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CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes.

Huysamen C, Willment JA, Dennehy KM, Brown GD - J. Biol. Chem. (2008)

Bottom Line: We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex.CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis.These data indicate that CLEC9A functions as an activation receptor.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, South Africa.

ABSTRACT
We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex. Expression of human CLEC9A is highly restricted in peripheral blood, being detected only on BDCA3(+) dendritic cells and on a small subset of CD14(+)CD16(-) monocytes. CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. CLEC9A possesses a cytoplasmic immunoreceptor tyrosine-based activation-like motif that can recruit Syk kinase, and we demonstrate, using receptor chimeras, that this receptor can induce proinflammatory cytokine production. These data indicate that CLEC9A functions as an activation receptor.

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Expression of hCLEC9A as a glycosylated dimer. A, flow cytometry of live NIH3T3 cells stably expressing HA-tagged hCLEC9A or mDectin-1, as indicated (gray lines), demonstrating expression of these receptors at the cell surface. The filled histograms indicate vector-only transduced controls. B, anti-HA Western blotting of lysates from these cells demonstrating that CLEC9A is expressed as a dimer of ∼100 kDa under nonreducing conditions, which can be resolved to two bands of ∼45 and 40 kDa under reducing conditions. Treatment of the cell lysates with PNGase, which removes N-linked glycosylation, reduced the mass of these bands to close to that expected from the predicted amino acid sequence, indicating that CLEC9A is N-glycosylated. The post-translation modification giving rise to the second band seen in these lysates was not due to O-glycosylation, but was also seen with mDectin-1, which is expressed as a monomer (32) and was included as a control for these experiments.
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fig2: Expression of hCLEC9A as a glycosylated dimer. A, flow cytometry of live NIH3T3 cells stably expressing HA-tagged hCLEC9A or mDectin-1, as indicated (gray lines), demonstrating expression of these receptors at the cell surface. The filled histograms indicate vector-only transduced controls. B, anti-HA Western blotting of lysates from these cells demonstrating that CLEC9A is expressed as a dimer of ∼100 kDa under nonreducing conditions, which can be resolved to two bands of ∼45 and 40 kDa under reducing conditions. Treatment of the cell lysates with PNGase, which removes N-linked glycosylation, reduced the mass of these bands to close to that expected from the predicted amino acid sequence, indicating that CLEC9A is N-glycosylated. The post-translation modification giving rise to the second band seen in these lysates was not due to O-glycosylation, but was also seen with mDectin-1, which is expressed as a monomer (32) and was included as a control for these experiments.

Mentions: To characterize the structural features of hCLEC9A, we cloned and expressed an HA-tagged version of the receptor in NIH3T3 fibroblasts. Murine Dectin-1 (18) was included as a control. By flow cytometry, we could demonstrate that hCLEC9A was expressed at the cell surface (Fig. 2A), suggesting that similar to the other receptors in the Dectin-1 cluster, CLEC9A did not require an adaptor protein for expression (8). Western blot analysis of lysates from the transfected NIH3T3 cells, under nonreducing conditions, indicated that hCLEC9A was expressed as protein of ∼100 kDa, more than twice its predicted mass (Fig. 2B). However, under reducing conditions, hCLEC9A resolved to two bands of mass of ∼40 and ∼45 kDa. Treatment of the transduced cell lysates with PNGase, which removes all N-linked glycosylation, further reduced the mass of both bands to ∼30 kDa (the predicted molecular mass of this protein) and ∼35 kDa, respectively. The presence of a second band in these cell lysates, which was also observed with Dectin-1, may be due to other post-translational modifications. However, this is unlikely to be O-glycosylation, as treatment with O-deglycosidase did not affect the mass of this protein. Thus these data indicate that hCLEC9A is expressed as a glycosylated dimer at the cell surface.


CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes.

Huysamen C, Willment JA, Dennehy KM, Brown GD - J. Biol. Chem. (2008)

Expression of hCLEC9A as a glycosylated dimer. A, flow cytometry of live NIH3T3 cells stably expressing HA-tagged hCLEC9A or mDectin-1, as indicated (gray lines), demonstrating expression of these receptors at the cell surface. The filled histograms indicate vector-only transduced controls. B, anti-HA Western blotting of lysates from these cells demonstrating that CLEC9A is expressed as a dimer of ∼100 kDa under nonreducing conditions, which can be resolved to two bands of ∼45 and 40 kDa under reducing conditions. Treatment of the cell lysates with PNGase, which removes N-linked glycosylation, reduced the mass of these bands to close to that expected from the predicted amino acid sequence, indicating that CLEC9A is N-glycosylated. The post-translation modification giving rise to the second band seen in these lysates was not due to O-glycosylation, but was also seen with mDectin-1, which is expressed as a monomer (32) and was included as a control for these experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562446&req=5

fig2: Expression of hCLEC9A as a glycosylated dimer. A, flow cytometry of live NIH3T3 cells stably expressing HA-tagged hCLEC9A or mDectin-1, as indicated (gray lines), demonstrating expression of these receptors at the cell surface. The filled histograms indicate vector-only transduced controls. B, anti-HA Western blotting of lysates from these cells demonstrating that CLEC9A is expressed as a dimer of ∼100 kDa under nonreducing conditions, which can be resolved to two bands of ∼45 and 40 kDa under reducing conditions. Treatment of the cell lysates with PNGase, which removes N-linked glycosylation, reduced the mass of these bands to close to that expected from the predicted amino acid sequence, indicating that CLEC9A is N-glycosylated. The post-translation modification giving rise to the second band seen in these lysates was not due to O-glycosylation, but was also seen with mDectin-1, which is expressed as a monomer (32) and was included as a control for these experiments.
Mentions: To characterize the structural features of hCLEC9A, we cloned and expressed an HA-tagged version of the receptor in NIH3T3 fibroblasts. Murine Dectin-1 (18) was included as a control. By flow cytometry, we could demonstrate that hCLEC9A was expressed at the cell surface (Fig. 2A), suggesting that similar to the other receptors in the Dectin-1 cluster, CLEC9A did not require an adaptor protein for expression (8). Western blot analysis of lysates from the transfected NIH3T3 cells, under nonreducing conditions, indicated that hCLEC9A was expressed as protein of ∼100 kDa, more than twice its predicted mass (Fig. 2B). However, under reducing conditions, hCLEC9A resolved to two bands of mass of ∼40 and ∼45 kDa. Treatment of the transduced cell lysates with PNGase, which removes all N-linked glycosylation, further reduced the mass of both bands to ∼30 kDa (the predicted molecular mass of this protein) and ∼35 kDa, respectively. The presence of a second band in these cell lysates, which was also observed with Dectin-1, may be due to other post-translational modifications. However, this is unlikely to be O-glycosylation, as treatment with O-deglycosidase did not affect the mass of this protein. Thus these data indicate that hCLEC9A is expressed as a glycosylated dimer at the cell surface.

Bottom Line: We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex.CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis.These data indicate that CLEC9A functions as an activation receptor.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, South Africa.

ABSTRACT
We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex. Expression of human CLEC9A is highly restricted in peripheral blood, being detected only on BDCA3(+) dendritic cells and on a small subset of CD14(+)CD16(-) monocytes. CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. CLEC9A possesses a cytoplasmic immunoreceptor tyrosine-based activation-like motif that can recruit Syk kinase, and we demonstrate, using receptor chimeras, that this receptor can induce proinflammatory cytokine production. These data indicate that CLEC9A functions as an activation receptor.

Show MeSH
Related in: MedlinePlus