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Immunosuppressive drugs affect high-mannose/hybrid N-glycans on human allostimulated leukocytes.

Pocheć E, Bocian K, Ząbczyńska M, Korczak-Kowalska G, Lityńska A - Anal Cell Pathol (Amst) (2015)

Bottom Line: The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs.Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response.This is the first study indicating that β1 and β3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA.

View Article: PubMed Central - PubMed

Affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Faculty of Biology and Earth Science, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland.

ABSTRACT
N-glycosylation plays an important role in the majority of physiological and pathological processes occurring in the immune system. Alteration of the type and abundance of glycans is an element of lymphocyte differentiation; it is also common in the development of immune-mediated inflammatory diseases. The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs. Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response. We evaluated the effects of cyclosporin A (CsA) and rapamycin (Rapa) on high-mannose/hybrid-type glycosylation in human leukocytes activated in a two-way mixed leukocyte reaction (MLR). CsA significantly reduced the number of leukocytes covered by high-mannose/hybrid N-glycans, and the synergistic action of CsA and Rapa led to an increase of these structures on the remaining leukocytes. This is the first study indicating that β1 and β3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA. Rapa taken separately and together with CsA changed the expression of β1 and β3 integrins and, by regulating the protein amount, increased the oligomannose/hybrid-type N-glycosylation on the leukocyte surface. We suggest that the changes in the glycosylation profile of leukocytes may promote the development of tolerance in transplantation.

No MeSH data available.


Related in: MedlinePlus

Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μg) were precipitated with 25 μL agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β-ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.
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fig2: Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μg) were precipitated with 25 μL agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β-ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

Mentions: Lectin blotting (LB) with biotinylated GNA disclosed changes in the oligomannose/hybrid-type glycosylation of some proteins in samples with CsA given together with Rapa; the reaction was more intense in bands 1 and 2, while reduction of GNA binding was seen in band 3 (Figure 2(a)). Based on the GNA reaction, bands found to have been altered in intensity by immunosuppressive agents were excised from the gels (Figure 2(b)) and their content was analyzed using MS/MS. This assay showed 62 possible proteins in band 1, 151 in band 2, and 126 in band 3. The ten identified proteins with the highest scores for each band are listed in Table 1. Among them are transmembrane proteins (β integrins, HLA class I histocompatibility antigen, LAMPs) and cytoplasmic proteins (enzymes of various metabolic pathways, coagulation factors). The molecular mass of the identified monomeric glycoproteins, calculated based on the amino acid sequences, was lower than the mass determined in SDS-PAGE, due to the presence of N-glycan components. The theoretical molecular weight of multimeric glycoproteins is higher than that determined based on Protein Ladder standards, because the samples were resolved in reducing conditions.


Immunosuppressive drugs affect high-mannose/hybrid N-glycans on human allostimulated leukocytes.

Pocheć E, Bocian K, Ząbczyńska M, Korczak-Kowalska G, Lityńska A - Anal Cell Pathol (Amst) (2015)

Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μg) were precipitated with 25 μL agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β-ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4538311&req=5

fig2: Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μg) were precipitated with 25 μL agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β-ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.
Mentions: Lectin blotting (LB) with biotinylated GNA disclosed changes in the oligomannose/hybrid-type glycosylation of some proteins in samples with CsA given together with Rapa; the reaction was more intense in bands 1 and 2, while reduction of GNA binding was seen in band 3 (Figure 2(a)). Based on the GNA reaction, bands found to have been altered in intensity by immunosuppressive agents were excised from the gels (Figure 2(b)) and their content was analyzed using MS/MS. This assay showed 62 possible proteins in band 1, 151 in band 2, and 126 in band 3. The ten identified proteins with the highest scores for each band are listed in Table 1. Among them are transmembrane proteins (β integrins, HLA class I histocompatibility antigen, LAMPs) and cytoplasmic proteins (enzymes of various metabolic pathways, coagulation factors). The molecular mass of the identified monomeric glycoproteins, calculated based on the amino acid sequences, was lower than the mass determined in SDS-PAGE, due to the presence of N-glycan components. The theoretical molecular weight of multimeric glycoproteins is higher than that determined based on Protein Ladder standards, because the samples were resolved in reducing conditions.

Bottom Line: The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs.Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response.This is the first study indicating that β1 and β3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA.

View Article: PubMed Central - PubMed

Affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Faculty of Biology and Earth Science, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland.

ABSTRACT
N-glycosylation plays an important role in the majority of physiological and pathological processes occurring in the immune system. Alteration of the type and abundance of glycans is an element of lymphocyte differentiation; it is also common in the development of immune-mediated inflammatory diseases. The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs. Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response. We evaluated the effects of cyclosporin A (CsA) and rapamycin (Rapa) on high-mannose/hybrid-type glycosylation in human leukocytes activated in a two-way mixed leukocyte reaction (MLR). CsA significantly reduced the number of leukocytes covered by high-mannose/hybrid N-glycans, and the synergistic action of CsA and Rapa led to an increase of these structures on the remaining leukocytes. This is the first study indicating that β1 and β3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA. Rapa taken separately and together with CsA changed the expression of β1 and β3 integrins and, by regulating the protein amount, increased the oligomannose/hybrid-type N-glycosylation on the leukocyte surface. We suggest that the changes in the glycosylation profile of leukocytes may promote the development of tolerance in transplantation.

No MeSH data available.


Related in: MedlinePlus