An antibody with specificity for surfactant protein C precursors: identification of pro-SP-C in rat lung.
Surfactant protein C (SP-C) is a lung-specific, hydrophobic peptide found in organic extracts of pulmonary surfactant. Alveolar SP-C (3.5 kD) is produced from proteolytic cleavage of a larger precursor molecule (pro-SP-C; 21 kD). While SP-C is synthesized by type II cells, the pathways for processing and secretion have remained elusive due, in part, to the lack of monospecific antibodies against SP-C or its precursors. This report describes production and characterization of a new antibody directed against pro-SP-C epitopes. Polyclonal antisera (anti-CPRO-SP-C) was prepared using a synthetic peptide corresponding to a portion of rat SP-C cDNA sequence (Ile26-Ser72). This contained amino acids 3-35 of mature SP-C plus additional C-terminal residues (His59-Ser72). On Western blots, anti-CPRO-SP-C competitively reacted to CPRO-SP-C but not to mature SP-C. Immunoblots of in vitro synthesized pro-SP-C confirmed that the antisera also recognized native protein. Immunocytochemistry with anti-CPRO-SP-C demonstrated staining for pro-SP-C peptides in isolated type II cells as well as in alveolar epithelial cells of rat lung sections. Pro-SP-C preferentially co-localized to cells that stained positive for Maclura pomifera antigen. Anti-CPRO-SP-C staining was not observed in lung interstitium, pulmonary vasculature, or several control tissues (brain, heart, and liver were negative). Western blotting of subcellular fractions demonstrated pro-SP-C peptides in plasma membrane (20 kD) and microsomal (20 and 21 kD) fractions with a 16 kD peptide present in lamellar bodies. No pro-SP-C peptides were detected in purified surfactant. These results demonstrate the use of a synthetic peptide to generate specific antiserum against more hydrophilic domains of pro-SP-C sequences and confirm that SP-C propeptides are unique to the lung.
Beers
M F
MF
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6068.
Wali
A
A
Eckenhoff
M F
MF
Feinstein
S I
SI
Fisher
J H
JH
Fisher
A B
AB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D007150:Immunohistochemistry; D008168:Lung / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011510:Proteolipids / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011663:Pulmonary Surfactants / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D010455:Peptides; D011498:Protein Precursors; D011510:Proteolipids; D011663:Pulmonary Surfactants; C077063:Sftpc protein, rat