Inhibition of the proteolytic activity of pregnancy-associated plasma protein-A by targeting substrate exosite binding.
The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.
Mikkelsen
Jakob H
JH
Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark.
Gyrup
Claus
C
Kristensen
Peter
P
Overgaard
Michael T
MT
Poulsen
Christine B
CB
Laursen
Lisbeth S
LS
Oxvig
Claus
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000737:chemistry; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008957:Models, Genetic; D019151:Peptide Library; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D018974:Insulin-Like Growth Factor Binding Protein 4; D019151:Peptide Library; D007334:Insulin-Like Growth Factor I; D011266:Pregnancy-Associated Plasma Protein-A