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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

Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β1 and β3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and 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. Protein extracts (15 μg) were digested with endo-β-N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus, SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti-β1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti-β3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.
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fig3: Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β1 and β3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and 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. Protein extracts (15 μg) were digested with endo-β-N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus, SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti-β1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti-β3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.

Mentions: In further analyses we focused on the β integrin subunits identified in the band 1 to verify the results obtained by MS/MS analysis. Immunodetection of the β1 subunit after releasing the high-mannose/hybrid-type N-glycans with Endo H revealed that the β1 precursor, with lower molecular mass, mainly carries these types of N-glycans (Figure 3), but the mobility shift in the control and immunosuppressive drug-treated MLR cells was comparable. Comparable loss of molecular weight between the control and tested cells after Endo H digestion was also found in the β3 subunit. We observed, however, a significant increase of the intensity of the β1 (both forms) and β3 subunits in MLR cells cultured in the presence of Rapa alone or combined with CsA. GNA precipitation confirmed the results from Endo H digestion of the β1 subunit (Figure 4). GNA recognized mainly the β1 precursor form; the mature subunit remained unprecipitated in supernatants. Here we also observed higher intensity of the β1 subunit in MLR treated with Rapa and the combination of both drugs. These results indicate that high-mannose/hybrid-type glycosylation did not change on a single β integrin molecule but that the increased binding of GNA resulted from enhancement of β integrin expression (Figures 3 and 4) and that it contributed to the overall increase of high-mannose/hybrid-type glycans on the cell surface in the presence of CsA and Rapa (Figure 1).


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)

Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β1 and β3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and 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. Protein extracts (15 μg) were digested with endo-β-N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus, SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti-β1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti-β3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β1 and β3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and 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. Protein extracts (15 μg) were digested with endo-β-N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus, SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti-β1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti-β3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.
Mentions: In further analyses we focused on the β integrin subunits identified in the band 1 to verify the results obtained by MS/MS analysis. Immunodetection of the β1 subunit after releasing the high-mannose/hybrid-type N-glycans with Endo H revealed that the β1 precursor, with lower molecular mass, mainly carries these types of N-glycans (Figure 3), but the mobility shift in the control and immunosuppressive drug-treated MLR cells was comparable. Comparable loss of molecular weight between the control and tested cells after Endo H digestion was also found in the β3 subunit. We observed, however, a significant increase of the intensity of the β1 (both forms) and β3 subunits in MLR cells cultured in the presence of Rapa alone or combined with CsA. GNA precipitation confirmed the results from Endo H digestion of the β1 subunit (Figure 4). GNA recognized mainly the β1 precursor form; the mature subunit remained unprecipitated in supernatants. Here we also observed higher intensity of the β1 subunit in MLR treated with Rapa and the combination of both drugs. These results indicate that high-mannose/hybrid-type glycosylation did not change on a single β integrin molecule but that the increased binding of GNA resulted from enhancement of β integrin expression (Figures 3 and 4) and that it contributed to the overall increase of high-mannose/hybrid-type glycans on the cell surface in the presence of CsA and Rapa (Figure 1).

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