S80 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

breakdown of proteins and peptides into their constituent amino acids. PE activity was decreased by 4550% in the frontal and medial temporal cortex of AD patients (mean age 67 f 5) as compared to age-matched controls (mean age 67 i 3). Combination of cytosolic fractions from neocortical tissue from AD patients and from controls did not result in decreases in the sum of activities, which excludes that the reduced enzyme activity results from the presence of 8 protease inhibitor in AD brains, but rather from protein damaging effects or reduced enzyme synthesis. Major AP activity was unaltered. Whether the decrease in PE activity is representative of other serine proteases is an interesting possibility which should be further investigated. The reduction in PE activity found in the present study suggests that changes in peptidase activity may be involved in the etiology of AD. We also measured the two peptidase activities in medial temporal cortex samples of AD patients who died between 55 and 90 years of age. PE activity was signifiiantly lower in those who died relatively young than in those who died relatively old. Major AP did not change with age. The age-dependence of PE activity underscores the notion that there may be heterogeneity in neurochemical changes in AD.

315 CLONING OF A DIFFERENTIALLY SPUCED SERINE PROTEASE FROM HUMAN FETAL BRAIN B. Meckelein and CR. Abraham. Boston University School of Medicine, Boston MA 02118. USA

The characterization of proteases which are involved in the pathological degradation of the amyloid precursor protein (APP) to yield amyloidogenic fragments or ‘mature’ p-protein is an important step towards the under- standino of the develooment of Alrheimer’s Disease (AD) and may provide promising targets for b therapeutical approach.

We were able to detect cathepsin G (cath G) immunoreactivity in aged monkey brain. Furthermore, we could purify a serine protease from monkey brain which is capable to cleave APP and crossreacts with cath G antibodies (Razzaboni et al., submitted for publication). We therefore used these antibodies to screen a Agtll-cDNA library (gift from R. Neve). which had been derived from human fetal brain RNA. Upon several screening rounds, three different phage clones were purified and their cDNA-inserts of 3.2, 1.3 and 0.7 Kb were subcloned into pUC18. DNA sequence analysis revealed that all three cDNA-inserts share an identical 6’.terminus but differ in their 3’. regions. The derived amino acid sequence of the common part of the three cDNAs contains a sequence motif which is highly conserved in all serine proteases and includes the active site serine. Although fusion proteins of Ihe cloned cDNAs linked to p-galactosidase sequences and expressed in E. co/i show a strong immunoreactivity with cath G antibodies, no further significant homology in DNA- or protein-sequence between the fetal brain clones and cath G, or any known protease. could be detected. We therefore conclude lhal we have cloned the cDNA of a hitherto unknown serine protease which is differentially spliced. Further studies now concentrate on the tissue localization of the differentially spliced RNAs by using Northern blot analysis of RNA from different human tissues, including normal adult. fetal and AD brain, and from cultured human neuroblastoma and glioblastoma cells.

Supported by NIH grant AG09905.

316 PURlFlCATlON AND CHARACTERUATION OF A CYSTEINE METAL DEPENDENT PROTEASE FROM ALZHEIMERS DISEASE BRAIN ABLE OF DEGRADING HUMAN APP. G. Papastoitsis. R. Siman’ and C.R. Abraham. Boston University School of Medicine, Arthritis Center, Boston 02118, USA and ‘Cephalon, Inc., West Chester. PA 19380. USA.

A 4.2 kDa polypeptide termed O-protein accumulates in senile plaques and blood vessel walls in Alzheimer’s disease (AD) and Down’s Syndrome (DS). It is widely believed that the &protein is the product of the abnormal degradation of a larger precursor protein, the l3-amyloid precursor protein (APP). The proteolytic processes involved in the generation of the O-protein are virtually unknown.

Recently, we have extensively purified and characterized a protease capable of degrading a 10 amino acid synthetic substrate flanking the N-terminus of O-protein at the Met-Asp bond. aspartic acid being the N-terminus of the 0. protein. The enzyme was purified through a series of ion exchange (DEAE- Trisacryl). hydrophobic interaction (Phenyl Sepharose), gel filtration (Sephacryl S-200). and chromatofocusing (PBE 94) chromatographies. The purified protease requires the presence of a reducing agent, e.g. dithiothreitol. for its activity. Its pH optimum is around physiological pH while the enzyme is inactive at acidic pH (below pH 5.0) and basic pH (over pH 8.0). The enzyme is strongly inhibited by N-ethylmaleimide (NEM). hydroxymercurybenzoate. 1 ,I 0 phenanthroline, EGTA and EDTA. Phenylmethylsulfonylfluoride (PMSF) and leupeptin have no effect on this activity. This protease is activated in the presense of mM concentrations of

