Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats.
We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3'-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.
Pérez de la Lastra
J M
JM
Complement Biology Group, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom.
Harris
C L
CL
Hinchliffe
S J
SJ
Holt
D S
DS
Rushmere
N K
NK
Morgan
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018958:CD55 Antigens / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D016466:CHO Cells; D016384:Consensus Sequence; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000276:immunology / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics / Q000276:immunology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020449:Repetitive Sequences, Amino Acid; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D013552:Swine; D013737:Testis / Q000276:immunology / Q000378:metabolism
D018958:CD55 Antigens; D018076:DNA, Complementary; D008565:Membrane Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger