Purification, characterization and immunolocalization of porcine surfactant protein D.
Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca(2+)-dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a band of approximately 53,000 MW in the reduced state and approximately 138,000 MW in the unreduced state. Porcine SP-D was sensitive to collagenase digestion and N-deglycosylation, which reduced the molecular mass to approximately 24,000 MW and approximately 48,000 MW respectively, in the reduced state. N-deglycosylation of the collagen-resistant fragment, reduced the molecular mass to approximately 21,000 MW showing the presence of an N-glycosylation site located in the CRD. Porcine SP-D bound to solid-phase mannan in a dose and Ca(2+)-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP-D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkuhn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.
Soerensen
C M
CM
Department of Veterinary Pathobiology, Laboratory of Pathology, The Royal Veterinary Agricultural University, Ridebanevej 3, 1870 Frederiksberg, Denmark.
Nielsen
O L
OL
Willis
A
A
Heegaard
P M H
PM
Holmskov
U
U
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001992:Bronchoalveolar Lavage Fluid / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D017364:Collagenases; D045647:DNA Glycosylases; D005260:Female; D007421:Intestine, Small / Q000276:immunology; D007765:Lacrimal Apparatus / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D037663:Pulmonary Surfactant-Associated Protein D / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D013552:Swine / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011134:Polysaccharides; D037663:Pulmonary Surfactant-Associated Protein D; D045647:DNA Glycosylases; D017364:Collagenases