Characterization of the 37-kDa/67-kDa laminin receptor as the cell surface receptor for the cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPSc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies (TSEs), which affect both, humans and animals. Human prion diseases occur in infectious, sporadic or genetic forms. The "protein only" hypothesis argues that the key event in the pathogenesis represents the conversion of the normal host protein, PrPc, into its pathogenic isoform PrPSc. Prion diseases have been associated with the accumulation of this abnormally folded protein and its neurotoxic effects. However, it is not known if PrPc loss of function is an important factor since the normal biological function of PrPc, a cell surface-anchored glycoprotein predominantly expressed in neuronal cells, and the cellular processes in which this protein is involved remain obscure. Recently, the human 37 kDa laminin receptor precursor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), was identified as a binding partner for the cellular prion protein in a yeast two-hybrid screen. In order to characterize the possible role of LRP/LR as a cell surface receptor for PrPc, cell culture studies were performed to investigate the cellular localization of PrP and LRP/LR and to analyse the binding and internalization behaviour of PrP depending on the presence of LRP/LR on the cell surface of neuronal and non-neuronal cells. Immunofluorescence analysis of non-permeabilized murine neuroblastoma cells demonstrated that PrP and LRP/LR co-localize on the surface of these cells. In addition, baby hamster kidney (BHK) cells transfected with recombinant Semlik-Forest virus RNAs overexpressed human PrP and human LRP at their cell surface, the latter one orientated as a type II transmembrane protein with its C-terminus outside and its N-terminus inside the cell. Co-localization of both proteins was observed on BHK cells co-transfected with LRP and PrP encoding recombinant SFV RNAs. Cell binding and internalization assays with recombinant human PrP demonstrated the LRP/LR-dependent binding and endocytosis of externally added human PrP. An increased, dose-dependent cell binding of recombinant PrP was demonstrated by BHK cells overexpressing full-length human LRP on their cell surface. Trypsin treatment of the cell surface revealed the LRP dependent internalization of GST-tagged and untagged, glycosylated PrP. In contrast to wild-type LRP, the expression of an LRP mutant lacking its transmembrane domain led to the secretion of this mutant from transfected BHK cells and totally abolished the binding and internalization of exogenous, recombinant PrP. This LRP mutant could function as a decoy recetor in therapy of TSEs. The strict LRP/LR specificity of the PrP binding to neuronal cells was verified by testing the displacement capacity of a series of different antibodies in the LRP-PrP binding reaction. Only LRP and PrP specific antibodies were able to block totally the binding of human GST-fused PrP to N2a and NT2 cells whereas various control antibodies used for competition showed no effect. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of cellular PrP to the 37-kDa/67-kDa LRP/LR. The relationship between the 37-kDa LRP and the 67-kDa high-affinity LR is unknown so far. Both forms were observed in plasma membrane fractions of N2a cells. We conclude from these data that the 37-kDa/67-kDa laminin receptor acts as the main cell surface receptor for PrP. High-level expression and purification of recombinant, glycosylated prion proteins in mammalian cells is essential for a better understanding of the physiological function of PrPc and biochemical processes responsible for prion diseases. Due to the presence of important organelles, membranes and other cellular cofactors which are necessary for the correct processing, trafficking and localization of prion proteins mammalian cell culture systems such as the Semliki-Forest virus (SFV) system allow the synthesis and characterization of wild-type as well as mutant PrP to get a better insight into the biology of these proteins. Therefore, the SFV system was used to generate recombinant highly glycosylated human wild-type and human disease-associated mutant prion proteins as well as FLAG-tagged human and bovine PrP in cultured BHK cells. Both mutated variants, which are related to the human prion diseases fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) reveal proteinase K (PK) resistance, one of the most typical biochemical properties characteristic for the infectious scrapie isoform of the prion protein. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP without any significant differences regarding the cellular distribution and expression level. In addition, FLAG-tagged prion proteins were expressed with high efficiency in BHK cells showing the typical glycosylation pattern allowing the rapid and simple purification via anti-FLAG antibody chromatography. PrP dimers could play an essential role in the PrPc to PrPSc conversion process and might be involved in PrP interspecies transmission. Recently, crystallization of the prion protein in a dimeric form was reported. Size exclusion chromatography showed that native soluble homogeneous FLAG tagged prion proteins from hamster, man and cattle expressed in the baculovirus system were predominantly dimeric. The PrP/PrP interaction was confirmed in rec. SFV-RNA transfected BHK cells co-expressing FLAG and oligohistidine tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. The identification of the 37-kDa/67-kDa laminin receptor as the receptor for the cellular prion protein might represent an important step for a better understanding of the molecular biology of prion diseases and might lead to the development of powerful therapeutics such as LRP/LR specific antibodies for the treatment of these unconventional diseases

The first report of RPSA polymorphisms, also called 37/67 kDa LRP/LR gene, in sporadic Creutzfeldt-Jakob disease (CJD)

