Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Aug:142:104955.
doi: 10.1016/j.nbd.2020.104955. Epub 2020 May 24.

Prion protein post-translational modifications modulate heparan sulfate binding and limit aggregate size in prion disease

Julia A Callender  1 , Alejandro M Sevillano  1 , Katrin Soldau  1 , Timothy D Kurt  1 , Taylor Schumann  1 , Donald P Pizzo  1 , Hermann Altmeppen  2 , Markus Glatzel  2 , Jeffrey D Esko  3 , Christina J Sigurdson  4
Affiliations

Prion protein post-translational modifications modulate heparan sulfate binding and limit aggregate size in prion disease

Julia A Callender et al. Neurobiol Dis. 2020 Aug.

Abstract

Many aggregation-prone proteins linked to neurodegenerative disease are post-translationally modified during their biogenesis. In vivo pathogenesis studies have suggested that the presence of post-translational modifications can shift the aggregate assembly pathway and profoundly alter the disease phenotype. In prion disease, the N-linked glycans and GPI-anchor on the prion protein (PrP) impair fibril assembly. However, the relevance of the two glycans to aggregate structure and disease progression remains unclear. Here we show that prion-infected knockin mice expressing an additional PrP glycan (tri-glycosylated PrP) develop new plaque-like deposits on neuronal cell membranes, along the subarachnoid space, and periventricularly, suggestive of high prion mobility and transit through the interstitial fluid. These plaque-like deposits were largely non-congophilic and composed of full length, uncleaved PrP, indicating retention of the glycophosphatidylinositol (GPI) anchor. Prion aggregates sedimented in low density fractions following ultracentrifugation, consistent with oligomers, and bound low levels of heparan sulfate (HS) similar to other predominantly GPI-anchored prions. Collectively, these results suggest that highly glycosylated PrP primarily converts as a GPI-anchored glycoform, with low involvement of HS co-factors, limiting PrP assembly mainly to oligomers. Since PrPC is highly glycosylated, these findings may explain the high frequency of diffuse, synaptic, and plaque-like deposits in the brain as well as the rapid conversion commonly observed in human and animal prion disease.

Keywords: ADAM10 cleavage; Amyloid; Glycans; Glycosaminoglycans; Glycosylation; Neurodegeneration; Prion strains; Protein misfolding.

PubMed Disclaimer

Conflict of interest statement

Declaration of Competing Interest

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. The T187N mutation introduces a third N-linked glycan to the globular C-terminal domain of PrPC.
A. The PrPC primary structure shows the positively charged segments (light blue), Cu2+-binding octapeptide repeats (dark grey), beta sheets (dark blue), alpha helices (purple), disulfide bonds (dotted line), and GPI-anchor signal sequence (yellow). The glycans are linked to PrP residues 180N and 196N, and to the mutated 187N residue. B. Three-dimensional structure of the mature PrPC protein shown in ribbon form (left) or as a charged surface (right) (PDB ID code 1AG2). N-linked glycans attached to N180, N196, and N187 are depicted in magenta, green, and orange, respectively (note: tetra-antennary, terminally-sialylated glycans are shown [30, 50]). C. Western blots (left) show total and ADAM10 cleaved PrPC protein levels in whole brain lysates from adult WT or Prnp187N mice. Graphs indicate relative percentages of each glycoform from total PrP: un- (green), mono- (purple), di- (grey), and tri-glycosylated (white) (mean ± SEM). Also quantified is ADAM10-cleaved PrP. N = 3 – 6 mice per genotype.
Figure 2.
Figure 2.. Prnp187N mice initially show a prolonged survival time and plaque-like prion deposits in the brain for select strains.
A. Survival curves reveal a delayed time to the onset of terminal disease on the first passage for all strains, which decreased on subsequent passages. B. HE and PrP immunostains revealed similar spongiform degeneration and diffuse PrPSc deposits in the RML and 22L-infected WT and Prnp187N mice (arrowheads). Plaque-like deposits (arrowheads) were significantly larger in the ME7- and mNS-infected Prnp187N mice. Graph indicates plaque diameter (N=3 mice per group, n ≥ 35 plaques). Note the peri-neuronal prion deposits in the ME7 and mNS-infected WT and Prnp187N brain (arrows). C. Lesion profiles indicate the severity of spongiform change, astrogliosis, and PrPSc deposition in 7 brain areas and are nearly superimposable for RML and 22L-infected WT and Prnp187N mice. ME7- and mNS-infected mice showed less severe histopathologic lesions on first passage as compared to WT mice, however lesion severity increased by second passage (1-dorsal medulla, 2-cerebellum, 3-hypothalamus, 4-medial thalamus, 5-hippocampus, 6-cerebral cortex, 7-cerebral peduncles). For panel C: n=4–6 mice/group. D. Prion-like ME7 and mNS plaques in the Prnp187N brains are largely non-congophilic, unlike the mouse-adapted CWD prion strain (mCWD) used as a positive control. Brain regions shown in B: RML and 22L, cerebral cortex; ME7 and mNS, thalamus, and (D) Congo red stain: mCWD, corpus callosum; ME7 and mNS, cerebral cortex. Scale bar = 50 μm (panel B). *P< 0.05, **P< 0.01, ***P< 0.001, Log-rank (Mantel-Cox) test (panel A), unpaired t-test (panel B), 2-way ANOVA with Bonferroni post (panel C).
Figure 3.
Figure 3.. Biochemical properties of 187N-PrPSc.
A. Electrophoretic mobility and glycoform profile of PrPSc from RML-, 22L-, ME7-, or mNS-prion-infected brain lysates (upper) show low levels of unglycosylated PrPSc in the Prnp187N mice brain. Lysates were treated with proteinase K (PK) where indicated to degrade PrPC. Quantification of glycoforms (lower) show percentage of PrPSc that contains zero (green), one (purple), two (grey), or three glycans (white). B. Representative western blots of ME7- or mNS-infected brain lysates from WT or Prnp187N mice show total (upper) or ADAM10-cleaved PrPSc shed from the cell surface (lower). Brain from mCWD-infected mice contain abundant ADAM10-cleaved PrPSc (positive control) [54]. Graphs show quantification (mean ± SE). C. Western blots show ME7-infected brain using antibodies against the octapeptide region of the PrP N-terminus (amino acids 53–88) (POM2), amino acids 95–100 (POM3), and a discontinuous epitope that includes the middle and distal C-terminus, which includes amino acid220 (POM19) [43]. D. Western blots from a sedimentation velocity of RML, mNS, or ME7 prion-infected WT or Prnp187N mice overlayed on a 4–24% Opti-prep™ step gradient, with the pellet fraction in the far right lane. Quantifications are shown below. N = 3 – 4 biological replicates per genotype for each strain, except for ME7-infected Prnp187N shown in panel B (N = 2).
Figure 4.
Figure 4.. Heparan sulfate binding properties of PrP187N.
A. ME7 and mNS plaque-like deposits in the WT and Prnp187N brain sections do not label for HS using the anti-HS antibody 10E4, in contrast to amyloid plaques in the mCWD-infected brain (note that heparinase treatment ablates the HS labelling). B. Levels (left) and composition (right) of HS bound to PrP, as measured by LC-MS. Quantified are N-acetylated (N-Ac), unsubstituted glucosamine (NH2), N-sulfated (N-S), 2O-sulfated, and 6O-sulfated. Brain regions shown for HS stain in panel (A): ME7: cerebral cortex (WT) and thalamus (Prnp187N); mNS: cerebral cortex (WT and Prnp187N). Scale bar = 50 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Aguilar-Calvo P, Sevillano AM, Bapat J, Soldau K, Sandoval DR, Altmeppen HC, Linsenmeier L, Pizzo DP, Geschwind MD, Sanchez H et al. (2019) Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions. Acta Neuropathol: Doi 10.1007/s00401-019-02085-x - DOI - PMC - PubMed
    1. Aguilar-Calvo P, Xiao X, Bett C, Erana H, Soldau K, Castilla J, Nilsson KP, Surewicz WK, Sigurdson CJ (2017) Post-translational modifications in PrP expand the conformational diversity of prions in vivo. Sci Rep 7: 43295 Doi 10.1038/srep43295 - DOI - PMC - PubMed
    1. Aguzzi A, Lakkaraju AK (2016) Cell Biology of Prions and Prionoids: A Status Report. Trends Cell Biol 26: 40–51 Doi 10.1016/j.tcb.2015.08.007 - DOI - PubMed
    1. Baiardi S, Rossi M, Capellari S, Parchi P (2019) Recent advances in the histo-molecular pathology of human prion disease. Brain Pathol 29: 278–300 Doi 10.1111/bpa.12695 - DOI - PMC - PubMed
    1. Baldwin MA, Stahl N, Reinders LG, Gibson BW, Prusiner SB, Burlingame AL (1990) Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein. Anal Biochem 191: 174–182 - PubMed

Publication types