Identification of a Binding Site in Protease Nexin I (PN1) Required for the Receptor Mediated Internalization of PN1-Thrombin Complexes

• Published in: The Journal of biological chemistry Vol. 272; no. 19; pp. 12261 - 12264
• Main Authors: Knauer, M F, Hawley, S B, Knauer, D J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 09-05-1997
• Subjects: Amyloid beta-Protein Precursor; Animals; Binding Sites; Carrier Proteins - metabolism; Cells, Cultured; Fibroblasts - metabolism; Mice; Protease Nexins; Protein Conformation; Receptors, Cell Surface; Serine Proteinase Inhibitors - metabolism; Serpins - metabolism; Thrombin - antagonists & inhibitors; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.19.12261

An overlapping synthetic peptide library was constructed representing most of the mature protease nexin I (PN1) sequence from
the amino terminus to the reactive center. This library, along with peptides from the heparin binding domain and from the
region carboxyl-terminal to the P1 residue of the cleavage site, was screened for the inhibition of 125 I-thrombin (Th)-PN1 complex binding and degradation. A peptide corresponding to residues Pro 47 –Ile 58 in the PN1 sequence was identified as a potent inhibitor of 125 I-Th-PN1 complex degradation, although it did not affect binding significantly. Pro 47 –Ile 58 was shown to competitively inhibit the low density lipoprotein receptor-related protein (LRP)/α 2 -macroglobulin receptor-mediated endocytosis of 125 I-Th-PN1 complexes in mouse embryo fibroblasts. Pro 47 –Ile 58 is an apparent transition sequence in PN1, separating sheet-6B and helix-B. The sequence of Pro 47 –Ile 58 , P HDNIVIS PHGI, suggests that it forms a loop structure defined by the seven underlined amino acids bordered by proline residues at
each end. These studies are the first to identify a putative binding site in a serine protease inhibitor that is required
for LRP-mediated internalization.