Mammalian Prion protein expression in yeast; a model for transmembrane insertion

The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the ER translocon and substrates in transit. Most inserted TM1 has its N-terminus in the ER lumen (Ntm orientation), as expected for arrest of normal secretion. However, 20% is found in inverted Ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased Ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between CtmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a C-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. However, secretion of PrP by the mammalian translocon requires the TRAP complex, absent in yeast, where essentially all PrP ends up as TM species, 85–90% Ntm and 10–15% Ctm. Although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on Ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. Electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and Ctm orientation. An increase in ΔCh from −5 to −7 caused a marked increase in Ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.     

Impaired transport of intrinsically disordered proteins through the Sec61 and SecY translocon; implications for prion diseases

The prion protein (PrP) is composed of two major domains of similar size. The structured C-terminal domain contains three alpha-helical regions and a short two-stranded beta-sheet, while the N-terminal domain is intrinsically disordered. The analysis of PrP mutants with deletions in the C-terminal globular domain provided the first hint that intrinsically disordered domains are inefficiently transported into the endoplasmic reticulum through the Sec61 translocon. Interestingly, C-terminally truncated PrP mutants have been linked to inherited prion disease in humans and are characterized by inefficient ER import and the formation of neurotoxic PrP conformers. In a recent study we found that the Sec61 translocon in eukaryotic cells as well as the SecY translocon in bacteria is inherently deficient in translocating intrinsically disordered proteins. Moreover, our results suggest that translocon-associated components in eukaryotic cells enable the Sec61 complex to transport secretory proteins with extended unstructured domains such as PrP and shadoo.     

Inter-oligomer interactions of the human prion protein are modulated by the polymorphism at codon 129

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in β-oligomer (β^O) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the β^O N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in β^O formation. Atomic force microscopy revealed distinctive 1.8 × 15 × 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for β^O with valine at position 129. This result provides evidence for conformational differences between the β^O allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.     

The residue 129 polymorphism in human prion protein does not confer susceptibility to Creutzfeldt-Jakob disease by altering the structure or global stability of PrPC

There are two common forms of prion protein (PrP) in humans, with either methionine or valine at position 129. This polymorphism is a powerful determinant of the genetic susceptibility of humans toward both sporadic and acquired forms of prion disease and restricts propagation of particular prion strains. Despite its key role, we have no information on the effect of this mutation on the structure, stability, folding, and dynamics of the cellular form of PrP (PrP(C)). Here, we show that the mutation has no measurable effect on the folding, dynamics, and stability of PrP(C). Our data indicate that the 129M/V polymorphism does not affect prion propagation through its effect on PrP(C); rather, its influence is likely to be downstream in the disease mechanism. We infer that the M/V effect is mediated through the conformation or stability of disease-related PrP (PrP(Sc)) or intermediates or on the kinetics of their formation.     

The presence of valine at residue 129 in human prion protein accelerates amyloid formation

The polymorphism at residue 129 of the human PRNP gene modulates disease susceptibility and the clinico-pathological phenotypes in human transmissible spongiform encephalopathies. The molecular mechanisms by which the effect of this polymorphism are mediated remain unclear. It has been shown that the folding, dynamics and stability of the physiological, alpha-helix-rich form of recombinant PrP are not affected by codon 129 polymorphism. Consistent with this, we have recently shown that the kinetics of amyloid formation do not differ between protein containing methionine at codon 129 and valine at codon 129 when the reaction is initiated from the alpha-monomeric PrP(C)-like state. In contrast, we have shown that the misfolding pathway leading to the formation of beta-sheet-rich, soluble oligomer was favoured by the presence of methionine, compared with valine, at position 129. In the present work, we examine the effect of this polymorphism on the kinetics of an alternative misfolding pathway, that of amyloid formation using partially folded PrP allelomorphs. We show that the valine 129 allelomorph forms amyloids with a considerably shorter lag phase than the methionine 129 allelomorph both under spontaneous conditions and when seeded with pre-formed amyloid fibres. Taken together, our studies demonstrate that the effect of the codon 129 polymorphism depends on the specific misfolding pathway and on the initial conformation of the protein. The inverse propensities of the two allelomorphs to misfold in vitro through the alternative oligomeric and amyloidogenic pathways could explain some aspects of prion diseases linked to this polymorphism such as age at onset and disease incubation time.     

Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms

Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases.     

Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C terminus

Prion protein (PrP) is synthesized at the membrane of the endoplasmic reticulum (ER) in three different topological forms as follows: a fully translocated one ((sec)PrP) and two with opposite orientations in the membrane ((Ntm)PrP and (Ctm)PrP). We asked whether other signal sequences exist in the PrP, other than the N-terminal signal sequence, that contribute to its topological diversity. In vitro translocation assays showed that PrP lacking its N-terminal signal sequence could still translocate into ER microsomes, although at reduced efficiency. Deletion of each of the two hydrophobic regions in PrP revealed that the C-terminally located hydrophobic region (TM2) can function as second signal sequence in PrP. Translocation mediated by the TM2 alone can occur post-translationally and yields mainly (Ctm)PrP, which is implicated in some forms of neurodegeneration in prion diseases. We conclude that, in vitro, PrP can insert into ER membranes co- and post-translationally and can use two different signal sequences. We propose that the unusually complex topology of PrP results from the differential utilization of two signal sequences in PrP.     

Role of N-terminal familial mutations in prion protein fibrillization and prion amyloid propagation in vitro

A self-perpetuating conformational conversion of the prion protein (PrP) is believed to underlie pathology and transmission of prion diseases. Here we explore the effects of N-terminal pathogenic mutations (P102L, P105L, A117V) and the residue 129 polymorphism on amyloid fibril formation by the human PrP fragment 23-144, an in vitro conversion model that can reproduce certain characteristics of prion replication such as strains and species barriers. We find that these amino acid substitutions neither affect PrP23-144 amyloidogenicity nor introduce barriers to cross-seeding of soluble protein. However, the polymorphism strongly influences the conformation of the amyloid fibrils, as determined by infrared spectroscopy. Intriguingly, unlike conformational features governed by the critical amyloidogenic region of PrP23-144 (residues 138-139), the structural features distinguishing Met-129 and Val-129 PrP23-144 amyloid fibrils are not transmissible by cross-seeding. While based only on in vitro data, these findings provide fundamental insight into the mechanism of prion-based conformational transmission, indicating that only conformational features controlling seeding specificity (e.g. those in critical intermolecular contact sites of amyloid fibrils) are necessarily transmissible by cross-seeding; conformational traits in other parts of the PrP molecule may not be "heritable" from the amyloid template.     

Unaltered prion protein expression in Alzheimer disease patients

The suggested role of cellular prion protein (PrP^C) in mediating the toxic effects of oligomeric amyloid β peptide (Aβ) in Alzheimer disease (AD) is controversial. To address the hypothesis that variable PrP^C expression is involved in AD pathogenesis, we analyzed PrP^C expression in the frontal and temporal cortices and hippocampus of individuals with no cognitive impairment (NCI), amnestic mild cognitive impairment (aMCI), mild AD (mAD) and AD. We found that PrP^C expression in all brain regions was not significantly altered among the various patient groups. In addition, PrP^C levels in all groups did not correlate with expression of methionine (M) or valine (V) at codon 129 of the PrP gene, a polymorphism that has been linked in some studies to increased risk for AD, and which occurs in close proximity to the proposed binding region for the oligomeric Aβ peptide. Our results indicate that, if PrP^C is involved in mediating the toxic effects of the oligomeric Aβ peptide, these effects occur independently of steady state levels of PrP or the codon 129 polymorphism.Key words: prion protein, PrP codon 129 polymorphism, Alzheimer disease, oligomeric Aβ, Alzheimer precursor protein     

Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.

To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.     

Molecular genetic and biochemical approaches for defining lipid-dependent membrane protein folding

We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long-range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually dependent on each other for functional organization of proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Dissociation of infectivity from seeding ability in prions with alternate docking mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrP(Sc), suggesting that these domains of cellular prion protein (PrP(C)) serve as docking sites for PrP(Sc) during prion propagation. To examine the role of polybasic domains in the context of full-length PrP(C), we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ～5 rounds of sPMCA, PrP(Sc) molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrP(Sc) prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrP(Sc) molecules. Remarkably, ΔC-PrP(Sc) and other polybasic domain PrP(Sc) molecules displayed diminished or absent biological infectivity relative to wild-type PrP(Sc), despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrP(Sc) prions interact with PrP(C) molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrP(Sc) propagation. Furthermore, polybasic domain deficient PrP(Sc) molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrP(Sc) molecules may not depend on a single stereotypic mechanism, but that normal PrP(C)/PrP(Sc) interaction through polybasic domains may be required to generate prion infectivity.     

Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel

The decisive events that direct a single polypeptide such as the prion protein (PrP) to be synthesized at the endoplasmic reticulum in both fully translocated and transmembrane forms are poorly understood. In this study, we demonstrate that the topological heterogeneity of PrP is determined cotranslationally, while at the translocation channel. By evaluating sequential intermediates during PrP topogenesis, we find that signal sequence-mediated initiation of translocation results in an interaction between nascent PrP and endoplasmic reticulum chaperones, committing the N terminus to the lumen. Synthesis of the transmembrane domain before completion of this step allows it to direct the generation of (Ctm)PrP, a transmembrane form with its N terminus in the cytosol. Thus, segregation of nascent PrP into different topological configurations is critically dependent on the precise timing of signal-mediated initiation of N-terminus translocation. Consequently, this step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated (Ctm)PrP caused by disease-associated mutations in the transmembrane domain. These results delineate the sequence of events involved in PrP biogenesis, explain the mechanism of action of (Ctm)PrP-favoring mutations associated with neurodegenerative disease, and more generally, reveal that translocation substrates can be cotranslationally partitioned into multiple populations at the translocon.     

The erythropoietin receptor transmembrane domain mediates complex formation with viral anemic and polycythemic gp55 proteins

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A-->P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region.     

Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.     

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.     

Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain.

We previously reported that truncation of the cytoplasmic domain of the macaque simian immunodeficiency virus SIVmac239 envelope glycoprotein enhanced its ability to induce cell fusion in a variety of cell lines. In the present study, we examined the expression of the full-length and truncated SIVmac239 envelope glycoprotein complex on cell surfaces. Using a membrane-impermeable reagent to biotinylate proteins on cell surfaces followed by immunoprecipitation, we found that under conditions in which the full-length TM protein could not be detected on the surfaces of CD4-positive or CD4-negative cell lines, the truncated TM protein was detected efficiently. In contrast, using a membrane-impermeable iodination reagent to label proteins on cell surfaces, we could detect both the full-length and truncated TM proteins. No difference between the full-length and truncated proteins was observed in the detection of the SU proteins in the biotinylation assay. Additionally, we used an assay in which SIV-specific antibodies are prebound to the native envelope proteins expressed on the cell surface and then the proteins are immunoprecipitated. Using this assay, we could not detect the truncated or full-length TM protein on the cell surface, whereas we could detect the SU subunits of both proteins. We also observed that the truncated TM protein formed more stable sodium dodecyl sulfate-resistant oligomers than the full-length TM protein did. These results indicate that truncation of the cytoplasmic domain of the SIVmac239 envelope glycoprotein affects the conformation of the external domain of the TM protein on the cell surface, even though the two proteins have no differences in the amino acid sequences of their external domains. This altered conformation could play a role in the enhanced fusion activity of the truncated SIV glycoprotein.     

The N-terminal, polybasic region is critical for prion protein neuroprotective activity

Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.     

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.