Prion protein regulates glutamate-dependent lactate transport of astrocytes.

Prion-related protein (PrP) is a neural cell adhesion molecule involved in neurite outgrowth, neuronal survival, and synaptic function. In search of novel binding partners for PrP, we identified the alpha2/beta2-Na+/K+-ATPase and showed that this astroglial ATPase interacts directly with the immunoglobulin superfamily adhesion molecule basigin. In cultured astrocytes, PrP is involved in regulating lactate transport via the astroglial monocarboxylate transporter 1 (MCT1) and in conjunction with alpha2/beta2-ATPase and basigin. Lactate transport via MCT1 is glutamate dependent and regulated by glutamate receptor 2 (GluR2)-containing AMPA receptors with which PrP interacts. The functional interplay between PrP, GluR2, alpha2/beta2-ATPase, basigin, and MCT1 in regulating lactate transport of astrocytes may be functional in the metabolic cross talk between astrocytes and neurons, most likely under stress.

Epitopic characterization of native bovine beta-lactoglobulin.

Two monoclonal antibodies (mAbs) (mAb 97 and mAb 117) selected from a panel of 52 mAbs directed against beta-lactoglobulin (BLG) have previously been used to develop a two-site enzyme immunometric assay (EIA) specific for the native form of the protein [J. Immunol. Methods 220 (1998) 25]. In the present work, the conformational epitopes recognized by these two mAbs and by the 50 others have been studied. Firstly, an epitope map was drawn using a surface plasmon resonance (SPR) biosensor: the epitopes were organized in a circle of 11 overlapping and 1 nonoverlapping antigenic regions. Secondly, 55 site-directed BLGA mutants were prepared and tested by ELISA and competitive immunoassay to localize these 12 antigenic regions on the protein molecule. Among them, 20 mutants showed a 10- to 7500-fold decrease in relative affinity for the mAbs of one or several neighbouring regions: their circular dichroism (CD) spectra were identical to the spectrum of wild-type (WT) BLGA. At least one mutant was found for each of the 11 overlapping antigenic regions which circled the molecule and for the nonoverlapping one which was localized near the entrance of the calyx. The two mAbs initially chosen were each directed towards very conformation-dependent epitopes and were thus suitable for monitoring native BLG in food products and manufacturing processes. Other mAb pairs could be used to follow the fate of specific regions of the molecule during denaturation or proteolytic digestion.

Use of DNA immunization to produce polyclonal antibodies against the native human neurokinin-1 receptor for substance P.

Antibodies against the native form of the human NK1 receptor (hNK1R) for the neuropeptide substance P (SP), an important immunoregulator, are difficult to produce using classical immunization techniques. We show here that mice immunized with a plasmid harboring hNK1R cDNA developed antibodies recognizing extracellular epitopes of native hNK1R expressed on CHO cell membranes, as shown by FACS and immunofluorescence analysis, some antibodies being specifically directed against the second extracellular loop (E2) of the receptor. This original strategy, DNA immunization, thus efficiently generated new immunological tools to further analyse the role of SP in the regulation of immune cell functions.

Enhancement of antibody responses in DNA vaccination using a vector encoding a universal T-helper cell epitope.

DNA vaccination appears as a very promising approach to raise protective antibodies against a variety of proteins from pathogens or tumor cells, but is often hindered by the low immunogenicity of the genetic vectors used for the immunizations. To enhance the humoral response through improvement of the antigenic presentation of newly synthesized proteins upon vaccination, we engineered a plasmid coding for a low immunogenic protein (an scFv, i.e. the single-chain Fragment variable of a well-characterized antibody) fused to a small-size universal T-helper cell epitope derived from tetanus toxin, whose efficiency in classical protein-based immunization protocols has already been demonstrated. We found that immunization of C57Bl/6 mice using this vector greatly enhanced the production not only of specific antibodies recognizing essentially conformational epitopes on the undenatured scFv protein but also of antibodies against linear epitopes on the denatured protein. Since this T-epitope is known to be accommodated by several haplotypes of H-2 molecules in mice, as well as by various class II MHC molecules in humans, the results reported here allow us to conclude that this method could be of general interest for future applications of genetic immunization, including DNA-based vaccinations in humans.

Pharmacological properties of peptides derived from an antibody against the tachykinin NK1 receptor for the neuropeptide substance P.

Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.

Human prion diseases: from antibody screening to a standardized fast immunodiagnosis using automation.

Demonstration of pathological prion protein accumulation in the central nervous system is required to establish the diagnosis of transmissible subacute encephalopathies. In humans, this is frequently achieved using prion protein immunohistochemistry in paraffin-embedded tissue, a technique that requires multiple epitope retrieval and denaturing pretreatments. In addition to being time-consuming, this procedure induces tissue alterations that preclude accurate morphological examination. The aim of this study was to simplify prion protein immunohistochemistry procedure in human tissue, together with increased sensitivity and specificity. We screened a panel of 50 monoclonal antibodies produced using various immunogens (human and ovine recombinant prion protein, prion protein peptides, denatured scrapie-associated fibrils from 263K-infected Syrian hamsters) and directed against different epitopes along the human prion protein sequence. A panel of different forms of genetic, infectious and sporadic transmissible subacute encephalopathies was assessed. The monoclonal 12F10 antibody provided a high specificity and fast immunodiagnosis with very limited denaturing pretreatments. A standardized and reliable fast immunostaining procedure was established using an automated diagnostic system (Nexes, Ventana Medical Systems) and allowed prion protein detection in the central nervous system and in tonsil biopsies. It was evaluated in a series of 300 patients with a suspected diagnosis of transmissible subacute encephalopathies and showed high sensitivity and specificity.

Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells.

Prion diseases are transmissible neurodegenerative disorders affecting humans and animals for which no therapeutic or prophylactic regimens exist. During the last three years several studies have shown that anti-PrP monoclonal antibodies (mAbs) can antagonize prion propagation in vitro and in vivo, but the mechanisms of inhibition are not known so far. To identify the most powerful mAbs and characterize more precisely the therapeutic effect of anti-PrP antibodies, we have screened 145 different mAbs produced in our laboratory for their capacity to cure cells constitutively expressing PrPSc. Our results confirm for a very large series of antibodies that mAbs recognizing cell-surface native PrPc can efficiently clean and definitively cure infected cells. Antibodies having a cleaning effect are directed against linear epitopes located in at least four different regions of PrP, suggesting an epitope-independent inhibition mechanism. The consequence of antibody binding is the sequestration of PrPc at the cell surface, an increase of PrPc levels recovered in cell culture medium, and an internalization of antibodies. Taken together these data suggest that the cleaning process is more likely due to a global effect on the PrP trafficking and/or transconformation process. Two antibodies, Sha31 and BAR236, show an IC50 of 0.6 nM, thus appearing 10-fold more efficient than previous antibodies described in the literature. Finally, five co-treatments were also tested, and only one of them, described previously (SAF34 + SAF61), lowered PrPSc levels in the cells synergistically.

Cellular prion protein status in sheep: tissue-specific biochemical signatures.

Expression of the cellular prion protein PrP(C) is sine qua none for the development of transmissible spongiform encephalopathy and thus for the accumulation of the illness-associated conformer PrP(Sc). Therefore, the tissue distribution of PrP(C) at the protein level in both quantitative and qualitative terms was investigated. PrP(C) was quantified using a two-site enzyme immunometric assay which was calibrated with purified ovine recombinant prion protein (rPrP). The most PrP(C)-rich tissue was the brain, followed by the lungs, skeletal muscle, heart, uterus, thymus and tongue, which contained between 20- and 50-fold less PrP(C) than the brain. The PrP(C) content of these tissues seems to be comparable between sheep. Other organs, however, showed different, but low, levels of the protein depending on the animal examined. This was also the case for tissues from the gastrointestinal tract. The tissue containing the lowest concentration of PrP(C) was shown to be the liver, where PrP(C) was found to be between 564- and 16000-fold less abundant than in the brain. PrP(C) was concentrated from crude cellular extracts by immunoprecipitation using several monoclonal and polyclonal anti-ovine PrP antibodies. Interestingly, it was observed that the isoform profile of PrP(C) was tissue-specific. The most atypical electrophoretic profile of PrP(C) was found in the skeletal muscle, where two polypeptides of 32 and 35 kDa were detected.

Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein.

It is commonly assumed that the physiological isoform of prion protein, PrP(C), is cleaved during its normal processing between residues 111/112, whereas the pathogenic isoform, PrP(Sc), is cleaved at an alternate site in the octapeptide repeat region around position 90. Here we demonstrated both in cultured cells and in vivo, that PrP(C) is subject to a complex set of post-translational processing with the molecule being cleaved upstream of position 111/112, in the octapeptide repeat region or at position 96. PrP has therefore two main cleavage sites that we decided to name alpha and beta. Cleavage of PrP(C) at these sites leads us to re-evaluate the function of both N- and C-terminus fragments thus generated.

Heterogeneity and regulation of cellular prion protein glycoforms in neuronal cell lines.

The normal cellular prion protein is a small sialoglycoprotein highly expressed in neurons, the physiological function of which is largely unknown. Due to extensive N-glycosylations with a wide range of oligosaccharides, the prion protein displays a complex glycosylation pattern that could be of relevance for its function. The cellular prion protein patterns in adult mouse and rat brain, and in neuronal cell lines, appeared highly heterogeneous, as distinct levels and glycoforms of cellular prion protein were revealed by immunoblotting of corresponding samples. Amongst neuronal cell lines, mouse N2a neuroblastoma cells expressed low levels of endogenous prion protein. Mouse hypothalamic GT1-7 cells and rat pheochromocytoma PC-12 cells expressed highly glycosylated forms of cellular prion protein that were found neither in adult mouse and rat brain, nor in mouse brain during development. In contrast, rat B104 neuroblastoma cells abundantly expressed N-glycosylated cellular prion protein forms similar to those observed in mouse and rat brain. In all these cell lines, the prion protein was normally exported to and expressed at the outer cell membrane. Our results suggest that B104 cells may represent an appropriate cell model to investigate the physiological role of cellular prion protein in further detail as they highly express the normal 'brain-like' cellular prion protein glycoforms. In addition, we observed that the various prion glycoforms in B104 cells were tightly regulated both as a function of cell density and during neuronal differentiation, implying a potential role of cellular prion protein in cell-cell interactions and differentiation.

Ionizing radiation triggers chromatin-bound kin17 complex formation in human cells.

The human DNA-binding (HSA)kin17 protein cross-reacts with antibodies raised against the stress-activated Escherichia coli RecA protein. We show here that (HSA)kin17 protein is directly associated with chromosomal DNA as judged by cross-linking experiments on living cells. We detected increased amounts of DNA-bound (HSA)kin17 protein 24 h after gamma irradiation, with 2.6-fold more (HSA)kin17 molecules after 6 Gy of irradiation (46,000-117,000 molecules). At this time we observed that highly proliferating RKO cells displayed the concentration and co-localization of (HSA)kin17 and replication protein A in nucleoplasmic foci. Our results suggest that 24 h post-irradiation (HSA)kin17 protein may localize at the sites of unrepaired DNA damages. RKO clones expressing an (HSA)KIN17 antisense transcript (RASK.5 and RASK.13 cells) revealed that reduced (HSA)kin17 protein levels are correlated with a decrease in clonogenic cell growth and cell proliferation, as well as an accumulation of cells in early and mid-S phase. Taken together our observations support the idea that (HSA)kin17 protein is a DNA maintenance protein involved in the cellular response to the presence of DNA damage and suggest that it helps to overcome the perturbation of DNA replication produced by unrepaired lesions.

Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation.

The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein.

The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein.

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.

Selective and efficient immunoprecipitation of the disease-associated form of the prion protein can be mediated by nonspecific interactions between monoclonal antibodies and scrapie-associated fibrils.

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads.

KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis.

