Sequence of oving la γ2 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 γ 1 and γ 2 heavy chain constant regions. Primers complementary to regions of C Hl 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 y positive clones selected with a C Hl conserved-region probe. Of these, γ 1 clones were positively selected and γ 2 clones negatively selected with a γ 1 hinge-specific probe. Ovine γ 2 cDNA has 93% identity of nucleotides with ovine γ 1. Both ovine y 1 and γ 2 C Hl 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. IgGl isotypes are typical, with hinges containing the C-terminal Cys-Pro motif, but deletion and replacement of nucleotides (in the ancestral gene) of ruminant γ 2 has shortened the IgG2 hinge, removing the Cys-Pro motif and the consensus high affinity Fcγ RI receptor motif at the start of C H2. An N-terminal glycosylation site and the peptide motif for complement Clq binding are present in C H2 of both isotypes. The hinge regions of γ 1 and γ 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.