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 α-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 Gramnegative 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 α-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.
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