Robert Steven Siegel

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid β-protein precursor

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimers amyloid β-protein precursor (AβPP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2AβPP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285–288 of AβPP (Ponte et al. 1988 Nature 311:525–527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2AβPP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2AβPP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2AβPP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2AβPP.

Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid β-protein precursor-like protein-2

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.

Specific patterns of amyloid β-protein precursor isoform secretion and proteolysis in cultured human cells

A characteristic neuropathologic feature of Alzheimer c disease is an abundance of brain-localized neuritic plaques. These plaques are identified as areas of degenerating nerve terminals surrounding cores of aggregated and insoluble fibrils of amyloid β-protein (Aβ). Aβ is generated by proteolytic cleavage of a large precursor protein, the amyloid β-protein precursor (AβPP). In this study, soluble AβPPs (sAβPPs) secreted by human cell lines originating from different tissues were characterized. Various human cell types were shown to secrete different characteristic isoforms of AβPP. Peripheral cells (i.e., fibroblasts and keratinocytes) secreted only the soluble derivative of the 770-amino acid isoform (sAβPP770). In contrast, cells derived from the central and peripheral nervous system (i.e., neumblastoma and glioblastoma) secreted the soluble derivative of the 751-amino acid isoform (sAβPP751), while the 695-amino acid isoform (sAβPP695) was secreted exclusively by neuronal-type cells. We have also dete...