The Alzheimer A beta peptide develops protease resistance in association with its polymerization into fibrils.

An intriguing property of the polypeptide constituents of amyloid is that they apparently can escape the proteolytic mechanisms that normally catalyze turnover and prevent abnormal tissue accumulation of polypeptides. Here, we demonstrate that the A beta peptide, the principal component of cerebrovascular amyloid deposits in Alzheimer's disease, becomes resistant to an array of proteases as a result of structural changes associated with its polymerization into amyloid fibrils. It is further demonstrated that fibril formation per se does not lead to protease resistance but probably structural changes associated with polymerization. The results suggest that higher order structural changes, regulated by the primary structure, enable amyloidogenic polypeptides to escape proteolytic degradation and accumulate in tissues.

Alzheimer's disease is associated with parenchymal and cerebrovascular amyloidosis (4). The principal component of the amyloid, the AP peptide (5)(6)(7), is generated through proteolytic processing of the Alzheimer AP amyloid precursor protein (APP),I a large multidomain protein composed of a short cytoplasmic domain, a single transmembrane domain, and a long intraluminal or extracellular domain. The Ab peptide corresponds to parts of the transmembrane and intraluminal or extracellular domains (8)(9)(10)(11)(12)(13)(14). APP can be degraded by at least two separate pathways. One leads to cleavage of the APP molecule within the AP domain (15) and precludes amyloidogenesis, whereas the other generates intact AP (16)(17)(18)(19). The principal structural variants of the AP peptide contain 39-43 amino acid residues (5-7, 20, 21). Incubation of synthetic AP peptide in physiological solution leads to formation of fibril-like structures similar to those seen in amyloid formed in uiuo. In addition to polymerization, fibril formation involves conformational changes such as adoption of &sheet structures (22)(23)(24). Polymerization and fibril formation involve a multitude of structural changes. We will therefore refer to all structural alterations associated with polymerization and fibril formation as "higher order structural changes." I n vitro, the AP peptide has been found to be sensitive to proteolytic enzymes (7,20,21). How it is able to escape the proteolytic mechanisms that catalyze degradation and turnover of proteins in the body and form deposits in the brain is still largely unknown. Here, we have studied if higher order structures may be important in regulating the susceptibility of AP to proteolysis.

EXPERIMENTAL. PROCEDURES
MateriaZs-SyntheticAP'40 was obtained from Dr. David Teplow, the Biopolymer Laboratory at Harvard University. All other AP-derived peptides were obtained from Bachem (Bubendorf, Switzerland). Trypsin was obtained from Worthington. All other enzymes were from Boehringer Mannheim (Bromma, Sweden).
Polymerization, Enzymatic Digestion, and Chromatographic Separation of AP Peptide-Synthetic was dissolved in 1,1,1,3,3,3hexafluoro-2-propanol and aliquoted in test tubes. The solvent was then removed by lyophilization. After addition of Tris-buffered saline (TBS), pH 7.4, containing 0.02% NaN,, the peptides were allowed to polymerize by incubation in a shaking water bath at 37 "C for 48 h. The final concentration of all polypeptides used was 200 p~. Following polymerization, proteases (50 pg/ml, unless otherwise indicated) were added and the peptides digested for 5 h at 37 "C in a total volume of 100 1. 11. In separate controls, trypsin was added directly, i.e. without prior polymerization. As an additional control, polymerized AD was depolymerized using 90% formic acid for 30 min. The acid was then removed by lyophilization before addition of trypsin. The enzymatic reaction was stopped by freezing. After lyophilization, the samples were dissolved in 90% formic acid, diluted 10 times with 0.1% trifluoroacetic acid in water, and separated on a Vydac C-4 RPLC column (0.21 x 15 cm) using a solvent system containing 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid, 100% acetonitrile (buffer B). Elution was monitored by measuring absorbance at 214 nm. The percentage of intact peptide remaining after cleavage was calculated according to the following equation: 100 x (amount of peptide after cleavage/amount of peptide before cleavage).
N-terminal Sequencing-The N-terminal sequence was determined by adsorptive biphasic column technology using an HP G1005A protein sequenator (Hewlett-Packard protein chemistry system, Palo Alto, CA). Electron Microscopy-Synthetic polypeptides were prepared for electron microscopy by placing 5 pl of the polymerized and non-polymerized peptide solutions on grids covered by a carbon-stabilized Formvar film and then adding 5 pl of freshly prepared 1.5% uranyl acetate in water. After 2-3 min, excess fluid was removed with a filter paper and the grids were air-dried. The negatively stained specimens prepared in this way were examined and photographed in a JEOL EM lOOCX a t 60 kV.