MgCh and CaC12 and has been shown to degrade recombinant human APP produced in the baculovirus system, as well as proteolytic fragments of the native protein at physiological pH. The protease is devoid of caseinolytic or gelatinase activities, as well as activities against Cathepsin B and Calhepsin L substrates, Z-Val-Lys-Lys-Arg-AFC and Z-Phe-Arg-AFC,respectively. Currently, efforts are underway to sequence the protease and produce antibodies against it. The antibodies will allow us lo examine the cellular and subcellular distribution of the protease and clone the gene coding for the enzyme by screening expression libraries. Once the role of the protease in Ihe abnormal proteolysis of APP is proved specific inhibitors aiming lo regulate its activity will be synthesized.

Supported by a grant from Cephalon Inc.

317 Identification of a Major Met-Asp Cleaving Peptidase

Activity from Human Brain as Metalloendopeptidase EC 3.4.24.15. Peter Seubert, Harry F. Dovey, Russ Blather, Varghese John, Timothy Colby, Ivan Lieberburg & Sukanto Sinha. Athena Neurosciences, Inc., South San Francisco, CA 94080.

We have isolated, purified and identified a major peptidase activity from soluble extracts of post-mortem human brain tissue which specifically cleaves a synthetic decapeptide, Acetyl- Ser-Glu-Val-Met-Asp-Ala-Glu-Phe-Arg (APP592-601). APP592- 601 was designed to span the Met596-Asp597 peptide bond, as proteolytic cleavage at this site is believed to be a first step in the formation of the aggregating B-peptide. The purified endopeptidase activity cleaves APP592-601 at Met596-Asp597, Asp597-Ala598, and Ala598-Glu599. The enzyme, a single-chain polypeptide of MW -75 kDa, requires low micromolar concentrations of calcium for activity, being completely inhibited by EDTA. The peptidase activity is not inhibited by other protease inhibitors, including l,lO-phenanthroline, phosphoramidon, disopro-pylfluorophosphate, 3,4- dichloroisocoumarin, leupeptin, chymostatin, aprotinin, al- proteinase inhibitor, or al-antichymotrypsin. The purified enzyme has a broad spectrum of peptidase activity, selectively cleaving a variety of biologically active small peptides, such as bradykinin, neurotensin, LH-RI-I, and dynorphin AI-S. Based on the cleavage specificity, metal-ion dependence and approximate molecular weight, we observed a striking similarity to a group of enzymes which have been referred to as metalloendopeptidase EC 3.4.24.15 or Pz-endopeptidase. Although the amino-terminus of the purified peptidase was blocked, internal peptide sequence data were obtained. Comparison of this data with the deduced protein sequence of the recently-cloned rat metalloendopeptidase EC 3.4.24.15 revealed striking homology in otherwise novel sequences, further confirming the identification of the human enzyme as metalloendopeptidase EC 3.4.24.15.

318 TWO-WAY CLEAVAGE OF fi-AMYLOID PRECURSOR PROTEIN BY MULTICATALYTIC PROTEINASE, Shin-ichi Kojima and Motoko Omori. Research Institute, Sumitomo Pharmaceuticals Co., Ltd., Osaka, 554 Japan

Alzheimer’s disease (AD) is pathologically characterized by extracellular deposits of B-amyloid peptide (B-API in senile plaques and cerebral vessels. B-AP is a small peptide fragment of 39 to 42 amino acids derived from a larger aayloid precursor protein (APP). There are at least three different isoforms of APP with 695, 751 and 710 amino acids, generated by alternative splicing of DRNA. fi-AP is encoded as an internal peptide at position 597-638 of APP695. The nOrma processing occurs within B-AP sequence preventing the generation of /j-AP. Therefore, the abundant generation of B-AP in Alzheimer brain suggests the abnormal processing of APP, but the molecular mechanisms of normal and abnormal processing of APP remain unclear.

