Prions
Mad Cow Home or Best Links

New CJD mutation disrupts glycosylation: T183A
Lehmann/Harris: blockade of glycosylation promotes scrapie
Summer/Fall 1997 Meeting Abstracts
Full length prion nmr structure
Full length prion monomer easily purified in quantity from cow
Codon use in prion gene versus bovine codon use overall
New human GPI protein found
Scary news: Cross-beta filaments formed by leucine-rich repeat peptides
Clarification of cross-species in vitro barriers
Both apolipoprotein E and A-I genes
Gene affecting early onset of Alzheimer's
Squirrels: a natural reservoir for TSEs?
New jungle species: dwarf marmoset, soala ox, dwarf and giant muntjacs

July 13    Boston, MA

Opening Plenary Session
         Prion Proteins
                  S. Prusiner, UCSF (Amgen Awardee)
                  K. Wuthrich, ETH, Zurich 
Concurrent Session I     
        Misfolded Proteins
                  S. Lindquist, Chicago
                  J. Kelly, Texas A&M
Aug. 25  FASEB San Francisco

Chaperone-mediated Conversion Of Cellular Prion Protein Prp-c To Its  Protease-resistant Form.
Program Number: 290, Poster Board Nr: C-60
S.K. Debburman, G.J. Raymond, B. Caughey And S.L. Lindquist.

Molecular Basis Of Alzheimer's Disease And Related Disorders 
Session Organized By: S.B. Prusiner

Mad Cows And Englishmen: The Prion Folding Problem. 
F.E. Cohen.

Solubilization And Crystallization Of The N-terminally Truncated Scrapie Prion Protein
Program Number: 675, Poster Board Nr: C-192
H. Wille And S.B. Prusiner.

In Vitro Analysis Of Recombinant Yeast Prion- Like Protein Sup35

M.M. Patino, J.R. Glover, A.S. Kowal, E.C. Schirmer, S.L. Lindquist
The cytoplasmically-inherited element Psi decreases translational fidelity in S. cerevisiae. Cells with a Psi phenotype show increased suppression of nonsense mutations. Genetic, biochemical and microscopic evidence indicates that Psi corresponds to an aggregated prion-like form of the Sup35 protein, an essential subunit of the translation termination factor. The chaperone protein Hsp104 governs the aggregation state of Sup35 and Hsp104 is essential for maintenance of Psi. Two functional domains have been identified in Sup35. The C-terminus is essential for translation termination.

The N- terminus has an unusual amino acid composition. It is required for the maintenance of Psi and can itself induce Psi when overexpressed. We are studying the aggregation properties of Sup35 and the effects of Hsp104 in vitro. Full length Sup35 and polypeptides that comprise amino acids 1 to 254, 1 to 124, 124 to 254 and the C-terminus, are being analyzed by electron microscopy, CD and light scattering.

The Fiber Forming Protein Tau And Its Role In Protein Folding Diseases

A.M. Giannetti, G.A. Lindwall, M. Chau, L.A. Kohlstaedt
Tau is a 45 Kd protein that bundles and assembles microtubles in the axons of neural cells. In Alzheimer's patients, tau aggregates and precipitates to form structures known as neurofibrillary tangles (NFT). We present data on the biophysical characterization of tau and its role in the formation of NFTs. X-ray fiber diffraction data on synthetic domains of tau resembles that seen for fibers from diseases such as familial amyloidotic polyneuropathy, amyloidosis, and NFTs isolated from Alzheimer's affected brains. These data imply that these proteins all assemble into similar cross-beta structures, suggesting a relevance for this structure among the several classes of protein folding diseases.

Amyloid deposition is delayed by deletion of serum amyloid P component gene.

Nat Med 1997 Aug;3(8):855-859 
Botto M, Hawkins PN, Bickerstaff MC, ..., Tennent GA, Walport MJ, Pepys MB
The tissue amyloid deposits that characterize systemic amyloidosis, Alzheimer's disease and the transmissible spongiform encephalopathies always contain serum amyloid P component (SAP) bound to the amyloid fibrils. We have previously proposed that this normal plasma protein may contribute to amyloidogenesis by stabilizing the deposits. Here we show that the induction of reactive amyloidosis is retarded in mice with targeted deletion of the SAP gene. This first demonstration of the participation of SAP in pathogenesis of amyloidosis in vivo confirms that inhibition of SAP binding to amyloid fibrils is an attractive therapeutic target in a range of serious human diseases.

