Molecular cloning and functional characterization of the rat analogue of human decay-accelerating factor (CD55).
We report here the cloning of cDNAs encoding two forms of the rat analogue of human decay-accelerating factor (DAF; CD55). Screening of a rat kidney cDNA library using a mouse DAF probe identified a partial cDNA encoding the 3' end of rat DAF. The 5' end of the cDNA was cloned using the rapid amplification of cDNA ends (RACE) technique. A second form of rat DAF was identified using 3'RACE. Cloning and sequencing of full length cDNAs for both forms showed that they were identical up to nucleotide 1143 except for a 51-bp insert in the ST-rich region of the second form. After nucleotide 1143, the two sequences diverged; the cDNA cloned from the library encoded a unique 112-amino acid "tail," whereas the second form, identified by 3'RACE, encoded an 18-amino acid hydrophobic stretch, which was predicted to be a glycosylphosphatidylinositol (GPI) anchor addition signal. Expression in the NIH-3T3 mouse fibroblast cell line confirmed that the short tail did encode a GPI-addition signal, whereas the longer tail caused the protein to be secreted. Northern blot analysis identified two distinct transcripts for the GPI form, as well as a variability in expression levels of the different transcripts in the panel of tissues screened. Southern blot analysis showed that both the GPI and secreted forms of rat DAF were expressed in a wide range of tissues. The GPI-linked form of rat DAF stably expressed in a murine fibroblast cell line reduced C3 deposition and conferred protection from lysis by rat serum.
Hinchliffe
S J
SJ
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom.
Spiller
O B
OB
Rushmere
N K
NK
Morgan
B P
BP
D016475:3T3 Cells / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018958:CD55 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D005091:Exons; D005784:Gene Amplification; D018628:Gene Dosage; D015723:Gene Library; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007438:Introns; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018958:CD55 Antigens; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D020033:Protein Isoforms; D012333:RNA, Messenger