Biomedical subjects

Man-Sun Sy

Publications and source records attributed to Man-Sun Sy.

At least 19 recordsLinked to original sources

PubMed2007

Aggregation of prion protein with insertion mutations is proportional to the number of inserts.

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.

Antibodies, Monoclonal↗

PubMed2006

Prion protein in milk.

BACKGROUND: Prions are known to cause transmissible spongiform encephalopathies (TSE) after accumulation in the central nervous system. There is increasing evidence that prions are also present in body fluids and that prion infection by blood transmission is possible. The low concentration of the proteinaceous agent in body fluids and its long incubation time complicate epidemiologic analysis and estimation of spreading and thus the risk of human infection. This situation is particularly unsatisfactory for food and pharmaceutical industries, given the lack of sensitive tools for monitoring the infectious agent. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an adsorption matrix, Alicon PrioTrap, which binds with high affinity and specificity to prion proteins. Thus we were able to identify prion protein (PrP(C))--the precursor of prions (PrP(Sc))--in milk from humans, cows, sheep, and goats. The absolute amount of PrP(C) differs between the species (from microg/l range in sheep to ng/l range in human milk). PrP(C) is also found in homogenised and pasteurised off-the-shelf milk, and even ultrahigh temperature treatment only partially diminishes endogenous PrP(C) concentration. CONCLUSIONS/SIGNIFICANCE: In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie infected sheep suffering from mastitis, the appearance of PrP(C) in milk implies the possibility that milk of TSE-infected animals serves as source for PrP(Sc).

Animals↗

PubMed2006

Altered prion protein glycosylation in the aging mouse brain.

The normal cellular prion protein (PrP(C)) is a glycoprotein with two highly conserved potential N-linked glycosylation sites. All prion diseases, whether inherited, infectious or sporadic, are believed to share the same pathogenic mechanism that is based on the conversion of the normal cellular prion protein (PrP(C)) to the pathogenic scrapie prion protein (PrP(Sc)). However, the clinical and histopathological presentations of prion diseases are heterogeneous, depending not only on the strains of PrP(Sc) but also on the mechanism of diseases, such as age-related sporadic vs. infectious prion diseases. Accumulated evidence suggests that N-linked glycans on PrP(C) are important in disease phenotype. A better understanding of the nature of the N-linked glycans on PrP(C) during the normal aging process may provide new insights into the roles that N-linked glycans play in the pathogenesis of prion diseases. By using a panel of 19 lectins in an antibody-lectin enzyme-linked immunosorbent assay (ELISA), we found that the lectin binding profiles of PrP(C) alter significantly during aging. There is an increasing prevalence of complex oligosaccharides on the aging PrP(C), which are features of PrP(Sc). Taken together, this study suggests a link between the glycosylation patterns on PrP(C) during aging and PrP(Sc).

Aging↗

PubMed2006

Test for detection of disease-associated prion aggregate in the blood of infected but asymptomatic animals.

We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP(Sc). However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

Animals↗

PubMed2006

Novel single chain antibodies to the prion protein identified by phage display.

A well defined structure is available for the carboxyl half of the cellular prion protein (PrP(c)), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrP(c) recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrP(c) as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised 1 predominant and 3 additional scFv-Ps that contained different V(H) and V(L) sequences and that bound specifically to the PrP(c) target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrP(c) residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrP(c) that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the amino-central region of PrP(c). In addition, the binding of MAbs to known linear epitopes within PrP(c) depends strongly on the endpoints of the target PrP(c) fragment used.

Amino Acid Sequence↗

PubMed2006

Prion proteins with insertion mutations have altered N-terminal conformation and increased ligand binding activity and are more susceptible to oxidative attack.

We compared the biochemical properties of a wild type recombinant normal human cellular prion protein, rPrP(c), with a recombinant mutant human prion protein that has three additional octapeptide repeats, rPrP(8OR). Monoclonal antibodies that are specific for the N terminus of rPrP(c) react much better with rPrP(8OR) than rPrP(c), suggesting that the N terminus of rPrP(8OR) is more exposed and hence more available for antibody binding. The N terminus of PrP(c) contains a glycosaminoglycan binding motif. Accordingly, rPrP(8OR) also binds more glycosaminoglycan than rPrP(c). In addition, the divalent cation copper modulates the conformations of rPrP(c) and rPrP(8OR) differently. When compared with rPrP(c), rPrP(8OR) is also more susceptible to oxidative damage. Furthermore, the abnormalities associated with rPrP(8OR) are recapitulated, but even more profoundly, in another insertion mutant, which has five extra octapeptide repeats, rPrP(10OR). Therefore, insertion mutants appear to share common features, and the degree of abnormality is proportional to the number of insertions. Any of these anomalies may contribute to the pathogenesis of inherited human prion disease.

Amino Acid Motifs↗

PubMed2006

Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.

Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K (PK)-resistant conformer, scrapie PrP (PrP(Sc)). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti-PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse-adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already-infected N2a cells of PrP(Sc). These antibodies were 6D11 (generated to PK-resistant PrP(Sc) and detecting PrP residues 93-109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130-140 and 143-155, respectively. Mabs were found to interact with PrP(Sc) formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrP(C). Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrP(Sc) levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans.

Animals↗

PubMed2005

KDEL-tagged anti-prion intrabodies impair PrP lysosomal degradation and inhibit scrapie infectivity.

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into the infectious scrapie isoform (PrPSc). We have recently demonstrated that anti-prion intrabodies targeted to the lumen of the endoplasmic reticulum provide a simple and effective means to inhibit the transport of PrPC to the cell surface. Here, we report that they completely block the traffic of mature full-length PrPC molecules, impair prion lysosomal degradation, and interfere with the early phase of scrapie formation. Since anti-prion intrabodies efficiently block PrPSc accumulation in vitro, we investigated whether they could also antagonize scrapie infectivity in vivo. We found that mice intracerebrally injected with KDEL-8H4-NGF-differentiated PC12 cells infected with scrapie neither develop scrapie clinical signs nor brain damage. Furthermore, no protease-resistant PrPSc is detectable in brains of inoculated animals. These results indicate that anti-prion intrabody strategy may be effective against prion infection.

Animals↗

PubMed2005

Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models.

Chronic wasting disease (CWD), a prion disease affecting free-ranging and captive cervids (deer and elk), is widespread in the United States and parts of Canada. The large cervid population, the popularity of venison consumption, and the apparent spread of the CWD epidemic are likely resulting in increased human exposure to CWD in the United States. Whether CWD is transmissible to humans, as has been shown for bovine spongiform encephalopathy (the prion disease of cattle), is unknown. We generated transgenic mice expressing the elk or human prion protein (PrP) in a PrP-null background. After intracerebral inoculation with elk CWD prion, two lines of "humanized" transgenic mice that are susceptible to human prions failed to develop the hallmarks of prion diseases after >657 and >756 d, respectively, whereas the "cervidized" transgenic mice became infected after 118-142 d. These data indicate that there is a substantial species barrier for transmission of elk CWD to humans.

Animals↗

PubMed2005

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.

Biomarkers↗

PubMed2005

Novel antibody-lectin enzyme-linked immunosorbent assay that distinguishes prion proteins in sporadic and variant cases of Creutzfeldt-Jakob disease.

We used different anti-prion protein (anti-PrP) monoclonal antibodies to capture either full-length or truncated PrP species and then used biotinylated lectin to compare the nature of the glycans on bound PrP species present in control, sporadic Creutzfeldt-Jakob disease (sCJD), or variant CJD (vCJD) brains. When full-length PrP species in these three groups were compared, no significant difference in the binding of concanavalin A or Aleuria aurantia lectin was detected. However, the binding of Ricinus communis agglutinin I (RCA) to sCJD and vCJD samples was significantly increased. In contrast, when only truncated PrP species were compared, only vCJD samples had more RCA binding activity. Therefore, while most of the RCA binding activity in sCJD is restricted to the full-length PrP species, the RCA binding activity in vCJD is associated with truncated and full-length PrP species. Furthermore, the RCA binding activity in sCJD and vCJD samples is mostly associated with proteinase K-resistant PrP species, a known signature of infectious prion. Therefore, PrP species in sCJD and vCJD have dissimilar lectin immunoreactivity, which reflects differences in their N-linked glycans. These differences may account for the distinct phenotypes of sCJD and vCJD.

Biotinylation↗

PubMed2005

An aggregation-specific enzyme-linked immunosorbent assay: detection of conformational differences between recombinant PrP protein dimers and PrP(Sc) aggregates.

The conversion of the normal cellular prion protein, PrP(C), into the protease-resistant, scrapie PrP(Sc) aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP(Sc) aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP(Sc) aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.

Animals↗

PubMed2005

Biochemical fingerprints of prion infection: accumulations of aberrant full-length and N-terminally truncated PrP species are common features in mouse prion disease.

Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.

Animals↗

PubMed2005

Characterization of the F198S prion protein mutation: enhanced glycosylation and defective refolding.

Prion diseases are associated with the accumulation of a misfolded, protease resistant form of the prion protein, PrPres. In humans there are a variety of different prion related diseases that are sporadic, inherited, or acquired by infection. Gerstmann-Straussler-Sheinker syndrome (GSS) is an inherited prion disease in which PrPres accumulates as amorphous aggregates as well as in amyloid plaques. GSS has been associated with a variety of point mutations in the prion protein: 102, 105, 117, 131, 145, 187, 198, 202, 212, 217, and 232. The F198S mutation was discovered in a large Indiana kindred. Previous studies in vitro have shown that the 198 mutation results in structural instability of the prion protein. In the current study, we demonstrate in a cell model that the F198S mutant protein can be folded properly in a cellular context, but is unable to refold to a native state after denaturation. Further, the F198S mutation significantly affects glycosylation of the mutant protein.

Amyloid↗

PubMed2004

Calpain and other cytosolic proteases can contribute to the degradation of retro-translocated prion protein in the cytosol.

PrP, a cell surface-localized N-linked glycoprotein, is required for the pathogenesis of prion diseases. Recent studies have revealed that prion protein (PrP) becomes neurotoxic and prone to aggregation when it is in the cytosol, suggesting that cytosolic PrP may play a role in the pathogenesis of prion disease. Retro-translocation of PrP from the endoplasmic reticulum to the cytosol for proteasome degradation offers a natural route for PrP to enter the cytosol, but whether PrP is subject to retrotranslocation is controversial. In this study, we investigated the metabolism of endogenous wild-type PrP in several cell lines and in primary mouse cortical neurons. Our results suggest that a portion of the endogenous wild-type PrP is retro-translocated to the cytosol and degraded by the proteasome. Moreover, we also found that calpain and other cytosolic proteases could degrade PrP in the cytosol when the proteasome activity is compromised. These results provide the foundation for the hypothesis that cytosolic PrP may be involved in the pathogenesis of prion disease.

Animals↗

PubMed2004

Trapping prion protein in the endoplasmic reticulum impairs PrPC maturation and prevents PrPSc accumulation.

The conversion of the normal cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP(C) and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP(C) we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP(C) and on PrP(Sc) formation. We found that ER-targeted anti-prion scFvs retain PrP(C) in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP(C), with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP(C) acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP(Sc) accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.

Animals↗

PubMed2004

Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/B' region.

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.

Alleles↗

PubMed2004

Interaction of Doppel with the full-length laminin receptor precursor protein.

Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions. Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc. However, the molecular basis for the protective effect of PrPc remains unclear. Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc. The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues. Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl. These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same. Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.

Animals↗