title: Sequence of ovine Ig gamma 2 constant region heavy chain cDNA and molecular modelling of ruminant IgG isotypes.
abstract: Ovine mesenteric lymph node mRNA was used for PCR amplification of DNA coding for immunoglobulin gamma 1 and gamma 2 heavy chain constant regions. Primers complementary to regions of CH1 conserved between ruminants were used for upstream priming, with downstream priming on the poly-A segment. PCR products of the appropriate length were cloned and gamma positive clones selected with a CH1 conserved-region probe. Of these, gamma 1 clones were positively selected and gamma 2 clones negatively selected with a gamma 1 hinge-specific probe. Ovine gamma 2 cDNA has 93% identity of nucleotides with ovine gamma 1. Both ovine gamma 1 and gamma 2 CH1 domains encoded two consecutive cysteine residues (Cys-127, -128, Kabat numbering), an arrangement which is deduced to form a pair of disulphide bridges, one to the L chain and one as an intra-chain bridge to the uppermost Cys of the hinge, as in rabbit and goat IgG. The majority of the differences between the isotypes occur in the hinge region and an evolutionary pattern for ruminant IgG hinges can now be identified. IgG1 isotypes are typical, with hinges containing the C-terminal Cys-Pro motif, but deletion and replacement of nucleotides (in the ancestral gene) of ruminant gamma 2 has shortened the IgG2 hinge, removing the Cys-Pro motif and the consensus high affinity Fc gamma RI receptor motif at the start of CH2. An N-terminal glycosylation site and the peptide motif for complement C1q binding are present in CH2 of both isotypes. The hinge regions of gamma 1 and gamma 2 and predicted structures for ovine IgG1 and IgG2 have been modelled. Close apposition of Fab and Fc in IgG2 produces steric hindrance at the normally accessible Fab/hinge/Fc interface; the structural differences between the ruminant isotypes form a basis for understanding some of the differences in their effector properties.
authors.affiliation: Laboratory of Structural Studies, Agricultural and Food Research Council, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
authors.forename: C A
authors.initials: CA
authors.lastname: Clarkson
authors.forename: D
authors.initials: D
authors.lastname: Beale
authors.forename: J W
authors.initials: JW
authors.lastname: Coadwell
authors.forename: D B
authors.initials: DB
authors.lastname: Symons
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D003001:Cloning, Molecular; D003198:Computer Simulation; D005091:Exons / Q000235:genetics; D052058:Hinge Exons; D007127:Immunoglobulin Constant Regions / Q000235:genetics* / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000235:genetics* / Q000276:immunology; D007142:Immunoglobulin gamma-Chains / Q000235:genetics* / Q000276:immunology; D008198:Lymph Nodes / Q000276:immunology; D008643:Mesentery / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012418:Ruminants / Q000276:immunology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep / Q000235:genetics* / Q000276:immunology
chemical_list: D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007142:Immunoglobulin gamma-Chains