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 [alpha]2-macroglobulin (720 kDa) under non-denaturing conditions, and generated trypsin-fragments consistent with ficolin [alpha] by MALDI-TOF mass spectrometry (MS). The subunit mass of ficolin [alpha] 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 [beta]. 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 [beta] by MALDI-TOF MS. Porcine neutrophils constitutively expressed ficolin [beta] mRNA, but not ficolin [beta] as determined by RT-PCR. Activation of isolated neutrophils with PMA induced secretion of ficolin [beta] but binding of these released forms to APP 5b was not detected. These studies demonstrate that pigs have distinct protein forms of ficolin [alpha] in plasma and ficolin [beta] in peripheral blood neutrophils. Ficolin [alpha] 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 [beta] 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 [alpha].