Prion and Doppel Gene Annotation
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Artefacts: 4-hydroxyproline at Pro 44, polypro II pre-repeat helix: Error Alert
3D modelling of prion glycans and GPI anchor: helix A left for dimer
Warwicker pdb model of domain-swapped dimer
6 new human prion mutations
Another case of intergenic splicing on human chr 1
Prion incubation time QTL in mouse
Iatrogenic CJD: the ultimate review article
PrPC in peripheral nervous system: neuroinvasion
Prion protein colocalizes with dystroglycan complex in brain
Diagnostics: tonsils, third eyelids, and plasminogen

Modelling of prion glycans and GPI anchor: helix A left for dilmer

Glycobiology 2000 Oct 1;10(10):959-974
J. Zuegg and J. Gready
(Color graphics not in the paper but kindly provided by the authors were recast here for the web)
Comment (webmaster): This is an excellent paper. Prior nmr structural determinations were all done on prion protein missing 5 known covalent modifications, notably the glycans and GPI, and in the absence of repeat metals. The two large, negatively charged glycans are too flexible and mobile to be resolved in any event with nmr. This leaves unresolved the nasty question of what the native structure really is: the technology may never be there to visualize structures of highly mobile constituent sugars.

However, computer modelling (here Amber force field, mainly electrostatic) has demonstrably improved to the point where investigators experienced in molecular dynamics can make informed suggestions, if not provide final answers. It helps of course to have a good baseline nmr structure of the globular prion domain. The basic conclusions of the 15-page paper are:


(1) the two glycans (here modelled nearly fully built-out with 3 sialic acids) do not interact that much with specific amino acid side chains nor shield them from solvent (except F198 and T199), though large patches of protein surface are assuredly shielded from any conceivable protein-protein interaction by these large glycan fur-balls; a stabilization effect of glycans, notably at the N197 loop, is attributed to solvent immobilization;

(2) the GPI anchor does not have much interaction with side chains either, mainly affecting terminal residues of helix C, and serving mainly to position the terminal serine perhaps 12 angstroms from the membrane bilayer, and orienting the protein either in the "lying down" with helix A facing the membrane or more plausibly "standing" with the GPI linearly extending helix C and the glycans facing outward;

(3) the standing orientation allows formation of a membrane-bound dimer created by 4 salt bridges in the anti-parallel alignment of helix A (which is about all that is left exposed in the diglycosylated form), a structure anticipated earlier by Warwicker and Morissey and Shaknovich [PNAS 96 11293-11298 1999], but not favored by general dimer theory (upside-down anti-parallel) or recent experiment, though the authors provide 5 literature citations to in-situ GPI multimers;

(4) recomputation of the electrostatic surface replaces the irrelevant published picture of recombinant protein (though uncertainty in the multiply positively charged mature amino terminus still leaves the situation unresolved). Only helix A is exposed; the main salt bridges are still intact in the diglycosylated form, namely E146-R208, R164-D178, and R156-E196; the middle one is disfavored slightly (less occupied) in the full protein.

Note D178N, R208H, and especially E196K cannot form this salt bridge and are CJD-causative (coincidence?). There are also helix A salt bridges H140-D144-R148-E152 and D147-R151 within the entirely hydrophilic and well-conserved wheel DYEDRYYRE; no mutations have been observed in these. These are classical examples of co-evolving non-local pairs that confound phylogeny software which assumes site independence.

In other portions of the paper, it is hard to muster much enthusiasm for modelling of prp-sc nucleation and the like, since this may takes place exclusively off-membrane in partialy deglycosylated proteolytic fragments; one wonders too what the effect on global structure calculations would be of using a copper-reconfigured amino terminus.

Still, the calculations are a good exercise; many people will want to borrow the superb graphics for their own papers (to replace third-rate cartoons). Glockshuber and Dyson may release both a prion with bound copper and adoppel nmr structure shortly; it will be necessary to experimentally determine the character of the attached glycans for doppel before the program here can be extended.

Earlier paper:

Molecular dynamics simulations of human prion protein: importance of correct
treatment of electrostatic interactions.
Biochemistry 1999 Oct 19;38(42):13862-76
Zuegg J, Gready JE

Warwicker pdb model of domain-swapped dimer

Modeling a Prion Protein Dimer: Predictions for Fibril Formation
BBRC 2000 Nov 30;278(3):646-652
Warwicker J
Comment (webmaster): In connection with a new prion dimer paper, Warwicker's group released model pdb coordinates on their web site. These links open directly in a browser window as interactive 3D, or at least they did for the webmaster: hold mouse down to get at image control menu. To get an initial orientation, it is best to set "display" to backbone and "color" to group. Monomers are postulated to have intercalating beta hairpins and an extra alpha helix N terminally that are domain-swapped. The models, while concrete and motivated, are not derived from experiment -- time will tell whether they are insightful or nonsensical.

Article highlights: Prion dimer model ... Human doppel model

Models of structural transition in prion protein (PrP) focus on the domain visualised by solution NMR. Accumulating evidence suggests that the adjacent and highly conserved nonpolar segment, as well as PrP-membrane interactions, should also be considered.

Calculations predict that membrane-induced structural destabilisation is mediated by stabilisation of the unfolded form. Comparative analysis of PrP structures leads to a model for PrP dimerisation that incorporates the nonpolar segment.

A prediction that PrP will interact with the PrP-like protein (Dpl) to form a heterodimer, but that Dpl will not form a homodimer, can be tested. Modelling is discussed in the context of ataxias associated with the expression of Dpl or truncated PrP in transgenic animals lacking wild-type PrP. A PrP(C) dimer model forms the basis for considering the geometry of PrP(Sc) fibril formation.

Earlier related papers

Warwicker J.
Modelling charge interactions in the prion protein: predictions for
pathogenesis.
FEBS Lett. 1999 Apr 30;450(1-2):144-8.

Warwicker J.
A hypothesis describing a potential link between molecular structure and TSE
strains.
Biochem Biophys Res Commun. 1997 Sep 8;238(1):185-90.

Warwicker J.
Species barriers in a model for specific prion protein dimerisation.
Biochem Biophys Res Commun. 1997 Mar 17;232(2):508-12.

Warwicker J, Gane PJ.
A model for prion protein dimerisation based on alpha-helical packing.
Biochem Biophys Res Commun. 1996 Sep 24;226(3):777-82.

6 new human prion gene mutations

Tue, 10 Oct 2000 correspondence
These 6 silent and neutral human prion gene mutations were found some time back by J-L Laplanche's group and kindly sent in to be added to the reference set of known point mutations. These had fallen through the cracks (not exciting enough for stand-alone publication) -- none of them had any association with CJD; 4 of the 6 were silent mutations did not change the coded amino acid. The hotspot effect of CpG dominated this new set, with all but 2 being canonical CpG transitions.

GeneBander tracks for the prion protein
 G+C content
 G+C blur r=3
 CpG all
 CpG observed
 CpG CJD sites
 domain structure
 non-CpG observed
 non-CpG CJD
 all CJD sites
 domain structure
 tyr-trp-phe
 pro
 arg lys/glu asp
 GeneBander
The total is now a respectable 42 distinct point mutations, 24 of them causative of familial CJD, 6 neutral (or modulatory), and 12 silent. More than one mutation occurs at 6 codons (with codon 188 having 4 distinct variants). CpG accounts for 40% of all observed changes (17/42), ie, it is the dominant mutational mechanism.

Silent mutations are no doubt under-reported; neutral mutations are often just alluded to in the text; while causative mutations usually make the abstract, they rarely get to GenBank. The mutations are not distributed at all uniformly across the gene -- only one change has been reported in the first 101 positions. In the bad old days, methodologies were designed to detect certain common changes such as P102L, D178N and E200K, which became a self-fulfilling prophecy.

Just because a mutation does not cause CJD, that does not mean that it is neutral to normal structure/function of the prion gene, prion mRNA, or prion protein.

However, Laplanche's first coding change, I138M or ile to met at codon 138, probably is in fact neutral at the protein function level: Leucine is the ancestral mammal amino acid at position138 -- isoleucine is restricted to great apes, rodents are all methionine, marsupial valine, phenylalanine is a rare sheep allele. Thus all possibilities in the the first column of the genetic code occur, suggesting that the I138M is near-neutral. [Birds and turtle have histidine or arginine here; doppel alignability does not begin until position 139.]

In contrast, the second coding change, G142S, is damaging to the protein in the webmaster's opinion. Serine is not a conservative change for this ancient glycine in terms of normal structure/function. The context, HFG, is a highly conserved region -- no glycine substitutions are seen in any sequenced species, suggesting a very small side chain is required here for packing or turning. Even the 5 doppel sequences align and are conserved as FG here. (Birds are all FD.) This substitution could be tested in vitro for loss of Fyn signaling capabilities.

