Characterization of porcine plasma ficolins that bind Actinobacillus pleuropneumoniae serotype 5B.
Ficolins are collagenous lectins implicated in resistance to infection by some micro-organisms with surfaces containing N-acetylglucosamine residues. Pigs have two known ficolin genes, alpha and beta, but ficolins in pig plasma appear in various electrophoretic forms, the origin and function of which are unclear. In this investigation the forms of ficolin in pig plasma that bind to the pneumonic pathogen Actinobacillus pleuropneumoniae serotype 5b (APP5) were compared with affinity-purified porcine plasma ficolins. APP5-bound ficolins consisted of two distinct multimeric (non-denatured) forms composed of subunits that migrated as multiple 38,40 and 42 kDa forms (apparent MW) with pI range 5.2-6.0. The APPS-binding forms of ficolin were consistent with ficolin alpha and were indistinguishable from affinity-purified plasma ficolins by peptide mass fingerprint analysis. Subunit bands separated by 2D-PAGE were consistent with porcine ficolin alpha. Affinity-purified plasma ficolins had a major subunit mass of 35081 Da by MALDI-TOF MS. Affinity-purified ficolins digested with N-glycosidase F retained APP5-binding activity and migrated faster as a single band of 38 kDa. These studies indicate that ficolin alpha is the major APPS-binding form in porcine plasma and suggest that N-glycosylation contributes to the apparent electrophoretic heterogeneity of porcine ficolins but is not required for APP5-binding activity.
Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Andrew S
AS
Brooks
Josepha P
JP
DeLay
M Anthony
MA
Hayes
D016977:Actinobacillus pleuropneumoniae / Q000145:classification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D037102:Lectins; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine / Q000097:blood*; D000097604:Ficolins
D002352:Carrier Proteins; D037102:Lectins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D021122:Protein Subunits; D000097604:Ficolins