Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1

Baral, Pravas Kumar; Wieland, Barbara; Swayampakula, Mridula; Polymenidou, Magdalini; Rahman, Muhammad Hafiz; Kav, Nat N. V.; Aguzzi, Adriano; James, Michael N.G.

doi:10.1107/s0907444912037328

Cited by 27 publications

(28 citation statements)

References 39 publications

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“…The asterisk to the right indicates the location of a cross-reactive proteinase K band. (C) rPrP-res was PK-digested and assayed by immunoblot with three anti-PrP antibodies: 8B4 (residues 37–44), POM1 (multiple residues between140–212, see [38]), and R20 (residues 218–231). Unless otherwise indicated, all samples were loaded without dilution.…”

Section: Resultsmentioning

confidence: 99%

“…This suggested that either some of the 16 kDa fragment was highly PK-resistant or that digestion with PK was removing the 6D11 antibody epitope at residues 93–109. To determine if other protease-resistant forms of abnormal rPrP were present in the product, immunoblots were also developed with the anti-PrP antibodies 8B4 (N-terminal residues 37–44), POM1 (multiple residues between 140–212, see [38]), and R20 (C-terminal residues 218–231). The 16 kDa rPrP-res fragment reacted with both POM1 and R20 but not with 8B4 (Figure 1C), indicating that it was truncated at the N-terminus and contained PrP sequence from approximately 93–230.…”

Section: Resultsmentioning

confidence: 99%

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Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

et al. 2013

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During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

et al. 2013

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“…PrP C exposures Recombinant mouse PrP C (amino acids 23-230) was expressed in BL21 Escherichia coli (Stratagene) and purified as previously described. 31 The PrP C gene contained a His 6 affinity tag at the C-terminus that was removed by a thrombin cleavage site after purification on a Ni-NTA agarose column (Qiagen). After re-folding, PrP C was dialyzed against dH 2 O and concentrated using Amicon Ultra centrifugal filters (3kDa, Millipore).…”

Section: Discussionmentioning

confidence: 99%

Can plants serve as a vector for prions causing chronic wasting disease?

Rasmussen

Gilroyed

Reuter

et al. 2014

Prion

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“…The monoclonal antibody ICSM18 recognizes huPrP(119–231) [63], VRQ14 Fab binds to ovine PrP C and to PrP Sc , [64] POM1 Fab binds PrP C from many species[65, 66], and nanobody Nb484 binds to full length human prion protein[67]. These antibodies recognize different epitopes, residues 143–156 for ICSM18, residues 140–147 and 204–212 for POM1, and 188–199 for VRQ14.…”

Section: Introductionmentioning

confidence: 99%

Conformational selection in amyloid-based immunotherapy: Survey of crystal structures of antibody-amyloid complexes

Zhao

Nussinov

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The dominant feature in neurodegenerative diseases is protein aggregations that lead to neuronal loss. Immunotherapies using antibodies or antibody fragments to target the aggregations are a highly perused approach. The molecular mechanisms underlying the amyloid-based immunotherapy are complex. Deciphering the properties of amyloidogenic proteins responsible for these diseases is essential to obtain insights into antibody recognition of the amyloid antigens. Scope of Review We systematically explore all available crystal structures of antibody-amyloid complexes related to neurodegenerative diseases, including antibodies that recognize the Aβ peptide, tau protein, prion protein, alpha-synuclein, huntingtin protein (mHTT), and polyglutamine. Major Conclusions We found that antibodies mostly use the conformational selection mechanism to recognize the highly flexible amyloid antigens. In particular, solanezumab bound to Aβ12–28 tripeptide motif conformation (F19F20A21), which is shared with the Aβ42 fibril. This motif, which is trapped by the antibody, may provide the missing link in amyloid formation. Water molecules often bridge between the antibody and amyloid, contributing to the recognition. General Significance This paper provides the structural basis for antibody recognition of amyloidogenic proteins. The analysis and discussion of known structures are expected to help in the design and optimization of antibodies in neurodegenerative diseases.

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Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1

Cited by 27 publications

References 39 publications

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

Can plants serve as a vector for prions causing chronic wasting disease?

Conformational selection in amyloid-based immunotherapy: Survey of crystal structures of antibody-amyloid complexes

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