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Jens Christian Jensenius

Studies of the pattern recognition molecule H-ficolin: specificity and purification

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Ficolins are pattern recognition molecules (PRMs) of the innate immune system. H-ficolin is found in plasma associated with mannan-binding-lectin-associated serine proteases (MASPs). When H-ficolin binds to microorganisms the MASPs are activated which in turn activate the complement system. H-ficolin is the most abundant ficolin in humans, yet its ligand binding characteristics and biological role remain obscure. We examined the binding of H-ficolin to Aerococcus viridans as well as to a more defined artificial target, i.e., acetylated bovine serum albumin (AcBSA). A strict dependence for calcium ions and inhibition at high NaCl concentration was found. The binding to AcBSA was inhibited by acetylsalicylic acid and sodium acetate as well as by N-acetylated glucosamine and galactosamine (GlcNAc and GalNAc), and glycine (GlyNAc). The binding to A. viridans was sensitive to the same compounds, but, importantly, higher concentrations were needed for inhibition. N-acetylated cystein was also inhibitory, but this inhibition was parallel with reduction in the oligomerization of H-ficolin and thus represents structural changes of the molecule. Based on our findings we developed a procedure for the purification of H-ficolin from serum, involving PEG precipitation, affinity chromatography on Sepharose derivatized with acetylated serum albumin, ion exchange chromatography and gel permeation chromatography (GPC). The purified H-ficolin was observed by GPC to elute at 700 kDa, similar to what we find for H-ficolin in whole serum. MASP-2 was co-purified with H-ficolin and the purified H-ficolin/MASP-2 complex could activate complement as measured by cleavage of complement factor C4. The present report extends our knowledge of the specificity of this PRM and the purified product will enable further studies.
Original languageEnglish
JournalJournal of Biological Chemistry
ISSN0021-9258
DOIs
Publication statusPublished - 2012

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