RESULTS
Prolonged Incubation of Ab1"* in TBS Leads to Increased Resistance against Thyptic Degradation-The principal Ab variant, contains 40 amino acid residues and can be sepa- pg/ml) for 5 h led t o degradation of more than 90% of the peptide (Fig. lB). In contrast, that had been incubated for 48 h in TBS prior to addition of trypsin displayed a high degree of resistance against the enzyme, and the bulk of the molecules remained intact (Fig. 1C). Formic acid treatment of that had been incubated in TBS for 48 h forced the peptide back to a trypsin-sensitive state (Fig. lD). Interestingly, previous findings indicated that formic acid is capable of dissociating Ab fibrils from Alzheimer brain (6). Two tryptic fragments eluting after -24.5 and -28 min are visible in Fig.   1, B-D. These were identified as Ab17- 28 and ApZMo by N-terminal sequencing. The sequence data show that Ap140 was cleaved after Lyslfi and Lys2*, which is in agreement with the predicted substrate specificity of trypsin (see legend to Fig. 4). Fig. 1E shows a representative experiment demonstrating that the trypsin resistance of AB developed in a time-dependent manner, increasing rapidly during the first 48 h of incubation and then marginally between 48 and 96 h. The findings that the trypsin-resistant state was (i) induced spontaneously during incubation without addition of enzymes or co-factors and (ii) reversed by treatment with formic acid indicate that it did not involve covalent modifications but reversible higher order structural changes of the peptide.
An important question was to investigate if the conditions used (e.g. substrate:enzyme ratio and incubation time) allowed the cleavage reaction to proceed to completion. Near maximal degradation of both incubated and control was obtained with 0.5 pg/ml trypsin (substrate:enzyme ratio, -96001, an enzyme concentration 100 times lower than that used in the earlier experiments (Fig. 2). Incubation of the substrate with trypsin for 16 h yielded essentially identical results as when incubated for 5 h (data not shown). A reasonable conclusion from these experiments is that the conditions used indeed allowed the reaction to proceed to completion. Moreover, since trypsin concentrations above 0.5 pg/ml and prolonged incubation time did not add to the degradation, only a limited number of were probably accessible to the enzyme. These nonresistant molecules may correspond to a population of API4', which had failed to adopt a trypsin-resistant structure.
Ultrastructure of Ab1"* and Ab-derived Peptides Incubated in TBS-Synthetic incubated in a buffer of physiological pH and ionic strength polymerizes into fibrillar structures with a morphology closely resembling that of amyloid fibrils. Electron microscopic examination revealed that the fibrils formed byAp140 during incubation in TBS for 48 h were typically a few rated by RPLC (Fig. M). Treatment of ApI4O with trypsin (50 hundred nm in length with a tendency to adhere both side to Bar, 100 nm. E, the indicated synthetic peptides, with the hydrophobic amino acid residues of the transmembrane domain within a box, were exposed to trypsin directly (not incubated) or after incubation in TBS for 48 h (incubated). The percentage of intact peptide remaining after tryptic cleavage was determined using RPLC. Data are expressed as mean values of the indicated number of experiments.
side and end to end, giving rise to loosely arranged aggregates of moderate size (Fig. 3A). A shorter peptide, AP"", corresponding to only the hydrophilic, intraluminal, or extracellular part of AP, polymerized and produced fibrils of a similar character, aggregating into thin bundles, often of considerable length (Fig. 3B ), An even shorter peptide, AP'2-2x, yielded large polymers of a different character. Even if occasional thin fibrils could be detected in this preparation, platelike structures (about 50-200 nm in width and up to 1 V M or more in length) with a diffuse fibrillar substructure predominated (Fig.  3 0 . Another short peptide, AP2' -' .' , covering a hydrophobic transmembrane part ofAP and a few amino acids in the intraluminal or extracellular part, likewise polymerized in TBS forming platelike structures of defined width and length (Fig. 3 0 ) . In controls in which AP'"' and the other peptides described above were prepared for electron microscopy immediately after being dissolved in TBS, no or only occasional short ( d o 0 nm) fibrillar structures could be detected (not shown). In conclusion, all polypeptides studied had the capacity to polymerize, although the polymers formed displayed different morphologies. 'IFypsin Sensitivity of Polymerized a n d Non-polymerized APderived Peptides-The same peptides that had been allowed to polymerize in TBS for 48 h and then examined by electron microscopy had at least one tryptic cleavage site and were subjected to tryptic cleavage. In contrast to AP'"", AP"'* that had polymerized into fibrils was only slightly more resistant to trypsin than the corresponding non-polymerized peptide (Fig.  3E). This indicates that fibril formation per se is insufficient to induce resistance. In contrast to AP' "' and API-", the two shorter peptides, AP12-2R and AP' "-' ' , did not form typical fibrils when incubated under the conditions used here. Nevertheless, the two latter peptides both developed trypsin resistance similar to that of AP'"". The AP' "35 molecule, which contains 7 amino acid residues from the transmembrane region ofAPP (9), was in fact quite resistant even without prior incubation in TBS. This peptide almost exclusively contains amino acids from the hydrophobic transmembrane domain of the APP molecule, suggesting that hydrophobic interactions possibly are important in protecting the single tryptic cleavage site present in this region of the APP molecule. The fact that AP'""' and AP2"-"5 did not form typical fibrils but still developed protease resistance is further evidence that fibril formation per se is not the actual cause of protease resistance. Instead, it is possible that conformational alterations closely associated with polymerization lead to protease resistance.
Polymerization of Aptno into Fibrils Leads to Increased Resistance against a Wide Array of Proteases-It was also investigated whether polymerized AP becomes insensitive to proteolytic enzymes other than trypsin. A number of mammalian and non-mammalian exo-and endopeptidases with varying substrate specificities (25) indicated in the legend to Fig. 4 were tested for their ability to degrade polymerized and control A/314n. Incubation in TBS and polymerization were associated with increased resistance to all enzymes tested (Fig.  4). Since a large number of potential cleavage sites is present within the peptide, structural changes throughout its entire length may be implicated in the development of protease resistance. The finding that the bacterial enzyme proteinase K was able to degrade the bulk of polymerized AP' "' strongly argues against Non-amyloidogenic products . If the peptide polymerizes and succeeds in developing protease resistance, it evades degradation (to the right in the figure). Polymerized peptides can serve as nucleation seeds driving newly formed monomeric AP into the same resistant structure. Eventually, the protease-resistant AP polymers accumulate in neuronal and vascular tissue forming amyloid deposits. that resistance was caused by formation of peptide aggregates too dense for the proteolytic enzymes to penetrate. DISCUSSION In summary, a novel and possibly pathogenic property of the AP peptide has been demonstrated here. The AP peptide can develop protease resistance, a postulated prerequisite for the final tissue deposition of amyloid, through higher order structural changes. Irrespective of the morphological appearances of the polymers formed (Le. fibrils or platelike structures), it is likely that conformational changes closely associated with polymerization determine whether the peptide will acquire protease resistance or not. Apparently, these conformational changes make putative cleavage sites inaccessible to the proteases. The conclusion that polymerization is associated with resistance to proteases is in part supported by the findings of Bush et al. (26), who showed that incubation of the AP peptide in the presence of ZnC1, enhances the formation of AP oligomers and polymers and increases resistance to tryptic cleavage at the a-secretase site.
Reportedly, polymerization of AP also affects its biological properties, making it toxic to neuronal cells (27). It is therefore possible that induced protease resistance is involved in this phenomenon, perhaps by allowing excessive intracellular accumulation of non-digestible Ap peptide endocytosed from the culture medium.
Lansbury and co-workers (28) have shown that polymers of the AP peptide can serve as seeds that increase the rate by which the monomeric peptide polymerizes into fibrils. This process has been termed nucleation-dependent polymerization and may increase the probability that an individual AP molecule secreted by a cell adopts protease resistance and thereby evades degradation. A schematic outline of the proposed fate of secreted AP peptide is given in Fig. 5. Notably, several other proteins polymerize into fibrils with a morphology similar to or indistinguishable from those formed by APlAo (29, 30). These proteins are also able to form amyloid in different tissues, apparently without being degraded. Hence, it is reasonable to assume that they become able to withstand proteolytic attacks in association with polymerization and fibril formation, perhaps through structural changes similar to those of AP. We therefore propose that the general mechanism described here, polymerization-associated protease resistance, may be extended to other proteins capable of forming amyloid in vivo.