Purification, characterization and immunolocalization of porcine surfactant protein D

C.M. Sørensen, Ove Lilholm Nielsen, A. Willis, Peter M. H. Heegaard, U. Holmskov

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    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 Ca2+-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 similar to53 000 MW in the reduced state and similar to138 000 MW in the unreduced state. Porcine SP-D was sensitive to collagenase digestion and N-deglycosylation, which reduced the molecular mass to similar to24 000 MW and similar to48 000 MW respectively, in the reduced state. N-deglycosylation of the collagen-resistant fragment, reduced the molecular mass to similar to21 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 Ca2+-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.
    Original languageEnglish
    JournalImmunology
    Volume114
    Issue number1
    Pages (from-to)72-82
    ISSN0019-2805
    DOIs
    Publication statusPublished - 2005

    Keywords

    • collectins
    • immunolocalization
    • immunology
    • surfactant protein D
    • innate immunity
    • porcine

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