I138M: man,57 yrs France, frontal dementia...non CpG transversion

G142S: man,69, multiple sclerosis
G142S: woman,25

N173N, clinical improvement--new site, non CpG transition, code conservation
T188T under usual CpG, hotspot
D202D two CpGs; V203I also canoncial CpG

Error alert: 4-hydroxyproline at Pro 44, polypro II repeat helix

 EMBO Journal, Vol. 19, No. 20 pp. 5324-5331, 2000
Andrew C. Gill, Mark A. Ritchie, ... Alexandre G.O. Rhie, Alan D. Bennett and James Hope
Question: Isn't it fascinating, after all these years, to see a new modified amino acid in the prion protein and a rigid polyproline II helical conformation for a domain supposedly a random coil?

Answer: Neither result has been established: artefacts adequately account for both conclusions. No one in their right mind ever thought that the pre-repeat region was a random coil: the rate of site evolution is 1000 times too slow, it is one of the most highly conserved regions in the whole protein. See the comma-delimited database of the repeat region. A polyproline II helix was ruled out years ago.

Question: Is collagen the normal substrate for prolyl 4-hydroxylase as the authors state?

Answer: No. This post-translational modification enzyme acts on monomeric procollagens. Collagen refers to mature protein, a hyper-modified and cross-linked trimeric triple coil. Collagen (like globin) is a generic term for any of a large, complex superfamily of genes (see 119 entries for collagen + human at SwissProt; there are 18 collagen genes just for alpha type) coding primarily for an imperfect 3 amino acid repeat, most typically PGK (the lysine is separately hydroxylated). Additionally, a large number of proteins have procollagen-like domains, by both descent and convergence, and are also substrates for prolyl 4-hydroxylase. In humans, some 17 different procollagen genes have been identified, scattered over many chromosomes.

A further absurdity here is that humans have a substantial superfamily of distinct prolyl 4-hydroxylase genes, again with representatives on various chromosomes. The level of amino acid identity is only 60% between the best studied members, indicating divergence times of several hundred million years. The number of relevant enzymes can be counted by tBlastn of known members against finished and unfinished human genome. Needless to say, in vivo modification (substrate specificity) of specific procollagens by specific prolyl 4-hydroxylases has not been sorted out.

Curiously the beta chain of prolyl 4-hydroxylase is protein disulfide isomerase, which may also have prolyl cis-trans isomerase activity. The multienzyme complex prolyl 4-hydroxylase acts as a chaperone during collagen synthesis in multicellular organisms.The 4-hydroxyproline is essential to folding and assembly of mature collagen and stabilizes the 3 strand triple helix by inter-chain hydrogen bonding. A collagen chain viewed in isolation within the 3 strand has PPII conformation (meaning all-trans proline peptide bonds and left-handed helices with 3.0 amino acids per turn). Glycine hydrogen bonds to the "next" residue inter-chain (various homologous chains can be take part). Only short stretches of PPII are seen in non-collagen globular proteins.

Question: Is Xaa-Pro-Gly a possible motif signature for "prolyl 4-hydroxylase" as the authors state?

Answer: No. First, a leading unspecified amino acid (Xaa) is completely vacuous in a putative recognition motif: no proteins (in particular, no procollagen begins with proline. Second, the real repeat in collagen is Gly-Xaa-Xaa (not relevant to prion protein); no other side chain is small enough for the trimeric collagen axis. For example, of 147 GxxG in human collagen alpha I(X), only 77 have a proline in position 3. It is these prolines that are modified.

Note that proline and glycine are very abundant amino acids, meaning that pro-gly is an extremely common dipeptide even excluding collagen domains. Yet 99.99% of these go unrecognized by prolyl 4-hydroxylases. Note that gonadotropin-releasing hormone, produced in hypothalamus, contains hydroxyproline [Neuroendocrinol 2000 Sep;12(9):919-26] even though brain is said not to be a generating site. Using "hydroxyproline homo BUTNOT collagen" turns up a variety of GenBank proteins lacking the alleged signature, eg, osteocalcin: qwlgapvpypd.

Looking at amino acid use at 11,310,862 residues in 23,477 distinct human proteins, we see human proteins are 6.7% glycine and 6.1% proline meaning, since dipeptide distributions show few anomalies, that pro-gly occurs roughly once every 246 residues, ie in nearly every protein (for example hemoglobin which of course has no hydroxyproline).

Therefore a wide spectrum of control peptides, including randomized order of the ones used, must be studied as controls in order to establish specificity. Unfortunately, the single control peptide run, which was missing 3 residues of the first octapeptide repeat, failed as substrate. In intact prion, these residues will be constrained by other duties.

But it gets worse. Pro-Gly, even in the context of a procollagen and polypro II helix, cannot suffice as a recognition site because 3-hydroxyproline is specifically formed instead at some of these sites. Thus from bioinformatics alone, it is clear that "Xaa-Pro-Gly" even in conjunction with polypro II cannot possibly define the substrate signature of these enzymes in vivo.

Question: But the circular dichroism proves that the prion peptide adopts a polypro II helix, right?

Answer: Wrong. Short polypeptides in solution rarely have a stable configuration. Instead, they transiently populate various states according to a Boltzmann distribution. The polypro II helix, an extended state, is much more widespread than realized initially; indeed Woody et al argue that much of "random coil" involves this structure instead. Thus the problem here is not that a small prion peptide can populate a polypro II helix but rather that just about any peptide can and does. Here, random peptides of the same composition and length were needed as controls.

If small peptides in isolation adopted the same conformation as in the intact protein, no one would study intact proteins. Instead, small structures would be quickly determined and assembled. Here, the only way they could get the result they wanted was by borrowing residues downstream that were already committed to the copper binding motif and hence unavailable in the native structure to support a polypro II helix. Question: How did they distinguish 3-hydroxyproline and 4-hydroxyproline in the paper, were cis and trans substituents distinguished, didn't they report an enantiomeric mixture?

Answer: First note that these four proline modifications have identical molecular mass and therefore cannot be distinguished in the mass spec fragments here. The HPLC column may or may not have resolved them -- unspecified standards were run but not this issue was not discussed. The 4-hydroxyproline was simply inferred from the mass whereas 3-hydroxyproline fit the data equally well but was not considered.

Enzymes in general and prolyl hydroxylases in particular are stereospecific, in the case of procollagens producing strictly the trans enantiomer of both hydroxyprolines. Therefore the racemic mix observed does not support the authors inference. Acid hydrolysis is known to mix the two states.

The other bizarre aspect of mass spectroscopy here was that the modified arginines, observed previously by 4 labs but never characterized, seem to have been absent. How was it possible that these modifications were not studied?

Question: Why did they express mouse prion in hamster ovary cells to study in vivo brain proline hydroxylation, and later incubate mouse protein in crude extracts of chicken?

Answer: No one knows. It is not a good idea to introduce gratuitous variables inherent to multiply heterologous systems. Chicken are remote from mammal; crystalline 4-prolyl-hydroxylase from the same mammalian species is readily obtainable, as are mouse cell lines. Here they picked a hamster cell line known to be a collagen hyper-producer and found every proline in sight was hydroxylated, and similarly for a chicken chicken hyper-producer. The artefactual nature of this was confirmed by a drop from 75% modified to "less than 1%" when they moved over to in vivo protein in brain. Note 0% is consistent with "less than 1%."

Question: Let's assume for the sake of argument that 4-hydroxyproline really occurs, can we guess at its purpose?

Answer: Yes, we can guess it has no purpose. This residue is modified for a reason in procollagens. That reason is anchoring by hydrogen bonds to the other two strands of triple coil mature collagen. Thus it makes no sense in sense to have 4-hydroxyproline in the prion protein because it cannot form a trimeric triple coil since it lacks the essential triphasic glycines. No other use of hydroxyproline is known.

Question: They write that a CD spectral shoulder is diagnostic for polypro II helices, citing an off-medline 1992 Woody paper on conformations of unordered polypeptides but not his major 1999 review of PPII in Protein Science. What's the problem?

Answer: Two problems: the first is the usual one of false positives and false negatives. Woody later inventoried xray structures of globular proteins known to contain a turn or two of PPII but there is no exhaustive study on CD spectra of these compared to CD spectra of proteins and polypeptides known not to have PPII. Circular dichroism is merely a blunt measure optical activity as a function of wavelength with very little spectral fine structure; what is attributed to PPII is a process of subtractive elimination. Each and every novel twisting structure of a fixed handedness will contribute in some unknown and non-calculable way to the CD spectrum. The second problem, according to Woody, is that PPII is just about everywhere in random coil or short peptides (eg, poly alanine), making the observation vaccuous even if valid:

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

Proteins 1999 Sep 1;36(4):400-6 
Sreerama N, Woody RW 
A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation.