<p>Abstract</p> <p>Background</p> <p>Although polymorphisms of <it>PRNP</it>, the gene encoding prion protein, are known as a determinant affecting prion disease susceptibility, other genes also influence prion incubation time. This finding offers the opportunity to identify other genetic or environmental factor (s) modulating susceptibility to prion disease. Ribosomal protein SA (<it>RPSA</it>), also called 37 kDa laminin receptor precursor (LRP)/67 kDa laminin receptor (LR), acts as a receptor for laminin, viruses and prion proteins. The binding/internalization of prion protein is dependent for LRP/LR.</p> <p>Methods</p> <p>To identify other susceptibility genes involved in prion disease, we performed genetic analysis of <it>RPSA</it>. For this case-control study, we included 180 sporadic Creutzfeldt-Jakob disease (CJD) patients and 189 healthy Koreans. We investigated genotype and allele frequencies of polymorphism on <it>RPSA </it>by direct sequencing or restriction fragment length polymorphism (RFLP) analysis.</p> <p>Results</p> <p>We observed four single nucleotide polymorphisms (SNPs), including -8T>C (rs1803893) in the 5'-untranslated region (UTR) of exon 2, 134-32C>T (rs3772138) in the intron, 519G>A (rs2269350) in the intron and 793+58C>T (rs2723) in the intron on the <it>RPSA</it>. The 519G>A (at codon 173) is located in the direct PrP binding site. The genotypes and allele frequencies of the <it>RPSA </it>polymorphisms showed no significant differences between the controls and sporadic CJD patients.</p> <p>Conclusion</p> <p>These results suggest that these <it>RPSA </it>polymorphisms have no direct influence on the susceptibility to sporadic CJD. This was the first genetic association study of the polymorphisms of <it>RPSA </it>gene with sporadic CJD.</p

Green Tea Polyphenol EGCG Sensing Motif on the 67-kDa Laminin Receptor

BACKGROUND: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K(166) and H(169), have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. CONCLUSIONS/SIGNIFICANCE: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions

Interactions of the 67 kDa laminin receptor and its precursor with laminin

The 67LR (67 kDa laminin receptor) enables cells to interact with components of the extracellular matrix. The molecule is derived from the 37LRP (37 kDa laminin receptor precursor); however, the precise molecular mechanism of this conversion is unknown. Recombinant 37LRP, expressed in and purified from Escherichia coli, bound to human laminin in a SPR (surface plasmon resonance) experiment. 67LR isolated from human breast-cancer-derived cells in culture was also shown to bind to laminin by SPR. However, the kinetics of association are qualitatively different. 37LRP, but not 67LR, binds to heparan sulfate. The binding of 37LRP to heparan sulfate did not affect the interaction of 37LRP with laminin. In contrast, heparan sulfate reduces the extent of binding of laminin to 67LR. Taken together, these results show that 37LRP has some of the biological activities of 67LR, even prior to the conversion event. However, the conversion affects the sites of interaction with both laminin and heparan sulfate

The N Terminus of the Prion Protein Mediates Functional Interactions with the Neuronal Cell Adhesion Molecule (NCAM) Fibronectin Domain

The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrPC function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrPC protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrPC co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrPC N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrPC N terminus as a dynamic and functional element responsible for protein-protein interaction

Antibody-based immunotherapeutic attempts in experimental animal models of prion diseases.

BACKGROUND: There has been a dramatic decrease in the risk of transmission of bovine spongiform encephalopathy to humans. In contrast, the risk of human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) via medical treatments became potentially high since 4 vCJD cases were reported to be possibly transmitted through blood transfusion in the UK. However, no treatments are yet available for curing prion diseases. OBJECTIVE: Conversion of the normal prion protein, PrP(C), to the amyloidogenic PrP, PrP(Sc), plays a pivotal role in the pathogenesis. Recently, certain anti-PrP or anti-37/67-kDa laminin receptor (LRP/LR) antibodies were shown to have the potential to cure chronically infected cells, clearing PrP(Sc) from the cells. This has raised the possibility of antibody based-immunotherapy for prion diseases. This article aims to introduce and discuss the recently published attempts of immunotherapy in prion diseases. METHODS: Bibliographic research was carried out using the PubMed database. Patent literature was searched using the UK Intellectual Property Office website. RESULTS/CONCLUSION: No satisfying consequences in animals could be detected with anti-PrP antibodies directly infused into the brains of animals by the intraventricular route or by anti-PrP or anti-LRP/LR single chain fragment antibodies directly delivered into the brain by virus vector-mediated gene transfer. This is probably because such delivery systems failed to deliver the antibodies to the neurons relevant for the treatments

The Cellular Prion Protein Interacts with the Tissue Non-Specific Alkaline Phosphatase in Membrane Microdomains of Bioaminergic Neuronal Cells

BACKGROUND: The cellular prion protein, PrP(C), is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C) in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C) acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C), we have described a neuronal specificity pointing to a role of PrP(C) in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C) signaling prompted us to search for PrP(C) partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C) with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT) and 1C11(NE) cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT) and 1C11(NE) bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C) partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C) and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C)-laminin interplay. The partnership between TNAP and PrP(C) in neuronal cells may provide new clues as to the neurospecificity of PrP(C) function

Inoculation of Scrapie with the Self-Assembling RADA-Peptide Disrupts Prion Accumulation and Extends Hamster Survival

Intracerebral inoculation of 263K Scrapie brain homogenate (PrPsc) with a self-assembling RADA-peptide (RADA) significantly delayed disease onset and increased hamster survival. Time of survival was dependent on the dose of RADA and pre-incubation with PrPsc prior to inoculation. RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice. In all PrPsc inoculated animals, clinical symptoms were observed ∼10 d prior to sacrifice and brains showed spongiform degeneration with Congo red positive plaques. A time-dependent increase in reactive gliosis was observed in both groups with more GFAP detected in RADA treated animals at all time points. The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red. We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation. This is the first demonstration that a self-assembling biomolecular peptide can interact with PrPsc, disrupt the course of Scrapie disease process, and extend survival

Extraribosomal functions associated with the C terminus of the 37/67 kDa laminin receptor are required for maintaining cell viability

The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G1 phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization