Congenital alveolar proteinosis (CAP) is a familial disorder fatal in full term newborns. We have identified a cause: deficiency of surfactant protein B (SP-B) one of four surfactant proteins secreted by the Type II pneumocyte (NEJM 328:406-410, 1993). In the first ten infants studied we identified two mutations in the SP-B gene - the more common, a frameshift mutation - 121ins2, or a 3' splice junction deletion (delta Ex 4) (J Pediatr: 125:43-50, 1994). Of great significance, our immunostaining of SP-B deficient lungs revealed an abnormal increase in Type II cell SP-C and alveolar luminal SP-A and SP-C; ultrastructure with immunogold localization showed accumulation of SP-A, SP-C and membranous (lipid) vesicles between Type II cells and their basement membrane indicating loss of Type II cell apical (polar) secretion of surfactant lipids and proteins. (Am J Respir Cell Mol Biol 11:230-239, 1994). These features provide unique insight into the importance of SP-B in Type II cell biology and suggest the hypotheses that SP-B: 1) is a regulatory factor in the synthesis, assembly and secretion of surfactant lipids and proteins SP-A and SP-C; 2) maintains the polarity of the cell essential for anatomical and structural integrity of the alveolar lining. To test these hypotheses we will use our immortalized, fully characterized, polarized, differentiated type II cell line T7, that secretes surfactant lipids and SP-A, SP-B, and SP-C, to: 1) produce an SP-B deficient cell line by infection with our SP-B anti-sense retroviral vector derived from Moloney murine leukemia virus; 2) compare in normal and SP-B deficient Type II cells (SP-B+, & SP-B-) the synthesis, assembly, polar secretion, reutilization of surfactant lipids and proteins SP-A, SP-B, and SP-C and the cell polarity, using radiolabelled choline and methionine incorporation with pulse-chase, phospholipid assay, immunoprecipitation, Western blotting and immunolocalization of surfactant with fluorescence, confocal and electron microscopy. These studies will greatly expand understanding of the role of SP-B in regulating surfactant metabolism and in maintaining the integrity of the alveolar epithelial and surfactant lining that are disrupted in many important diseases of the newborn and many forms of lung injury.
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