Article highlights:

The importance of the highly flexible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion-binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP37-53, showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa-Pro-Gly consensus sequence, it may act as a substrate for the collagen-modifying enzyme prolyl 4-hydroxylase.

short incubation period mouse AAC02804 254 aa
  1 MANLGYWLLA LFVTMWTDVG LC KKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGTWGQ 
 59 PHGGGWGQ PHGGSWGQ PHGGSWGQ PHGGGWGQ GGGTHNQWNK PSKPKTNLKH VAGAAAAGAV
  121 VGGLGGYMLG SAMSRPMIHF GNDWEDRYYR  ENMYRYPNQV YYRPVDQYSN QNNFVHDCVN
  181 ITIKQHTVTT TTKGENFTET DVKMMERVVE  QMCVTQYQKE SQAYYDGRRS SSTVLFSSPP
  241 VILLISFLIF LIVG
Direct evidence for this modification was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrPSc from the brains of scrapie-infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.

...The importance of the N-terminal region has largely been overlooked because it does not appear to be strictly necessary for replication of prions. However, most, if not all, PrPSc that accumulates in affected brain includes the mature PrPC N-terminal sequences and the role of this domain of PrP in prion diseases has recently become more widely appreciated...

In this paper, we show by CD spectroscopy, mass spectrometry and Edman degradation that: (i) a synthetic peptide from this region of PrP can form a PPII structure in aqueous buffers; (ii) this sequence is a signal for proline 4-hydroxylation; (iii) almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of recombinant murine PrP secreted from Chinese hamster ovary (CHO) cells; and (iv) the same modification is found, at lower levels, in PrPSc extracted from the brains of scrapie-infected mice. This post-translational modification may provide an epigenetic control mechanism for the cross-species transmission of prions and/or the control of normal PrPC function.

Recombinant [mouse] prion protein secreted into the culture medium of stably transfected CHO [hamster] cells was purified and analysed by HPLC MS. The relative heights of the peaks indicate that 75% of the protein is oxidized.... The site of oxidation was localized to a peptide spanning residues 32-56 by capillary HPLC-MS analysis of peptides released by chymotryptic digestion of purified recPrP (Figure 4). This peptide was purified by reversed-phase HPLC and subjected to sequencing by Edman degradation. This technique indicated a modification to Pro44 of recPrP and conclusively identified it as 4-hydroxyproline by comparison with standards. The modification is specific for this proline residue and is the first identification of a 4-hydroxyproline residue in PrP.

In order to confirm its biological significance, we looked for this hydroxylation in PrPSc purified from the brains of VM mice infected with the 87V strain of scrapie. The denatured, alkylated protein was digested with chymotrypsin and the target peptide containing Pro44 was purified by HPLC and subjected to Edman degradation. 4-hydroxyproline was also identified in this protein. Two peaks corresponding to the different isomers of 4-hydroxyproline are labelled, eluting on either side of those resulting from histidine and alanine, and, although small, are clearly and reproducibly detectable at Pro44 and not in the other proline residues within this peptide.

The peak corresponding to unmodified proline is also labelled and is of greater intensity, indicating that only a small amount of 4-hydroxyproline is present, estimated to be less than 1%. The low incidence of 4-hydroxyproline in protein extracted from brain, and its presence in the part of PrPSc routinely cleaved from the protease-resistant core by proteinase K, may explain why previous studies have not identified this modification.

Pro44 is within a short sequence of PrP that has high homology to collagen [Not so. It is the composition of the repeat region proper that elicits false Blastp matches. -- webmaster]. Procollagen chains are hydroxylated in vivo within the consensus sequence Xaa-Pro-Gly [not so -- webmaster] by the action of prolyl 4-hydroxylase. To date, no other enzyme has been identified that produces 4-hydroxyproline from proline and this modification is difficult to effect non-enzymatically.

Pro44 of PrP occurs in the sequence Ser-Pro-Gly and so fits the consensus sequence for hydroxylation by prolyl 4-hydroxylase.

In addition to the requirement for a consensus sequence, however, the enzyme has also been reported to require formation of a PPII helix and possibly a beta-turn in the substrate. It has previously been proposed that PPII structure can form in the octarepeat region of PrP which starts 10 amino acids C-terminal to the hydroxylation site. To investigate whether PPII structure is likely to occur in the residues around Pro44, two short peptides spanning this residue have been synthesized and their conformations probed by CD spectroscopy.

Figure 6 shows the CD spectra of PrP peptide 1 [residues 37-53 RYPGQGSPGGNRYPPQG -- note the last 3 residues are part of the first repeat -- webmaster] at various temperatures. The spectrum at 0įC shows a weak positive A shorter peptide (PrP peptide 2, residues 41≠48) also incorporating the hydroxylation site was analysed and was found to have a random coil conformation (data not shown).The spectrum is typical of peptides possessing at least some PPII helical conformation.

...PPII helices are stabilized primarily by hydrogen bonding between side chains and the peptide backbone [Impossible -- the methylene side chains of proline and the glycine hydrogen are incapable of hydrogen bonding in the canonical polyproline PPII], and CaCl2 has been shown to disrupt PPII helices by binding preferentially to amino acid side chains [CaCl2 is a readily dissolved salt -- webmaster], thereby preventing such interactions...

Proline-rich peptides with the Xaa-Pro-Gly motif are bound at the N-terminal region of the alpha-subunit of prolyl 4-hydroxylase and need an extended, principally PPII conformation to bind (Myllyharju and Kivirikko, 1999), and a partial beta-turn in order to extend the Xaa-Pro-Gly into the catalytic site for hydroxylation. The PPII helix is an extended left-handed helix with three residues per turn, the average dihedral angles of phi and psi being approximately -75 and 145. Our findings that the N-terminal peptide of PrP containing the sequence Ser-Pro44-Gly-Gly can form a PPII structure in aqueous buffers, and that this sequence, and not others, can act as a substrate for prolyl 4-hydroxylation in CHO cells and in brain cells of mice infected with prions, strongly suggest that the polypeptide forms an extended PPII structure in vivo. [No, it does not suggest anything beyond a very local structure and weak artificial substrate. -- webmaster]

... The hydroxylated Pro44 of PrP occurs in a short stretch of sequence that has high homology to collagen; indeed, the PrP sequence Gly42-Ser-Pro-Gly-Gly46 also occurs in many types of collagen. Intriguingly, lyophilization or long-term storage of chicken PrP containing the tandem six-amino-acid repetitive peptides induces the formation of trimeric oligomers and partial resistance to proteolysis has also been found in the repeat region (Marcotte and Eisenberg, 1999). Both findings may be explained by the formation of collagen-like triple helices by the N-terminal regions of different chicken PrP molecules. It remains to be investigated whether such structures have a function, perhaps stabilized by prolyl 4-hydroxylation, and whether they exist in vivo in mammals or birds.

Neurons normally lack prolyl 4-hydroxylase (Blass et al., 1994) and neuronal PrPC is unlikely to be hydroxylated. The low percentage of PrPSc molecules containing 4-hydroxyproline in scrapie-affected mouse brain may derive from astrocytes or microglia; alternatively, and more provocatively, this hydroxylated PrP may be an indicator of prion-infected neurons. In contrast to neurons, normal CHO cells actively produce collagen and express high levels of prolyl 4-hydroxylase. This may be why almost all PrP secreted from these cells is specifically hydroxylated at Pro44 [the authors finally admit that their result is an artifact -- webmaster].

From this perspective, other fibroblastic and non-neuronal cells synthesizing PrP may also do so in a hydroxylated form. However, there may be other explanations. For example, we note that our recombinant PrP lacked sites for its normal post-translational glycosylation and glypiation [Medline shows only 5 previous uses of this word, most recently 1995. It means GPI attachment here. -- webmaster], and so the prolyl 4-hydroxylation of this molecule, while indicative of PPII structure, could be due to increased cell stress or abnormal protein trafficking.

At least two testable ideas spring from our observations and are currently under investigation within our laboratory. The first idea predicts that prolyl 4-hydroxylation of PrP is cell-type specific and that the percentage of hydroxylated PrPSc in infected tissues is an indicator of the cell type in which prion replication takes place. A second scenario to be tested views hydroxylation of PrP as an unnatural event that takes place as a general or specific response to mis-trafficking of this protein in a sick, infected or otherwise stressed cell. In collaboration with Blanch and colleagues ("Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, there is no evidence for any increase in beta-structure." J. Mol. Biol., 2000, 301, 553-563) we have now directly confirmed by Raman optical activity spectroscopy that full-length, recombinant sheep PrP contains significant amounts of PPII structure.

The Cellular Prion Protein Colocalizes with the Dystroglycan Complex in the Brain

Journal of Neurochemistry, Vol. 75, No. 5, 2000 1889-1897  Received March 28, 2000; revised manuscript received July 3, 2000; accepted July 7, 2000.
Gilmor Keshet, Osnat Bar-Peled, David Yaffe, Uri Nudel and Ruth Gabizon 
Comment (webmaster): This interesting paper follows up in good detail various earlier reports of prion protein localized to pre-synaptic cholesterol-rich membrane rafts. The main technique here, co-immunoprecipitation of prion protein with known raft components as detergent is varied, is however incapable of establishing stable or signficant physical association (hetero-oligomer formation) -- for this cross-linking reagents are needed. Indeed, these are likely to fail because the prion protein is not available to strictly integral membrane proteins or those facing the cytoplasmic side because only the non-specific GPI anchor is accessible for interaction.

There is really no exhaustive protein inventory nor functional understanding for either the rafts or the dystroglycan complex, though perhaps the more abundant components have been identified. Membrane-bound multi-protein complexes are very difficult to study even in E. coli, primarily because properties and associations do not survive the reductionist approach of solubilization. Thus results here, while suggestive and worth pursuing, do not establish direct binding of prion protein to proteins mentioned here, but rather a correlation of raft properties. Copper binding is not easily reconciled with properties of other proteins reported here.

Because prion disease occurs primarily in the brain, it is easy to think that normal prion protein function is uniquely or somehow specialized to brain. What then is native prion protein doing on erythrocytes and lymphocytes? Since these cells have are cleared from circulation in a matter of weeks, it is not surprising that pathology needing decades to accrue is absent. Indeed, the SAGE profile of prion gene expression in a wide variety of cell types is incompatible with strictly neurological normal prion function. Any function has to be viable for all cell types in which the prion protein is expressed (unless multiple distinct functions are envisioned).

Highlights of paper:

The function of PrPC, the cellular prion protein (PrP), is still unknown. Like other GPI-anchored proteins, PrP resides on Triton-insoluble, cholesterol-rich membranous microdomains, termed rafts. We have recently shown that the activity and subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) are impaired in adult PrP0/0 mice as well as in scrapie-infected mice.

In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions.

Using this detergent-dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophin- glycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein.

It is still unknown whether the function of PrPC is fulfilled during prion infection as well as if the absence of such function may accelerate the symptoms of the disease. We have recently shown that in both scrapie-infected and adult PrP0/0 mice, the activity and the normal cellular localization of the neuronal isoform of nitric oxide synthase (nNOS) were impaired (Keshet 1999 J. Neurochem. 72, 1224 -1231). These results are consistent with possibility that PrP is inactive in prion diseases.

However, even if functionally related, PrP and nNOS cannot bind directly. Although both these proteins colocalize in cholesterolrich rafts, PrP, like other GPI-anchored glycoproteins, is attached to the extracellular plasma membrane, whereas nNOS, although 60% of its activity is membrane-bound, presents only intracellularly.

nNOS synthesizes the neurotransmitter nitric oxide, which mediates diverse functions in neuronal signaling and is expressed in skeletal muscle and in brain . In the skeletal muscle, nNOS was found to interact with dystrophin, which in its mutated form causes Duchenne's muscular dystrophy. In the brain, nNOS was found to interact with 1-syntrophin, a component of the dystrophin- glycoprotein complex. These facts and the association of PrP with nNOS render the components of the DGC as candidates for interaction with PrP.

We have used immunoprecipitation methods with diverse antibodies and evaluated the strength of the identified interactions by testing their sensitivity to different detergents. We report here that PrP and NOS colocalized in the molecular layer of the cerebellum as well as coimmunoprecipitated in the presence of NP-40, a detergent that does not solubilize rafts, with antibodies against either PrP or nNOS.

In addition to nNOS, anti-PrP antibodies immunoprecipitated in the presence of NP-40 other intracellular proteins, such as dystroglycan, Dp71, which is the major isoform of dystrophin in the brain, syntrophin, alpha-tubulin, glutamic acid decarboxylase (GAD), and synaptophysin. The first three are known to belong to the DGC. Only the interaction of PrP with dystroglycan was resistant to dissociation by Sarkosyl, suggesting that dystroglycan may be the transmembrane protein that connects the extracellular GPI-anchored PrP to intracellular components, such as nNOS.

Antibodies: 3F4, rabbit anti-PrP serum RO73 for immunoblotting, Mab MANDRA1 raised against the C terminus of dystrophin which contains Dp71, Mabs against beta-dystroglycan, syntrophin, synaptophysin, alpha-tubulin, beta-actin, nNOS, postsynaptic density 95 (PSD95), annexin II.

Double immunofluorescence as viewed by the confocal microscope demonstrated colocalization of PrP and nNOS in the cerebellar basket cells in the molecular layer. PrP and nNOS coimmunoprecipitate: Cold TX100 extracts of Syrian hamster brains were incubated with 3F4 and nNOS monoclonal , or an anti-annexin II. Annexin II is an unrelated raft-associated protein. It is not surprising that nNOS immunoprecipitates with PrP only in the TX100-insoluble fraction because PrP is an established raft protein.

PrP interacts with proteins from the DGC in brain: To identify additional proteins that associate with PrP and especially to find a membrane protein that could serve as a link between PrP and nNOS, we tested proteins that are known to interact with nNOS as candidates for coimmunoprecipitation with PrP in the presence of various detergents.. To test whether these proteins interact with PrP, we immunoprecipitated a brain extract of normal Syrian hamsters with a protein A-Sepharose resin cross-linked to a monoclonal anti-PrP antibody (3F4). The immunoprecipitated samples were boiled in SDS and subsequently immunoblotted with antibodies directed against proteins of the DGC.

Dp71, the major isoform of dystrophin in the brain, syntrophin, and beta-dystroglycan precipitated with the anti-PrP antibody resin. It is interesting that dystroglycan precipitated with the resin even in the presence of 1% Sarkosyl, which totally solubilizes membrane lipids, including rafts, in contrast to NP-40, which leaves the rafts intact. These results suggest that dystroglycan, a transmembrane protein, associated strongly with PrP and may act as the link between PrP and intracellular molecules. A non-GDC protein found in this work to immunoprecipitate with PrP is GAD, the enzyme that synthesizes GABA from glutamate. We will show later that the membrane fraction of all these molecules is targeted to rafts.

Not all raft-associated proteins immunoprecipitate with PrP antibodies. Annexin II, a raft brain protein, is not a component of the immunoprecipitated complex. In addition to testing candidate proteins for their interaction with PrP, we systematically looked in the fraction immunoprecipitated with anti-PrP antibodies for abundant and specific protein bands, as revealed by Coomassie Blue staining. Two such bands were chosen and sequenced after being excised from the gel and digested with trypsin . The sequences corresponded to beta-actin and alpha-tubulin, respectively. We suggest that actin and tubulin have a strong interaction with the transmembranal dystroglycan and therefore appear as if they are bound directly to PrP.

It has been suggested recently that PrP is mostly a presynaptic protein. Whereas synaptophysin, a well-established presynaptic protein, precipitates with an anti-PrP antibody (Fig. 6a), this is not so for PSD95, associated postsynaptically with nNOS, which can be either pre- or postsynaptic. Cholesterol rafts are insoluble in TX100 and float to the top of such a Nycodenz density gradient. We found that a fraction of all proteins that were found to associate with PrP indeed cofractionate, as PrP, to the top fractions of the gradient. The fact that the immunoreactivity of these proteins was distributed across the gradient shows that, as expected, the proteins are distributed between a soluble and a raft fraction.

The fact that membrane-associated proteins might coimmunoprecipitate only owing to their colocalization on an insoluble membrane fragment is often ignored. In this study, we took advantage of this limitation to separate between interactions of different strengths. Beta-dystroglycan precipitated with a PrP antibody even in the presence of 1% digitonin (data not shown), a detergent reported to fully solubilize the DGC.

The DGC is much less defined in brain than it is in muscle, and not all the DGC components are necessarily present in all brain cells. Different neurons may contain only a portion of the potential DGC components. Except for dystroglycan, which is transmembranal, all the proteins shown in this work to interact with PrP are not integral membrane proteins.

