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Boon-Seng Wong

Publications and source records attributed to Boon-Seng Wong.

13 recordsLinked to original sources
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↗
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↗
PubMed2004

Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice.

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.

Animals↗
PubMed2004

Epitope scanning reveals gain and loss of strain specific antibody binding epitopes associated with the conversion of normal cellular prion to scrapie prion.

We used anti-prion (PrP) monoclonal antibodies (Mabs) in different combinations to scan changes in the availability of antibody binding epitopes--using an epitope scanning assay--in brain homogenates from normal mice, and from mice infected with either ME7 or 139 A strains of infectious scrapie prion (PrPSc). In ME7-infected brains, the epitope detected by the Mab pair 8B4/8H4 is reduced, while the epitope detected by the Mab pair 8F9/11G5 is increased. Mab 8F9/11G5 detect a conformational epitope on PrPSc because the rise in Mab 8F9/11G5 binding is sensitive to a denaturing agent but resistant to proteinase K (PK). While the increase in Mab 8F9/11G5 binding correlates with the presence of PK-resistant PrP and clinical signs of infection, the reduction in Mab 8B4/8H4 binding is detected earlier. Fractionation of the ME7-infected brain homogenate in sucrose gradient revealed that the PrPSc species detected by the epitope scanning assay are heterogeneous in size, with a molecular mass of approximately > or = 2000-kDa. We also investigated whether these findings were applicable to two other strains of PrPSc, namely 87 V and 22 L. We found that the decrease in Mab 8B4/8H4 binding detected in ME7-infected brains was also detected in 87 V-infected brains but not in 22 L-infected brains. In contrast, the increase in Mab 8F9/11G5 binding detected in ME7- and 139 A-infected brains was also detected in 22 L-infected brains but not in 87 V-infected brains. Therefore, each prion strain has its unique conformation, and we can monitor the conversion of normal cellular prion (PrPC) to PrPSc based on the changes in the antibody binding patterns. The epitope can be decreased or increased, linear or conformational, detected late or early during infection, in a strain specific manner.

Alanine↗
PubMed2004

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite 'scavenger'?

Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to stimulate N-methyl-D-aspartate (NMDA) receptors to enhance long-term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 microm. Considerably lower H2S levels are reported in the brains of Alzheimer's disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitrite-mediated damage to biomolecules and to cultured human SH-SY5Y cells. H2S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of alpha1-antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 microm). H2S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.

Cell Line↗
PubMed2003

On the same cell type GPI-anchored normal cellular prion and DAF protein exhibit different biological properties.

Normal cellular prion protein (PrP(C)) and decay-accelerating factor (DAF) are glycoproteins linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Both PrP(C) and DAF reside in detergent insoluble complex that can be isolated from human peripheral blood mononuclear cells. However, these two GPI-anchored proteins possess different cell biological properties. The GPI anchor of DAF is markedly more sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) than that of PrP(C). Conversely, PrP(C) has a shorter cell surface half-life than DAF, possibly due to the fact that PrP(C) but not DAF is shed from the cell surface. This is the first demonstration that on the surface of the same cell type two GPI-anchored proteins differ in their cell biological properties.

CD55 Antigens↗
PubMed2003

Guanidine hydrochloride extraction and detection of prion proteins in mouse and hamster prion diseases by ELISA.

Current detection of transmissible spongiform encephalopathy (TSE) relies on the proteolytic generation of a protease-resistant core from the scrapie isoform of prion protein (PrP(Sc)) followed by immunoblotting. This process is non-quantitative, time-consuming, and technically demanding. Recently, an alternative in vitro test for TSE based on the differential extraction of brain homogenates using guanidine hydrochloride followed by DELFIA (Dissociation Enhanced Lanthanide FluoroImmunoAssay) has been developed. In the present study, this approach was adopted using a panel of anti-PrP monoclonal antibodies (MAbs) in conventional sandwich enzyme-linked immunosorbent assay (ELISA) to investigate hamster and two distinct strains of mouse prion diseases. Although PrP species were present in both soluble and insoluble fractions from normal as well as TSE samples, only the PrP species in the insoluble fractions from the latter samples were protease-resistant. In addition, certain anti-PrP MAb pairs could distinguish the PrP species in infected brains from those in the normal samples. The ability to differentiate disease-associated PrP isoforms without proteinase K digestion could serve as a panacea for developing a reliable and rapid diagnostic test for prion diseases.

Animals↗
PubMed2002

Cell-surface prion protein interacts with glycosaminoglycans.

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs.

Amino Acid Motifs↗
PubMed2002

Intercellular transfer of the cellular prion protein.

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated whether PrP(C) can move from one cell to another cell in a cell model. Little PrP(C) transfer was detected when a PrP(C) expressing human neuroblastoma cell line was cultured with the human erythroleukemia cells IA lacking PrP(C). Efficient transfer of PrP(C) was detected with the presence of phorbol 12-myristate 13-acetate, an activator of protein kinase C. Maximum PrP(C) transfer was observed when both donor and recipient cells were activated. Furthermore, PrP(C) transfer required the GPI anchor and direct cell to cell contact. However, intercellular protein transfer is not limited to PrP(C), another GPI-anchored protein, CD90, also transfers from the donor cells to acceptor cells after cellular activation. Therefore, this transfer process is GPI-anchor and cellular activation dependent. These findings suggest that the intercellular transfer of GPI-anchored proteins is a regulated process, and may have implications for the pathogenesis of prion disease.

Blotting, Western↗
PubMed2002

Human prion diseases.

Transmissible spongiform encephalopathy (TSE) is a group of rare, sub-acute, fatal neurodegenerative diseases in humans and animals. TSE includes Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia, and Kuru (a disease confined to the Fore linguistic group, a tribe in Papua-New Guinea). From the onset, it was recognized that some of these spongiform diseases occur in clusters, in an inherited, familial manner. This article describes these diseases, current treatment modalities, and suggests directions for future research.

Adult↗
PubMed2002

Heterogeneity of normal prion protein in two- dimensional immunoblot: presence of various glycosylated and truncated forms.

The common use of one-dimensional (1-D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrP(C) ) comprises few and simple forms. In this study we used two-dimensional (2-D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrP(C) present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrP(C) species based on combinations of different molecular weights and isoelectric points (pIs). The PrP(C) heterogeneity is due to the presence of a full-length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N-terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full-length and truncated PrP(C) forms differ in their structure and ratios of the glycoforms. The truncation of PrP(C) and the heterogeneity of the linked glycans may play a role in regulating PrP(C) function. Furthermore, the presence of relatively large quantities of different PrP(C) species may provide additional mechanisms by which the diversity of prion strains could be generated.

Antibodies, Monoclonal↗