The prion-like protein Doppel fails to interact with itself, the prion protein and the 37 kDa/67 kDa laminin receptor in the yeast two-hybrid system

The prion-like protein termed Doppel (Dpl) shows approx. 25% sequence identity with all known prion proteins (PrP). We recently showed that the cellular PrP is dimeric under native conditions, a finding which was confirmed by the investigation of its crystal structure. Human PrP further interacts with its cellular receptor, the 37 kDa/67 kDa laminin receptor (LRP/LR). Here we report that human Doppel fails to interact with (i). itself, (ii). the human 37 kDa/67 kDa LRP/LR, and (iii). the human cellular prion protein (huPrP) in the yeast two-hybrid system. Our findings suggest that Dpl and PrP are not related or are only marginally related with respect to their ligand binding behaviour.     

Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins

The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-DeltaSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-DeltaSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules.     

A trans-dominant negative 37kDa/67kDa laminin receptor mutant impairs PrP(Sc) propagation in scrapie-infected neuronal cells

The 37kDa/67kDa laminin receptor (LRP/LR) has been identified as a cell surface receptor for cellular and infectious prion proteins. Here, we show that an N-terminally truncated LRP mutant encompassing the extracellular domain of the LRP/LR (LRP102-295::FLAG) reduces the binding of recombinant cellular huPrP to mouse neuroblastoma cells, and infectious moPrP27-30 to BHK cells, and interferes with the PrP(Sc) propagation in scrapie-infected neuroblastoma cells (N2aSc(+)). A cell-free binding assay demonstrated the direct binding of the LRP102-295::FLAG mutant to both PrP(c) and PrP(Sc). These results, together with the finding that endogenous LRP levels remain unaffected by the expression of the mutant, indicate that the secreted LRP102-295::FLAG mutant may act in a trans-dominant negative manner as a decoy by trapping PrP molecules. The LRP mutant might represent a potential therapeutic tool for the treatment of TSEs.     

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

We identified the 37kDa/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrP^c) and the infectious prion protein (PrP^Sc). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrP^Sc content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrP^Sc propagation. In addition, we observed a 1.8-fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrP^Sc propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrP^Sc propagation in vivo.Key Words: 37kDa/67kDa laminin receptor, LRP/LR, prion, PrP, TSE-therapy     

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.     

Knock-Down of the 37-kDa/67-kDa Laminin Receptor in Mouse Brain by Transgenic Expression of Specific Antisense LRP RNA

The 37-kDa/67-kDa laminin receptor (LRP/LR) plays a major role in the propagation of PrPSc, the abnormal form of the prion protein. In order to ablate the expression of LRP/LR in mouse brain we generated transgenic mice ectopically expressing antisense LRP RNA in the brain under control of the neuron-specific enolase (NSE) promoter. Hemizygous transgenic mice TgN(NSEasLRP)2 showed a significant reduction of LRP/LR protein levels in hippocampal and cerebellar brain regions. These mice might act as powerful tools to investigate the role of the laminin receptor in scrapie pathogenesis.     

Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor.

Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144-179] as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.     

The 67 kDa laminin receptor: structure, function and role in disease

The 67LR (67 kDa laminin receptor) is a cell-surface receptor with high affinity for its primary ligand. Its role as a laminin receptor makes it an important molecule both in cell adhesion to the basement membrane and in signalling transduction following this binding event. The protein also plays critical roles in the metastasis of tumour cells. Isolation of the protein from either normal or cancerous cells results in a product with an approx. molecular mass of 67 kDa. This protein is believed to be derived from a smaller precursor, the 37LRP (37 kDa laminin receptor precursor). However, the precise mechanism by which cytoplasmic 37LRP becomes cell-membrane-embedded 67LR is unclear. The process may involve post-translational fatty acylation of the protein combined with either homo- or hetero-dimerization, possibly with a galectin-3-epitope-containing partner. Furthermore, it has become clear that acting as a receptor for laminin is not the only function of this protein. 67LR also acts as a receptor for viruses, such as Sindbis virus and dengue virus, and is involved with internalization of the prion protein. Interestingly, unmodified 37LRP is a ribosomal component and homologues of this protein are found in all five kingdoms. In addition, it appears to be strongly associated with histones in the eukaryotic cell nucleus, although the precise role of these interactions is not clear. Here we review the current understanding of the structure and function of this molecule, as well as highlighting areas requiring further research.     

Scrapie-Infected Transgenic Mice Expressing a Laminin Receptor Decoy Mutant Reveal a Prolonged Incubation Time Associated with Low Levels of PrPres

The 37-kDa/67-kDa laminin receptor (LRP/LR) was identified as a cell surface receptor for prion proteins. The laminin receptor mutant LRP102-295∷FLAG interfered with PrP^Sc propagation in murine neuronal cells presumably acting as a decoy in a transdominant negative fashion by trapping PrP molecules in the extracellular matrix. Here, we generated hemizygous transgenic mice expressing LRP102-295∷FLAG in the brain. Scrapie-infected transgenic mice exhibit a significantly prolonged incubation time in comparison to scrapie-infected wild-type (FVB) mice. At the terminal stage, transgenic mice revealed significantly reduced proteinase-K-resistant PrP levels by 71% compared to wild-type mice. Our results recommend the laminin receptor decoy mutant as an alternative therapeutic tool for treatment of transmissible spongiform encephalopathies.     

Cell Localization and Redistribution of the 67 kD Laminin Receptor and α6βl Integrin Subunits in Response to Laminin Stimulation: An Immunogold Electron Microscopy Study

The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa polypeptide (37LRP), previously identified as the 67LR precursor, is abundantly present in the cytoplasm and has been implicated in polysome formation. To better understand the cellular localization of the 67LR and its precursor, transmission electron microscopic studies of human melanoma A2058 cells were carried out using immunogold labeling and a variety of antibodies: (a) affinity purified antibodies directed against 37LRP cDNA-derived synthetic peptides; (b) anti-67LR monoclonal antibodies raised against intact human small cell lung carcinoma cells; and (c) monoclonal antibodies against the subunits of the integrin laminin receptor, α6β1. Double-labeling immunocyto-chemistry revealed that anti-67LR monoclonal antibodies as well as anti-37 LRP antibodies recognized antigens that were localized in the cytoplasm in electron dense structures. As expected, cell membrane labeling was also observed. Surprisingly, α6 and β1 integrin subunits were detected in the same cytoplasmic structures positive for the 67LR and the 37LRP. After addition of soluble laminin to A2058 cells in suspension, the number of labeled cytoplasmic structures increased especially in the vicinity of the plasma membrane, and were exported onto the cell surface. Neither fibronectin nor BSA induced such an effect. The data demonstrate that the 67 LR and the 37 LRP antibodies detect colocalized antigens that are in cytoplasmic structures with α6β1 integrin. These laminin binding protein rich structures could potentially form a supply of receptors that are exported to the surface upon exposure of the cells to laminin, with a consequent increase in the number of binding sites for the ligand. This system could define a mechanism through which cancer cells modulate their interaction with laminin.     

Structural and mechanistic commonalities of amyloid-β and the prion protein

Amyloid β (Aβ) is a major causative agent of Alzheime disease. This neurotoxic peptide is generated as a result of the cleavage of the Amyloid-Precursor-Protein (APP) by the action of beta secretase and gamma secretase. The neurotoxicity was previously thought to be the result of aggregation. However, recent studies suggest that the interaction of Aβ with numerous cell surface receptors such as N-methyl-D-aspartate (NMDA), receptor for advanced glycosylation end products (RAGE), P75 neurotrophin receptor (P75NTR) as well as cell surface proteins such as the cellular prion protein (PrP^c) and heparan sulfate proteoglycans (HSPG) strongly enhances Aβ induced apoptosis and thereby contributes to neurotoxicity. This review focuses on the molecular mechanism resulting in Aβ-shedding as well as Aβ-induced apoptotic processes, genetic risk factors for familial Alzheimer disease and interactions of Aβ with cell surface receptors and proteins, with particular emphasis on the cellular prion protein. Furthermore, comparisons are drawn between Alzheimer disease and prion disorders and the role of laminin, an extracellular matrix protein, glycosaminoglycans and the 37 kDa/67 kDa laminin receptor (LRP/LR) have been highlighted with regards to both neurodegenerative diseases.     

