Laminin Receptor
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Two possible receptors found for prion protein: Lancet
Will discovery of prion receptors lead to therapy: Nature Medicine
Abstracts released 6 days after press release
Laminin binding and Alzheimer
Clue to possible strategy to block mad-cow disease: NY Times
Backgrounder on laminin receptor: webmaster
Mutations in Fanconi anemia gene similar to prion
Red deer and camel prion sequences updated
Antibodies distinguishing sheep alleles
INSERM Meeting?
Are prion deletions a CJD risk?
CJD special issue of Canada Med Assoc J

Two possible receptors found for prion protein

11.28.97 Lancet Jane Bradbury

Two groups report this week that they may have found a cellular receptor for prion protein. The groups have homed in on different proteins but this advance may still be an important step towards a cure for nvCJD.

Prions, the infectious agents that cause transmissible spongiform encephalopathies including nvCJD, act by entering cells and causing a conformational change in the cellular prion protein, probably in the cell's endosomes. Prion internalisation seems to need a receptor.

Ricardo Brentani (Ludwig Institute for CancerResearch, Sao Paulo, Brazil) and colleagues used a technique known as complementary hydropathy to generate a peptide that they hoped would mimic that part of the receptor responsible for prion binding (Nat Med 1997; 3: 1376-82). Antibodies raised against this peptide recognise a 66 kDa protein on the surface of mouse neurons. In addition, the antibodies and the peptide itself can prevent a neurotoxic peptide derived from human PrP killing neurons in culture. "Such reagents might therefore have therapeutic applications", say the researchers.

Stefan Weiss (Laboratorium fuer MolekulaireBiologie-Genzentrum Institut f¸rBiochemie der LMU M¸nchen, Munich, Germany) and co-workers used a technique known as the yeast two-hybrid screen to demonstrate that PrPC interacts with a 37 kDa laminin receptor precursor. The scientists report that the amount of this protein correlates with abnormal PrP accumulation in the brain, spleen, and pancreas of scrapie-infected mice and hamsters. In addition, the researchers show that the laminin receptor precursor is present on the surface of neuronal cells where PrPC is located and they speculate that the protein could act as a receptor for PrP on mammalian cells (Nat Med 1997;3: 1383-88).

Discovery of prion receptors may lead to therapy

December 1997 Nature Medicine
Prion diseases such as Creutzfeldt-Jakob, bovine spongiform encephalopathy (BSE or `mad cow disease') and scrapie occur when a normal cellular protein known as PrPC is converted into an abnormal disease-causing protein, PrPSc. Two papers in the December issue of Nature Medicine report the discovery of possible cellular receptors thought to be necessary for this transformation process.

Using a technique called complementary hydropathy to develop a peptide that binds to PrPC, a team of Brazilian researchers has developed a protein that recognizes a distinct site on mouse neurons which is thought to be the transforming receptor. The peptide and serum from mice injected with the peptide, were able to block neurotoxic effects of PrPSc, suggesting a possible therapy for prion diseases.

In a second paper, a German group led by Stefan Weiss, has identified a laminin receptor precursor protein that interacts with PrPC, which they believe to be the transforming receptor. They show that the receptor is found in all the organs associated with prion propagation and that levels of the receptor rise as disease-causing protein, PrPSc, accumulates in animal tissues.

In an accompanying photo story it is explained that these two receptors are distinct proteins and although it is not yet possible to conclude that either one is the physiological receptor for PrPC, insight into the binding and behavior of the mysterious protein is an important advance.

The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells

Nature Medicine Vol 3 Number 12 - December 1997 
[Puzzling over prion partners ...commentary by Alison Mitchell page 1312 is offline]  
Roman Rieger, Frank Edenhofer, Corinne Ida Lasmezas & Stefan Weiss
Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies. According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPc into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs. 1‚3).

Although the physiological role of PrPc is poorly understood, studies with PrP knockout mice demonstrated that PrPc is required for the development of prion diseases5. Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPc. [Nine clones, or 20%, carried LRP, apparently all pseudo-gene versions. -- webmaster]

Mapping analysis of the LRP‚PrP interaction site in S. cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP. [The authors did not characterize which part of the prion molecule is involved in binding. LRP domains were characterized in JBC 266:20440-6 (1991)-- webmaster]

The LRP‚PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7).

The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice. In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation. LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of scrapie-infected hamsters.

These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters. The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.

[The authors observe that LRP is a known receptor for Sindbis virus on mammalian cells, citing J.Virol 66:4992-5001 (1992)

Complementary hydropathy identifies a cellular prion protein receptor

Nature Medicine Vol 3 Number 12 - December 1997 
Vilma R. Martins, Edgard Graner, JosÈ Garcia-Abreu, ... Vivaldo Moura Neto & Ricardo R. Brentani 
Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. [The authors assert 40 examples of this form of lignad characterization in cites. The drawback is gene identification: small amounts of protein must be sequenced, whereas the yeast method yields DNA to be sequenced. -- webmaster

Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. [However, only 6% of the binding activity corresponded to this. The rest was attributed to a 60k cytoplasmic rotein that was not characterized further. -- webmaster] Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide [namely 106-126] toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.

Clue discovered to possible strategy to block mad-cow disease in brain

November 25, 1997 The New York Times SANDRA BLAKESLEE [correspondent summary]
NEW YORK-- A story about the papers in the December issue of Nature Medicine by researchers in Brazil and Germany, who say they have found receptors on the surface of brain cells that may allow prions to enter the cells.

The findings suggest that when prions latch on to these brain-cell receptors, the infectious proteins work their way into the interior of the cells and eventually destroy them. If the discoveries hold up, the researchers were cited as saying, it may be possible to treat or halt prion diseases by blocking the action of the receptors.

Dr. Patrick Bosque, a prion expert and neurologist at the University of San Francisco School of Medicine, was cited as saying that the reports were interesting but that the techniques used to find the receptors had many problems that could lead to false results, adding, "It's not at all clear that their findings are relevant."

The Brazilian researchers, led by Dr. Ricardo Brentani at the Ludwig Institute for Cancer Research in Sao Paulo, found their receptor using a technique called complementary hydropathy. They identified a protein fragment from a prion that appears to be particularly toxic to brain cells in culture and reasoned that it might play an important role in disease.

By making an artificial copy of the fragment, they were able to find a receptor site on normal brain cells that binds the prion fragment. The German group, led by Dr. Ernst Winnacker, director of the Gene Center at the University of Munich, used a technique called a yeast two hybrid screen to identify proteins that interact with prions. They found one, identified as laminin receptor precursor, that is also found on brain cell membranes. In animal studies, they found that the laminin receptor increased along with infectious prion proteins.

The researchers stressed that it was impossible to say whether either of these molecules was the critical receptor for prion infection. But it makes sense to look for such molecules, given that prions would need a way to get into brain tissue.

Commentary on laminin receptors

1 Dec 97 Webmaster commentary and review
"25 Nov 97 NY Times....Dr. Patrick Bosque, a prion expert and neurologist at the University of San Francisco School of Medicine, was cited as saying that the reports were interesting but that the techniques used to find the receptors had many problems that could lead to false results, adding, "It's not at all clear that their findings are relevant."

Hmmm. Other people would say that the yeast two-hybrid reporter system is quite solid, used by hundreds of labs, with many independent verifications [924 medline cites]. Curious coincidence too that Brentani's unidentified protein is 66k whereas Weiss' mature laminin receptor protein (LRP) is 67k. (Don't be fooled by the smaller 37k precursor.) In an odd twist, Brentani worked quite a bit on laminin-alpha 6 beta1 integrin in past years. Did two labs using very different methods fish out same prion-binding protein? Brentani could test this quickly using anti-LRP in a western blot.

Who is Bosque anyway and what is the agenda? This would be the PJ Bosque with a single publication [all topics] back in 1992 . That publication is fine, a clinician-type foray into sequencing, 5 doctors run 2 gels, no experiments. They liked their data so much that they published it twice. I am not seeing any demonstated expertise in eukaryotic membrane receptors nor the techniques used to study them. Certainly not taught at any US medical school or learned in neurology residency. Possibly some carry-forward from neurotransmitter work but I don't see representation there either.

Nature Medicine has not actually published its December issue as of 5 days past the Times' story (nothing online as of Dec 1 GMT) and does not publish full text in any event nor fax it to reporters. The fledgingly journal is not of the same caliber as Nature itself. Neither abstract has appeared at Medline.

