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cDNA, deduced polypeptide structure and chromosomal assignment of human pulmonary surfactant proteolipid, SPL(pVal)
S W Glasser1, T R Korfhagen, T E Weaver
1University of Cincinnati, College of Medicine, Department of Pediatrics, Ohio 45267-0541.
Insights
Researchers identified key hydrophobic surfactant proteins (SPLs) crucial for lung function in premature infants. These proteins, particularly SPL(pVal), are vital for preventing respiratory distress by aiding lung surfactant activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Pulmonary Medicine
Background:
- Hyaline membrane disease in premature infants is caused by surfactant deficiency, leading to lung collapse and respiratory distress.
- Hydrophobic surfactant proteins (Mr = 5,000-14,000) are known to enhance phospholipid spreading and surface tension reduction in mammalian lung surfactant.
- Understanding these proteins is critical for developing treatments for neonatal respiratory disorders.
Purpose of the Study:
- To characterize the amino-terminal amino acid sequences of pulmonary proteolipids from various mammalian species.
- To identify and isolate the complementary DNA (cDNA) and genomic DNA encoding the human surfactant proteolipid SPL(pVal).
- To determine the primary structure, precursor protein size, and gene locus of human SPL(pVal).
Main Methods:
- Amino-terminal sequencing of pulmonary proteolipids from bovine, canine, and human surfactant extracts.
- Oligonucleotide probing using a valine-rich sequence to isolate human cDNA and genomic DNA.
- Nucleotide sequence analysis to deduce the primary structure of the precursor protein.
- Hybrid-arrested translation and immunoprecipitation to confirm precursor protein size and processing.
- Northern blot analysis to assess mRNA size and abundance.
- Gene mapping to determine the chromosome location of the SPL(pVal) gene.
Main Results:
- Two distinct hydrophobic peptides, SPL(pVal) and SPL(Phe), were identified in mammalian surfactant.
- The human SPL(pVal) gene was isolated, revealing a precursor protein of 20,870 daltons.
- Proteolytic processing generates the active hydrophobic peptide (Mr = 5,000-6,000) from the precursor.
- Two classes of cDNAs encoding SPL(pVal) were identified, with mRNA of approximately 900 bases.
- Human SPL(pVal) mRNA levels were higher in adult than fetal lung tissue.
- The SPL(pVal) gene locus was assigned to chromosome 8.
Conclusions:
- The hydrophobic surfactant protein SPL(pVal) plays a critical role in lung surfactant function.
- The identified precursor protein and its processing pathway are key to producing active surfactant peptides.
- Understanding the genetic basis and expression of SPL(pVal) provides insights into neonatal respiratory distress syndrome.
- The gene for SPL(pVal) is located on chromosome 8, contributing to the genetic map of pulmonary surfactant components.
Abstract:
In hyaline membrane disease of premature infants, lack of surfactant leads to pulmonary atelectasis and respiratory distress. Hydrophobic surfactant proteins of Mr = 5,000-14,000 have been isolated from mammalian surfactants which enhance the rate of spreading and the surface tension lowering properties of phospholipids during dynamic compression. We have characterized the amino-terminal amino acid sequence of pulmonary proteolipids from ether/ethanol extracts of bovine, canine, and human surfactant. Two distinct peptides were identified and termed SPL(pVal) and SPL(Phe). An oligonucleotide probe based on the valine-rich amino-terminal amino acid sequence of SPL(pVal) was utilized to isolate cDNA and genomic DNA encoding the human protein, termed surfactant proteolipid SPL(pVal) on the basis of its unique polyvaline domain. The primary structure of a precursor protein of 20,870 daltons, containing the SPL(pVal) peptide, was deduced from the nucleotide sequence of the cDNAs. Hybrid-arrested translation and immunoprecipitation of labeled translation products of human mRNA demonstrated an Mr = 22,000 precursor protein, the active hydrophobic peptide being produced by proteolytic processing to Mr = 5,000-6,000. Two classes of cDNAs encoding SPL(pVal) were identified. mRNA of approximately 900 bases was identified on Northern analysis of fetal and adult RNA. Human SPL(pVal) mRNA was more abundant in the adult than in fetal lung. The SPL(pVal) gene locus was assigned to chromosome 8.