Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor.
Protease nexin-2 (PN-2) is a protease inhibitor that is synthesized and secreted by a variety of extravascular cells including human fibroblasts. It forms sodium dodecyl sulfate-stable complexes with trypsin, the epidermal growth factor binding protein and the gamma-subunit of nerve growth factor. Recently we reported that PN-2 is the secreted form of the amyloid beta-protein precursor (APP) and is a potent inhibitor of chymotrypsin. Here we describe a two-step procedure to purify PN-2/APP using a monoclonal antibody immunoaffinity column. We also quantitated the protease inhibitory properties of purified PN-2/APP on a number of serine proteases. PN-2/APP was a potent inhibitor of coagulation factor XIa with a Ki = 2.9 x 10(-10). The inhibition of factor XIa by PN-2/APP was augmented by heparin and resulted in a Ki = 5.5 x 10(-11) M. Trypsin and chymotrypsin were also effectively inhibited with a Ki = 4.2 x 10(-10) and 1.6 x 10(-9), respectively. PN-2/APP also inhibited the epidermal growth factor binding protein, the gamma-subunit of nerve growth factor, and chymase and plasmin to a lesser extent. In view of recent findings that PN-2/APP is contained in alpha-granules of platelets and is secreted upon platelet activation, the potent inhibition of factor XIa suggests that PN-2/APP may play a regulatory role in the coagulation pathway at vascular wound sites. In addition, these studies define biochemical activities of PN-2/APP which may be involved in regulating proteases that lead to the generation and deposition of the beta-protein in neurodegenerative lesions associated with Alzheimer's disease and Down's syndrome.
Van Nostrand
W E
WE
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Wagner
S L
SL
Farrow
J S
JS
Cunningham
D D
DD
D000595:Amino Acid Sequence; D000682:Amyloid / Q000302:isolation & purification* / Q000494:pharmacology; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000302:isolation & purification*; D058255:Protease Nexins; D011498:Protein Precursors / Q000302:isolation & purification* / Q000494:pharmacology; D011956:Receptors, Cell Surface; D012867:Skin / Q000378:metabolism; D013379:Substrate Specificity
C545487:APP protein, human; D000682:Amyloid; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D011480:Protease Inhibitors; D058255:Protease Nexins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D010447:Peptide Hydrolases