Characterization of four new monoclonal antibodies against the distal N-terminal region of PrP^c

Abstract

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein

“…Intact N-terminal fragments resulting from these cleavage events were not detectable ( SI Appendix , Fig. S6 C ), perhaps due to disruption of the EB8 antibody epitope (residues 26 to 34 ( 41 )) by additional processing even closer to the N terminus. As for the other fragments produced by recDPP4, we identified a cleavage site after Gly91, which is slightly C-terminal of the OR domain ( Fig.…”

Characterization of prion protein function by focal neurite stimulation

“…To address this question, PrP C was probed with three different monoclonal antibodies (mAbs) to test their ability to bind distinct epitopes situated in the distal region of both the PrP C N-and C-terminus. The localization of PrP C was investigated by immunostaining with W226 (Petsch et al, 2011;binding epitope: 145-155), SAF34 (Perrier et al, 2004;binding epitope: 59-89) and EB8 (Didonna et al, 2015;binding epitope: 26-34). All three mAbs recognized the PrP C in wt mouse hippocampal neuronal culture, showing similar staining patterns (Fig.…”

“…W226 (Petsch et al, 2011) binds to C-terminal domain of PrP C (epitope: 145-155), whereas SAF34 (Perrier et al, 2004) and EB8 (Didonna et al, 2015;Snajder et al, 2012) bind to N-terminal domain of PrP C (epitope: 59-89 and 26-34, respectively). Anti-NCAM antibodies (AB5032 from Millipore) were used in local delivery and immunocytochemical experiments.…”

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils

“…Membranes were blocked in 5% non-fat milk in TBST (Tris 200 mM, NaCl 1.5 mM, 1% Tween-20, Sigma-Aldrich) for 1 h at RT. The following primary antibodies were incubated overnight at 4 °C: anti-PrP W226 1:1000 [ 41 ] and anti-PrP EB8 1:1000 [ 43 ]. After three washes in TBST, membranes were incubated with goat anti-mouse IgG conjugated with horse-radish peroxidase for 1 h at RT and developed using Immobilon Classico Western HRP substrate (Millipore).…”