Polymorphism of Fecundity Genes (FecB,FecX, and FecG) in the Indian Bonpala Sheep

The 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR, also known as ribosomal protein SA, RPSA) has been reported to be involved in cancer development and prion internalization. Previous studies have shown that the LRP/LR is expressed in a wide variety of tissues. In particular, expression of LRP/LR mRNA may be closely related to the degree of PrP^Sc propagation. This study presents a detailed investigation of the LRP/LR mRNA expression levels in eleven normal ovine tissues. Using real-time quantitative PCR, the highest LRP/LR expression was found in neocortex (p < 0.05). Slightly lower levels were found in the heart and obex. Intermediate levels were seen in hippocampus, cerebellum, spleen, thalamus, mesenteric lymph node, and the lowest levels were present in liver, kidney, and lung. In general, the LRP/LR mRNA levels were much higher in neuronal tissues than in peripheral tissues. The observation that differences in LRP/LR mRNA expression levels are consistent with the corresponding variation in PrP^Sc accumulation suggests that the 37-kDa/67-kDa laminin receptor may be involved in the regulation of PrP^Sc propagation.     

Biosynthesis of the 67 kDa high affinity laminin receptor.

High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone and obtained by in vitro translation of hybrid-selected laminin receptor mRNA, has been immunologically identified in cancer cell extracts as the putative precursor of the 67 LR. In this study, we used affinity purified antibodies developed against cDNA-deduced 37 LRP synthetic peptides in pulse chase experiments and demonstrated a precursor-product relationship between the 37 LRP and the 67 LR. Immunoblot, pulse chase and immunofluorescence experiments showed that transient transfection of the full length 37 LRP cDNA clone induced a dramatic increase in the synthesis of the 37 LRP but not of the mature 67 LR. We propose that the 67 LR results from the association of two gene products: the 37 LRP and a polypeptide yet to be identified.     

Cloning and characterization of full-length coding sequence (CDS) of the ovine 37/67-kDa laminin receptor (<Emphasis Type="Italic">RPSA</Emphasis>)

The 37-kDa Laminin Receptor Precursor (LRP)/67-kDa Laminin Receptor (LR), also known as ribosomal protein SA (RPSA), had been identified as a putative cell surface receptor for prions. Herein, we isolated the full-length coding sequence (CDS) of the ovine 37/67-kDa LRP/LR gene and submitted it to the GenBank under accession number EF649775. The open reading frame (ORF) of the 37/67-kDa LRP/LR CDS is 885 bp in length, containing six exons encoding a protein of 295 amino acids. The nucleotide sequence presented here is well coincided with the whole ovine genome of the 37/67-kDa LRP/LR previously published. Moreover, we identified four novel single nucleotide polymorphism sites (SNPs) at position 324 in exon 4, positions at 809, 875, and 881 in exon 7, respectively. Further, based on the deduced amino acid sequence alignment of the 37/67-kDa LRP/LR from human, cattle, mice, pig, chicken, and sheep, we also identified three polymorphic amino acid sites (PAAs) at residues 241, 272, and a novel site at residue 270 in the putative indirect prion protein (PrP) interaction region (180–285) on 37/67-kDa LRP/LR. Prediction of protein secondary structure further indicated that PAAs at residues 241, 270 and 272 may cause protein conformation changes as predicted, which may affect on the binding with prion protein. In addition, multiple-tissues RT-PCR results revealed that 37/67-kDa LRP/LR mRNA is expressed in all the 11 selected ovine tissues.     

In Vitro Inhibition of Angiogenesis by Antibodies Directed against the 37kDa/67kDa Laminin Receptor

The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.     

Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells

Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.     

67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism causing meningitis, and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for penetration into the central nervous system. We have reported previously that cytotoxic necrotizing factor 1 (CNF1) contributes to E. coli K1 invasion of HBMEC and interacts with 37-kDa laminin receptor precursor (37LRP) of HBMEC, which is a precursor of 67-kDa laminin receptor (67LR). In the present study, we examined the role of 67LR in the CNF1-expressing E. coli K1 invasion of HBMEC. Immunofluorescence microscopy and ligand overlay assays showed that 67LR is present on the HBMEC membrane and interacts with CNF1 protein as well as the CDPGYIGSR laminin peptide. 67LR was up-regulated and clustered at the sites of E. coli K1 on HBMEC in a CNF1-dependent manner. Pretreatment of CNF1+ E. coli K1 with recombinant 37-kDa laminin receptor precursor reduced the invasion rate to the level of Deltacnf1 mutant, and the invasion rate of CNF1+ E. coli K1 was enhanced in 67LR-overexpressing HBMEC, indicating 67LR is involved in the CNF1+ E. coli K1 invasion of HBMEC. Coimmunoprecipitation analysis showed that, upon incubation with CNF1+ E. coli K1 but not with Deltacnf1 mutant, focal adhesion kinase and paxillin were recruited and associated with 67LR. When immobilized onto polystyrene beads, CNF1 was sufficient to induce internalization of coupled beads into HBMEC through interaction with 67LR. Taken together, this is the first demonstration that E. coli K1 invasion of HBMEC occurs through the ligand-receptor (CNF1-67LR) interaction, and 67LR promotes CNF1-expressing E. coli K1 internalization of HBMEC.     

