title: Use of site-specific mutagenesis and monoclonal antibodies to map regions of CD46 that interact with measles virus H protein.
abstract: Researchers at our laboratory have been dissecting the binding domains of the receptor for the Edmonston laboratory strain of measles virus (CD46) through site-specific mutagenesis. We initially substituted most of the hydrophilic amino acids in the two external short consensus regions (SCRI and SCRII) of CD46 with the amino acid alanine [Hsu et al. (1997) J. Virol. 71:6144-6154] and found that the glutamic-arginine residues at positions 58 and 59 were particularly sensitive to change. Here we consider the roles of hydrophobic amino acids in the binding between measles virus H protein and CD46. Hydrophobic amino acids in the SCRI and SCRII domains of CD46 were systematically replaced with serine. The effects of these changes were monitored through the interaction of Sf9 insect cells expressing the H protein and mouse OST-7 cells synthesizing the mutant CD46 molecules. Binding was quantified through a colorimetric assay for beta-galactosidase that was also produced by the insect cells. Our results indicate that E45, Y54, 58E/R59, Y68, F69, Y101, I102, R103, D104, and Y117 seem to be critical residues for the binding of CD46 to measles virus H protein. The hydrophilic amino acid R59 in SCR1 and hydrophobic residues Y101, I102, and Y117 in SCR2 seem to be especially important for interaction between H protein and CD46. In addition, we mapped the antigenic epitopes of five monoclonal antibodies that are known to inhibit the binding between H protein and CD46. Three of these antibodies recognized regions in SCR1, and two reacted with amino acids in SCR2. For the most part, the determinants recognized by the monoclonal antibody corresponded to the amino acids that were most sensitive to change in the binding process. The SCR1 and SCR2 domains of CD46 were modeled from an analogous region in another complement regulatory protein, factor H, whose three-dimensional structure has been previously reported. Amino acids implicated in binding seem to lie on one planar face of the SCR1 and SCR2 domains. These studies serve as a prelude to understanding the structural interactions that occur between CD46 and the measles virus H protein.
authors.affiliation: Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada.
authors.forename: E C
authors.initials: EC
authors.lastname: Hsu
authors.forename: S
authors.initials: S
authors.lastname: Sabatinos
authors.forename: F J
authors.initials: FJ
authors.lastname: Hoedemaeker
authors.forename: D R
authors.initials: DR
authors.lastname: Rose
authors.forename: C D
authors.initials: CD
authors.lastname: Richardson
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D005434:Flow Cytometry; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008459:Measles virus / Q000378:metabolism*; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C497857:CD46 protein, human; D018985:Epitopes, B-Lymphocyte; D006389:Hemagglutinins, Viral; C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D011991:Receptors, Virus; C032397:hemagglutinin protein G, measles virus