Comparative study of the human ficolins reveals unique features of Ficolin-3 (Hakata antigen)
Introduction
In humans, four initiators of the lectin pathway of complement have been described: mannose-binding lectin (MBL), Ficolin-1 (also called M-ficolin or Ficolin/P35-related protein), Ficolin-2 (also called L-ficolin or Ficolin/P35) and Ficolin-3 (also called H-ficolin or Hakata antigen). Ficolin-3 is, as Ficolin-2 and MBL, found in serum, whereas Ficolin-1 is not considered to be a serum protein, but is expressed by monocytes and granulocytes (Endo et al., 1996, Lu et al., 1996, Teh et al., 2000, Liu et al., 2005). The molecules bind surface structures on different classes of microorganisms and are able to activate the complement system and are thereby important in the initiation of the innate immune response. The proteins share certain structural elements, including an N-terminal domain that contains cysteine residues, a collagen-like domain with typical Gly-Xaa-Yaa repeats of varying length and a C-terminal carbohydrate recognition domain (CRD) in MBL, and fibrinogen-like binding domains (FGB) in the ficolins (Ohashi and Erickson, 1998, Yokota et al., 1995, Yae et al., 1991). They are further assembled into larger multimeric structures composed of four or more trimers attached together with N-terminal disulphide bonds forming C-terminal globular domain that enables the proteins to bind carbohydrates such as GlcNAc (Matsushita et al., 1996, Sugimoto et al., 1998, Teh et al., 2000, Turner, 1996). The collagen-like domain is associated with the mannose-binding lectin serine proteases (MASPs), which activates C2, C3, and C4 of complement (Matsushita et al., 2002, Matsushita et al., 2000, Matsushita et al., 1995).
Although the ficolins together with MBL are very similar both in terms of structure and function it is likely that these proteins have diverse roles in immunity, however, this has not yet been clarified. In the present study we compared the mRNA level of the FCN1, FCN2, FCN3, and MBL2 in different human tissues and we demonstrated a unique tissue distribution of FCN3. Furthermore, the oligomeric structures of the ficolins were compared and we could show that Ficolin-3 was able to activate the lectin pathway of complement to an even higher degree than Ficolin-1, Ficolin-2 and MBL. Ficolin-3, but not Ficolin-1 and Ficolin-2, was highly resistant to collagenase-treatment, which is the first described unique property of Ficolin-3 compared with the other initiators of the lectin complement pathway.
Section snippets
Materials
Restriction enzymes, Hybond ECL nitrocellulose membrane, donkey peroxidase-conjugated anti-rabbit, anti-mouse and anti-rat Ig, streptavidin-horseradish peroxidase conjugate (RPN 1231), and the fast protein liquid chromatography system ÄKTA3 were from Amersham Biosciences. Human tissue cDNA panels, liver cDNA and pSV2neo were from Clontech (Palo Alto, CA). KOD DNA polymerase was from Novagen. ABI 7700 platform, TaqMan Gene expression assays, TaqMan Universal PCR master mix, BigDye terminator
Real-time quantitative (RQ)-PCR analysis
Real-time PCR was performed at human cDNA panels. Considerable differences in tissue mRNA levels between the molecules were observed (Fig. 1). FCN1 was primarily expressed in peripheral blood leukocytes and bone marrow but minor expression of the gene was also observed in the spleen and the lung. FCN2 and MBL2 were mainly expressed in the liver. However, FCN3 was highly transcripted both in the liver as well as in the lung, but expression was also detected in heart, kidney, spleen, pancreas,
Discussion
The ficolins and MBL are known as the initiator molecules of the lectin complement pathway (Endo et al., 2007). Although Ficolin-3 has been known as Hakata antigen for many years, the biological knowledge and relevance and its mutual biological characteristics compared with the other molecules is poorly understood. At present, it is not known if the ficolins and MBL have redundant of different biologic functions and only a few ligand studies have been reported. It has been shown that Ficolin-2
Acknowledgements
We thank Ms Vibeke Witved for technical assistance. This work was supported by grants from the Danish Medical Research Council, The Danish Rheumatism Association, The Novo Nordisk Foundation, Lundbeck Foundation, Ther Benzon Foundation, Rigshospitalet and EU (QLRT-CT-2001-01039), which are greatly appreciated. Tina Hummelshoj is a University of Copenhagen research fellow.
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