So where did Sandra Blakeslee of the NY Times get Bosque's phone number, why would she think that he had access to full text and had already read both articles, who represented him to her as an expert on this subject amongst all the hundreds who have actually published in this area? Cannot have been Nature Medicine that put his name on the bottom of the press release -- they would have hand-picked someone who would comment favorably.

My working hypothesis: Blakeslee got a press release, called up the Prusiner lab reasonably enough for comment, someone there had reviewed both papers many months ago, the phone got handed to Bosque who has perhaps started on a similar project, they were upset at getting scooped, or were concerned about the status of prion binding proteins the lab had recently reported in an obscure journal.

What a nightmare if laminin receptor turns out to be "protein X" -- people would naturally just cite Weiss et al and Brentani et al. Could this be the agenda, preventing a 'misconception' about priority from developing?

Stefan Weiss and Ernst L Winnacker are known entities -- top-drawer molecular biologists with a hundred publications, including creative and cutting-edge studies relevent to the present endeavor.

However, on their last go-round with prions and yeast two-hybrid screen, I recall them in J Virol 1996 70(7):4724-4728 pulling out just the molecular chaparone heat shock protein 60. Some people pooh-poohed the signficance of this, saying HSP60 binds anything and everything, as befits a chaparone. However it was localized specifically to residues 180-210 of the prion molecule, though the method was anaemic compared to the 2-step regilonal identification process used in the recent Oesch 15B3 paper.

But where was laminin receptor precursor in this earlier fishing expedition and why is it just 'surfacing' now?

Laminin itself is not exactly an obscure protein. It is a basement membrane component invaded as the first step in tumor formation, accounting for its 8,356 cites; it mediates interactions with cell surface receptors such as alpha 6 beta 1 integrin and cell-matrix interactions generally. It is comprised of B1 and B2 chains 220k each associating via disulphides with a 440k chain in an asymmetric cross-structure.

Laminin receptor precursor (LRP) is such a can of worms that it left me wondering exactly what molecular species is supposed to bind prions:

"This molecule appears to be very peculiar since so far only a full-length gene encoding a 37 kD precursor protein has been isolated and the mechanism by which the precursor reaches the mature form is not understood.... acylation of the precursor is the key mechanism in maturation of the 67 kD form... Interactions are mediated by different cell surface molecules, including the monomeric 67 kD laminin receptor. The function of the membrane receptor is to stabilize the binding of laminin to cell surface integrins, acting as an integrin-accessory molecule, although homology of the gene encoding the receptor precursor with other genes suggests additional functions...." J Cell Biochem 1997 Nov 1;67(2):155-165
"Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2... permitted us to isolate the active 37LRP/p40 human gene. This gene contains seven exons and six introns. Ribonuclease protection experiments suggest multiple transcription start sites. The promoter area does not bear a TATA box but contains four Sp1 sites. The first intron is also GC rich containing five Sp1 sites. Intron 4 contains the full sequence of the small nuclear RNA E2 and two Alu sequences are found in intron 3." Biochim Biophys Acta 1996 Feb 7;1305(1-2):98-104
"To date, the active 37LRP/p40 gene has never been identified in any species due to the existence of multiple pseudogenes in most vertebrates genomes. The chicken genome was selected to undergo this characterization because it is the only known vertebrate that bears a single 37 LRP/p40 gene copy. The 37 LRP/p40 active gene is composed of 7 exons and 6 introns and bears features characteristic of a ribosomal protein gene ... precursor of the metastasis-associated 67-kD laminin receptor (67LR) and as a cytoplasmic ribosomal-associated protein.

On the basis of gene structure and extensive protein evolutionary study, we found that the carboxyterminal domain of the protein is a conserved lock-and-key structure/function domain that could be involved in the biosynthesis of the higher-molecular-weight 67-kD laminin receptor in vertebrates, whereas the central core of the protein would be responsible for the ribosome associated function" DNA Cell Biol 1996 Dec;15(12):1009-1023

"The yeast genes, designated YST1 and YST2, encode proteins with over 95% amino acid sequence identity with one another and over 60% identity with the human p40/laminin receptor precursor. "
U43901 on chromosome 3p21.3; J02870; X94368, and U33756 are the relevent GenBank entries.

RR Brentani has surfaced out of "nowhere" with the N171S human prion allele in Nature last week and now this. The idea with "complementary hydropathy" is that the mold of a mold has the same shape as the original object. So, starting with a prion, anything that docks with it would have a 'complementary' surface in the chemical sense. An antibody to that would look again like the prion surface. Except now the antibody can be used to pull out the unknown docking protein, whereas the prion couldn't.

