Abstract
Membrane cofactor protein (MCP), a regulatory molecular of the complement system with cofactor activity for the factor I-mediated inactivation of C3b and C4b, is widely distributed, being present on leukocytes, platelets, endothelial cells, epithelial cells, and fibroblasts. MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation. An oligonucleotide probe based on this sequence was used to identify a clone from a human monocytic (U937) cDNA library. Nucleotide sequencing showed a 43-bp 5'-untranslated region, an open reading frame of 1,152 bp, and a 335-bp 3'-untranslated region followed by a 16-bp poly(A) track. The deduced full-length MCP protein consists of a 34-amino acid signal peptide and a 350-amino acid mature protein. The protein has, beginning at the NH2 terminus, four approximately 60-amino acid repeat units that match the consensus sequence found in a multigene family of complement regulatory proteins (C3b-receptor or CR1, C3d-receptor or CR2, decay-accelerating factor, C4-binding protein, and factor H), as well as several other complement and non-complement proteins. The remainder of the MCP protein consists of 25 amino acids that are rich in serine and threonine (probable site of heavy O-linked glycosylation of MCP), 17 amino acids of unknown significance, and a 23-amino acid transmembrane hydrophobic region followed by a 33-amino acid cytoplasmic tail. The MCP gene was localized to human chromosome 1, bands 1q31-41, by analysis of human x rodent somatic cell hybrid clones and by in situ hybridization. This same genetic region contains the multigene family of complement-regulatory proteins, which is thereby enlarged to include the functionally and structurally related MCP.
Highlights
The DNA sequence ofthis cDNA clone was determined by dideoxychain termination sequencing of both strands and contains a long open reading frame encoding 384 amino acids beginning with an initiation methionine codon (Fig. 1)
The polypeptide encoded by this cDNA clone matches several other properties for Membrane cofactor protein (MCP): (a) typical structure for a membrane protein, with a signal peptide at the NH2 terminus, and a region ofhydrophobic amino acids near the COOH terminus to serve as the transmembrane domain; (b) overall size of 39 kD, in agreement with the precursors seen in biosynthetic labeling experiments [40]; (c) three sites for N-linked glycosylation and multiple sites for 0-linked glycosylation, consistent with the oligosaccharide structure of MCP [40]; and (d) tandemly arranged NH2-terminal homologous consensus repeat units characteristic of other C3b/C4b-binding proteins
MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation
Summary
T cell line HSB2 by our previously reported procedure [19] using NP-40 solubilization followed by sequential chromatography on chromatofocusing, hydroxylapatite, C3 (methylamine)$epharose, and Mono Q columns. 20 Rg of protein was run on a SDS-10% polyacrylamide gel, electroeluted, and electrodialyzed as described [26]. This material was divided in half and subjected to automated Edman degradation on polybrenecoated glass filters in an Applied Biosystems, Inc . RNA was isolated from the U937 cell line by the quanidinium isothiocyanate/CsCl method [27]. Poly(A)' RNA was purified by oligo(dT)-cellulose chromatography [28]. A cDNA library was prepared from 5 ug poly(A)'
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