Human anti-IgG1 hinge autoantibodies reconstitute the effector functions of proteolytically inactivated IgGs.

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.

The N-terminal epidermal growth factor-like domain in factor IX and factor X represents an important recognition motif for binding to tissue factor.

Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal gamma-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIa*TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa*TF complex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIX(WT)), FIX(Q50P), FIX(PCEGF1) (EGF1 domain replaced with that of protein C), FIX(DeltaEGF1) (EGF1 domain deleted), FX(WT), and FX(PCEGF1). Complexes of FVIIa with TF as well as with soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa*TF or FVIIa*sTF activated each mutant significantly more slowly than the FIX(WT) or FX(WT). Importantly, in ligand blot assays, FIX(WT) and FX(WT) bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments revealed that the affinity of the mutants for sTF was reduced 3-10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF with K(i) of approximately 60 microm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FX(WT) and FX(PCEGF1) normally, which is interpreted to mean that the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.