Glycosylation of the amyloid peptide precursor containing the Kunitz protease inhibitor domain improves the inhibition of trypsin

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.     

In Vivo Neuronal Synthesis and Axonal Transport of Kunitz Protease Inhibitor (KPI)-Containing Forms of the Amyloid Precursor Protein

Abstract: We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.     

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.     

The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage

beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product.     

Structural and mutational analyses of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.     

Abnormal and deficient processing of beta-amyloid precursor protein in familial Alzheimer's disease lymphoblastoid cells

Western blot analysis showed abnormal processing of beta-amyloid precursor protein (APP) in lymphoblastoid cell lines (LCLs) of familial Alzheimer's disease (FAD). Antibody raised against central APP751 revealed that media of early and late-onset FAD LCLs had highly increased amounts of a 120 kD long-lived. SDS-stable, heat-labile complex of the Kunitz protease inhibitor domain of secreted APP and a approximately 70 kD FAD-specific, yet unidentified serine protease. Antibody against the beta A4-cytoplasmic domain showed a slower APP processing and increased amounts of 16 kD C-terminal preamyloid in lysates of early and late-onset FAD LCLs, first indicating a deficient intra-beta A4 proteolysis in FAD as a possible cause of abundant amyloid deposits in AD brain.     

F-spondin interaction with the apolipoprotein E receptor ApoEr2 affects processing of amyloid precursor protein

A recent study showed that F-spondin, a protein associated with the extracellular matrix, interacted with amyloid precursor protein (APP) and inhibited beta-secretase cleavage. F-spondin contains a thrombospondin domain that we hypothesized could interact with the family of receptors for apolipoprotein E (apoE). Through coimmunoprecipitation experiments, we demonstrated that F-spondin interacts with an apoE receptor (apoE receptor 2 [ApoEr2]) through the thrombospondin domain of F-spondin and the ligand binding domain of ApoEr2. Full-length F-spondin increased coimmunoprecipitation of ApoEr2 and APP in transfected cells and primary neurons and increased surface expression of APP and ApoEr2. Full-length F-spondin, but none of the individual F-spondin domains, increased cleavage of APP and ApoEr2, resulting in more secreted forms of APP and ApoEr2 and more C-terminal fragments (CTF) of these proteins. In addition, full-length F-spondin, but not the individual domains, decreased production of the beta-CTF of APP and Abeta in transfected cells and primary neurons. The reduction in APP beta-CTF was blocked by receptor-associated protein (RAP), an inhibitor of lipoprotein receptors, implicating ApoEr2 in the altered proteolysis of APP. ApoEr2 coprecipitated with APP alpha- and beta-CTF, and F-spondin reduced the levels of APP intracellular domain signaling, suggesting that there are also intracellular interactions between APP and ApoEr2, perhaps involving adaptor proteins. These studies suggest that the extracellular matrix molecule F-spondin can cluster APP and ApoEr2 together on the cell surface and affect the processing of each, resulting in decreased production of Abeta.     

Hydrogen peroxide promotes Abeta production through JNK-dependent activation of gamma-secretase

Accumulation of senile plaques composed of amyloid beta-peptide (Abeta) is a pathological hallmark of Alzheimer disease (AD), and Abeta is generated through the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase. Although oxidative stress has been implicated in the AD pathogenesis by inducing Abeta production, the underlying mechanism remains elusive. Here we show that the pro-oxidant H(2)O(2) promotes Abeta production through c-Jun N-terminal kinase (JNK)-dependent activation of gamma-secretase. Treatment with H(2)O(2) induced significant increase in the levels of intracellular and secreted Abeta in human neuroblastoma SH-SY5Y cells. Although gamma-secretase-mediated cleavage of APP or C99 was enhanced upon H(2)O(2) treatment, expression of APP or its alpha/beta-secretase-mediated cleavage was not affected. Silencing of the stress-activated JNK by small interfering RNA or the specific JNK inhibitor SP600125 reduced H(2)O(2)-induced gamma-secretase-mediated cleavage of APP. JNK activity was augmented in human brain tissues from AD patients and active JNK located surrounding the senile plaques in the brain of AD model mouse. Our data suggest that oxidative stress-activated JNK may contribute to senile plaque expansion through the promotion of gamma-secretase-mediated APP cleavage and Abeta production.     

Amyloid Precursor Protein Is Synthesized by Retinal Ganglion Cells, Rapidly Transported to the Optic Nerve Plasma Membrane and Nerve Terminals, and Metabolized

Abstract: We have investigated the synthesis, axonal transport, and processing of the β-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f_1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [³⁵S]- methionine, [³⁵S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

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

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) 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-2/A beta PP. 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 A beta PP (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-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.     

Detection of soluble forms of the beta-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.     

Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations

Immunotherapy against beta-amyloid peptide (Abeta) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Abeta immunization reduces Abeta plaque pathology and improves cognitive function. However, the biological mechanisms by which Abeta antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Abeta antibodies decreases levels of intracellular Abeta. Antibody-mediated reduction in cellular Abeta appears to require that the antibody binds to the extracellular Abeta domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Abeta antibodies protects against synaptic alterations that occur in APP mutant neurons.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.