Use of the GlycanID software made relatively rapid analysis of complex glycan LC-MS-MS(2) data feasible. The results demonstrate that the complexity of glycan LC-MS-MS(2) data can be used as an asset to increase the reliability of the identifications.
Asunto(s)
Cromatografía Liquida , Espectrometría de Masas , Polisacáridos , Antígenos de Superficie/química , Fibroblastos/química , Humanos , Polisacáridos/análisis , Polisacáridos/química , Reproducibilidad de los ResultadosRESUMEN
Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by ß3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.
Asunto(s)
Sangre Fetal/citología , Galectina 3/metabolismo , Sistema del Grupo Sanguíneo I/metabolismo , Células Madre Mesenquimatosas/citología , Amino Azúcares/metabolismo , Biomarcadores/análisis , Diferenciación Celular , Epítopos/química , Sangre Fetal/metabolismo , Citometría de Flujo , Galectina 3/genética , Perfilación de la Expresión Génica , Humanos , Ligandos , Espectrometría de Masas , Células Madre Mesenquimatosas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Nicho de Células MadreRESUMEN
Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.
Asunto(s)
Bifidobacterium/aislamiento & purificación , Fucosiltransferasas/genética , Intestinos/microbiología , Electroforesis en Gel de Poliacrilamida , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
BACKGROUND: Although ischemia-reperfusion (I/R) injury represents a major problem in posttransplant organ failure, effective treatment is not available. The acute phase protein alpha-1-acid glycoprotein (AGP) has been shown to be protective against experimental I/R injury. The effects of AGP are thought to be mediated by fucose groups expressed on the AGP protein inhibiting neutrophil infiltration. However, the precise mechanism of protection remains to be established. We therefore studied the effects of exogenous human AGP (hAGP) in a mouse model of ischemic acute renal failure. METHODS: Mice were subjected to renal I/R and treated with hAGP, fucose-depleted hAGP, or control treated. Also, transgenic mice over-expressing rat AGP or wild-type controls were subjected to renal I/R. RESULTS: Treatment was with hAGP as well as fucose-depleted hAGP protected mice against I/R-induced acute renal failure. Surprisingly, AGP-over-expressing mice were not protected against I/R injury. Both natural and fucose-depleted hAGP inhibited the activation of the complement system, as determined by renal C3 deposition and influx of neutrophils measured by immunohistochemistry and myeloperoxidase-enzyme-linked immunoadsorbent assay. Tubular epithelial cell structure (actin cytoskeleton) and cell-cell interaction (tight-junction architecture) were completely preserved in AGP-treated mice. Also, epithelial caspase activation and apoptotic DNA cleavage were prevented by AGP treatment. CONCLUSIONS: Both natural and fucose-depleted hAGP protect against renal I/R injury by preservation of tubular epithelial structure and inhibition of apoptosis and subsequent inflammation. Therefore, hAGP can be regarded as a potential new therapeutic intervention in the treatment of acute renal failure, as seen after transplantation of ischemically injured kidneys.
Asunto(s)
Apoptosis/efectos de los fármacos , Inflamación/prevención & control , Orosomucoide/farmacología , Circulación Renal , Daño por Reperfusión/prevención & control , Animales , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Relación Dosis-Respuesta a Droga , Fucosa/metabolismo , Humanos , Ratones , Ratones Transgénicos , Orosomucoide/administración & dosificación , Orosomucoide/genética , Orosomucoide/metabolismo , Ratas , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatologíaRESUMEN
Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis and other nonoral infections in human patients. The serotype a-specific polysaccharide antigen of A. actinomycetemcomitans contains solely 6-deoxy-D-talose and its O-2 acetylated modification. This polysaccharide is synthesized from the donor GDP-6-deoxy-D-talose with the relevant talosylation enzyme(s). In the synthesis of GDP-6- deoxy-D-talose, GDP-D-mannose is first converted by GDP-mannose-4,6-dehydratase (GMD) to GDP-4-keto-6-deoxy-D-mannose and then reduced to GDP-6-deoxy-D-talose by GDP-6-deoxy-D-talose synthetase (GTS). In this study, we cloned and overexpressed in Escherichia coli the A. actinomycetemcomitans GTS enzyme responsible for the synthesis of GDP-6-deoxy-D-talose. The recombinant A. actinomycetemcomitans GTS enzyme expressed in E. coli converted the GDP-4-keto-6-deoxy-intermediate to a novel GDP-deoxyhexose. The synthesized GDP-deoxyhexose was shown to be GDP-6-deoxy-D-talose by HPLC, MALDI-TOF MS, and NMR spectroscopy. The functional expression of gts provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which can be used as donors for the corresponding glycosyltransferases.
Asunto(s)
Aggregatibacter actinomycetemcomitans/genética , Carbohidrato Epimerasas/genética , Aggregatibacter actinomycetemcomitans/enzimología , Secuencia de Aminoácidos , Carbohidrato Epimerasas/metabolismo , Línea Celular , Cromatografía Líquida de Alta Presión , Clonación Molecular , Desoxiazúcares/biosíntesis , Desoxiazúcares/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Hexosas/biosíntesis , Hexosas/química , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Espectrometría de Masa Bombardeada por Átomos Veloces/métodosRESUMEN
Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosa contains d-rhamnosylated O-antigen. The synthesis of GDP-D-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-D-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-D-mannose, and then reduced to GDP-D-rhamnose by GDP-4-keto-6-deoxy-D-mannose reductase (RMD). Here, we describe the enzymatic characterization of P. aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S. cerevisiae allowed us to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. Thus, coexpression of the Helicobacter pylori gmd and P. aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose. This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy. The functional expression of P. aeruginosa RMD in S. cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.