In an attempt to investigate the APP-processing enzyme and the fi -AP-releasing enzyme in the rat brain, ve used peptide AP33 (SEVKMD~FGRDSCFEVRR~KLVFFAEDVGSNK). synthesized according to the sequence (residues 592-624) of rat APP695. The peptide consists of the extracellular domain of fi-AP, i.e., from Asp(l) to Lys(28), and

THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE S81

five amino acids upstream from the N-terminus of fl-AP, i.e., Sar(-5) to nett-11. The enzyme activity YBS determined by the AP33 fragmentation assay. Ue investigated the cleavage site of AF’P in the rat brain using AP33 and found that the cleave occurred at a middle site between Arg(l3) and Phe(20). mainly at the ~ln(151-~ys(16) bond, but not at the net.-l)-Asp(l) bond. We purified the main APP- processing enzyme, which cleaved B-AP at the ~ln(151-~ys(l6) bond, from the rat brain by gel-filtration chromatography and anion- exchange chromatography, and identified it to be a macropain-like multicatalytic proteinase (also known as proteasome or ingensinl. It is important to note that the purified APP-processing enzyme cleaved the Gln(l5)-Lys(l6) bond of B-AP, but altered to cleave at the N- terminus of B-AP to release the extracellular domain of B-AP in the presence of Ca’+. These findings suggest that the multicatalytic proteinase iS a

candidate for both the APP-processing enzyme and a-AP-releasing enzyme, and the functional change in this multicatalytic proteinase may result in abnormal processing of APP. leading to the generation of B-AP.

319 BIOGENESIS AND METABOLISM OF APP IN PRIMARY NEURONAL CELL CULTURES, M. Dichgansl, R. Sandbrinlcl, U. Manning1.G. W. Rebeckz, C. L. Master@, K. Beyreutherl, Center for Molecular Biology Heidelberg, University of Heidelberg, D-6900 Heidelberg Germany’; Dept. of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA2 ; Dept. of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia3

It has been previously sugge.sted that APP 695 is tbe predominantly expressed form in the developing cenaal nervous system.

To examine neuronal APP biosynthesis we established primary cell cultures of murine brain. Analysis of the APP mRNA by polymerase chain reaction showed that APP695 is indeed the predominantly expressed form in neurons, independend of brain region and days in cultun.

We have further analyzed the biogenesis of transmembrane and secreted forms. The secre.ted forms arc identical with the forms found in cerebrospinal fluid. Large amounts of C-terminal APP fragments remain to be associated with the cells. However, by comparison of APP expression of neuronal cells derived from different brain regions significant differences in APP biogenesis were observed.

Future experiments will show whether these differences reflect the different susceptibility of neurons of those brain regions that are vulnerable to pathological events in AD in viva.

Acknowledgements. GWR is supported by the Fulbright commission, KB by the DFG (SFB’s 258 and 317). Bm. Metropolitan Life Foundation, and CLM by NHC and MRC of Australia and the Victorian Health Promotion Foundation.

320 THE AMYLOID PROTEIN PRECURSOR OF AJZHEIMER’S DISEASE BINDS TO CHICK BRAIN EXTRACELLULAR MATRIX AND IS RELEASED BY AN ACETYLCHOLINESTERASE- ASSOCIATED PROTEASE, +D.H. Small, ‘V. Nurcombe, +R. Moir, +S. Michaelson, #D. Monard, “K. Beyreuther & ‘CL. Masters. Depts. of ‘Pathology and ‘Anatomy, University of Melbourne, Parkville, Victoria, Australia, *Friedrich Meischer Institut, Base& Switzerland and OCentre for Molecular Biology, University of Heidelberg, Heidelberg, Germany. Previous studies have suggested that the amyloid protein precursor (APP) of Alzheimer’s disease may be associated with protein components of the extracellular matrix (ECM). In the present study, we examined the ability of APP to bind to ECM produced by dissociated cultures of embryonic chick brain cells. APP was found to bind saturably to the chick ECM with an equilibrium dissociation constant (K,,) of 60 nM. The binding of APP to ECM was not inhibited by either 10 &ml heparin or heparan sulfate. Pretreatment of cells with the proteoglycan biosynthesis inhibitor 4. methylumbelliferyl-!3-D-xyloside (10 PM), resulted in a 20% loss of APP binding sites on the ECM. This suggested that high affinity binding sites may be associated with the core protein of a heparan sulfate proteoglycan, as well as to other protein components of the matrix. Acidic fibroblast growth factor (aFGF) also bound to ECM. When ECM was incubated with a protease previously demonstrated to be associated with the enzyme acetylcholinesterase, APP and aFGF were released intact from the matrix. The acetylcholinesterase-associated protease was at least 100-fold more potent in releasing APP from ECM than other trypsin-like proteases such as trypsin, plasmin, thrombin. The action of the AChE-AP was inhibited by glia-derived nexin (Protease nexin I or PN

I) and by human brain APP at low nanomolar concentrations. These results suggest that in an AChE-AP may cleave ECM proteins to regulate the availability of soluble APP or other factors bound to the ECM.

