| Co-Investigator(Kenkyū-buntansha) |
SOHMA Hitoshi Sapporo Medical University School of Medicine, Department of Biochemistry, Assoc, 医学部, 講師 (70226702)
KUROKI Yoshio Sapporo Medical University School of Medicine, Department of Biochemistry, Assoc, 医学部, 助教授 (70161784)
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| Research Abstract |
Pulmonary surfactant is a mixture of lipids and proteins in which dipalmitoylphosphatidylcholine (DPPC) is a most abundant component. It stabilizes lung alveoli at the air-liquid interface. In addition to this essential function, it has been revealed recently that lung surfactant present in the alveoli exhibits other functions, and surfactant apoproteins, especially hydrophilic surfactant apoproteins, SP-A and SP-D are involved in the expression of these functions by their receptor binding to alveolar type II cells or lipid specific binding. Therfore, the present study focusses upon to analyze the roles of SP-A and SP-D structures for expression of the functions.
1. The SP-A and SP-D possess similar structures as members of the mammalian C-type lectin supre family. Both proteins are composed of four characteristic domains which are NH_2-terminal domaincollagenous domain, neck domain and C-terminal carbohydrate recognition domain (CRD). The domains of SP-A which mediate its functions hav
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e not been fully mapped. The binding of SP-A to its high affinity receptor on alveolar type II cells is thought to be dependent on a CRD.To exposure the role of CRD in the interaction of SP-A with type II cells and lipids, we prepared chimeric molecules of SP-A and SP-D,and introduced mutations into the cDNA focussing on Arg^<197>. As the results, the binding of SP-A to its receptor and inhibition of surfactant secretion were critically dependent on the CRD region, and the impotance of Arg^<197> in the CRD region was demonstrated. The interaction with DPPC was shown to attribute to the hydrophobic region of the SP-A molecule. Furthermore, its has been shown thet the neck and CRD domains of SP-D play a role in specific bindings of lipids, phosphatidylinositol (PI) and glucosylceramide, and that the CRD domain is essential for PI binding.
2. To investigate the regulation of surfactant metabolism by Ca^<2+>, we identified the Ca^<2+>-dependent binding proteins to both lamellar bodies and SP-A using lamellar body-and SP-A-conjugated Sepharose 4B,respectively, and Mono Q.Four annexins (III,IV,V and VIII) were identified as the lamellar body-binding proteins. Annexins III,V and VIII bound mainly to phospholipids in the lamellar bodies, while annexins IV was the major component of the SP-A-binding protein. The present study also revealed that interaction between the C-terminal domein of SP-A and the C-terminal domain of annexin IV is important for the biological activities.
3. In the alveoli of patients with alveolar proteinosis, abnormal multimerized form of SP-A are accumelated, and this unusual subpopulation of SP-A oligomer exhibits abnormal functions on tublar myelin formation and on biological activities induced by its receptor binding to alveolar type II cells. Less
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