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Mouse ficolin B has an ability to form complexes with mannose-binding lectin-associated serine proteases and activate complement through the lectin pathway.

Endo Y, Iwaki D, Ishida Y, Takahashi M, Matsushita M, Fujita T - J. Biomed. Biotechnol. (2012)

Bottom Line: Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity.Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine.Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1295, Japan. yendo@fmu.ac.jp

ABSTRACT
Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

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Characterization of FcnB in the mouse bone marrow. (a) Western blotting of FcnB in the supernatant (lane 1) and precipitate (lane 2) of bone marrow tissue, and plasma (lane 3) (left panel). Two μL of the supernatant/precipitate equivalent to the original tissue and 2 μL of plasma from FcnA−/− mice were subjected to western blotting under reducing conditions. FcnB in the supernatant (4 μL equivalent to the original tissue) was treated with endoglycosidase F (endoF) or neuraminidase plus endo-α-N-acetylgalactosaminidase (o-gly) and subjected to western blotting (right panel).—, not treated. (b) Superose 6 gel chromatography of FcnB in the bone marrow supernatant from FcnA−/− mice. An aliquot of each fraction was subjected to western blotting under reducing conditions. Arrowheads depict the eluted positions of the molecular weight markers (661 kDa, thyroglobulin; 440 kDa, ferritin; 230 kDa, catalase; 67 kDa, BSA).
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fig1: Characterization of FcnB in the mouse bone marrow. (a) Western blotting of FcnB in the supernatant (lane 1) and precipitate (lane 2) of bone marrow tissue, and plasma (lane 3) (left panel). Two μL of the supernatant/precipitate equivalent to the original tissue and 2 μL of plasma from FcnA−/− mice were subjected to western blotting under reducing conditions. FcnB in the supernatant (4 μL equivalent to the original tissue) was treated with endoglycosidase F (endoF) or neuraminidase plus endo-α-N-acetylgalactosaminidase (o-gly) and subjected to western blotting (right panel).—, not treated. (b) Superose 6 gel chromatography of FcnB in the bone marrow supernatant from FcnA−/− mice. An aliquot of each fraction was subjected to western blotting under reducing conditions. Arrowheads depict the eluted positions of the molecular weight markers (661 kDa, thyroglobulin; 440 kDa, ferritin; 230 kDa, catalase; 67 kDa, BSA).

Mentions: To detect the FcnB protein in the bone marrow, the tissue supernatants and precipitates were subjected to western blotting. As shown in Figure 1(a), a 38 kDa band was observed in the supernatant under reducing conditions, suggesting that FcnB is secreted into the mouse bone marrow fluid as a soluble protein. FcnB was also detected as a 37 kDa band at high levels in the precipitate. This suggests that FcnB in bone marrow cells is slightly small due to incomplete processing prior to secretion. When FcnB in the supernatant was treated with endoglycosidase F, its molecular weight reduced from 38 to 34 kDa, whereas treatment with endo-α-N-acetylgalactosaminidase resulted in either no or a smaller reduction in molecular weight (Figure 1(a)). To determine the size distribution of oligomeric FcnB, the supernatant was subjected to gel chromatography. FcnB was recovered from fractions corresponding to the elution positions of marker proteins ranging from 100 to >1000 kDa with a peak around 600 kDa (Figure 1(b)), indicating a heterogeneous structure composed mainly of 12–18-mers.


Mouse ficolin B has an ability to form complexes with mannose-binding lectin-associated serine proteases and activate complement through the lectin pathway.

Endo Y, Iwaki D, Ishida Y, Takahashi M, Matsushita M, Fujita T - J. Biomed. Biotechnol. (2012)

Characterization of FcnB in the mouse bone marrow. (a) Western blotting of FcnB in the supernatant (lane 1) and precipitate (lane 2) of bone marrow tissue, and plasma (lane 3) (left panel). Two μL of the supernatant/precipitate equivalent to the original tissue and 2 μL of plasma from FcnA−/− mice were subjected to western blotting under reducing conditions. FcnB in the supernatant (4 μL equivalent to the original tissue) was treated with endoglycosidase F (endoF) or neuraminidase plus endo-α-N-acetylgalactosaminidase (o-gly) and subjected to western blotting (right panel).—, not treated. (b) Superose 6 gel chromatography of FcnB in the bone marrow supernatant from FcnA−/− mice. An aliquot of each fraction was subjected to western blotting under reducing conditions. Arrowheads depict the eluted positions of the molecular weight markers (661 kDa, thyroglobulin; 440 kDa, ferritin; 230 kDa, catalase; 67 kDa, BSA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306798&req=5

fig1: Characterization of FcnB in the mouse bone marrow. (a) Western blotting of FcnB in the supernatant (lane 1) and precipitate (lane 2) of bone marrow tissue, and plasma (lane 3) (left panel). Two μL of the supernatant/precipitate equivalent to the original tissue and 2 μL of plasma from FcnA−/− mice were subjected to western blotting under reducing conditions. FcnB in the supernatant (4 μL equivalent to the original tissue) was treated with endoglycosidase F (endoF) or neuraminidase plus endo-α-N-acetylgalactosaminidase (o-gly) and subjected to western blotting (right panel).—, not treated. (b) Superose 6 gel chromatography of FcnB in the bone marrow supernatant from FcnA−/− mice. An aliquot of each fraction was subjected to western blotting under reducing conditions. Arrowheads depict the eluted positions of the molecular weight markers (661 kDa, thyroglobulin; 440 kDa, ferritin; 230 kDa, catalase; 67 kDa, BSA).
Mentions: To detect the FcnB protein in the bone marrow, the tissue supernatants and precipitates were subjected to western blotting. As shown in Figure 1(a), a 38 kDa band was observed in the supernatant under reducing conditions, suggesting that FcnB is secreted into the mouse bone marrow fluid as a soluble protein. FcnB was also detected as a 37 kDa band at high levels in the precipitate. This suggests that FcnB in bone marrow cells is slightly small due to incomplete processing prior to secretion. When FcnB in the supernatant was treated with endoglycosidase F, its molecular weight reduced from 38 to 34 kDa, whereas treatment with endo-α-N-acetylgalactosaminidase resulted in either no or a smaller reduction in molecular weight (Figure 1(a)). To determine the size distribution of oligomeric FcnB, the supernatant was subjected to gel chromatography. FcnB was recovered from fractions corresponding to the elution positions of marker proteins ranging from 100 to >1000 kDa with a peak around 600 kDa (Figure 1(b)), indicating a heterogeneous structure composed mainly of 12–18-mers.

Bottom Line: Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity.Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine.Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1295, Japan. yendo@fmu.ac.jp

ABSTRACT
Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

Show MeSH