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9-O-Acetylation of sialomucins: a novel marker of murine CD4 T cells that is regulated during maturation and activation.

Krishna M, Varki A - J. Exp. Med. (1997)

Bottom Line: In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells.However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O-acetylation, primarily in the mucin glycoprotein component.Thus, 9-O-acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation.

View Article: PubMed Central - PubMed

Affiliation: Glycobiology Program, UCSD Cancer Center, the Department of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Terminal sialic acids on cell surface glycoconjugates can carry 9-O-acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O-acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8-10% carry 9-O-acetylation; the great majority of these are the more mature PNA-, HSA-, and TCRhi medullary cells. While low levels of 9-O-acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80- 85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O-acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O-acetylation is also present on 90-95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O-acetylation. Among mature peripheral CD4 T lymphocytes, the highly O-acetylated cells are Mel 14(hi), CD44(lo), and CD45R(exon B)hi, features typical of naive cells. Digestions with trypsin and O-sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O-acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O-acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O-acetylated gangliosides in human T cells. Tethering 9-O-acetylated mucins with the Influenza C probe with or without secondary cross-linking did not cause activation of CD4 T cells. However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O-acetylation, primarily in the mucin glycoprotein component. Thus, 9-O-acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation.

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Sialomucins on  CD8 T cells can mask the recognition of 9-O-acetylated gangliosides by CHE-FcD probe. (A)  Mouse spleen cells were stained  with CHE-FcD before or after  OSGPase or trypsin treatment  and staining profiles were traced  from gated CD4 and CD8 subpopulations. Trypsin treatment  was done on CD4 and CD8 T  cells that were pre-sorted on a  FACSorter®. Loss of binding of  anti-Mel14 was a positive control for trypsinization (data not  shown). (B) Mouse thymocytes  were treated with OSGPase as  described earlier, washed and  stained with anti-CD4, CD8,  and CHE-FcD. Sham treated  cells were incubated with an  equal volume of buffer under  identical conditions. 100,000 live  events were acquired and CD4  and CD8 SP cells were gated  from a CD4/CD8 dot plot.  CHE-FcD binding within those  populations was plotted in the  histograms. The dotted line  shows background staining with  the second antibody.
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Figure 10: Sialomucins on CD8 T cells can mask the recognition of 9-O-acetylated gangliosides by CHE-FcD probe. (A) Mouse spleen cells were stained with CHE-FcD before or after OSGPase or trypsin treatment and staining profiles were traced from gated CD4 and CD8 subpopulations. Trypsin treatment was done on CD4 and CD8 T cells that were pre-sorted on a FACSorter®. Loss of binding of anti-Mel14 was a positive control for trypsinization (data not shown). (B) Mouse thymocytes were treated with OSGPase as described earlier, washed and stained with anti-CD4, CD8, and CHE-FcD. Sham treated cells were incubated with an equal volume of buffer under identical conditions. 100,000 live events were acquired and CD4 and CD8 SP cells were gated from a CD4/CD8 dot plot. CHE-FcD binding within those populations was plotted in the histograms. The dotted line shows background staining with the second antibody.

Mentions: The level of 9-O-acetylation detected on mature CD8 T cells was considerably lower than on CD4 T cells. We considered the possibility that this might be due to differential expression of sialylated substrates for 9-O-acetylation on the respective cell types. Given the preponderance of 9-O-acetylated sialomucins on CD4 T cells we looked for their presence on CD8 T cells. As shown in Fig. 10 A, removal of the sialomucins by OSGPase actually gives an increase in CHE-FcD binding to CD8 T cells, i.e., the opposite of the result with CD4 T cells. The same result was confirmed by treating cells with trypsin (Fig. 10 A) or by a combination of trypsin followed by OSGPase (data not shown) indicating that detection of protease resistant 9-O-acetylated gangliosides can be masked by the presence of sialomucins on CD8 cells. This pattern of increased CHE-FcD binding after trypsin or OSGPase treatment was also reproduced on CD8 SP thymocytes (Fig. 10 B). These results demonstrate the presence of sialomucins on CD8 T cells, and indicate that unlike in CD4 cells, these molecules are not substrates for 9-O-acetylation.


