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 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-B 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 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-B delta C, SP-BN and SP-BC constructs 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 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.
Lin
S
S
Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, OH 45229-3039, USA.
Phillips
K S
KS
Wilder
M R
MR
Weaver
T E
TE
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011510:Proteolipids / Q000096:biosynthesis / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins