Protease nexin-I(PN-I) is representative of a class of cell-secreted protein inhibitors collectively referred to as the Protease Nexins(PN's). It has a molecular weight of 43,000 Daltons, and is related to, but distinct from the plasma-borne protease inhibitors, heparin cofactor-II(HC-II) and antithrombin-III(AT-III). PN-I forms covalently-linked complexes with thrombin, plasmin, urokinase and trypsin, and mediates their inactivation, binding, internalization and degradation by cells. Thus, PN-I is likely to be a significant regulatory element in a number of important biological processes where these proteases are involved. The interaction of PN-I with cells will be examined. The receptor that mediates the binding and internalization of PN-I:protease complexes will be characterized at the molecular level, purified, and monoclonal antibodies against it will be generated. The molecular characterization will include chemical crosslinking of 125I-Protease:PN-I complexes to receptors on human fibroblasts. The receptor will be purified from human placental membranes using standard biochemical approaches. Either partially purified or homogeneous receptor will be used to generate monoclonal antibodies. These antibodies will be used to study the dynamics and regulation of the PN-I receptor. This will largely be accomplished by employing a combination of metabolic labeling with 35S-methionine followed by immunoprecipitation. PN-I also interacts with cells via cell-surface and extracellular matrix glycosaminoglycans. This leads to an acceleration of linkage formation between PN-I and thrombin on fibroblasts. The heparin binding domain of PN-I will be identified, purified and sequenced using a combination of proteolytic digestion followed by separation on reverse-phase high performance liquid chromatography. Peptides of 25 amino acid or less will be synthesized that overlap within the identified heparin binding domain of PN-I and antibodies will be raised against them. These antibodies will be used to verify the physiological relevance of the identified heparin binding domain by assessing their ability to block heparin binding and/or augment protease inhibitory activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034001-06
Application #
3284342
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1984-07-01
Project End
1993-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J (2002) Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV. J Biol Chem 277:47285-91
Crisp, R J; Knauer, D J; Knauer, M F (2000) Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or intern J Biol Chem 275:19628-37
Knauer, D J; Majumdar, D; Fong, P C et al. (2000) SERPIN regulation of factor XIa. The novel observation that protease nexin 1 in the presence of heparin is a more potent inhibitor of factor XIa than C1 inhibitor. J Biol Chem 275:37340-6
Knauer, M F; Crisp, R J; Kridel, S J et al. (1999) Analysis of a structural determinant in thrombin-protease nexin 1 complexes that mediates clearance by the low density lipoprotein receptor-related protein. J Biol Chem 274:275-81
Mentz, S; de Lacalle, S; Baerga-Ortiz, A et al. (1999) Mechanism of thrombin clearance by human astrocytoma cells. J Neurochem 72:980-7
Kridel, S J; Knauer, D J (1997) Lysine residue 114 in human antithrombin III is required for heparin pentasaccharide-mediated activation. J Biol Chem 272:7656-60
Knauer, M F; Kridel, S J; Hawley, S B et al. (1997) The efficient catabolism of thrombin-protease nexin 1 complexes is a synergistic mechanism that requires both the LDL receptor-related protein and cell surface heparins. J Biol Chem 272:29039-45
Knauer, M F; Hawley, S B; Knauer, D J (1997) Identification of a binding site in protease nexin I (PN1) required for the receptor mediated internalization of PN1-thrombin complexes. J Biol Chem 272:12261-4
Kridel, S J; Chan, W W; Knauer, D J (1996) Requirement of lysine residues outside of the proposed pentasaccharide binding region for high affinity heparin binding and activation of human antithrombin III. J Biol Chem 271:20935-41
Smith, J W; Dey, N; Knauer, D J (1990) Heparin binding domain of antithrombin III: characterization using a synthetic peptide directed polyclonal antibody. Biochemistry 29:8950-7

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