Structure of "full-length" prion protein

Two important new papers by the Glockshuber and Kurt Wuthrich groups on the "full-length" prion protein have appeared in FEBS Letters, which has a pathetic web site, just tables of contents, no abstracts much less pay-for-fulltext. Abstracts and reprints are not yet available. The structure coordinates for 212-231 will be released on September 30 by the Brookhaven Protein Database. 1AG2 Tracking Number: T11143

The mature protein begins at 23 and ends at 240 (not 231):

MANLG YWLLA LFVTM WTDVG LC:  mouse signal peptide (short incubation)
KKRPKPGGWNTGGSRYPGQGSPGGNRYP: residue 23 on, pre-repeat region
PQGGTWGQPHGGGWGQPHGGSWGQPHGGSWGQPHGGGWGQGGG: repeat region
THNQWNKPSKPKTNFKHVAGAAAAGA: post-repeat region
VVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITIKQHT
VVTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDG: original region studied by NMR
RRSSSTVLF:  232 on, not studied, site of attachment of GPI anchor
SSPPVILLISFLIFLIVG: GPI region cleaved in mature protein
Prof. Glockshuber writes:
"I have just read the "Kyodo newspaper story [below]. It is not true that we have solved the structure of PrPsc isolated from bovine brain. We have performed and initial characterization of the 3D-strucutre of the recombinant full-length mouse prion protein, PrP(23-231). We obtained very convincing data that residues 23-120 in the full-length protein are flexibly disordered, while the C-terminal domain, PrP(121-231) has exactly the same structure in the full-length protein as the isolated C-terminal domain."

Recombinant full-length murine prion protein, mPrP(23-231):
Purification and spectroscopic characterization

FEBS Lett. 413, 277-281 1997.  (Number 2, 18 August 1997) 
Hornemann, S., Korth, C., Oesch, B., Riek, R., Wider, G., WŸthrich, K. and Glockshuber, R. 

NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231)

FEBS Letter. 413, 282-288 1997 Riek, R., Hornemann, S., Wider, R., Glockshuber, R. and WŸthrich, K.

Three-dimensional NMR structure of a self-folding domain of the prion protein PrP.

Trends Biochem Sci 1997 Jul;22(7):241-242 
Glockshuber R, Hornemann S, Riek R, Wider G, Dilleter M, Wuthrich K

Protein structure responsible for mad cow disease found

 August 15, 1997   Kyodo News Int'l
GENEVA, Aug. 14 (Kyodo) _ A team of Swiss researchers reported Wednesday the discovery of a three-dimentional moleoular structure in a malformed protein, which they blamed for mad cow disease and its fatal human equivalent.

Scientists said the achievement could shed light on the cause of mad cow disease, or bovine spongiform encephalopathy (BSE), and its human equivalent, called Creutzfeldt-Jakob disease (CJD). The team at the Institute for Molecular Biology and Biophysics at the Swiss Federal Institute of Technology said they are the world's first to decode such a structure of an intact prion protein.

Kurt Wuethrich and Rudi Glockshuber said that they and their colleagues at the institute found that a malformed protein taken from the brain of a cow with BSE consists of 98 amino acid residues without the mobile functions as seen in a normal protein. Wuethrich and Glockshuber said this indicates that:

''the conversion of the normal to the disease-inducing form of the prion protein may occur much more easily than has been assumed so far, by way of a new fold in the flexible part of the normal protein.''
The discovery could pave the way for uycovering why a normal prion protein converts into its disease-inducing variant without any genentic mutation.

BetAbetafilaments formed by leucine-rich repeat peptides

webmaster 20 Aug 1997
The authors observe that a very common peptide motif called a leucine-rich repeat (LRR), found in all manner of proteins, can form that cross-beta fibril familiar from known conformational diseases (such as CJD). Although LRR are 'not at present correlated with degenerative disease, the implication is that they will be sooner or later. There is a growing realization that "a propensity for betAbetafilament formation is a common feature of protein sub-domains."

The really interesting observation is the importance of glutamine and asparagine amides to cross beta at least for LRR. For your convenience, I appended the human prion sequence so you can see where its N and Q are, and also the 1991 prediction that the octapeptide repeat of prion protein forms beta structure.

Gln and Asn in human prion protein:

    
     MANLGCWMLV LFVATWSDLG LCKKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP
     HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA
     VVGGLGGYML GSAMSRPIIH FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV
     NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYQRGS SMVLFSSPPV
     ILLISFLIFL IVG
Disease mentioned, rightly or wrongly, as cross-beta structure formers:
Alzheimer, Alzheimer, atrial amyloidosis, atrial amyloidosis, cerebral amyloid angiopathy, cerebral amyloid angiopathy, familial amyloid polyneuropathy I, familial amyloid polyneuropathy I, familial amyloid polyneuropathy III, familial amyloid polyneuropathy III, Finnish hereditary systemic amyloidosis, Finnish hereditary systemic amyloidosis, hemodialysis-related amyloidosis, hemodialysis-related amyloidosis, hereditary renal amyloidosis, injection-localized amyloidosis, injection-localized amyloidosis, medullary thyroid carcinoma, medullary thyroid carcinoma, non-neuropathic systemic amylodosis, primary systmatic amyloidosis, primary systmatic amyloidosis, secondary systmatic amyloidosis, secondary systmatic amyloidosis, spongiform encepalopathies, spongiform encepalopathies, type II diabetes, type II diabetes

Filaments formed by a leucine-rich repeat peptide: structural similarity to the amyloid fibrils of prions and Alzheimer's disease betAbetaprotein.

