A Novel Chicken Membrane-Associated Complement Regulatory Protein: Molecular Cloning and Functional Characterization

Abstract

A cDNA encoding a membrane-associated complement (C) regulatory protein was identified here for the first time in an oviparous vertebrate, chicken. This protein, named Cremp, possessed five short consensus repeats (SCRs) and one SCR-like domain followed by a transmembrane domain and a cytoplasmic tail. SCR1/SCR2 of Cremp were 43.6% identical with SCR2/SCR3 of human decay-accelerating factor (CD55), and SCR3/SCR4 were 45.3% identical with those of human membrane cofactor protein (CD46). Cremp is likely to be an ancestral hybrid protein of human decay-accelerating factor and membrane cofactor protein rather than a homolog of rodent C receptor 1-related protein y, which structurally resembles human CR1 (CD35). Chinese hamster ovary cells transfected with Cremp were efficiently protected from chicken C but not from human or rabbit C in both classical and alternative pathways. Thus, chicken Cremp is a membrane C regulator for cell protection against homologous C. Cremp mRNA was seen as a doublet comprised of a faint band of 2.2 kb and a thick band of 3.0 kb on RNA blotting analysis. An Ab against chicken Cremp recognized a single band of 46.8 kDa on immunoblotting. mRNA and protein of Cremp were ubiquitously expressed in all chicken organs tested. Minute amounts of dimer were present in some tissues. Surface expression of Cremp was confirmed by flow cytometry and immunofluorescence analysis. These results suggested that even in nonmammals a C regulatory membrane protein with ubiquitous tissue distribution should be a prerequisite for protection of host cells from homologous C attack.

A Short Consensus Repeat-Containing Complement Regulatory Protein of Lamprey That Participates in Cleavage of Lamprey Complement 3

“…Rabbit anti-Lacrep polyclonal Ab (anti-Lacrep Ab) was produced by the method established in our laboratory (9). Briefly, RK13 cells (1 ϫ 10 7 ) were transiently transfected with a pFlag CMV-Lacrep-Hisϫ6 full-length Lacrep construct using Lipofectamine Plus reagent (Invitrogen Life Technologies).…”

“…These cells were transfected with expression vectors. Lamprey proteins were harvested from the culture supernatant and cell lysate as described previously (9). For RNA and protein blot analysis, total RNA and proteins were obtained from various lamprey tissues and stored at Ϫ80°C until use.…”

“…A partial lamprey SCR protein (Lacrep; accession no. AB061219) cDNA fragment was obtained by nested PCR using degenerated primers, the vector T3 and SK primers (first and second primers, respectively, in Table I) as previously described (9). A degenerated primer was designed from the junctional sequence of SCR3 and SCR4 of human Mcp (CD46) as described previously for molecular cloning of mouse Mcp (19).…”

“…From an evolutionary point of view, a nonmammalian, membrane-anchored, C regulatory protein (8) was first identified in the chicken: chicken C regulatory membrane protein bears structural and functional similarity to mammalian Mcp and DAF (9). A nonmammalian soluble-form of C regulator named sand bass protein 1 (SBP1) has been cloned from a bony fish, Parablax nebulifer (sand bass) (10), but no such regulator has been identified to date in invertebrates.…”

A CD46-like Molecule Functional in Teleost Fish Represents an Ancestral Form of Membrane-Bound Regulators of Complement Activation

“…Loci of each group form a compact cluster, and, in the human genome, the group 1 cluster and the group 2 loci are also located in proximity, forming a RCA gene cluster (20)(21)(22). Genomic arrangement of the RCA genes and their clustering are diverse among species analyzed to date, such as mice (23,24), chicken (25,26), frog (27), and teleost, which is the most common group of the ray-finned bony fish (28)(29)(30)(31)(32)(33)(34).…”