A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme

Abstract

experiments in Fig. 5, the cultured microglia (see Supplementary Figure) that had been preincubated with or without ATP (50 mM) were injected intrathecally in normal rats (see Supplementary Methods for full details). ImmunohistochemistryTransverse L5 spinal cord sections (30 mm) were cut and processed for immunohistochemistry with anti-P2X4R antibody (Alomone). Identification of the type of P2X 4 R-positive cells was performed with the following markers: for microglia, OX42 (Chemicon) and iba1 (a gift from S. Kohsaka); for astrocytes, GFAP (Boehringer Mannheim); for spinal cord neurons, NeuN (Chemicon) and MAP2 (Chemicon). To assess immunofluorescence staining of cells quantitatively, we measured the immunofluorescence intensity of the P2X 4 R or OX42 as the average pixel intensity within each cell (see also Supplementary Methods). Western blottingWestern blot analysis of P2X 4 R expression in the membrane fraction from L4-L6 spinal cord was performed with anti-P2X4R polyclonal antibody (Oncogene) as described in detail in the Supplementary Methods. Microglial cultureRat primary cultured microglia were prepared in accordance with the method described previously 28 . In brief, mixed glial culture was prepared from neonatal Wistar rats and maintained for 10-16 days in DMEM medium with 10% fetal bovine serum. Immediately before experiments, microglia were collected by a gentle shake as the floating cells over the mixed glial culture. The microglia were transferred to coverslips or to Eppendorf tubes for subsequent intrathecal administration. StatisticsStatistical analyses of the results were made with Student's t-test, Student's paired t-test or the Mann-Whitney U-test. Lancet 353, 1959Lancet 353, -1964Lancet 353, (1999. 2. Woolf, C. J. & Salter, M. W. Neuronal plasticity: Increasing the gain in pain. Science 288, 1765Science 288, -1769Science 288, (2000. 3. Bo, X., Zhang, Y., Nassar, M., Burnstock, G. & Schoepfer, R. A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett. 375, 129-133 (1995). 4. Buell, G., Lewis, C., Collo, G., North, R. A. & Surprenant, A. An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J. 15, 55-62 (1996). 5. Seguela, P., Haghighi, A., Soghomonian, J. J. & Cooper, E. A novel neuronal P2X ATP receptor ion channel with widespread distribution in the brain. J. Neurosci. 16, 448-455 (1996). 6. Soto, F. et al. P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc. Natl Acad. Sci. USA 93, 3684-3688 (1996). 7. Wang, C. Z., Namba, N., Gonoi, T., Inagaki, N. & Seino, S. Cloning and pharmacological characterization of a fourth P2X receptor subtype widely expressed in brain and peripheral tissues including various endocrine tissues. Biochem. Biophys. Res. Commun. 220, 196-202 (1996 Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues 1-3 , often with serious pathological consequences. Non-neuropathic systemic...

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies

“…In order to carry out detailed studies of such processes in vitro, it is therefore necessary to increase considerably the rates at which they occur. For globular proteins, one way of achieving this objective is to employ conditions that favour the formation of at least partially unfolded states, for example low pH values [25], high temperatures [26], low to moderate concentrations of strong denaturants [27,28], or the presence of organic solvents [13,29]. Second, the structural characterisation of the various species formed during the process of fibril formation is complicated by their heterogeneity and by their transient or insoluble nature.…”

“…Antibodies could mediate their inhibitory effect by directly binding to the regions of a peptide or a protein where the conformational changes or the aggregation processes are initiated [64,74]. It has been shown, however, that they can also act through a more subtle mechanism [28]. Thus, we have recently reported that a single-domain fragment of a camelid antibody (V H H) raised against wild type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H (Fig.…”

“…Four single-point mutants (I56T, F57I, D67H and W64R) and a double mutation (F57I/T70N) of human lysozyme are associated with systemic amyloid disease [114][115][116]. The effects of the D67H mutation on the properties of the lysozyme molecule have been studied at the molecular level using a variety of techniques including pulse labelling hydrogen/deuterium exchange analysed by mass spectrometry and NMR spectroscopy [28,117]. In the electrospray mass spectra of the D67H variant pulse labelled in the absence of the antibody fragment, two peaks are observed (Panel a), whereas the spectrum of the wild type protein under similar conditions shows a single peak [117].…”

“…Tests based on the detection of protease resistant forms of PrP: These tests are based on the observation that the C-terminal region of PrP (usually denoted as PrP [27][28][29][30] ) is resistant to a treatment with the proteinase K in PrP Sc but not in PrP C . Thus, the PrP C species is first eliminated from the sample by a proteolytic treatment.…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…The fact that nanobodies exhibit a high stability is especially important for their use as molecular probes to study the process of amyloid fibril formation because denaturing conditions and/or long incubation time (up to several weeks) need generally to be used to trigger the in vitro aggregation of target proteins. Finally, their high stability allows most of nanobodies to be easily concentrated to 1e15 mg mL À1 in standard buffers [46] and to be freeze-dried without losing their functionality [37,69].…”