There may be more than one mechanism by which soluble proteins associate with rafts. Some proteins have been shown to carry a C-terminally attached fatty acid, and others may interact with transmembrane raft proteins.

Depending on the exact function of PrP in copper metabolism, there are several possible roles for PrP in the DGC. PrP may serve as a copper chelator, thereby protecting the complex from the toxic effects of high copper levels. Indeed, copper was found to inhibit nitric oxide synthase activity. PrP, acting as a copper chelator, may prevent such inhibition at certain conditions. Otherwise, PrP might supply copper to proteins requiring this metal for their normal function. A putative functional interaction of PrP with members of the DGC and in particular with dystroglycan remains to be elucidated.

PrPC expression in the peripheral nervous system is a determinant of prion neuroinvasion

J Gen Virol. 2000 Nov;81(Pt 11):2813-2821
Glatzel M, Aguzzi A.
Comment (webmaster): This article has some thought-provoking findings on how peripheral prion neuroinvasion may proceed differently depending on expression levels. The domino model (locally converted protein converts centripedally adjacent protein in situ) is favored over vessicular axonal transport.

Reportedly the J Miller lab at ARS which oversees CWD intracerebral transmission tests on cattle at Ames has found so far three of 13 cattle have come down with CWD within three years, though at least one and possibly all three didn't show clinical signs until the end. The brains look fine; abnormal prions gathered instead along neural pathways. So the CWD took on an unexpected peripheral nervous system form; pre-clinical deer/elk might be affected in the same way..

Article highlights:

Transmissible spongiform encephalopathies are often propagated by extracerebral inoculation. The mechanism of spread from peripheral portals of entry to the central nervous system (neuroinvasion) is complex: while lymphatic organs typically show early accumulation of prions, and B-cells and follicular dendritic cells are required for efficient neuroinvasion, actual entry into the central nervous system occurs probably via peripheral nerves and may utilize a PrPC-dependent mechanism.

This study shows that transgenic mice overexpressing PrPC undergo rapid and efficient neuroinvasion upon intranerval and footpad inoculation of prions. These mice exhibited deposition of the pathological isoform of the prion protein (PrPSc) and infectivity in specific portions of the central and peripheral sensory pathways, but almost no splenic PrPSc accumulation. In contrast, wild-type mice always accumulated splenic PrPSc, and had widespread deposition of PrPSc throughout the central nervous system even when prions were injected directly into the sciatic nerve.

These results indicate that a lympho-neural sequence of spread occurs in wild-type mice even upon intranerval inoculation, while overexpression of PrPC leads to substantial predilection of intranerval over lymphoreticular spread. The rate of transport of infectivity in peripheral nerves was ca. 0.7 mm per day, and prion infectivity titres of sciatic nerves were much higher in tga20 than in wild-type mice, suggesting that overexpression of PrPC modulates the capacity for intranerval transport.

Oral administration is most probably involved in kuru, BSE and the nvCJD, while parenteral administration of growth hormone and gonadotropins has resulted in iatrogenic CJD. Transport of the infectious agent from the site of entry into the body to the central nervous system (CNS) is of crucial importance; both the lymphoreticular system (LRS) and the peripheral nervous system (PNS) are involved in scrapie neuroinvasion. Transport along the PNS was suggested by experiments showing that intranerval (i.n.) injection of infectivity could bypass the need for extraneural replication of the agent (Kimberlin et al., 1983). Also, the first pathological lesions and replication of infectivity after peripheral administration of the scrapie agent occur in spinal cord and medulla oblongata: these sites are consistent with entry via peripheral nerves like the vagal or splanchnic nerves.

The LRS clearly plays an important role in the transport of the scrapie agent. In several animal models, including hamsters and mice, lymphoid organs such as the spleen are early sites of accumulation and replication of the agent following intraperitoneal inoculation. Although B-lymphocytes are required for efficient neuroinvasion of the agent, they do not need to express PrPC. It appears that their role in neuroinvasion consists -- at least in part -- of lymphotoxin -mediated induction of follicular dendritic cell maturation. In order to replicate prions within lymphatic tissues follicular dendritic cells may need to express PrPC.

Although various components of the immune system play a pivotal role in scrapie neuroinvasion, there is substantial evidence that the PNS may be important for neuroinvasion of prions as well (Lasmezas et al., 1996<#REF25>). Adoptive bone marrow transfer of PrPC-expressing cells into PrPC knockout mice restored accumulation and replication of prions in the lymphatic tissue, yet not transport of the agent to the brain (Blšttler et al., 1997). These results indicated that a non-haematopoetic PrPC-expressing tissue is required for efficient neuroinvasion. Further experiments using Prnp knockout mice expressing transgenic PrPC under a neuron-specific promoter provided evidence that this tissue may be the PNS (Race et al., 2000<#REF31>).

Here we show that transgenic mice overexpressing PrPC under the control of its own regulatory sequences support rapid neuroinvasion upon i.n. and footpad (f.p.) inoculation of the infectious agent. The route of neuroinvasion was consistent with direct intranerval spread in all transgenic mice, and in only one subset of i.n. inoculated wild-type mice. The use of two different routes of inoculation in transgenic and wild-type mice enabled us to calculate the actual rate of spread of the infectious agent in the PNS of tga20 mice as a function of PrPC expression.

Mice were inoculated with a 1 % homogenate of heat- and sarcosyl-treated brain prepared from mice infected with the Rocky Mountain Laboratory (RML) scrapie strain. Previous studies have shown that tga20 mice overexpress PrPC in the CNS about tenfold). The expression of PrPC in the PNS of tga20 mice was determined to be five to seven times higher than in wild-type mice. In addition to the different expression level there is a marked difference between the various PrPC glycotypes as assessed by the electrophoretic pattern of PrPC between wild-type and tga20 nerves. In tga20 mice the strongest band is the high molecular mass band (diglycosylated PrPC), whereas in wild-type nerves a lower molecular mass band (monoglycosylated PrPC) gives the strongest signal....

In wild-type mice accumulation of PrPSc in the LRS occurs very early following i.p. injection . To examine the role of the spleen in neuroinvasion following i.n. and f.p. injection of tga20 and wild-type mice, we performed Western blots of selected spleens from i.n. and f.p. injected mice. In all of the tested wild-type mice we could detect sizeable PrPSc accumulation in spleens, whereas PrPSc could not be detected in spleens of tga20 mice, or was present in very low amounts.

This unexpected finding may, in principle, point to lower expression levels of PrPC in lymphoreticular organs of tga20 transgenic mice: we therefore determined the expression levels of PrPC in spleens and in inguinal lymph nodes of tga20 mice. However, similarly to what was observed in other tissues, PrPC was massively overexpressed in these tissues. We conclude that i.n. and f.p. injection of peripheral nerves overexpressing PrPC facilitates intranerval spread so extensively that lymphoinvasion of prions becomes marginal or absent.

The fact that we did not find any PrPSc by Western blot analysis of the sciatic nerves of wild-type and transgenic mice may surprise, especially in view of the infectivity readily detectable by bioassay of the same samples. However, considerable amounts of infectivity that are not associated with detectable PrPSc deposits have been observed before (Manson et al., 1999), and are probably due to the limited sensitivity of the Western blot technique.

Using the difference in incubation times of the f.p. and the i.n. inoculated tga20 mice we attempted to estimate the velocity of transport of infectivity in the PNS. The distance between the footpad and the mid sciatic nerve, where the i.n. injection is performed, is 2.1 cm on average, and the difference in incubation times of the i.n. and f.p. injected mice is about 30 days. Because tga20 mice transport primarily in the PNS after inoculation at either of these two sites, we calculated the speed of transport in the PNS by dividing the distance between the different sites of inoculation by the difference in incubation time. The calculated rate of spread of infectivity is 0.7 mm per day. This velocity is similar to that reported for wild-type mice where the rate of spread was calculated to be around 1 to 2 mm per day (Kimberlin et al., 1983). Neither of these values correspond to fast axonal transport or to slow axonal transport, whereas PrPC was reported to be transported with fast axonal transport with a velocity of about 1 cm/h (Borchelt et al., 1994).

The bioassay data gathered in this study provide intriguing insights into the kinetics of intranerval spread. Prion infectivity titres of tga20 sciatic nerves were up to 1.8 log higher than those observed in wild-type mice, yet the velocity of transport was similar in wild-type and in transgenic mice. Therefore, PrPC availability in the nerve modulates the capacity of intranerval spread, but does not affect its velocity. Perhaps the significantly higher titres in the sciatic nerves of tga20 mice are indicative of a mode of transport in which PrPC localized on the PNS is converted into PrPSc in a 'domino' fashion centripetally towards the CNS.

Besides confirming a central role of PrPC in the PNS in prion neuroinvasion, the present study provides surprising evidence that mice which overexpress PrPC can effect strictly intranerval neuroinvasion and bypass LRS pathogenesis. One may wonder whether similar phenomena may underlie neuroinvasion of BSE prions in cows, which also appear to bypass the LRS. Because overexpression of PrPC leads to increased intranerval prion titres, PrPC may well be rate-limiting for prion spread. This hypothesis bears some relevance to the prospect of post-exposure prophylaxis for prion diseases.

Prion incubation time QTL in mouse

Genomics 2000 Oct 1;69(1):47-53
Stephenson DA, Chiotti K, Ebeling C, Groth D, DeArmond SJ, Prusiner SB, Carlson GA 
Comment (webmaster): This paper turned out to be very instructive. There is good coverage of conflicting literature on additional genetic loci in the introduction, detailed strain and passage histories, clear exposition of how they arrived at lod scores, and a thorough but not excessive level of gene candidate discusssion at the end.

Quantitative trait loci (QTL) refers to any continuously varying trait (like blood pressure) due to contributions from several to many genes. Monogenic disorders (such as familial CJD) are much easier to map, though the feasibility of both types of mapping has benefited immensely from the last years of genomic sequencing. QTL mapping is more of a frontier, not something to embark on lightly if the plan is to eventually pin down the actual genes. Two papers in the 7 Nov 00 issue of PNAS provide the latest developments (1, 2).

Possible QTL traits in prion disease include incubation time, accumulation of prp-sc, infectious titre, and so on. The idea is to find auxillary genes beyond prion and doppel that influence the disease process; this has been very productive in other amyloidoses such as Alzheimer (presenilins) and yeast sup35 (20 genes).

Here they went after incubation time (the same mouse cross may work for quantitative prp-sc as well, possibly yielding other loci). It amounts to crossing two strains of inbred mice representing disjoint values of the QTL, here the CAST/Ei at 172(6) days and SLJ/J at 105(4). Crossing mice takes some getting used to: some die for no good reason, samples don't get taken or are lost, others are lost and found as the lab moves, quite a few did not have intermediate values of incubation period (shorter than SLJ/J) -- all of this is honestly reported.

They ended up with 43 mice at incubation extremes that were scored against a panel of 153 microsatellites. A chromosome 9 locus near D9M91 emerged with a peak lod of 6.35, as well as a broader peak on chr 11 near D11Mit219. These are called prion incubation determinants pid2 and pid3 and accounted for 13% and 17% of the variance, respectively. Note pid1 was used back in 1983 for a chr 17 locus: Kingsbury et al. J Immunon 131:491-96 but not supported here (which means nothing as the authors note because different strains were used). There may be more than one relevant loci at these sites.

Veteran mouse geneticist George Carlson kindly answered some webmaster questions:

Q1. What was going on with the19 mice that had shorter incubation periods than pure SJL/Js? On page 5 it says the shortest incubation times went with homogeneous S alleles on both chr 9 and chr 11 which the SJL/Js were already, yet these had a very narrow incubation span. Could there be another QTL model included, beyond additive, dominant, and recessive, say negative dominant? It seems like QTL is predicated on intermediate incubation times.

A1. At present, we can't say what's responsible for the very short incubation time mice among the F2s. One possibility is the existence of alleles in SJL/J that prolong, rather than shorten incubation times. Certainly this is seen in other systems-- Ward Wakeland's lupus model where resistance genes are found in susceptible strains and vice versa. It may also be revealing something about the biology of scrapie-- remember Alan Dickinson's original observation of "overdominance" in heterozygotes with some scrapie strains. Epistatic interactions could also account for the distribution in incubation times, but the cross is really too small to evaluate this aspect. In short, I don't know the answer, but the initial linkage analysis (free association) does not assume any particular mode of inheritance.

Q2. What would be the expected effect in sharpening the lod score chr 9 peak (figure 2 ) if all the relevant lost-in-lab-move mice (2 short, 16 long) could now be genotyped, in addition to the 43? That is, would it help all that much in narrowing the chromosomal locations to have more mice or would it mostly just further statistically validate a result already seen? Suppose additional microsatellites in the D9M91 region were used -- does this sharpen the location or are too many mice needed to get recombination? It seems like 10cM could easily correspond to 5 megabases which might have 50 genes. How it will get narrowed down enough to pin down the gene.

A2. QTL analysis is a very poor way to determine the exact position of a gene, so the additional mice wouldn't help that much. My feeling is, at this stage, a fairly large interval is preferable. We are isolating each locus in congenic strains so we can treat each QTL as a monogenic trait and narrow the location to a centimorgan or less.

Q3. The paper mentions Prp-Sc concentration as a further possibility for QTL. Can this be done with the existing set of samples or would it need a fresh start with different strains chosen for initial differentiation?

A3. Our preliminary results suggest that we can get useful information from this cross and we are starting the measurements.

Q4. With human F198S, there is said to be a 3000 member Indiana kindred. It seems like there would be an opportunity for QTL here too on age of onset. Could there not be synergy with mouse work given the synteny, in the sense of the lod peaks perhaps intersecting in a smaller genomic region?

A4. The mouse data can be useful in prioritizing the analysis. Much higher resolution can be achieved in the human than in the mouse, due to very high density of SNPs and the fact that humans are outbred. Genes can be localized by association or identity by descent rather than by segregation analysis.

Q5. Unlike human chr 20, in which 80/80 markers map to mouse chr2, mouse chr 9 is syntenic to human chr 3, 11, 15, 19, 6, and 14 in decreasing order. Now ACRV1 (acrosomal vesicle protein) on human 11q23-q24 (accession NC_001044 ) is the nearest known human marker to D9Mit91 (accession NC_001198) using the Jackson build-a-map. The laminin receptor is on human chr 3 rather than chr 11 -- is there data putting D9Mit91 not syntenic with human chr 11?

A5. The Lamr1 gene is probably on distal Chr 9 (the region homologous with human 3p21) -- this is a region that showed marginally significant linkage using composite interval analysis. D9Mit91 is indeed linked QTL in the region homologous to human Chromosome 11. There may be at least 2 QTLs (one highly significant that is linked to D9Mit91 (Human Chr 11) and one that is marginally significant on the distal end of the Chromosome in the D9Mit212 to D9Mit52 interval (Human Chr 3).

Intergenic splicing example on human chr 1

Chromosomal Location and Genomic Structure of the Human Translin-Associated
Factor X Gene (TRAX; TSNAX) Revealed by Intergenic Splicing to DISC1
Genomics vol 47#1 pp. 69-77 July 2000
Kirsty Millar et al. 
Comment (webmaster):

These loci on human chromosome 1 turned out to be a very nasty region of the human genome to annotate despite intensive experimental effort. In addition to the complex intergenic splicing between seemingly functionally unrelated ssprawling genes, possibly producing fused proteins lacking the terminal and initial coding exons of the pair, there is a long anti-sense non-coding gene on the opposite strand running under some of the intermediate exons and introns of the second gene.

Evidence for an anti-sense gene for Prnp was found some years back but bizarrely never pursued, other than to show it was still present in a prnp mouse knockout. However, it may be locatable as the genome projects reach completion.

It has proven difficult to find other examples of exon-skipping as documented in prion-doppel, though it may not be rare in tight tandem duplications because otherwise the second copy initially has no promoter of its own. The details of this chr 1 gene complex are best provided in this and a set of accompaning papers, below are the results of their literature searches on intergenic splicing and anti-sense genes:

"Splicing together of exons from separate genes (intergenic splicing) has been observed very rarely in normal mammalian cells. To our knowledge, only four well-characterized examples have been reported:

(1) human MDS1 and EVI1 are separated by 170≠400 kb of genomic DNA and, when spliced together, specify a novel transcription factor (Fears et al., 1996) with activity antagonistic to that of EVI1.

Soderholm, J., Kobayashi, H., Mathieu, C., Rowley, J. D., and Nucifora,G.
The leukemia-associated gene MDS1/EVI1 is a new type of GATA-binding transactivator. 
Leukemia 11: 352≠358 (1997)
(2) Human GALT and IL-11R alpha are separated by 4 kb. Intergenic splicing between these two genes generates a transcript translated to produce a novel fusion protein lacking any detectable activity of the proteins normally produced separately from the genes.
Magrangeas, F., Pitiot, G.et al.
Cotranscription and intergenic splicing of human galactose-1-phosphate uridylyltransferase
and interleukin-11 receptor alpha-chain genes generate a fusion
mRNA in normal cells. Implication for the production of multidomain proteins during evolution. 
J. Biol. Chem. 273: 16005≠16010 (1998)
(3) Human HHLA1 and OC90 are separated by less than 10 kb in the genome. Intergenic splicing between these genes and an HERV-H endogenous retrovirus generates transcripts that could, in theory, but apparently not in practice, be translated to produce a fusion protein
Kowalski, P. E., Freeman, J. D., and Mager, D. L. 
Intergenic splicing between a HERV-H endogenous retrovirus and two adjacent human genes. 
Genomics 57: 371≠379 (1999).

