328회 Examination of the Physio-Chemical Properties of the Human Lung S…

328회

연사 : 최 성 현 , University of Oxford

제목:  Examination of the Physio-Chemical Properties of the Human Lung Surfactant Proteins, SP-A and SP-D

Abstract

Lung surfactant protein A (SP-A) is a protein which belongs to the collectin group of mammalian C-type lectins, which contain collagen-like regions and carbohydrate recognition domains within their structures, and are found in lung surfactant, serum and mucosal secretions. SP-A is hexamer of a trimeric subunit (octadecamer) and it has a shape of a bouquet of tulips. It is known that SP-A recognises carbohydrate (sugar) in calcium-dependent manner and also that SP-A interacts with phospholipids, especially of the di-saturated species. In this research, self-aggregation of SP-A in the presence of Ca2+ ions has been extensively studied. It has been found that SP-A in the presence of Ca2+ ions shows aggregation and forms aggregates large enough to cause precipitation. Also, it has been found that this aggregation is reversible and that dissociation of the aggregates is mediated by the calcium-chelating substance, EDTA, or the chemicals such as citrate, containing a COO- group, in the presence of detergents. An important finding in the thesis is that SP-A alters its hydrophobic properties in the presence and absence of Ca2+ ions. The purification of SP-A, from lung lavage obtained from alveolar proteinosis patients, was carried out by exploiting these properties of SP-A. The collagenase resistant fragment of SP-A (CRF-A), has been produced by digesting-off both the N-terminal and the collagen-like regions of SP-A by collagenase. The self-aggregation properties of native SP-A were compared with that of the CRF-A. The speculation that a motif involved in the mechanism of self-aggregation may be located in the collagen-like domain has been made indirectly, by showing that the CRF-A does not aggregate in the presence of Ca2+ ions. Also, surfactant protein D (SP-D) from lung lavage and a recombinant fragment of SP-D (Neck and CRD) expressed and secreted from yeast were purified by using columns packed with non-sugar derivitised resins, such as Sephacryl-400 and Sepharose 4B, instead of using a column packed with mannose- or maltose-agarose resin, for affinity chromatography. The behaviour of SP-D in columns packed with non-sugar derivitised resins was compared with that of SP-A. In addition, it was found that an extra cysteine residue is present, in a defined ratio, at the N-terminal ends of the chains of mature SP-A preparations, isolated from the lavage of 5 different alveolar proteinosis patients. The appearance of the dimer band (66KDa) of SP-A, which is seen in reducing SDS-PAGE conditions, has been studied and molecular mechanisms for the presence of this band have been put forward. The results obtained from the experiments in the thesis show that the dimer band of SP-A seen in reducing SDS-PAGE can be converted to the monomer by treatment of the SP-A sample with an alkylating chemical or methanolic NaOH prior to examination by a reducing SDS-PAGE system. It was therefore speculated that SP-A may have reducible cross-link (bond) which is more complex than simple disulfide bond between the SP-A chains. Furthermore, the ratio of the products of the SP-A1 and SP-A2 genes has been estimated, by amino acid sequence analysis of SP-A preparations from three alveolar proteinosis patients, to be approximately four to one (SP-A2: SP-A1).