Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein.
CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glycosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was less than 50% sequence homology of these peptides with the cDNA-derived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8), similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps & Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins.
Persson
A
A
Department of Pathology, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
Chang
D
D
Rust
K
K
Moxley
M
M
Longmore
W
W
Crouch
E
E
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001466:Barium Sulfate; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007525:Isoelectric Focusing; D008168:Lung / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011663:Pulmonary Surfactants / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D007507:Therapeutic Irrigation
D000596:Amino Acids; D007106:Immune Sera; D010446:Peptide Fragments; D011663:Pulmonary Surfactants; D001466:Barium Sulfate; D003488:Cyanogen Bromide