The problem is that there really isn't a reliable prescription for taking a primary sequence and determining from it what the 'complementary' surfaces should look like. The complementary hydropathy scheme based on the anti-gene sequence is less than persuasive, yet sometimes it has given results, due to the duality structure of the genetic code vis-a-vis hydrophobicity. That is evidently what happened here, they are reporting recovering something with some interesting properties.

Brentani RR, et al.
Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach.
Proc Natl Acad Sci U S A. 1988 Jan 1; 85(2): 364-367.
"It has been shown that a significant correlation is seen when the hydropathy scores of amino acids encoded by the coding strand of double-helical DNA are plotted against those of the noncoding strand. Thus, peptides encoded by complementary DNA strands might form amphiphilic structures and bind one another. "
Determination of the putative binding site for fibronectin on platelet
glycoprotein IIb-IIIa complex through a hydropathic complementarity approach.
J Biol Chem 1989 Aug 25;264(24):14566-14570
Pasqualini R, Chamone DF, Brentani RR
"We have applied the principle of complementary hydropathy to the prediction of the binding site for fibronectin (FN) and for the alpha-chain of fibrinogen in the platelet receptor complex glycoprotein"
A monoclonal autoantibody against a complementary peptide recognizes interstitial collagenase.
Immunomethods 1994 Oct;5(2):172-176
de Souza SJ, Madaio MP, Juliano Neto L, Brentani RR
"A monoclonal autoantibody (mab 16) against the complementary peptide TKKTLRT, which was deduced from the collagenase-sensitive site in collagen, is described. "

There are also a large number of laminin and alpha6 beta1-integrin with an RR Brentani as author, such as:

Functional hypotheses for aberrant glycosylation in tumor cells.
Braz J Med Biol Res 1994 Feb;27(2):505-507
Chammas R, Jasiulionis MG, Cury PM, Travassos LR, Brentani RR
"... Our group has been focusing on the role of glycosylation in cell:matrix interactions, such as adhesion, spreading and migration on defined substrata (e.g., laminin and fibronectin). Animal lectins, such as galaptins, also seem to be involved in these processes.

Having now seen the full texts, I could agree with Bosque that a lot of work could remain in demonstrating relevence. Perhaps LRP is sticky and binds lots of proteins. Does it bind to prion protein in vivo and what would be the functional point? Is this to be a hetero-oligomer, with prion protein modulating the normal cell adhesion role of LRP? Does this have anything to do in getting LRP from 37k to 67k? Or is LRP envisioned as some sort of phagocytotic scavenger involved in uptake of bad extra-cellular proteins, such as exogenous rogue conformer? Correlations in cellular distributions and amounts of LRP might be supportive but are hardly definitive. Curious if 106-126 region is in fact the binding site as it has quite a history. Be interesting to see what becomes of this -- there has been quite a history of prion-other protein interactions that have gone nowhere.

1a.  Vnencak-Jones CL, et al.
Am J Hum Genet. 1992 Apr 1; 50(4): 871-872.

1b.  A PrP gene codon 178 base substitution and a 24-bp interstitial deletion in familial CJD.
Neurology 1992 Oct;42(10):1864-1870
Bosque PJ, Vnencak-Jones CL, Johnson MD, Whitlock JA, McLean MJ

Laminin binding and Alzheimer

Prog Neurobiol 1996 Dec;50(5-6):493-503
"Apolipoprotein E might be involved in maintaining synaptic integrity after injury and during aging. ApoE might help maintain the integrity of the synaptodendritic complex if it: (1) stabilizes the neuronal cytoskeleton; (2) plays an important role in transporting esterified cholesterol to neurons undergoing reinnervation where it is taken up by the low density lipoprotein receptor-related protein pathway and used as a precursor for the synthesis of new synaptic terminals; (3) regulates interactions between neurons and the extracellular matrix (e.g. laminin); and (4) regulates levels of intracellular calcium. "

