title: Physiological concentration of calcium inhibits elastase-induced cleavage of a functional recombinant fragment of surfactant protein D.
abstract: Surfactant protein D (SP-D) plays an important role in lung host defence. SP-D levels have been shown to be depleted in cystic fibrosis (CF) patients. A recombinant fragment of the human SP-D (rfhSP-D) which consist of a hydrophobic neck and a CRD has been shown to be active in vivo and partially reverses the symptoms of the SP-D deficiency in the lungs when administered to SP-D knock-out mice. In this paper we studied the in vitro effect of different proteolytic enzymes commonly found in CF patients lungs, such as neutrophil elastase, cathepsin G and protease 3 as well as Pseudomonas elastase, on rfhSP-D. It was also shown that cleavage was inhibited by physiological concentration of calcium. When Western blot was compared with ELISA, we show that an anti-SP-D ELISA is a not a reliable assay of functional SP-D levels since non-functional fragments of SP-D are also detected. Thus, ELISA cannot be used as a reliable "diagnostic" tool for SP-D deficiency. Finally, we observe that SP-D is not cleaved in control patients but is degraded in about half the samples from cystic fibrosis patients, indicating that degradation of endogenous SP-D, by enzymes present in CF bronchioalveolar lavage fluid (BALF), may lead to deficiency of the protein as seen in CF and therefore rfhSP-D may be a useful future therapy.
authors.affiliation: MRC Immunochemistry Unit, Biochemistry Dept, University of Oxford, South Parks Road, OX1 3QU Oxford, UK.
authors.forename: Annelyse
authors.initials: A
authors.lastname: Duvoix
authors.forename: Rose-Marie
authors.initials: RM
authors.lastname: Mackay
authors.forename: Neil
authors.initials: N
authors.lastname: Henderson
authors.forename: Eamon
authors.initials: E
authors.lastname: McGreal
authors.forename: Anthony
authors.initials: A
authors.lastname: Postle
authors.forename: Kenneth
authors.initials: K
authors.lastname: Reid
authors.forename: Howard
authors.initials: H
authors.lastname: Clark
mesh_terms: D000293:Adolescent; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D002648:Child; D002675:Child, Preschool; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D037663:Pulmonary Surfactant-Associated Protein D / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D037663:Pulmonary Surfactant-Associated Protein D; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D002118:Calcium