Characterization of the porcine multicopy ribosomal protein SA/37-kDa laminin receptor gene family

Prions represent a new class of infectious agents. The pathogenic prion protein (PrPSc) is known as the trigger of bovine transmissible spongiform encephalopathy (TSE). By contrast, an oral transmission of PrPSc and an ensuing infection seems to be blocked in non-ruminants such as pigs. Several investigations postulate that the ribosomal protein SA (RPSA) previously named 37-kDa laminin receptor precursor (LRP)/67-kDa laminin receptor (LR) is the candidate for binding and internalization of externally added cellular prion protein in the gut. We isolated a porcine ribosomal protein SA cDNA that consists of 1064 bp with an open reading frame of 885 bp encoding a 295 aa protein. The alignment of vertebrate ribosomal protein SA sequences displayed interspecies differences between cattle and pigs at positions 241 and 272 in the putative indirect PrP interaction site (aa 180-285) on RPSA. A PAC library screen revealed the existence of two processed ribosomal protein SA pseudogenes (RPSAP1 and RPSAP3) and of one non-processed pseudogene (RPSAP2). The pseudogenes have been assigned to SSC6 and SSC1 by hybrid panel analyses and FISH. Compared with the porcine cDNA 3, 7, and 13 insdels, 36, 25, and 57 single nucleotide exchanges and 6, 10, and 8 premature stop codons have been deciphered for RPSAP1, RPSAP2, and RPSAP3. In the 5', 3', and intron like regions, 2 (RPSAP1), 10 (RPSAP2), and 4 (RPSAP3) repeats have been detected. Basically, the repeats belong to one of the class/family LINE/L1, SINE/tRNA-Glu and DNA/MER1_type. We conclude that the pig genome contains multiple copies of the RPSA sequence probably as a consequence to maintain the multifunctionality of the mature protein.     

Identification of cellular proteins binding to the scrapie prion protein

The scrapie prion protein (PrPSc) is an abnormal isoform of the cellular protein PrPc. PrPSc is found only in animals with scrapie or other prion diseases. The invariable association of PrPSc with infectivity suggests that PrPSc is a component of the infectious particle. In this study, we report the identification of two proteins from hamster brain of 45 and 110 kDa (denoted PrP ligands Pli 45 and Pli 110) which were able to bind to PrP 27-30, the protease-resistant core of PrPSc on ligand blots. Pli 45 and Pli 110 also bound PrPC. Both Pli's had isoelectric points of approximately 5. The dissociation rate constant of the Pli 45/PrP 27-30 complex was 3 x 10(-6) s-1. Amino acid and protein sequence analyses were performed on purified Pli 45. Both the composition and the sequence were almost identical with those predicted for mouse glial fibrillary acidic protein (GFAP). Furthermore, antibodies to Pli 45 reacted with recombinant GFAP. The identification of proteins which interact with the PrP isoforms in normal and diseased brain may provide new insights into the function of PrPC and into the molecular mechanisms underlying prion diseases.     

Structural and functional analysis of the ovine laminin receptor gene (RPSA): Possible involvement of the LRP/LR protein in scrapie response

Scrapie is a prion disease affecting sheep and goats. Susceptibility to this neurodegenerative disease shows polygenic variance. The involvement of the laminin receptor (LRP/LR) in the metabolism and propagation of prions has previously been demonstrated. In the present work, the ovine laminin receptor gene (RPSA) was isolated, characterized, and mapped to ovine chromosome OAR19q13. Real-time RT-PCR revealed a significant decrease in RPSA mRNA in cerebellum after scrapie infection. Conversely, no differences were detected in other brain regions such as diencephalon and medulla oblongata. Association analysis showed that a polymorphism reflecting the presence of a RPSA pseudogene was overrepresented in a group of sheep resistant to scrapie infection. No amino acid change in the LRP/LR protein was found in the 126 sheep analyzed. However, interesting amino acid positions (241, 272, and 290), which could participate in the species barrier to scrapie and maybe to other transmissible spongiform encephalopathies, were identified by comparing LRP/LR sequences from various mammals with variable levels of resistance to scrapie.