title: Analysis of a structural determinant in thrombin-protease nexin 1 complexes that mediates clearance by the low density lipoprotein receptor-related protein.
abstract: We recently identified a synthetic peptide, Pro47-Ile58, derived from the mature protease nexin 1 (PN1) sequence, that inhibited the low density lipoprotein receptor-related protein (LRP)-mediated internalization of thrombin-PN1 (Th-PN1) complexes. Presently, we have analyzed this sequence in Th-PN1 complex catabolism using two independent approaches: 1) An antibody was generated against Pro47-Ile58, which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface heparins. This places the structural determinant in PN1 mediating complex internalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His48, or two Ala substitutions, one at His48 and another at Asp49, were expressed in Sf9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15%, respectively, when compared with the catabolic rate of native Th-PN1 complexes. This is the first analysis of a structural determinant in a serine protease inhibitor (SERPIN) required for LRP-mediated internalization and in part may explain the cryptic nature of this site in the unreacted serine protease inhibitor.
authors.affiliation: Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, California 92697, USA. mfknauer@uci.edu
authors.forename: M F
authors.initials: MF
authors.lastname: Knauer
authors.forename: R J
authors.initials: RJ
authors.lastname: Crisp
authors.forename: S J
authors.initials: SJ
authors.lastname: Kridel
authors.forename: D J
authors.initials: DJ
authors.lastname: Knauer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016564:Amyloid beta-Protein Precursor; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D058255:Protease Nexins; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism*; D058258:Serpin E2; D018411:Spodoptera; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011973:Receptors, LDL; C545486:SERPINE2 protein, human; D058258:Serpin E2; D006493:Heparin; D013917:Thrombin