Identification of candidate proteins binding to prion protein.
Neurobiol Dis 1997;3(4):339-355  [totally offline journal]
Yehiely F, Bamborough P, Da Costa M, Perry BJ, Thinakaran G, Cohen FE, Carlson GA, Prusiner SB
" To search for PrPC binding proteins, we fused PrP with alkaline phosphatase (AP) to produce a soluble, secreted probe. PrP-AP was used to screen a lambda gt11 mouse brain cDNA library, and six clones were isolated. Four cDNAs are novel while two clones are fragments of Nrf2 (NF-E2 related factor 2) transcription factor and Aplp1 (amyloid precursor-like protein 1). The observation that PrP binds to a member of the APP (amyloid precursor protein) gene family is intriguing, in light of possible relevance to Alzheimer's disease. Four of the isolated clones are expressed preferentially in the mouse brain and encode a similar motif."
J Psychiatry Neurosci 1996 Mar;21(2):128-134
"The coordinated expression of apoE and its main receptor, the apoE/apoB (LDL) receptor, appears to regulate the transport of cholesterol and phospholipids during the different phases of the reinnervation process."
Exp Neurol 1995 Dec;136(2):251-257
"The extracellular matrix protein laminin profoundly affects neuronal adhesion, spreading, differentiation, and growth by binding integrin-type cell surface receptors. Laminin binds other basement membrane components, including heparan sulfate proteoglycans. Apolipoprotein E (apoE) also binds basement membrane and heparan sulfate proteoglycans and colocalizes with s-laminin in the neuromuscular junction. We sought to determine whether apoE interacts with laminin and whether, as a result, apoE alters the regulation of neuronal adhesion and differentiation by laminin. We demonstrate high-avidity interaction between apoE and laminin in vitro."
Proc Natl Acad Sci U S A 1992 Apr 1;89(7):3040-3044
"Peptides from the three type I repeats of human endothelial cell thrombospondin, containing the consensus sequence -Trp-Ser-Xaa-Trp-, bind to sulfated glycoconjugates including heparin and sulfatide. The peptides are potent inhibitors for the binding of thrombospondin, laminin, or apolipoprotein E to these ligands... thrombospondin sequence containing the previously identified adhesive motif Val-Thr-Cys-Gly inhibit heparin-dependent binding of thrombospondin or laminin to human melanoma cells."

Sequence variation in the Fanconi anemia gene

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13051-13056, November 1997 
Orna Levran, Tamar Erlich, ... Arleen D. Auerbach
The gene for Fanconi anemia (FAA) encodes a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function. We screened genomic DNA from 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry. A total of 85İvariant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common. . Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. Slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

The close association of short direct repeats and homonucleotide tracts with microdeletions/microinsertions as seen in FAA has been reported to be common in human genes, including cystic fibrosis transmembrane conductance regulator, beta-globin, factor IX, hypoxanthine phosphoribosyltransferase, and many cancer-related genes including p53, retinoblastoma, adenomatous polyposis coli, Wilms tumor, and breast cancer (BRCA1). Such deletions also are induced by mutagens. The repeat units that are associated with microdeletions usually are between 2İand 8İbp. These deletions/insertions can be explained by a slipped-strand mispairing mechanism initially proposed by Streisinger et al., in which one DNA strand of a repeat can be misaligned by chance with the downstream repeat of the complementary strand. The resulting loop subsequently is excised, fixing the deletion before the next round of replication. This slippage-misalignment mechanism has been proposed to be a ubiquitous mechanism of mutagenesis and is responsible for a significant proportion of mutations in mammalian cells. The FAA coding sequence and flanking intronic sequences contain many such hypermutable repetitive sequences.

Our results show that the single bp substitutions identified in FAA are associated with three major motifs: CpG motifs (20%), homonucleotide tracts (20%), and short direct repeats (33%); the remainder of the point mutations do not appear to fit into any of these categories. DNA methylation is considered responsible for bp substitutions at CpG sites, and it was shown that CpG mutation hot spots in BRCA1 (26), NF1 (30), and p53 (31), for example, are methylated. Further study is needed to learn if CpG sites associated with mutations in FAA also are methylated. We propose that misalignment-mediated errors during DNA synthesis and methylation-induced mutagenesis account for the majority of the mutations described in this analysis.

