Molecular cloning of the rat analogue of human CD59: structural comparison with human CD59 and identification of a putative active site.
We have previously described the purification and partial characterization of the rat analogue of the human complement regulatory molecule CD59 [Hughes, Piddlesden, Williams, Harrison and Morgan (1992) Biochem. J. 284, 169-176]. We present here the molecular cloning and full sequence analysis of this molecule. A PCR-based approach utilizing primers designed from the amino-terminal protein sequence was used to isolate a full-length cDNA clone from a rat kidney cDNA library. This clone encoded a 92 bp 5'-flanking sequence, a 66 bp signal peptide and a 315 bp coding region containing putative glycosylation and GPI-anchor signals. The 3' untranslated flanking region was approximately 1.1 kbp long and included the poly-A tail and a CATA repeating sequence. The coding region was 58% identical with the human cDNA at the nucleotide level and 44% identical at the amino acid level. Despite this relatively low overall sequence conservation, several highly conserved stretches were apparent, particularly in the N-terminal portion of the molecule, in the cysteine-rich region immediately preceding the site of glycolipid attachment and in the C-terminal peptide removed during glycolipid attachment. An N-glycosylation site was identified at Asn-16 and a putative glycosylphosphatidylinositol anchor addition site at Asn-79, indicating that the mature processed protein was two residues longer than human CD59. Comparison of the sequences of rat and human CD59, together with consideration of the published three-dimensional structure of human CD59 and functional data, implicates specific regions of the protein in interactions with C-8 and/or C-9.
Rushmere
N K
NK
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, U.K.
Harrison
R A
RA
van den Berg
C W
CW
Morgan
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D018957:CD59 Antigens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D016415:Sequence Alignment; D017421:Sequence Analysis; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018957:CD59 Antigens; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins