III. Reversed phase high pressure liquid chromatography for the identification and purification of neuropeptides

Reversed phase HPLC has wide applications in studies on neuropeptides. It provides a fast and effective technique for assessing the purity of synthetic peptides and for purifying mg amounts of synthetic peptides (examples: angiotensins II and III and analogues; neurohypophysial hormones). Due to the very small quantities of peptides which can usually be safely recovered after HPLC, the method is also useful in the isolation, purification and sequencing of peptides from biological sources (examples: urotensins I and II), and in the identification of neuropeptides in tissues when coupled with radioligand-binding displacement assays (example: [arginine⁸]vasotocin in the anterior ganglia of $\text{Aplysia}$$\text{california}$).     

Direct regulation of rat testicular steroidogenesis by neurohypophysial hormones. Divergent effects on androgen and progestin biosynthesis

The direct regulation of testis androgen and progestin biosynthesis by neurohypophysial hormones was investigated in a primary culture of rat testis cells. Treatment with arginine vasotocin (AVT; 10(-6) M) over a 10-day period inhibited the human chorionic gonadotropin (hCG)-stimulated testosterone accumulation while enhancing hCG-stimulated progesterone accumulation. Furthermore, treatment with increasing doses (10(-11) - 10(-6) M) of AVT by itself led to dose-dependent increases in the accumulation of pregnenolone (ED50: 8.0 +/- 0.2 X 10(-9) M) and progesterone (ED50: 1.6 +/- 0.3 X 10(-8) M) but not testosterone. Under blockade of pregnenolone metabolism using cyanoketone and spironolactone, AVT, like hCG, stimulated pregnenolone accumulation with an ED50 dose of 5.8 +/- 0.3 X 10(-9) M. Similar effects were observed with several related neurohypophysial hormones, but not with nine unrelated peptides. AVT, arginine vasopressin, and lysine vasopressin were about 100-fold more potent than mesotocin, valitocin, and oxytocin. Pressor (but not antidiuretic or oxytocic)-selective agonists of the neurohypophysial hormones also exerted dose-dependent stimulation of pregnenolone accumulation. Potent pressor (but not oxytocic)-selective antagonistic analogs of the neurohypophysial hormones prevented the AVT-stimulated accumulation of pregnenolone. Thus, the neurohypophysial hormones may exert a direct stimulatory effect on testis pregnenolone and progesterone biosynthesis via putative, pressor-selective recognition sites, and this progestin-stimulatory activity may be partly due to stimulation of steroidogenic steps preceding pregnenolone formation. Since the effective doses of neurohypophysial hormones in vitro are higher than the serum hormone levels, the present results suggest an intratesticular paracrine role for these peptides.     

Molecular evolution of the neurohypophysial hormones: The active peptides of a primitive bony fish Polypterus bichir

Neurohypophysial hormones have been so far identified in Neopterygii and Crossopterygii but not in species of the bird sub-class of bony fishes, the Palaeopterygii. Isolation and chemical characterization of the active principles of a primitive bony fish, Polypterus bichir, have been performed. Isotocin (Ser(4)-Ile(8)-oxytocin) and arginine vasotocin (Arg(8)-oxytocin) have been identified. Because the same peptides were found in the recent Neopterygii, it can be deduced that neurohypophysial hormones have displayed a peculiar stability in the course of the evolution of bony fishes. However isotocin and vasotocin are replaced by oxytocin and vasopressins in mammals and therefore might be regarded as "old" molecules.     

Hydrophobic Effects on Antibacterial and Channel-forming Properties of Cecropin A–Melittin Hybrids

The design of cecropin–melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile⁸ in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH₂, [CA(1–7)M(2–9)NH₂] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile⁸ reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile⁸ by a hydrophobic leucine maintained good activity and Ala⁸ was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser⁸ strongly reduced potency against all five organisms. Deletion of Leu¹⁵ decreased activity, but addition of Thr¹⁶ maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic α-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in α-helicity with an increase in hexafluoro 2-propanol concentration.     

Phylogeny of neurohypophyseal hormones in birds: microidentification of mesotocin and vasotocin in the ostrich (Struthio camelus)

Ostrich (Struthio camelus) neurohypophysial hormones have been isolated from 5 freeze-dried posterior pituitary glands. Purification has involved three steps: a first molecular sieving on Sephadex G-75 for eliminating proteins, a second molecular sieving on Bio-Gel P4 for separating the two active principles and a high pressure reverse-phase liquid chromatography (HPLC) on Nova-Pak C 18 with an 10 mM acetate-acetonitrile gradient for isolating each hormone. The active peptides have been identified by their retention time in HPLC and their amino acid composition. Mesotocin and vasotocin have thus been characterized. Although the phylogeny of Ratites is disputed, in particular their possible common origin with Carinates, which include most of the living birds, species of the first sub-class seem to have the same neurohypophysial hormones as those of the second.     

Neurohypophyseal hormones and behavior: effects of intracerebroventricularly injected hormone analogs in mice.

Neurohypophyseal hormones evoke spontaneous behavioral changes in mice. This study compares the potency of four naturally occuring neurohypophyseal hormones and of ten analogs with amino acid residue replacements selected in such a manner as to cover each residue position of the hormones with the exception of the cystine residue. Peptides were administered intraventricularly and the sum of foraging, scratching and squeaking, recorded at 30 second intervals during a 30 min session, was measured as a function of peptide dose. The most potent group of peptides is represented by the neurohypophyseal hormones as well as the five analogs [Hly⁸] vasopressin, [Δ³-Pro⁷]AVP, [Thi³]LVP, [Abu⁴]AVP and [Abu⁴]LVP. [Leu⁴]LVP showed significant activity but was far less potent than the natural hormones. None of the remaining analogs enhanced activity with an increase in peptide dose. This group included both peptides with C-terminal modifications and those in which the tyrosine (position 2) or the asparagine residue (position 5) of the hormones were substituted by alanine. The neurohypophyseal hormone-induced behavioral results of this study reveal a structure-function relationship, which is in its most important conclusions, identical to the conformation-activity model proposed for endocrine activities of neurohypophyseal peptides.     

Triple helix–coil transition of covalently bridged collagenlike peptides

The thermal triple helix–coil transition of covalently bridged collagenlike peptides with repeating sequences of (Ala-Gly-Pro)_n, n = 5–15, was studied optically. The peptides were soluble in water/acetic acid (99:1) and were found to form triple-helical structures in this solvent system beginning with n = 8. The thermodynamic analysis of the transition equilibrium curves for n = 9–13 yielded the parameters ΔH°_s = ?7.0 kJ per tripeptide unit, ΔS°_s = ?23.1 J deg^?1 mol^?1 per tripeptide unit for the coil-to-helix transition, and the apparent nucleation parameter σ ? 5 × 10^?2. It was suggested through double-jump temperature experiments that the rate-limiting step during refolding is not only influenced by the difficulties of nucleation, but also by cis–trans isomerization of the Gly-Pro peptide bond.     

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (π_e) were clearly lower than the biological membrane pressure (24–30 mN m^− 1) and the HIV-1 FP (35 mN m^− 1). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G^E). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive G^E values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP–lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Structure and function relationships of three novel hGHRH-GGC analogs

Growth hormone releasing hormone (GHRH) is one of the hypothalamus hormones. For its potential applications in agriculture and medicine, GHRH analog with higher activity and longer half-life has been looked for. By using the fusion expression with unique acid labile linker Asp-Pro and biochemical purification, the three novel GHRH peptides, Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys, Pro-hGHRH(1–44)-Gly-Gly-Cys, and ¹Pro-GHRH(2–44)-Gly-Gly-Cys, were obtained. The peptide molecular weight with 5,455, 5,373 or 5,210 Da measured by EIS-MS is coincident with the actual values. The peptides at 0.1–10 μg/ml increased rat pituitary GH releases in a dose-dependent manner and at 5 μg/ml increased human pituitary GH releases. The activity comparisons showed that at 10 μg/ml there were significant between ¹Pro-hGHRH(2–44)-Gly-Gly-Cys and Pro-Pro-hGHRH(1–44)-Gly-Gly-Cys or Pro-hGHRH(1–44)-Gly-Gly-Cys, ¹Pro-hGHRH(2–44) (P < 0.05). The ¹Pro-hGHRH(2–44)-Gly-Gly-Cys showed the highest GH release from rat pituitary. The activity results showed that the N-terminal Pro modulations and the C-terminal Gly-Gly-Cys extension regulate GH release from pituitary. The results showed that the three peptides had good GH release, function-selectivity and species specificity.     

Neurophpophysial hormones and the emission of semen in rabbits.

To elucidate the importance of the neurohypophysial hormones for the emission of semen, several neurohypophysial peptides were tested in male rabbits and the sperm density in the ejaculates was determined. Besides oxytocin and vasopressin, vasotocin and one oxytocin analogue (de-amino1-oxytocin) were used. Only vasopressin, in a dose as low as 10 mU, increased the number of spermatozoa in the ejaculates. It is suggested that vasopressin is of physiological significance for the emission of semen, at least in rabbits.     

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β-peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ-related peptides, under acute conditions, potently inhibit K⁺-evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ-mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ_1–40 (10^?8M) potently inhibited veratridine-evoked endogenous ACh release from rat hippocampal slices and also decreased the K⁺-evoked release potentiated by the nitric oxide-generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ_1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10^?14 to 10^?6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10^?6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.     

Modified adipokinetic peptides containing two tryptophan residues and their activities in vitro and in vivo in Locusta

Locusta migratoria has three adipokinetic hormones, adipokinetic hormone-I, II and III. Adipokinetic hormone-III (50=1.33·10^-10 mol·l^-1) compared with other adipokinetic hormones (EC₅₀5.33·10^-10 mol·l^-1) at inhibiting acetate uptake into locust fat body in vitro, especially so when it is only moderately potent in mobilizing lipid in vivo. The Trp⁷ in adipokinetic hormones-III, alongside the Trp⁸ characteristic of adipokinetic hormones, is not seen in any other adipokinetic hormones. To test whether this is responsible for the high potency of adipokinetic hormone-III in the assay in vitro, novel peptides were synthesised to include or remove this structural motif. Thus, 7-Locusta-adipokinetic hormone-I or [Gly^8a-Thr^8b]-Locustra-adipokinetic hormone-III); 9-Thr¹⁰]-Locusta-adipokinetic hormone-I or Asn⁷-Locusta-adipokinetic hormone-III); 7-Locusta-adipokinetic hormone-II) and 7-Acheta-adipokinetic hormones) were tested both in vitro and in vivo. Except for Trp⁷-adipokinetic hormone-I in the acetate uptake assay, each of these analogues is less potent then its respective parent, irrespective of the assay. However, the acetate uptake response is highly tolerant of peptides containing Trp⁷-Trp⁸, whereas this motif markedly reduces potency in the lipid assay. The different responses exploited in these assays may be exerted through different receptor populations.Abbreviations AKH adipokinetic hormones - BSA bovine serum albumin - cAMP cyclic adenosine monophosphate - EC ₅₀ effective concentration giving 50% of effect - FA fatty acid(s) - HPLC high performance liquid chromatography - RPCH red pigment-concentrating hormone     

Synthesis and conformational study of peptides possessing helically arranged ΔZPhe side chains

Z-Dehydrophenylalanine (Δ^zPhe) possessing four oligopeptides, Boc-(L -Ala-Δ^zPhe-Aib)_n-OCH₃ (n = 1–4: Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by ¹H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. ¹H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L -Ala-Δ^zPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2–4 have a 4 → 1 hydrogen-bonding pattern characteristic of 3₁₀-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the Δ^zPhe chromophores. The corresponding CD spectra of the peptides n = 2–4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2–4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3₁₀-helix that contains three residues per turn, and that their side chains of Δ^z Phe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be ～ 5.5 Å. The chemical shifts of the Δ^zPhe side-chain protons (H^β and aromatic H) for the peptides n = 2–4 indicated anisotropic shielding effect of neighboring phenyl group(s); the effect also supports a regular arrangement of the Δ^z Phe side chains along the helical axis. © 1993 John Wiley & Sons, Inc.     

Specificity of anti-peptide antibodies elicited against synthetic peptides mimicking conserved regions of HIV1 envelope glycoprotein

Comparison of HIV1_Bru and HIV2_Rod external envelope glycoprotein sequences enabled us to select ten highly conserved peptide sequences. The corresponding peptides were chemically synthesized, then coupled to bovine serum albumin before injection in rabbits. Although all peptides were immunogenic, only antibodies directed against peptides P1 (amino acid residues 33–55), P22 (418–462), P8 (487–508) and P21 (487–534) were able to interact with significant affinity (K_0.5 about 10⁻⁶ and 10⁻⁸ M) with the native glycoprotein by radioimmunoassay. Noteworthy was the capacity of anti-P1 antibodies to also recognize the glycoprotein of HIV2. Anti-peptide antibodies were tested for their ability to interfere with the gp120-CD4 interaction, membrane fusion and virus replication. Preincubation of gp 120 with antibodies directed to the region previously described as the putative CD4-binding site, P22 (418–462), did not abolish gp120 binding to CD4-positive cells.     

Osmoregulatory Functions of Neurohypophysial Hormones in Fishes and Amphibians

The renal effects of neurohypophysial hormones in fishes andamphibians are discussed. Injections of arginine vasotocin (AVT)elicit diuresis in fishes, but antidiuresis in amphibians. However,the physiological significance of these hormonal responses remainsto be demonstrated. Studies with bioassays and radio-immunoassayson circulating levels of AVT indicate that hypovolemia may bea very potent stimulus for the release of the hormone. Hyperosmoticstimuli may not be as important. In anurans, mesotocin has aglomerular diuretic effect. This neurohypophysial hormone appearsto dilate, while AVT constricts, the afferent glomerular circulationin bullfrogs, Rana catesbeiana.     

Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

Perlecan Binds to the β-Amyloid Proteins (Aβ) of Alzheimer's Disease, Accelerates Aβ Fibril Formation, and Maintains Aβ Fibril Stability

Abstract: Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar β-amyloid (Aβ) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth-Holm-Swarm tumor was used to define perlecan's interactions with Aβ and its effects on Aβ fibril formation. Using a solid-phase binding immunoassay, freshly solubilized full-length Aβ peptides bound immobilized perlecan at two sites, representing both high-affinity [K_D = ～5.8 × 10^?11M for Aβ (1–40); K_D = ～6.5 × 10^?12M for Aβ (1–42)] and lower-affinity [K_D = 3.5 × 10^?8M for Aβ (1–40); K_D = 4.3 × 10^?8M for Aβ (1–42)] interactions. An increase in the binding capacity of Aβ (1–40) to perlecan correlated with an increase in Aβ amyloid fibril formation during a 1-week incubation period. The high-capacity binding of Aβ (1–40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin-4-sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of Aβ (1–40) amyloid fibril formation, causing a significant increase in Aβ fibril assembly over a 2-week incubation period at 1 h (2.8-fold increase), 1 day (3.6-fold increase), and 3 days (2.8-fold increase) in comparison with Aβ (1–40) alone. Perlecan also initially accelerated the formation of Aβ (1–42) fibrils within 1 h and maintained significantly higher levels of Aβ (1–42) thioflavin T fluorescence throughout a 2-week experimental period in comparison with Aβ (1–42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on Aβ (1–40) fibril formation and maintenance of Aβ (1–42) fibril stability occurred in a dose-dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of Aβ fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, α₁-antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of Aβ, but also accelerates Aβ fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of Aβ amyloidosis in Alzheimer's disease.