Forms and bacterial-binding functions of porcine ficolins

Ficolins are evolutionarily conserved collagenous lectins putatively involved in innate defence against micro-organisms with outer surfaces containing N-acetylglucosamine (GlcNAc) or related N-acetamido saccharides. Porcine plasma was previously shown to contain various forms of ficolins that can bind to Actinobacillus pleuropneumoniae serotype 5 (APP 5) in a GlcNAc-dependent manner. The objectives of this thesis were to further characterize the forms and bacterial-binding functions of ficolins in porcine plasma and leukocytes. Toyopearl AF-Epoxy 650M, without a coupled ligand, was used for efficient purification of APP serotype 5b-binding forms of porcine plasma ficolins. Two multimeric forms that migrated as multiple diffuse 38–42 kDa subunits (pI range 5.2–6.1) under reducing conditions were consistently purified. However, the electrophoretic forms produced were heterogeneous, and the molecular binding target on Toyopearl AF-Epoxy 650M was uncertain, so Toyopearl AF-Amino 650M, to which ficolins initially did not bind, was used to study test ligands. This matrix was converted into a ficolin-binding target by N-acetylation with acetic anhydride. Acetamide dissociated bound ficolin from this matrix and acetate, acetamide, and GlcNAc were similarly active in dissociating bound ficolin from intact APP 5b. The bacterial-binding forms of plasma ficolins eluted from intact APP 56 were indistinguishable from ficolins purified by affinity chromatography. Both consisted of two multimeric forms that migrated above human α2-macroglobulin (720 kDa) under non-denaturing conditions, and generated trypsin-fragments consistent with ficolin α by MALDI-TOF mass spectrometry (MS). The subunit mass of ficolin α is 35081 Da and removal of N-glycans by digestion of purified ficolin with N-glycosidase F reduced the number of electrophoretic forms but did not reduce the bacterial-binding activity. N-terminal amino acid sequences obtained from plasma ficolins suggested a minor amount (<10%) might be ficolin β. Comparisons of various cellular fractions from porcine blood revealed that neutrophils contain a distinct immunoreactive form of ficolin that migrated in the pI range 6.4–7.4. Trypsin-fragments of neutrophil ficolin were consistent with porcine ficolin β by MALDI-TOF MS. Porcine neutrophils constitutively expressed ficolin β mRNA, but not ficolin β as determined by RT-PCR. Activation of isolated neutrophils with PMA induced secretion of ficolin β but binding of these released forms to APP 5b was not detected. These studies demonstrate that pigs have distinct protein forms of ficolin α in plasma and ficolin β in peripheral blood neutrophils. Ficolin α binds the acetamido constituent of GlcNAc and may recognize patterns of acetamido sugars on microbial surfaces, such as those demonstrated in the capsular polysaccharides of APP serotype 5b. Ficolin β is a neutrophil protein that is likely secreted in acute inflammation, but it may not bind to the bacteria or other structures recognized by ficolin α.