9-O-Acetylation of sialomucins: a novel marker of murine CD4 T cells that is regulated during maturation and activation.

Krishna M, Varki A - J. Exp. Med. (1997)

Sialomucins on  CD8 T cells can mask the recognition of 9-O-acetylated gangliosides by CHE-FcD probe. (A)  Mouse spleen cells were stained  with CHE-FcD before or after  OSGPase or trypsin treatment  and staining profiles were traced  from gated CD4 and CD8 subpopulations. Trypsin treatment  was done on CD4 and CD8 T  cells that were pre-sorted on a  FACSorter®. Loss of binding of  anti-Mel14 was a positive control for trypsinization (data not  shown). (B) Mouse thymocytes  were treated with OSGPase as  described earlier, washed and  stained with anti-CD4, CD8,  and CHE-FcD. Sham treated  cells were incubated with an  equal volume of buffer under  identical conditions. 100,000 live  events were acquired and CD4  and CD8 SP cells were gated  from a CD4/CD8 dot plot.  CHE-FcD binding within those  populations was plotted in the  histograms. The dotted line  shows background staining with  the second antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196344&req=5

Figure 10: Sialomucins on CD8 T cells can mask the recognition of 9-O-acetylated gangliosides by CHE-FcD probe. (A) Mouse spleen cells were stained with CHE-FcD before or after OSGPase or trypsin treatment and staining profiles were traced from gated CD4 and CD8 subpopulations. Trypsin treatment was done on CD4 and CD8 T cells that were pre-sorted on a FACSorter®. Loss of binding of anti-Mel14 was a positive control for trypsinization (data not shown). (B) Mouse thymocytes were treated with OSGPase as described earlier, washed and stained with anti-CD4, CD8, and CHE-FcD. Sham treated cells were incubated with an equal volume of buffer under identical conditions. 100,000 live events were acquired and CD4 and CD8 SP cells were gated from a CD4/CD8 dot plot. CHE-FcD binding within those populations was plotted in the histograms. The dotted line shows background staining with the second antibody.
Mentions: The level of 9-O-acetylation detected on mature CD8 T cells was considerably lower than on CD4 T cells. We considered the possibility that this might be due to differential expression of sialylated substrates for 9-O-acetylation on the respective cell types. Given the preponderance of 9-O-acetylated sialomucins on CD4 T cells we looked for their presence on CD8 T cells. As shown in Fig. 10 A, removal of the sialomucins by OSGPase actually gives an increase in CHE-FcD binding to CD8 T cells, i.e., the opposite of the result with CD4 T cells. The same result was confirmed by treating cells with trypsin (Fig. 10 A) or by a combination of trypsin followed by OSGPase (data not shown) indicating that detection of protease resistant 9-O-acetylated gangliosides can be masked by the presence of sialomucins on CD8 cells. This pattern of increased CHE-FcD binding after trypsin or OSGPase treatment was also reproduced on CD8 SP thymocytes (Fig. 10 B). These results demonstrate the presence of sialomucins on CD8 T cells, and indicate that unlike in CD4 cells, these molecules are not substrates for 9-O-acetylation.

Bottom Line: In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells.However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O-acetylation, primarily in the mucin glycoprotein component.Thus, 9-O-acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation.

View Article: PubMed Central - PubMed

Affiliation: Glycobiology Program, UCSD Cancer Center, the Department of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Terminal sialic acids on cell surface glycoconjugates can carry 9-O-acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O-acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8-10% carry 9-O-acetylation; the great majority of these are the more mature PNA-, HSA-, and TCRhi medullary cells. While low levels of 9-O-acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80- 85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O-acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O-acetylation is also present on 90-95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O-acetylation. Among mature peripheral CD4 T lymphocytes, the highly O-acetylated cells are Mel 14(hi), CD44(lo), and CD45R(exon B)hi, features typical of naive cells. Digestions with trypsin and O-sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O-acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O-acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O-acetylated gangliosides in human T cells. Tethering 9-O-acetylated mucins with the Influenza C probe with or without secondary cross-linking did not cause activation of CD4 T cells. However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O-acetylation, primarily in the mucin glycoprotein component. Thus, 9-O-acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation.

Show MeSH
Related in: MedlinePlus