Kowalski, P. E., and Mager, D. L.
 A human endogenous retrovirus suppresses translation of an associated fusion transcript, PLA2L. 
J. Virol. 72: 6164≠6168 (1998)
(4) The prion protein gene Prnp and the newly identified downstream gene Prnd, which encodes doppel, are approximately 16 kb apart (Moore et al., 1999). Intergenic splicing between these two genes produces a complex series of transcripts in mice and rats. These transcripts skip the single coding exon of Prnp, but include the single coding exon of Prnd, and therefore encode only doppel. In some cases noncoding exons located in the region separating the two genes are also included. The authors make the intriguing observation that Prnp/ Prnd intergenic splicing and expression of Prnd are upregulated in mice where the final exon of Prnp, including the splice acceptor sequence, has been disrupted. Here we demonstrate intergenic splicing between DISC1 and the gene encoding TRAX.
Moore, R. C., ...Westaway, D.
Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel. 
J. Mol. Biol. 292: 797≠817  (1999)

Genomic anti-sense transcripts

A second gene, DISC2, is antisense to DISC1 and is also disrupted by the same translocation (Millar et al., 2000). Like several other antisense genes, DISC2 may regulate expression of DISC1. The antisense gene DISC2 may also play a regulatory role.
Millar,J.K. ... and Porteous D.J.
Disruption of two novel genes by a translocation cosegregating with schizophrenia.
Hum. Mol. Genet. 9: 1415≠1423(2000)

Constancia, M., Pickard, B., Kelsey, G., and Reik, W.
Imprinting mechanisms. 
Genome Res. 8: 881≠900.(1998)

Dolnick, B. J.
Naturally occurring antisense RNA. 
Pharmacol Ther. 75: 179≠184 (1997)

Knee, R., and Murphy, P. R.  
Regulation of gene expression by natural antisense RNA transcripts. 
Neurochem. Int. 31: 379≠392 (1997).

Detectable abnormal prion in nvCJD: plausible incubation periods and cautious inference.

J Epidemiol Biostat 2000;5(4):209-19
Cooper JD, Bird SM, de Angelis D Medical Research Council Biostatistics Unit
Comment (webmaster): It is proving very difficult to get at the overall size of the impending epidemic because of so many uncertainties of what positive tonsils, or for that matter, negative tonsils mean.

Article highlights:

Both small and large new variant Creutzfeldt Jakob disease (nvCJD) epidemics are consistent with the current observed incidence. Uncertainty in nvCJD projections could potentially be reduced by incorporating information on the prevalence of the infectious agent in persons incubating nvCJD. The prospect of nvCJD prevalence studies has been raised by detection of abnormal prion protein, thought to be the infectious agent, in appendices and tonsils removed from nvCJD patients.

Although unlinked anonymous testing of stored operative tissues for abnormal prion protein is very appealing, the design and interpretation of such prevalence studies is complicated by the lack of information on how early in the incubation period of nvCJD the abnormal prion protein becomes detectable.

We simulate a range of nvCJD epidemics, consistent with the limited available information on the incidence of nvCJD, to illustrate some of the potential problems encountered when interpreting the results from prevalence studies of detectable abnormal prion protein. We assume plausible incubation period distributions and dietary exposure patterns.

We demonstrate, in the context of our simulated epidemics, that prevalence studies of detectable abnormal prion protein would require the testing of tens of thousands of operative specimens and, even then, that unlinked anonymous testing positives would be unexpected.

Binding of disease-associated prion protein to plasminogen.

Nature 2000 Nov 23;408(6811):479-83
Fischer MB, Roeckl C, Parizek P, Schwarz HP, Aguzzi A
Comment (webmaster): This paper needed to be published in longer format than Nature allowed -- the text is very compressed. The authors state, but provide no data or reference, that the highly touted Prionics monoclonal does not bind specifically to the abnormal form. No persuasive rationale for plasminogen binding is provided -- it seems just good fortune. The authors leave development of an actual diagnostic test to supporting pharmaceuticals.

Abstract:

Transmissible spongiform encephalopathies are associated with accumulation of PrP(Sc), a conformer of a cellular protein called PrP(C). PrP(Sc) is thought to replicate by imparting its conformation onto PrP(C) (ref. 1), yet conformational discrimination between PrP(C) and PrP(Sc) has remained elusive. Because deposition of PrP(Sc) alone is not enough to cause neuropathology, PrP(Sc) probably damages the brain by interacting with other cellular constituents.

Here we find activities in human and mouse blood which bind PrP(Sc) and prion infectivity, but not PrP(C). We identify

plasminogen
, a pro-protease implicated in neuronal excitotoxicity, as a PrP(Sc)-binding protein. Binding is abolished if the conformation of PrP(Sc) is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I-III) retains this binding activity, and binding can be competed for with lysine.

Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.

Analysis of EEG and CSF 14-3-3 proteins as aids to the diagnosis of CJD

Neurology 2000;55:811-815
I. Zerr and 25 others
Comment (webmaster): This focuses on clinical diagnosis of sporadic CJD. The spectrum of familial CJD strongly suggestst that genotyping was inadequate (or inadequately reported), only 4 were found and kindreds were not investigated: P102L, n = 5 E200K, n = 13 V210I, n = 15 D178N-129M, n = 15

"Pooled data on initial and final diagnostic classification of suspected CJD patients were accumulated, including results of investigations derived from a coordinated multinational study of CJD. Prospective analysis for a comparison of clinical and neuropathologic diagnoses and evaluation of the sensitivity and specificity of EEG and 14-3-3 CSF immunoassay were conducted.

Data on 1,003 patients with suspected CJD were collected using a standard questionnaire. After follow-up was carried out, complete clinical data and neuropathologic diagnoses were available in 805 cases. In these patients, the sensitivity of the detection of periodic sharp wave complexes in the EEG was 66%, with a specificity of 74%. The detection of 14-3-3 proteins in the CSF correlated with the clinical diagnosis in 94% (sensitivity). The specificity (84%) was higher than that of EEG. A combination of both investigations further increased the sensitivity but decreased the specificity.

Incorporation of CSF 14-3-3 analysis in the diagnostic criteria for CJD significantly increases the sensitivity of case definition. Amended diagnostic criteria for CJD are proposed."

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue.

J Clin Microbiol 2000 Sep;38(9):3254-3259
O'Rourke KI, Baszler TV, Besser TE, Miller JM, Cutlip RC, Wells GA, Ryder SJ,
Parish SM, Hamir AN, Cockett NE, Jenny A, Knowles DP
Comment (webmaster): This live-animal eyelid test seems to be progressing well and moving towards validation. Blood testing remains as far off on the horizon as ever, so this is what we have to determine the scope of scrapie. The false positive/false negative results below strongly suggest that 60/60 Vermont sheep testing negative strongly implies that positive western blots were in error.

"Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing.

In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy.

The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97.6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie."

Iatrogenic Creutzfeldt≠Jakob disease at the millennium

Neurology 2000;55:1075-1081
P. Brown, MD, M. Preece, J.-P. Brandel, T. Sato, L. McShane, PhD, I. Zerr, A. Fletcher, R. G. Will, FRCP, M. Pocchiari,
N. R. Cashman, J. H. d'
Aignaux, L. CervenŠkovŠ, J. Fradkin, MD, L. B. Schonberger, MD and S. J. Collins 
Comment (webmaster):

This is a first-class review of information very difficult to pull together from other sources. It is clearly written, thorough in coverage, very modern, and strikes a good balance in the discussion of future risks. Growth hormone was managed in the UK like vaccine: use up existing warehouse stocks even if they were sourced in BSE herds:

"In the United Kingdom, chromatography purification was inaugurated around 1980, but because of supply shortages, hormone prepared by the earlier method remained in use, ..."

