Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B)
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- 作者：Sui ; Lin ; Phillips ; Karen S. ; Wilder ; Melinda R. ; Weaver ; Timothy E.
- 关键词：Pulmonary surfactant ; Intracellular trafficking ; Secretion ; Propeptide
- 刊名：Biochimica et Biophysica Acta (BBA)/Molecular Cell Research
- 出版年：1996
- 出版时间：July 24, 1996
- 年：1996
- 卷：1312
- 期：3
- 页码：177-185
- 全文大小：1001 K
文摘
Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-BΔC, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-BΔN, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-BΔC, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-BΔN) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.