Coagulation Factor XIa Cleaves the RHDS Sequence and Abolishes the Cell Adhesive Properties of the Amyloid β-Protein

Susan M Saporito-Irwin,William E Van Nostrand

doi:10.1074/jbc.270.44.26265

Abstract

Amyloid beta-protein (A beta) is the major constituent of senile plaques and cerebrovascular amyloid deposits in Alzheimer's disease and is proteolytically derived from its transmembrane parent protein the amyloid beta-protein precursor (A beta PP). Although the physiological role(s) of secreted A beta PPs are not fully understood, several potential functions have been described including the regulation of hemostatic enzymes factors XIa and IXa and a role in cell adhesion. In the present study, we investigated the proteolytic processing of A beta PP by factor XIa (FXIa). Incubation of the human glioblastoma cell line U138 stably transfected to overexpress the 695 isoform of A beta PP with FXIa (2.5-5 nM) resulted in proteolytic cleavage of secreted A beta PP. Higher concentrations of FXIa (> 25 nM) resulted in loss in cell adherence. Coincubation of FXIa with purified, recombinant Kunitz protease inhibitor domain of A beta PP blocked both the proteolytic processing of A beta PP and the loss of cell adhesion. The RHDS cell adhesion site of A beta PP resides within residues 5-8 of the A beta domain. Incubation of synthetic A beta 1-40 peptide with increasing concentrations of FXIa resulted in cleavage of A beta between Arg5 and His6 within the cell adhesion domain of the peptide. FXIa-digested A beta 1-40 or A beta PP695 lost their abilities to serve as cell adhesion substrates consistent with cleavage through this cell adhesion site. Together, these results suggest a new potential biological function for FXIa in the modulation of cell adhesion. In addition, we have shown that FXIa can proteolytically alter A beta and therefore possibly modify its physiological and perhaps pathological properties.

Highlights

Deposition of the amyloid ␤-protein (A␤)1 in senile plaques in the neuropil and in the walls of cerebral blood vessels is a pathologic feature of Alzheimer’s disease
These data suggest a new potential biological function for Factor XIa (FXIa) in the modulation of cell adhesion. These findings indicate that the Kunitztype serine protease inhibitors (KPI) domain of amyloid ␤-protein precursor (A␤PP) can regulate the activity of a protease that can process the A␤ domain, altering its potential physiological and pathological properties
FXIa Induces Proteolytic Processing of Secreted A␤PP in Cultured U138/695 Cells—We examined the effects of FXIa on A␤PP processing in cultured U138/695 cells that have previously been shown to overproduce the A␤PP695 isoform by 80fold over untransfected glioblastoma cells [35]

Summary

The abbreviations used are

A␤, amyloid ␤-protein; A␤PP, amyloid ␤-protein precursor; KPI, Kunitz protease inhibitor; PN-2, protease nexin-2; FXIa, factor XIa; mAb, monoclonal antibody; U138/695 cells, human glioblastoma U138 cells stably transfected to overexpress the A␤PP695 isoform; PAGE, polyacrylamide gel electrophoresis. Secreted forms of A␤PP that contain the KPI domain have been shown to inhibit several different serine proteases, which include trypsin, chymotrypsin, and coagulation Factors XIa and IXa [12, 25,26,27,28,29] Through inhibition of these latter two proteases, it has been suggested that the KPI-containing isoforms of A␤PP may play a role in regulating hemostasis by acting as an anticoagulant [30]. These data suggest a new potential biological function for FXIa in the modulation of cell adhesion These findings indicate that the KPI domain of A␤PP can regulate the activity of a protease that can process the A␤ domain, altering its potential physiological and pathological properties

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION