INTRACHAIN DISULFIDE BOND IN THE CORE HINGE REGION OF HUMAN IGG4

IgG is a tetrameric protein composed of two copies each of the light a nd heavy chains. The four-chain structure is maintained by strong nonc ovalent interactions between the amino-terminal half of pairs of heavy -light chains and between the carboxyl-terminal regions of the two hea vy chains. In addition, interchain disulfide bonds link each heavy-lig ht chain and also link the paired heavy chains. An engineered human Ig G4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetram ers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide b onded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rath er than serine, CDP571(S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded hal f-IgG tetramers were observed. Trypsin digest reverse-phase HPLC pepti de mapping studies of CDP571 and CDP571(gamma 1) with on-line electros pray ionization mass spectroscopy supplemented with Edman sequencing i dentified the chemical factor preventing inter-heavy chain disulfide b ond formation between half-IgG molecules: the two cysteines in the IgG 4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capab le of forming an intrachain disulfide bond. Conformational modeling st udies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded ener gy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constrai nts caused by the extra proline. These modeling results suggest a reas on why a larger fraction of intrachain bonds are observed in IgG4 rath er than IgG1 molecules: the serine in the core hinge region of IgG4 al lows more hinge region flexibility than the proline of IgG1 and thus m ay permit formation of a stable intrachain disulfide bond more readily .