title: Murine C4b-binding protein. Mapping of the ligand binding site and the N-terminus of the pre-protein.
abstract: We have constructed cell surface-bound forms of murine C4b-binding protein (mC4BP) that allowed us to monitor the binding of mC4BP to C4b with relatively simple erythrocyte rosette assays. We used two types of surface-bound mC4BP: one in which segments of mC4BP were fused directly to a peptide containing the transmembrane and cytoplasmic domains of human complement receptor CR2 (BPR1-type); and a second in which the same segments were fused to a longer peptide containing the five membrane-proximal short consensus repeats (SCR) of CR2 as well as the transmembrane and cytoplasmic domains (BPR2-type). COS cells transfected with either construct carrying all six mC4BP SCR rosetted with C4b-bearing EAC14 cells but not with C4b-lacking EAC1 cells; and rosetting was inhibited by excess inactivated C4 but not inactivated C3. COS cells transfected with BPR2 constructs carrying only SCR 1-3 or 1-4 gave similar rosetting behavior. However, rosetting was not observed with BPR2 constructs carrying only SCR 1-2 or 2-6, or with BPR1 constructs carrying only SCR 1-2, 1-3, 1-4, or 2-6. Finally, we found that alteration of the AUG sequence 56 triplet codons upstream of the putative N-terminus of mature mC4-BP eliminates rosetting whereas alteration of a second AUG sequence 13 codons upstream has no effect on rosetting. These results indicate that 1) SCR 1-3 of mC4BP are necessary and adequate for binding to C4b, 2) steric effects close to the cell surface may interfere with binding, and 3) mC4BP has an extraordinarily long 56 amino acid residue signal peptide.
authors.affiliation: Medical Biology Institute, La Jolla, CA 92037.
authors.forename: R T
authors.initials: RT
authors.lastname: Ogata
authors.forename: P
authors.initials: P
authors.lastname: Mathias
authors.forename: B M
authors.initials: BM
authors.lastname: Bradt
authors.forename: N R
authors.initials: NR
authors.lastname: Cooper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011951:Receptors, Complement / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011951:Receptors, Complement; D015935:Complement C4b