A variety of mutation hot spot consensus sequences have been reported in the literature. The sequence CCTG (CAGG), first identified as a homologous recombination hot spot in the murine major histocompatibility complex (32), has been observed to be a mutation hot spot in a large number of human genes (19), especially when it occurs near direct repeat sequences (17). We have found this motif in the vicinity of many of the mutations, both microdeletions/microinsertions and point mutations, described in this study. The TTC repeat motif, described in the Chinese hamster APRT gene as a hot spot for spontaneous deletions also was identified near several of the microdeletions/microinsertions in FAA

Red deer and camel prion sequences updated

2 Dec 97 webmaster 
Cervus elaphus and camel went up on GenBank as Y09761 and Y09760 dated Nov 30, 1997, which might represent some editing changes as the sequences have been out for a while.

In the associated full text, Gene 199 (1-2), 283-286 (1997), they studied at 15 unrelated red deer. There was one heterozygous animal with a point substitution M208I (CVNITVKQHTVTTTTKGENFTETDIK M MERVVEQMCIT) and one silent G to C at codon 63. M208I is a common change seen in many species such as goat, addax, rabbit, and marsupial and is M205 in human numbering, so not a CJD site. Red deer, though farmed in England, have not acquired scrapie, BSE, or chronic wasting disease; their sequence would hardly seem to suggest a species barrier.

Fallow deer sequence [Dama dama] is supposed to be released soon.

Antigenic features of prion proteins of sheep and of other mammalian species.

J Immunol Methods 1997 Aug 22;207(1):89-101
Groschup MH, Harmeyer S, Pfaff E
Pathological prion protein (PrPSc) which is a conformational isoform of a host-encoded protein designated (PrPC) serves as a specific marker protein for the immunochemical diagnosis of transmissible spongiform encephalopathies (TSE). The generation of suitable antibodies to PrPSc therefore underlies the specificity and sensitivity of diagnostic assays. However, most antibodies reported to date are directed to a limited number of epitopes only.

PrPC is a highly conserved cell membrane protein in all mammalian species studied to date. In an attempt to generate antibodies to further regions of PrP we raised antisera in rabbits and chicken against sixteen synthetic peptides which represent the complete aminoacid sequence of ovine PrP. By this approach immunotolerance was overcome and immunoblot-reactive antibodies were stimulated to epitopes at almost any site of ovine PrPC and PrPSc. A large number of different antibodies cross-reacted also with affinity-purified PrPCs from other mammalian species including cow, goat, pig, man, dog, cat, mink, mouse, hamster and guinea pig.

No epitope, however, was recognized exclusively on the pathological or cellular isoform of PrP indicating that both isoforms occur in highly denatured conformations on the immunoblots. Antibodies to the amino-terminus are suitable for immunoprecipitation of PrP. The availability of rabbit and chicken anti-peptide antibodies to PrP will greatly improve immunochemical diagnosis and pathogenetic studies on these diseases.

Allele identification using immobilized mismatch binding protein: detection and identification of sheep susceptibility to scrapie.

Nucleic Acids Res 1997 Dec 1;25(23):4825-4830 
Debbie P, Young K, Pooler L, Lamp C, Marietta P, Wagner R
A novel method for detection and identification of specific alleles has been developed utilizing immobilized mismatch binding protein (IMBP). The assay involves the use of biotin-labeled probes, which are prepared by PCR amplification of cloned fragments with known sequence. The use of probes avoids many of the problems associated with the extreme sensitivity of IMBP assays to errors in PCR amplification. The method can be used to monitor PCR fidelity and to genotype both diploid and haploid organisms and has been used to detect and distinguish two alleles of the sheep prion protein gene involved in susceptibility to scrapie.

Molecular mechanisms of infection and pathology in prion diseases

16-20 November 1997  Organised by Insitute National de la Sante et la Recherche Medicale as one of the Philip Laudat
INSERM Meeting
Steve Dealler writes:
This seems to be a major conference and has all the major speakers from around the world on the subject. I would say that by the end of September it is almost impossible to even get in contact with the people organising the conference and the web site appears to have been removed. The inner circle? Various groups have found it impossible to come to the meeting in France on the 16-20th of November. 'Full up' is the reason that has been given but this is not at all clear and worry is appearing that certain researchers are simply not wanted.
Application closing date 28 April 1997 Further information not available: Web site is dead.
Conferencess Philippe Laudat-Inserm
101 rue de Tolbiac
75654 Paris Cedex 13 France

Are prion deletions a CJD risk?

Tue, 2 Dec 1997 webmaster

Like with the N171S Brazilian allele, it is experimentally difficult to resolve the "neutrality" issue of the shorter repeat insertions and deletions. Cervenakova et al made a really excellent contribution to the deletion situation by carefully sequencing a series of 718(!) patients and controls; the article has only appeared so far in a symposium proceedings [ref 1]. The preceding article has a useful compilation of insertion by Goldfarb et al, who rule out 'anticipation' (ever-younger generational onset of poly-gln CAG disorders) for prion insertion disease.

They found a new fifth type of single deletion, R2c R3-R4 which has an R3R4 type deletion plus a CCT to CCC transition at codon 68 in R2. Deletion end points can only be specified up to an ambiguity zone determined by sequence internal repeats (complementarity). The deletion event is sometimes accompanied by a 'point mutation' that appears to be simple remnant of the DNA slippage mechanism rather than a replication error. To account for the point mutation here, the new deletion would represent a re-start slippage event on the template strand.

I should point out that Alex Bosser's earlier grievance with Swiss mouse prion numbering originates with a repeat deletion associated with smaller length scale triplet TGG repeats (poly-glycine) within the main repeat. This is another very unstable region of the prion gene; similar changes are found in many phylogenetic lineages (as with single octapeptide indels). There can also be insertions/deletions of a run of a single nucleotide provided a frameshift does not result. DNA slippage accounts for these events at all length scales.

These deletions are showing up in some odd diagnostic backgrounds such as Machado-Joseph, Unvericht-Lundborg-Lafora, presenilin-1 Alzheimer, atypical dementia, speech disorders, and in conjunction with D178 and E200K CJD, as well as sporadic, iatrogenic, and conjugal CJD. Codon 129 met/val affects outcomes and makes it hard to collect sufficient data. Prion repeat deletions thus "may predispose to spongiform encephalopathy or affect the phenotypic expression of other neurological conditions." The authors say they are "currently at an early stage of data accumulation." As I read the paper, the sequencing focus was 5', so the data doesn't all go into the pool for determining population incidence of N171S.

If only a specific mutation is sought, the primer and detection situation can be optimized. In other diseases, it is nothing to see 200,000 people screened. In prion diseases, this wouldn't be so informative if the clinical side is lost; no single mutation is of overriding concern.

The fact that 1-2% of the human population has an octapeptide deletion in the repeat region doesn't by itself make this a neutral allele. Deletion of two repeats has never been observed even though the same deletion mechanism could generate these at good frequencies; this should not be lethal in utero given that a knockout of both alleles is not, yet the patient class might be very young and not part of the usual senior adult dementia series. Here, they safely conclude that the data provide "a reasonable doubt that the deletions are not as neutral as sometimes regarded."

Single octapeptide insertions have only been seen once [ref 2], an extra R2. Almost every insertion is different and equally likely, after adjusting for distant familial relationships, unlike with deletions. As with double insertions, there is a weak association with mental disturbance. Correlation isn't causality, of course. No one has gone to transgenic rodent models; the lifespan may be too short and effects too subtle for this to yield results.

There is a superb online review of the full spectrum of causes and frequencies of human polymorphisms in the 25 Nov 97 PNAS, except that the article is about Franconi's anemia (and its 43 exons). Just replace FAA with CJD using a text editor and everything will be fine.


1. Transmissible Subacute Spongiform Encephalopathies: Prion Diseases;
L.Court, B.Dodet, eds., Elsevier, Paris 1996, pg 433-444.

2. JL Laplanche et al., Hum Mol Gen 1995 4:1109-1111

CJD special issue of Canada Med Assoc J

3 Dec 97 CMAJ Full text is online, English or French.
     Ricketts MN. İİİİİİİ 
       Is Creutzfeldt-Jakob disease transmitted in blood? Is the absence of evidence of risk
       evidence of the absence of risk? 
       CMAJ. 1997 Nov 15; 157(10): 1367-1370. 

     Stratton E. İİİİİ 
       Creutzfeldt-Jakob disease: the Canadian situation. 
       CMAJ. 1997 Nov 15; 157(10): 1405-1408.  

     Caulfield T, et al. İİİ 
       Notifying patients exposed to blood products associated with Creutzfeldt-Jakob
       disease: integrating science, legal duties and ethical mandates. 
       CMAJ. 1997 Nov 15; 157(10): 1389-1392. 

     Cashman NR. İİİİİ 
       A prion primer. 
       CMAJ. 1997 Nov 15; 157(10): 1381-1385. 

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