Biochemical characterization and bacterial binding functions of porcine ficolins

DeLay, Josepha
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University of Guelph

These studies were aimed at identification of porcine plasma proteins with carbohydrate-dependent binding to surface polysaccharides of ' Actinobacillus' spp. bacterial pathogens of swine. A multimeric (600-800 kDa) plasma protein composed of heterogeneous 38, 40 and 42 kDa subunits with N-terminal amino acid sequence identity with porcine ficolins a ( ~ 85%) and b (5-18%) was the major protein isolated by affinity to epoxy-activated Sepharose 6B conjugated with lipopolysaccharide (LPS) of 'Actinobacillus suis' or 'A. pleuropneumoniae'. Similar porcine plasma ficolins with kDa (38-42 kDa) and pI (4.98-5.90) bound N-acetyl-D-glucosamine (GlcNAc) coupled to epoxy-activated Toyopearl. Ficolins corresponding to those of LPS- and GlcNAc-affinity preparations were the major plasma proteins that bound in a GlcNAc-dependent manner to surfaces of cultured intact serotype O5 'A. pleuropneumoniae' isolates which have GlcNAc in their capsular polysaccharides. By comparison serotype O7 'A. pleuropneumoniae ' strains which lack capsular GlcNAc bound minimal plasma ficolin. Polyclonal rabbit antiserum to LPS-affmity purified pig plasma ficolins identified immunohistochemical reactivity within pulmonary alveolar macrophages and in intestinal crypt epithelial cells of pigs of various ages. By western blots, porcine lung lavage fluid contained mostly the 40 kDa ficolin band whereas intestinal mucosal scrapings contained mostly the 38 kDa ficolin band. Monolayer cultures of porcine hepatocytes secreted 35S-labelled bands corresponding to the 38-42 kDa bands, and amounts produced were not increased by prior exposure of pigs to doses of 'E. coli' LPS that induced hepatocellular synthesis of various acute phase proteins. Ficolin immunoreactivity was not detected in focal areas of suppurative pneumonia induced by infection with 'A. pleuropneumoniae', but was prominent in macrophages and surfactant fluid in adjacent areas of lung. These studies suggest that ficolins might be involved in host recognition of organisms with surface GlcNAc-containing polysaccharides.

identification, porcine, plasma proteins, carbohydrate-dependent binding, surface polysaccharides, Actinobacillus spp., bacterial pathogens, swine