Rame of the PrP gene, VPSPr is associated with a PrPres that bears three of the characteristics of inherited rather than sporadic prion diseases. First, the diglycosylated PrPSc in VPSPr is virtually undetectable, as it is also with PrPres in fCJDV180I and fCJDT183A [3,4,7]. Second, VPSPr is characterized by the presence in the brain of more than three 25033180 PrPres fragments including a ,7 kDa fragment, a characteristic of GSS [2,7]. However, in marked contrast to PrPres in GSS, PrPres in VPSPr is preferentially detected with the 1E4 antibody BTZ-043 instead of the widely used 3F4 antibody, forming a pathognomonic five-step ladder-like PrP electrophoretic profile [7]. Finally, in some VPSPr cases, a positive family history of cognitive impairment was observed [6,7]. Clearly, the PrPSc associated with VPSPr is distinct from the prion strains associated with other sporadic prion diseases. The molecular mechanism underlying the formation of the peculiar prion in VPSPr has yet to be determined. Compared to PrP in the most common sporadic CJD (sCJD), a significant decrease in the ratio of diglycosylated PrP to monoglycosylated PrP treated with or without PK was reported in fCJDT183A previously [3]. This is because the T183A PrP mutation completely abolishes the first N-linked glycosylation site at residue 181 (N181) [9?1] and the detected diglycosylated PrP 
is derived only from wild-type PrP (3, the current study). In contrast, the PrP glycoforms in VPSPr appear typical prior to PKtreatment; however, there is no detectable diglycosylated PrPSc after PK-treatment. As with VPSPr, the molecular mechanism underlying the absence of the diglycosylated PrP in fCJDV180I is unclear [4]. Using a combination of in vivo and in vitro assays, our current study indicates that the absence of the diglycosylated PrPSc in both VPSPr and fCJDV180I results from a glycoform-selective prion formation pathway associated with the inability of the diand mono-glycosylated PrPC at N181 to convert into PrPSc in the brain.Figure 1. Detection of PK-treated and untreated PrP with 3F4. (A) Brain homogenates from three fCJDV180I (one 129MM and two 129MV, lanes 2?) and three VPSPr-129MM cases (lanes 5?) were treated with PK at 10 mg/ml prior to Western blotting with 3F4. A sCJDMM2 case was used as a control (lane 1). (B) PrP in brain homogenates without PK-treatment from fCJDT183A, fCJDV180I, VPSPr, sCJD and non-CJD was examined by Western blotting. doi:10.1371/journal.pone.0058786.gResults Both inherited CJDV180I and sporadic VPSPr exhibit no diglycosylated PrPresIn contrast to sCJD, both fCJDV180I and VPSPr exhibit monoand un-glycosylated PK-resistant PrP bands but virtually no diglycosylated PrP when probed with the 3F4 antibody (Fig. 1A). However, in the samples that were not treated with PK (Fig. 1B), diglycosylated PrP was readily detectable not only in sCJD and non-CJD but also in fCJDV180I and VPSPr. The fCJDT183A exhibited a very faint diglycosylated PrP band that 16574785 was visible in over-exposed blots and is from the wild-type allele as reported previously [3].Lack of diglycosylated PrPres is attributable to loss of glycosylation at the first N-linked glycosylation site in fCJDV180I and Gracillin site VPSPrTo investigate whether and how the two individual N181 and N197 sites are associated with the lack of the diglycosylated PrPres in fCJDV180I and VPSPr, we probed PrP treated with PK or PK plus PNGase F using V14 and Bar209 antibodies that have been demonstrated to distinguish mono181 and 
mono197 b.Rame of the PrP gene, VPSPr is associated with a PrPres that bears three of the characteristics of inherited rather than sporadic prion diseases. First, the diglycosylated PrPSc in VPSPr is virtually undetectable, as it is also with PrPres in fCJDV180I and fCJDT183A [3,4,7]. Second, VPSPr is characterized by the presence in the brain of more than three 25033180 PrPres fragments including a ,7 kDa fragment, a characteristic of GSS [2,7]. However, in marked contrast to PrPres in GSS, PrPres in VPSPr is preferentially detected with the 1E4 antibody instead of the widely used 3F4 antibody, forming a pathognomonic five-step ladder-like PrP electrophoretic profile [7]. Finally, in some VPSPr cases, a positive family history of cognitive impairment was observed [6,7]. Clearly, the PrPSc associated with VPSPr is distinct from the prion strains associated with other sporadic prion diseases. The molecular mechanism underlying the formation of the peculiar prion in VPSPr has yet to be determined. Compared to PrP in the most common sporadic CJD (sCJD), a significant decrease in the ratio of diglycosylated PrP to monoglycosylated PrP treated with or without PK was reported in fCJDT183A previously [3]. This is because the T183A PrP mutation completely abolishes the first N-linked glycosylation site at residue 181 (N181) [9?1] and the detected diglycosylated PrP is derived only from wild-type PrP (3, the current study). In contrast, the PrP glycoforms in VPSPr appear typical prior to PKtreatment; however, there is no detectable diglycosylated PrPSc after PK-treatment. As with VPSPr, the molecular mechanism underlying the absence of the diglycosylated PrP in fCJDV180I is unclear [4]. Using a combination of in vivo and in vitro assays, our current study indicates that the absence of the diglycosylated PrPSc in both VPSPr and fCJDV180I results from a glycoform-selective prion formation pathway associated with the inability of the diand mono-glycosylated PrPC at N181 to convert into PrPSc in the brain.Figure 1. Detection of PK-treated and untreated PrP with 3F4. (A) Brain homogenates from three fCJDV180I (one 129MM and two 129MV, lanes 2?) and three VPSPr-129MM cases (lanes 5?) were treated with PK at 10 mg/ml prior to Western blotting with 3F4. A sCJDMM2 case was used as a control (lane 1). (B) PrP in brain homogenates without PK-treatment from fCJDT183A, fCJDV180I, VPSPr, sCJD and non-CJD was examined by Western blotting. doi:10.1371/journal.pone.0058786.gResults Both inherited CJDV180I and sporadic VPSPr exhibit no diglycosylated PrPresIn contrast to sCJD, both fCJDV180I and VPSPr exhibit monoand un-glycosylated PK-resistant PrP bands but virtually no diglycosylated PrP when probed with the 3F4 antibody (Fig. 1A). However, in the samples that were not treated with PK (Fig. 1B), diglycosylated PrP was readily detectable not only in sCJD and non-CJD but also in fCJDV180I and VPSPr. The fCJDT183A exhibited a very faint diglycosylated PrP band that 16574785 was visible in over-exposed blots and is from the wild-type allele as reported previously [3].Lack of diglycosylated PrPres is attributable to loss of glycosylation at the first N-linked glycosylation site in fCJDV180I and VPSPrTo investigate whether and how the two individual N181 and N197 sites are associated with the lack of the diglycosylated PrPres in fCJDV180I and VPSPr, we probed PrP treated with PK or PK plus PNGase F using V14 and Bar209 antibodies that have been demonstrated to distinguish mono181 and mono197 b.