title: The alpha-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids.
abstract: We have expressed the carbohydrate recognition domains (CRDs) of human lung surfactant protein D (SP-D) in Escherichia coli as a trimeric structure held together by the alpha-helical neck region of the molecule. The DNA sequence coding for the neck-region peptide and the CRD of SP-D was subcloned and expressed as a fusion protein containing the E. coli maltose binding protein (MBP). After removal of the MBP, the recombinant structure, containing three CRDs of SP-D, was found to be comparable to native SP-D in terms of carbohydrate binding specificity, the binding to lipopolysaccharides (LPSs) of Gram-negative bacteria, and interaction with phospholipids. The CRD of SP-D, without the neck region peptide, was also expressed and shown to behave as a monomer that showed a very weak affinity for maltose-agarose, LPSs and phospholipids. The alpha-helical neck region on its own showed affinity for phospholipids and thus might contribute to the binding of SP-D to these structures. However, the possibility that hydrophobic patches, which are exposed only in the isolated neck region and not in the intact SP-D, plays a role in neck region-phospholipid interaction, cannot be excluded. The results confirm the importance of the neck region as a trimerizing agent in bringing together three CRDs and suggest that multivalency is important in the strong binding of SP-D to carbohydrate targets.
authors.affiliation: Department of Biochemistry, University of Oxford, U.K.
authors.forename: U
authors.initials: U
authors.lastname: Kishore
authors.forename: J Y
authors.initials: JY
authors.lastname: Wang
authors.forename: H J
authors.initials: HJ
authors.lastname: Hoppe
authors.forename: K B
authors.initials: KB
authors.lastname: Reid
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002241:Carbohydrates / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006090:Gram-Negative Bacteria; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D008168:Lung / Q000378:metabolism; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D010743:Phospholipids / Q000378:metabolism*; D017433:Protein Structure, Secondary; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D002352:Carrier Proteins; D006023:Glycoproteins; D008070:Lipopolysaccharides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010743:Phospholipids; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins