Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is transmissible through iatrogenic routes due to abundant infectious prions [misfolded forms of the prion protein (PrPSc)] in the central nervous system (CNS). Some epidemiological studies have associated sCJD risk with non-CNS surgeries. We explored the potential prion seeding activity and infectivity of skin from sCJD patients. Autopsy or biopsy skin samples from 38 patients [21 sCJD, 2 variant CJD (vCJD), and 15 non-CJD] were analyzed by Western blotting and real-time quaking-induced conversion (RT-QuIC) for PrPSc. Skin samples from two patients were further examined for prion infectivity by bioassay using two lines of humanized transgenic mice. Western blotting revealed dermal PrPSc in one of five deceased sCJD patients and one of two vCJD patients. However, the more sensitive RT-QuIC assay detected prion seeding activity in skin from all 23 CJD decedents but not in skin from any non-CJD control individuals (with other neurological conditions or other diseases) during blinded testing. Although sCJD patient skin contained ~103- to 105-fold lower prion seeding activity than did sCJD patient brain tissue, all 12 mice from two transgenic mouse lines inoculated with sCJD skin homogenates from two sCJD patients succumbed to prion disease within 564 days after inoculation. Our study demonstrates that the skin of sCJD patients contains both prion seeding activity and infectivity, which raises concerns about the potential for iatrogenic sCJD transmission via skin.
The polypeptide N-alpha-acetylgalactosaminyltransferases (ppGalNAcTs) play a key role in mucin-type O-linked glycan biosynthesis by installing the initial GalNAc residue on the protein scaffold. The preferred substrates and functions of the >20 isoforms in mammals are not well understood. However, current data suggest that glycosylated mucin domains are created by the successive, often hierarchical, action of several specific ppGalNAcTs. Herein we analyzed the glycopeptide substrate preferences of several ppGalNAcT family members using a library screening approach. A 56-member glycopeptide library designed to reflect a diversity of glycan clustering was assayed for substrate activity with ppGalNAcT isoforms using an azido-ELISA. The data suggest that the ppGalNAcTs can be classified into at least four types, which working together, are able to produce densely glycosylated mucin glycoproteins.
A large family of uridine 5'-diphosphate (UDP)-alpha-N-acetylgalactosamine (GalNAc):polypeptide N-acetylgalactosaminyl transferases (ppGalNAc Ts) initiates mucin-type O-glycan biosynthesis at serine and threonine. The peptide substrate specificities of individual family members are not well characterized or understood, leaving an inability to rationally predict or comprehend sites of O-glycosylation. Recently, a kinetic modeling approach demonstrated neighboring residue glycosylation as a major factor modulating the O-glycosylation of the porcine submaxillary gland mucin 81 residue tandem repeat by ppGalNAc T1 and T2 [Gerken et al. (2002) J. Biol. Chem. 277, 49850-49862]. To confirm the general applicability of this model and its parameters, the ppGalNAc T1 and T2 glycosylation kinetics of the 80+ residue tandem repeat from the canine submaxillary gland mucin was obtained and characterized. To reproduce the glycosylation patterns of both mucins (comprising 50+ serine/threonine residues), specific effects of neighboring peptide sequence, in addition to the previously described effects of neighboring residue glycosylation, were required of the model. Differences in specificity of the two transferases were defined by their sensitivities to neighboring proline and nonglycosylated hydroxyamino acid residues, from which a ppGalNAc T2 motif was identified. Importantly, the model can approximate the previously reported ppGalNAc T2 glycosylation kinetics of the IgA1 hinge domain peptide [Iwasaki, et al. (2003) J. Biol. Chem. 278, 5613-5621], further validating both the approach and the ppGalNAc T2 positional weighting parameters. The characterization of ppGalNAc transferase specificity by this approach may prove useful for the search for isoform-specific substrates, the creation of isoform-specific inhibitors, and the prediction of mucin-type O-glycosylation sites.
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