Genotoxic agents deform DNA structure thus eliciting a complex genetic response allowing recovery and cell survival. The Kin17 gene is up-regulated during this response. This gene encodes a conserved nuclear protein that shares a DNA-binding domain with the bacterial RecA protein. The KIN17 protein binds DNA and displays enhanced expression levels in proliferating cultured cells, suggesting a role in nuclear metabolism. We investigated this by studying the expression profile of KIN17 protein during mouse spermatogenesis. As expected, the expression level of Kin17 is higher in proliferating than in differentiated cells. KIN17 is selectively extracted from this tissue by detergents and a fraction was tightly associated with the nuclear matrix. Germinal cells ubiquitously express Kin17 and the protein is located mainly in the nucleus except in elongated spermatids where cytoplasmic staining is also observed. Sertoli and germ cells that are no longer mitotically active express KIN17, suggesting a general role in all testicular cell types. In adult testis a significant proportion of KIN17 co-purifies with polyadenylated RNA. KIN17 directly binds RNA, preferentially poly(G) and poly(U) homopolymers. These results together with the identification of KIN17 as a component of the human spliceosome indicate that this protein may participate in RNA processing.

The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa.

The prion-like Doppel protein (Dpl) has many biochemical and structural properties in common with the cellular prion protein (PrP(c)), and the physiological role of neither protein is known. Experimental data suggest either direct or indirect interaction between the two proteins. In this study, we investigated the expression pattern and biochemical characteristics of Dpl in human tissues and in Chinese hamster ovary cells transfected with wild-type or variant human Dpl gene constructs. Human Dpl appears to be a glycosylphosphatidylinositol-anchored glycoprotein with N- and O-linked sugars. It was found on Sertoli cells in the testis, on the flagella of epididymal and mature spermatozoa, and in seminal plasma. Dpl coexists only with N-terminally truncated isoforms of PrP(c) on mature spermatozoa. The localization of human Dpl on both Sertoli cells (somatic cells) and spermatozoa (germinal cells) strongly suggests that this protein may play a major role in human male fertility. Finally, our data indicate that spermatozoa are thus an interesting model for studies of the potential interaction between Dpl and PrP(c).

Global genome repair is required to activate KIN17, a UVC-responsive gene involved in DNA replication.

UV light provokes DNA lesions that interfere with replication and transcription. These lesions may compromise cell viability and usually are removed by nucleotide excision repair (NER). In humans, inactivation of NER is associated with three rare autosomal recessive inherited disorders: xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. The NER earliest step is lesion recognition by a complex formed by XPC and HHR23B proteins. In a subsequent step, XPA protein becomes associated to the repair complex. Here we investigate whether XPA and XPC proteins, involved in global genome repair, may contribute to a signal transduction pathway regulating the response to UVC-induced lesions. We monitored the expression of several UVC-induced genes in cells deficient in either a transduction pathway or mutated on an NER gene. Expression of the KIN17 gene is induced after UVC irradiation independently of p53 and of activating transcription factor 2. However, in human cells derived from XPA or XPC patients the UVC-induced accumulation of KIN17 RNA and protein is abolished. Our results indicate that the presence of functional XPA and XPC proteins is essential for the up-regulation of the KIN17 gene after UVC irradiation. They also show that the integrity of global genome repair is required to trigger KIN17 gene expression and probably other UVC-responsive genes.

The cellular prion protein PrPc is expressed in human enterocytes in cell-cell junctional domains.

The physiological function of PrPc, the cellular isoform of prion protein, still remains unclear, although it has been established, in vitro or by using nerve cells, that it can homodimerize, bind copper, or interact with other proteins. Expression of PrPc was demonstrated as necessary for prion infection propagation. Considering the importance of the intestinal barrier in the process of oral prion infectivity, we have analyzed the expression of PrPc in enterocytes, which represent the major cell population of the intestinal epithelium. Our study, conducted both on normal human intestinal tissues and on the enterocytic cell line Caco-2/TC7, shows for the first time that PrPc is present in enterocytes. Interestingly, we found that this glycosylphosphatidylinositol-anchored glycoprotein was localized in cholesterol-dependent raft domains of the upper lateral membranes of enterocytes, beneath tight junctions, in cell-cell junctional domains. We observed that PrPc, E-cadherin, and Src co-localized in adherens junctions and that PrPc was co-immunoprecipitated with Src kinase but not with E-cadherin. Alteration of cell polarity after cholesterol depletion or loosening of the cell-cell junctions after EGTA treatment rapidly impaired membrane targeting of PrPc. Overall, our results point out the signaling of cell-cell contacts as a putative role for PrPc in epithelial cells.