Constitutive secretion of beta-trace protein by cultivated porcine choroid plexus epithelial cells: elucidation of its complete amino acid and cDNA sequences.
Primary porcine choroid plexus epithelial cells cultivated in chemically defined medium maintain their epithelial characteristics and form confluent monolayers. They produce a fluid the composition of which resembles cerebrospinal fluid. The present study demonstrates constitutive secretion of large amounts of beta-trace protein. This intrathecally synthesized protein is a prominent polypeptide constituent of natural cerebrospinal fluid. According to the identity of amino acid sequences it has previously been tentatively identified as a prostaglandin-D synthase and as a member of the lipocalin protein family. beta-Trace was purified from cell culture supernatants and was subjected to tryptic digestion and amino acid sequencing of the resulting peptides. The complete primary structure of the protein was obtained by additional isolation of the cDNA from cultured epithelial cells. The porcine 163-amino acid polypeptide showed 69% identity with the human beta-trace and contained two N-glycosylation sites occupied by complex-type oligosaccharides as is the case for the human protein. The amino acid sequences around the N-glycosylation sites of mammalian beta-trace proteins (porcine, human, murine, and rat) were highly conserved. The nucleotide sequence was found to be less conserved; the porcine cDNA had a strikingly high GC-content (67%). The constitutive secretion of beta-trace protein from the in vitro cultivated porcine choroid plexus epithelial cells demonstrates that the cells have retained their major in vivo physiological properties: secretion of cerebrospinal fluid proteins. Therefore, this in vitro culture system may be used as a versatile tool for studying the regulation of the formation of cerebrospinal fluid.
Gesellschaft für Biotechnologische Forschung, Department of Molecular Biology, Braunschweig, Germany.
A
A
Hoffmann
U
U
Gath
G
G
Gross
J
J
Lauber
R
R
Getzlaff
S
S
Hellwig
H J
HJ
Galla
H S
HS
Conradt
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001609:Beta-Globulins / Q000134:cerebrospinal fluid / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D017077:Culture Media, Conditioned / Q000737:chemistry; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000737:chemistry; D054834:Lipocalins; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D013552:Swine
D001609:Beta-Globulins; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D017931:DNA Primers; D018076:DNA, Complementary; D054834:Lipocalins; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase