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CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

Yang CY, Chen JB, Tsai TF, Tsai YC, Tsai CY, Liang PH, Hsu TL, Wu CY, Netea MG, Wong CH, Hsieh SL - PLoS ONE (2013)

Bottom Line: We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells.Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells.This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/-) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

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The expression and distribution of CLEC4F.(A) Tissue distribution of CLEC4F transcripts were analyzed by qRT-PCR. F4/80 is the pan macrophage marker. (B) The protein expression of CLEC4F was examined by Western blot. GAPDH was used as internal control. (C) CLEC4F is a glycoprotein. The pFLAG-CMV-2/CLEC4F-transfected 293T cells were treated with tunicamycin at 1.5 µg/ml for indicated time periods to inhibit N-linked glycosylation, and the molecular weight of CLEC4F was analyzed by Western blot. DMSO is the vehicle control. Mock indicates 293T cells transfect with pFLAG-CMV-2. mLN, mesenteric lymph node; iLN, inguinal lymph node; KC, Kupffer cells; PBL, peripheral blood leukocytes; BM, bone marrow cells; BMDM, bone marrow derived macrophages.
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pone-0065070-g001: The expression and distribution of CLEC4F.(A) Tissue distribution of CLEC4F transcripts were analyzed by qRT-PCR. F4/80 is the pan macrophage marker. (B) The protein expression of CLEC4F was examined by Western blot. GAPDH was used as internal control. (C) CLEC4F is a glycoprotein. The pFLAG-CMV-2/CLEC4F-transfected 293T cells were treated with tunicamycin at 1.5 µg/ml for indicated time periods to inhibit N-linked glycosylation, and the molecular weight of CLEC4F was analyzed by Western blot. DMSO is the vehicle control. Mock indicates 293T cells transfect with pFLAG-CMV-2. mLN, mesenteric lymph node; iLN, inguinal lymph node; KC, Kupffer cells; PBL, peripheral blood leukocytes; BM, bone marrow cells; BMDM, bone marrow derived macrophages.

Mentions: In contrast to F4/80, quantitative RT-PCR analysis revealed that CLEC4F mRNA is only expressed in liver and freshly isolated Kupffer cells, but not in all other tissues. In addition, CLEC4F mRNA is not detectable in bone marrow cells, bone marrow derived macrophages (BMDMs), peripheral blood cells (PBLs) or murine macrophage cell lines RAW264.7 and J774 (Figure 1A). In order to confirm CLEC4F expression at the protein level, recombinant CLEC4F was used to immunize BALB/c mice to generate anti-CLEC4F mAbs. Western blot analysis demonstrated the anti-CLEC4F mAb can recognize a 100 kDa protein in liver, which is 40 kDa more than predicted, but not the rest of tissues (left panel, Figure 1B). Moreover, CLEC4F is only detectable in fresh isolated Kupffer cells, but not in bone marrow or peripheral blood leukocytes, which contain monocytes (right panel, Figure 1B). In pFLAG-CMV-2/CLEC4F-transfected 293T cells (human embryonic kidney cells), anti-CLEC4F mAb detected polypeptides ranging from 100 kDa to 61 kDa, and addition of tunicamycin reduced the molecular weight of CLEC4F to 61 kDa (Figure 1C), indicating that CLEC4F is a glycoprotein with 40 kDa N-linked glycan, while the 70 kDa is the incompletely glycosylated form in CLEC4F-transfected 293 T cells.


CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

Yang CY, Chen JB, Tsai TF, Tsai YC, Tsai CY, Liang PH, Hsu TL, Wu CY, Netea MG, Wong CH, Hsieh SL - PLoS ONE (2013)

The expression and distribution of CLEC4F.(A) Tissue distribution of CLEC4F transcripts were analyzed by qRT-PCR. F4/80 is the pan macrophage marker. (B) The protein expression of CLEC4F was examined by Western blot. GAPDH was used as internal control. (C) CLEC4F is a glycoprotein. The pFLAG-CMV-2/CLEC4F-transfected 293T cells were treated with tunicamycin at 1.5 µg/ml for indicated time periods to inhibit N-linked glycosylation, and the molecular weight of CLEC4F was analyzed by Western blot. DMSO is the vehicle control. Mock indicates 293T cells transfect with pFLAG-CMV-2. mLN, mesenteric lymph node; iLN, inguinal lymph node; KC, Kupffer cells; PBL, peripheral blood leukocytes; BM, bone marrow cells; BMDM, bone marrow derived macrophages.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675125&req=5

pone-0065070-g001: The expression and distribution of CLEC4F.(A) Tissue distribution of CLEC4F transcripts were analyzed by qRT-PCR. F4/80 is the pan macrophage marker. (B) The protein expression of CLEC4F was examined by Western blot. GAPDH was used as internal control. (C) CLEC4F is a glycoprotein. The pFLAG-CMV-2/CLEC4F-transfected 293T cells were treated with tunicamycin at 1.5 µg/ml for indicated time periods to inhibit N-linked glycosylation, and the molecular weight of CLEC4F was analyzed by Western blot. DMSO is the vehicle control. Mock indicates 293T cells transfect with pFLAG-CMV-2. mLN, mesenteric lymph node; iLN, inguinal lymph node; KC, Kupffer cells; PBL, peripheral blood leukocytes; BM, bone marrow cells; BMDM, bone marrow derived macrophages.
Mentions: In contrast to F4/80, quantitative RT-PCR analysis revealed that CLEC4F mRNA is only expressed in liver and freshly isolated Kupffer cells, but not in all other tissues. In addition, CLEC4F mRNA is not detectable in bone marrow cells, bone marrow derived macrophages (BMDMs), peripheral blood cells (PBLs) or murine macrophage cell lines RAW264.7 and J774 (Figure 1A). In order to confirm CLEC4F expression at the protein level, recombinant CLEC4F was used to immunize BALB/c mice to generate anti-CLEC4F mAbs. Western blot analysis demonstrated the anti-CLEC4F mAb can recognize a 100 kDa protein in liver, which is 40 kDa more than predicted, but not the rest of tissues (left panel, Figure 1B). Moreover, CLEC4F is only detectable in fresh isolated Kupffer cells, but not in bone marrow or peripheral blood leukocytes, which contain monocytes (right panel, Figure 1B). In pFLAG-CMV-2/CLEC4F-transfected 293T cells (human embryonic kidney cells), anti-CLEC4F mAb detected polypeptides ranging from 100 kDa to 61 kDa, and addition of tunicamycin reduced the molecular weight of CLEC4F to 61 kDa (Figure 1C), indicating that CLEC4F is a glycoprotein with 40 kDa N-linked glycan, while the 70 kDa is the incompletely glycosylated form in CLEC4F-transfected 293 T cells.

Bottom Line: We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells.Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells.This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/-) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

Show MeSH
Related in: MedlinePlus