Identification of an epitope in antithrombin appearing on insertion of the reactive-bond loop into the A beta-sheet.
Previous work has shown that insertion of the reactive-bond loop of antithrombin into the main beta-sheet of the inhibitor, the A sheet, leads to exposure of epitopes that are not present in intact antithrombin. Identical epitopes are exposed in antithrombin-proteinase complexes, inferring that the reactive-bond loop is inserted into the A beta-sheet also in these complexes. Loop insertion thus presumably is involved in the mechanism of inhibition of target proteinases. In this work, we have identified a linear epitope in bovine antithrombin that reacts with antibodies specific for loop-inserted forms of the inhibitor. This epitope is a hexapeptide sequence comprising residues 342-347, Glu-Asp-Leu-Phe-Ser-Pro, and is located on the surface of the protein just carboxy-terminal of helix I. The Phe residue of this epitope is highly conserved in members of the serpin superfamily and appears to stabilize the region of the epitope in antithrombin and other serpins by interacting with the protein core. The conformational change involving expansion of the A beta-sheet following insertion of the reactive-bond loop is presumably transmitted through this Phe residue to the epitope region, thereby rendering this region accessible to antibodies.
Nordling
K
K
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Sweden.
Björk
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000737:chemistry* / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D003488:Cyanogen Bromide; D000939:Epitopes / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D013917:Thrombin / Q000737:chemistry
D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; C101078:antithrombin (342-347); D000990:Antithrombin III; D013917:Thrombin; D003488:Cyanogen Bromide