Dura mater and growth hormone are old topics , how about this megavitamin:

"A 71-year-old American woman experienced a progressive gait disorder and mental deterioration beginning in the autumn of 1989. Neurologic examination confirmed a severe ataxia with features of both cerebellar and basal ganglion dysfunction, global disorientation, and memory loss. She died 18 months after the onset of illness, and in view of the biopsy diagnosis, no autopsy was performed. The patient' s family noted that she had for many years regularly taken a megavitamin preparation that, on investigation, was found to contain large quantities of imported beef liver and bone meal. The country of origin of these imported tissues was not specified. "

The article does not specifically address whether cornea, organ, and tissue donations should be halted from individuals already excluded from blood donation by the same logic. There is a "distinction" between organs and tissues in America because organ transplants require permission from the donor and go to the recipient with considerable regulatory oversight, whereas tissue removal are taken, not donated, without permission or notification or regulation (doesn't matter if donor had CJD) and is wholly commercial (though laundered at front and back ends by "non-profits").

Vaccines sourced on fetal calf serum from herds with BSE are a serious concern given two proven massive TSE vaccine accidents have occurred already.

Article highlights:

"The causes and geographic distribution of 267 cases of iatrogenic Creutzfeldt≠Jakob disease (CJD) are here updated to July 2000. Small numbers of still-occurring cases result from disease onsets after longer and longer incubation periods following infection by cadaveric human growth hormone or dura mater grafts manufactured and distributed before the mid-1980s.

The proportion of recipients acquiring CJD from growth hormone varies from 0.3 to 4.4% in different countries, and acquisition from dura mater varies between 0.02 and 0.05% in Japan (where most cases occurred). Incubation periods can extend up to 30 years, and cerebellar onsets predominate in both hormone and graft recipients (in whom the site of graft placement had no effect on the clinical presentation).

Homozygosity at codon 129 of the PRNP gene is over-represented in both forms of disease; it has no effect on the incubation period of graft recipients, but may promote shorter incubation periods in hormone cases. Knowledge about potential high-risk sources of contamination gained during the last quarter century, and the implementation of methods to circumvent them, should minimize the potential for iatrogenic contributions to the current spectrum of CJD. [Note it is rarely if ever known what the donor had at codon 129, since valine is rarer, methionine was likely more common, and so by the like-like principle, methionine is expected to be more affected in the recipient. The authors note that "infected pituitaries in the UK might by chance have come mainly from valine-homozygous individuals, with a resulting predilection for similarly homozygous hormone recipients." -- webmaster]

Nearly 30 years have passed since the first case of iatrogenic Creutzfeldt≠Jakob disease (CJD) was recognized in the recipient of a corneal graft from a donor who had died of unsuspected CJD. Two further possible cases of corneal graft≠related CJD have occurred in the last decade: one in a patient with verified CJD who had received a graft 16 months earlier from a donor whose cause of death was unknown, and one in a patient with clinically typical CJD who had received a graft 30 years earlier from a patient with verified CJD. The few cases resulting from contaminated stereotactic intracerebral EEG needles or neurosurgical instruments identified in the 1970s have not grown in number.

The two most important causes of iatrogenic disease are contaminated cadaveric human growth hormone and dura mater grafts, first recognized as risks in the mid-1980s. The original hope for a limited number of cases that would quickly diminish over time has vanished, as cases continue to occur each year, with current tallies of 139 (growth hormone) and 114 (dura mater). Table 1 summarizes cases of iatrogenic disease from all causes, and table 2 shows their international distribution. More than half of all growth hormone≠related cases have occurred in France, and more than half of all dura-related cases have occurred in Japan.

The frequencies of CJD in growth hormone recipients shown in table 3 make no assumptions about risk factors other than the absence of risk in subjects treated after mid-1985 with either recombinant hormone (United States, United Kingdom, and New Zealand) or urea-treated native hormone (France), who were excluded from the treated population denominator. However, in the United States, all cases of CJD have so far occurred in individuals who began treatment before 1977, when the purification protocol was changed to include a column chromatography step. If this new purification protocol contributed to the disappearance of new cases, then a more appropriate calculation of risk in the United States would use only cases in the pre-1977 treated population, with a resulting frequency of CJD of 0.8%. Similarly, instead of the 5 cases in 184 patients treated in New Zealand with hormone prepared in the United States, the denominator would decrease to 46 individuals treated before 1977, and yield a frequency of 11%. No plausible reason has yet been advanced to explain this discrepancy between CJD incidence in United States and New Zealand hormone recipients.

In the United Kingdom, chromatography purification was inaugurated around 1980, but because of supply shortages, hormone prepared by the earlier method remained in use, and all of the CJD cases in the United Kingdom received at least some of this hormone during treatment. Thus, no limiting dates can be used for making an alternative risk calculation. In France, all cases have thus far shared a treatment period between the beginning of 1983 and mid-1985, which has been proposed as the most likely limiting period of risk.8 Using the population under treatment during this period (1260), the calculated frequency of CJD rises to nearly 6%....No cases of CJD appear to have resulted from the administration of commercially prepared hormone, which may explain why Japan, which used commercial hormone exclusively, has had no cases among 5000 treated subjects.

Japan, however, had a different problem -- that of dura mater grafts. Although nearly all contaminated dura mater grafts were produced before 1987 by a single German company, the absence of precise information about the extent of graft usage prevents reliable risk calculations. Based on a consensus estimate that in Japan about 20,000 grafts were used each year,9 it is possible to estimate a cumulative incidence of 0.025% in neurosurgical patients receiving grafts during the 13-year period 1979 to 1991, and an incidence of 0.053% during the peak years of 1984 to 1985. The extraordinary use of dura mater grafts in Japan almost certainly exceeded its use in any other country in the world (estimated usage in the United States, for example, was fewer than 4000 grafts per year, of which less than 10% were supplied by the German company). Thus, even with a random international distribution of infected grafts, the number of cases in Japan would be expected to far exceed that in any other country.

Thus, most of these cases have comparatively long incubation periods that are at least in part the result of a very small infecting dose (which cannot now be quantified), administered by a suboptimal route of infection....

Anecdotal observations. All proven or probable cases of iatrogenic CJD have resulted from the transmission of disease either from tissues or instruments contaminated by brain, or tissues proximate to the brain. Nevertheless, the literature includes a few anecdotal reports of patients with CJD in whom peripheral tissue grafts or transplants had been performed. As examples, we briefly reiterate two published case reports and add one unpublished observation.

The first report was of a 54-year-old man who developed visual and mental abnormalities 4 years after having had a tympanic membrane repair using a pericardial graft from a patient undergoing open heart surgery, who died postoperatively. The second report was of a 57-year-old woman with the onset of cerebellar symptoms 2 years after receiving a liver transplant from a 42-year-old man who died from rupture of a cerebral aneurysm. The recipient of a renal transplant from the same donor remained in good health at the time of writing. During the liver transplant operation, the patient also received a small amount of albumin from a large plasma pool that included a contribution from a donor who 3 years later died of sporadic CJD. Contamination from this source can probably be excluded on the basis of experimental animal model studies showing the absence of infectivity in albumin.

The unpublished observation is of a 63-year-old man whose illness began with memory loss and nominal aphasia 4 years after receiving a bone graft from a previously healthy 46-year-old man who had died of a myocardial infarct. Bone from the same patient was used in grafts for 27 other patients, of whom 13 were reported in good health up to 6 years afterwards (14 recipients could not be reached). Both corneas were also harvested and both recipients have remained in good health.

In each of these three examples, the tissue recipients were verified by postmortem neuropathologic examination to have died of CJD, and although none of the donors was known to have a primary neurologic illness, none had undergone autopsy.

In only one of the three cases -- that of the liver transplant recipient -- was there a cerebellar onset of illness, which is the proportion that would be expected for sporadic CJD, but not for iatrogenic CJD contracted by peripheral route infection. Whereas it is likely that these (and other) cases represent the chance occurrence of sporadic CJD in individuals with preceding transplantation procedures, their occurrence should not be ignored, as any similar future cases will shift the balance in favor of an iatrogenic cause, just as happened after the initial case reports of CJD in recipients of growth hormone and dura mater grafts. Attention should also be given to anecdotal cases of CJD in individuals exposed to other sources of human or animal tissues, including commercial dietary supplements such as vitamin tablets compounded in tissue powders, illustrated by the following case.

A 71-year-old American woman experienced a progressive gait disorder and mental deterioration beginning in the autumn of 1989. Neurologic examination confirmed a severe ataxia with features of both cerebellar and basal ganglion dysfunction, global disorientation, and memory loss. She subsequently developed visual loss, complex involuntary movements, and myoclonus. EEG was abnormal but showed no periodic activity. Brain biopsy of the frontal cortex showed spongiform change (no plaques were seen). She died 18 months after the onset of illness, and in view of the biopsy diagnosis, no autopsy was performed. The patient's family noted that she had for many years regularly taken a megavitamin preparation that, on investigation, was found to contain large quantities of imported beef liver and bone meal. The country of origin of these imported tissues was not specified."

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