STRUCTURAL REQUIREMENTS FOR INTRACELLULAR-TRANSPORT OF PULMONARY SURFACTANT PROTEIN-B (SP-B)

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 e xtremely hydrophobic and flanked by propeptides of 200 and 102 amino a cids at its NH2- and COOH-termini, respectively. The purpose of this s tudy was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To thi s end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-Bh(Delta C), in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP -B-Delta N, in which residues 28-200 were deleted), NH2-terminal prope ptide (SP-B-N), mature SP-B (SP-B-M) and COOH-terminal propeptide (SP- B-C), into the mammalian expression vector pcDNA3. The resulting expre ssion constructs were characterized by DNA sequencing and in vitro tra nscription/translation and subsequently transfected into Chinese hamst er ovary cells. 48 h after transfection, cells were labeled with [S-35 ]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiog raphy. Proteins encoded by SP-B, SP-Bh(Delta C) SP-B-N and SP-B-C cons tructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B-Delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rap idly 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 intra cellular trafficking of the mature peptide.