321 EXTRACTION "FAMILIAL"

AND CHARACTERIZATION OF AMYLOID FROM JAPANESE AMYLOID ANGIOPATHY PATIENT BRAIN

WITH ANTI-CYSTATIN C MONOCLONAL ANTIBODY A. Nagai, S.Kobayashi, K.Shimode, S. Fujihara, and T.Tsunematsu. The Third division of Internal Medicine, Shimane Medical University, 693 Izumo, Japan.

In hereditary cerebral hemorrhage with amyloidosis (HCHWA) in Iceland, cerebral hemorrhage is caused by deposition of amyloid fibrils in cerebral blood vessels. These are composed of a variant cystatin C, cysteine proteinase inhibitor. We characterized cystatin C deposited in a Japanese HCHWA case(HCHWA-J) using anti-cystatin C monoclonal antibody. This monoclonal antibody was produced by fusing antigen immunized BALB/c mice spleen with myeloma cells. The antigen was a crude extraction from the frozen brain of a HCHWA-J Datient in which cvstatin C deoosition on the cerebral blood vessels was confirmed by immunostaining. With the enzyme-linked immunosorbent assay(ELISA), we screened the hybridomas for production of antibodv against normal cystatin C(Protogen AG, Switzerland). The monoclonal antibody produced by the hybridoma (NAl) was of the IgM class. NAl cells were cloned, cultured and injected into the peritoneal cavity of pristane- primed BALB/c mice. We separated the IgM fraction of mouse ascites by gel filtration on Sephacryl S-200 (Pharmacia) column equilibrated in phosphated buffer saline(pH 7.4). This antibody reacted only with cystatin C by inhibition test with ELISA and by Western blotting, and did not react with other kinds of amyloid proteins including B-protein.

The monoclonal antibody was coupled to Sepharose 4B(Pharmacia) and incubated with crude amyloid fibrils of HCHWA-J. The bound fraction was eluted with O.lM glycine-HCl pH2.8, dialyzed against 1OmM Na phosphate and injected into rHPLC system. This fraction gave a single peak at about I minutes, and it was shown to have a M.W. of 14000 on the Western blotting using the NAl monoclonal antibody. The normal cystatin C(Protogen AG) elutes only at 18 minutes on rHPLC, although it has similar M.W. These results suggest that the amino acid sequence of HCHWA-J amyloid may be different from that of normal cystatin C.

322 CHARACTERIZATION OF FIBRONECTIN BINDING TO ALZHEIMER’S BETA AMYl,OID PRECURSOR PROTEINS, R. Kisilevsky. S. Narindrasorasak, R.A. Altman. M.B. Fairbanks. P GonTalez-DPWhitt and B. Crrrnbrrg. Depts of Pathology end Riorhemistry, Queen’s liniversity, KingsIon, Ontario, Canada and the Upjohn Co., K;llamozoo, Michigan, (J.S.A. Several extracelllllar matrix (ECM) proteins and the Alehrimer amyloid precursor protein:. (APP) are found as components of orurit ic pl.aque and cerebrnvascul;~t omyloid. We have previously shown high affinity internctious brt.wecn APP-695, -751 and 770 .~nd heptiran -.r!lT.itc, protec~glycan (HSPC) or laminin as the I j gand::. Wirh HSPG this lnterarl ion is primarily through protein:pro!.pin binding, is inhibited by heparin and drxtran-

SO4 1 but not chondroirin SO!+ or dery+tan S04. With laminin the binding is markedly activated by 7.n and dlthiothreitol but is no+ affected by glycosaminoglycans. There is also a specific order of addil ion of HSPG and laminin lo the APPs. Fibronectin (PN) has also been identified in neuritir plaques, and in manipulatable models of amyloid deposit.ion is co- deposited with the amyloidogrnic protein. Quantitative analyses have therefore been carried out on binding data between PN and APP-695, -751, and -770. These analyses suggested that, for FN, !hrrr is but one c_lgss of binding sites on each of the APP’s with J. Kd of I x 10 M. The Bmax was of the order of O.OZng/ng APP o~+~ppro~jmatpl~+ O.OO$+mol/mol APP. No significant effect of Mg , Kn , Ca ) %n or the, p”nt a-pvpt idp GRGDS was observed. Dextran sulfntr, but not chondroitin-6-SO4 nor hrperin inhibited FN binding to the APPs. Dexlran sulfate increased the Kd E-10 fold. Dithiothreitol activated FN binding to APP decreasing the Kd 3-4 fold but N-ethyl maleimide had no