Purification, characterization and cDNA cloning of human lung surfactant protein D.
Human pulmonary surfactant protein D (SP-D) was identified in lung lavage by its similarity to rat SP-D in both its molecular mass and its Ca(2+)-dependent-binding affinity for maltose [Persson, Chang & Crouch (1990) J. Biol. Chem. 265, 5755-5760]. For structural studies, human SP-D was isolated from amniotic fluid by affinity chromatography on maltose-Sepharose followed by f.p.l.c. on Superose 6, which showed it to have a molecular mass of approx. 620 kDa in non-dissociating conditions. On SDS/PAGE the human SP-D behaved as a single band of 150 kDa or 43 kDa in non-reducing or reducing conditions respectively. The presence of a high concentration of glycine (22%), hydroxyproline and hydroxylysine in the amino acid composition of human SP-D indicated that it contained collagen-like structure. Collagenase digestion yielded a 20 kDa collagenase-resistant globular fragment which retained affinity for maltose. Use of maltosyl-BSA as a neoglycoprotein ligand in a solid-phase binding assay showed that human SP-D has a similar carbohydrate-binding specificity to rat SP-D, but a clearly distinct specificity from that of other lectins, such as conglutinin, for a range of simple saccharides. Amino acid sequence analysis established the presence of collagen-like Gly-Xaa-Yaa triplets in human SP-D and also provided sequence data from the globular region of the molecule which was used in the synthesis of oligonucleotide probes. Screening of a human lung cDNA library with the oligonucleotide probes, and also with rabbit anti-(human SP-D), allowed the isolation of two cDNA clones which overlap to give the full coding sequence of human SP-D. The derived amino acid sequence indicates that the mature human SP-D polypeptide chain is 355 residues long, having a short non-collagen-like N-terminal section of 25 residues, followed by a collagen-like region of 177 residues and a C-terminal C-type lectin domain of 153 residues. Comparison of the human SP-D and bovine serum conglutinin amino acid sequences indicated that they showed 66% identity despite their marked differences in carbohydrate specificity.
Lu
J
J
Department of Biochemistry, University of Oxford, U.K.
Willis
A C
AC
Reid
K B
KB
D000595:Amino Acid Sequence; D000653:Amniotic Fluid / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007231:Infant, Newborn; D008168:Lung / Q000502:physiology*; D003012:Microbial Collagenase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D004247:DNA; D003012:Microbial Collagenase