FEBS Lett 1997 Jul 28;412(2):397-403
Symmons MF, Buchanan SG, Clarke DT, Jones G, Gay NJ
The development of neuro-degenerative diseases often involves amyloidosis, that is the formation of polymeric fibrillar structures from normal cellular proteins or peptides. For example, in Alzheimer's disease, a 42 amino acid peptide processed from the amyloid precursor protein forms filaments with a betAbetasheet structure. Because of this, the structure and dynamics of polymeric peptide filaments is of considerable interest. We showed previously that a 23 amino acid peptide constituting a single leucine-rich repeat (LRRN) polymerises spontaneously in solution to form long filaments of a betAbetasheet structure, a property similar to that of Alzheimer's betAbetaamyloid and prion peptides.

Here we report that a variant of LRRN in which a highly conserved asparagine residue is replaced by aspartic acid does not form either filaments or beta structure. By contrast, a variant which replaces this asparagine residue with glutamine forms filaments ultrastructurally indistinguishable from those of LRRN.

Electron micrographs of LRRN filaments show that many consist of two interleaved strands which appear to have a ribbon-like morphology. X-ray diffraction patterns from oriented LRRN fibres reveal that they are composed of long betAbetasheet arrays, with the interstrand hydrogen bonding parallel to the filament axis. This 'cross-beta' structure is similar to that adopted by betAbetaamyloid and prion derived fibres.

Taken together, these results indicate that the LRR filaments are stabilised by inter- or intrstrand hydrogen bonded interactions comparable to the asparagine ladders of betAbetahelix proteins or the 'glutamine zippers' of poly-glutamine peptides.

We propose that similar stabilising interactions may underlie a number of characterised predispositions to neuro-degenerative diseases that are caused by mutations to amide residues. Our finding that amyloid-like filaments can form from a peptide motif not at present correlated with degenerative disease suggests that a propensity for betfilament formation is a common feature of protein sub-domains. [The only human mutant applicable here is at 178, the GSS mutation just upstream of helix 2, and indeed an extra asparagine is created from an aspartic acid, the new protein forming disease-causing cross-beta. However, the change at codon 217 goes the wrong way, a loss of glutamine (to arginine) is the disease state. -- webmaster]

The complete mature bovine prion protein highly expressed in Escherichia coli

FEBS Lett 1997 Jul 28;412(2):359-364
Negro A, De Filippis V, Skaper SD, James P, Sorgato MC
[The abstract does not indicate whether the 5 or 6 octapeptide bovine prion was used. The signal peptide ends at residue 24 in both. The GPI anchor comes at 242 in the short bovine and at 249 in the long bovine. Evidently the latter was used. The question is, why wasn't this scheme done 15 years ago???--webmaster]

According to the 'protein only' hypothesis, modification of the 3-dimensional fold of the constituent cellular protein, PrP(C), into the disease-associated isoform, PrP(Sc), is the cause of neurodegenerative diseases in animals and humans. Here we describe the high-level synthesis in Escherichia coli, and purification in the monomeric form, of a histidine-tagged full-length mature PrP (25-249) of bovine brain, termed His-PrP.

Based on biochemical and spectroscopic data, His-PrP displays characteristics expected for the PrP(C) isoform. The reported expression system should allow the production of quantities of bovine PrP(C) sufficient to permit 3-dimensional structure determinations.

Codon use in prion gene versus bovine codon use overall

Bos taurus : 1,106 coding DNA sequences comprising 437,180 codons 
Codon Useage
Fields show frequency per thousand and total number of codons of that type seen 
when all bovine proteins in GenBank are analysed in a non-redundant fashion. 
The GC content is 53.83%, with  1st letter GC 55.97%, 2nd letter GC 41.60%, 3rd letter GC 63.91%

UUU 15.8 (6913)  UCU 12.4 (5424)  UAU 11.5 (5044)  UGU  9.7 (4229)
UUC 24.5 (10715) UCC 17.6 (7713)  UAC 19.6 (8553)  UGC 13.8 (6053)
UUA  5.0 (2165)  UCA  9.0 (3953)  UAA  0.7 ( 323)  UGA  1.3 ( 550)
UUG 11.1 (4851)  UCG  4.8 (2078)  UAG  0.5 ( 239)  UGG 13.9 (6056)

CUU 10.8 (4739)  CCU 14.6 (6381)  CAU  7.8 (3392)  CGU  4.0 (1768)
CUC 20.9 (9116)  CCC 20.4 (8906)  CAC 14.5 (6343)  CGC 11.2 (4877)
CUA  5.3 (2306)  CCA 14.1 (6182)  CAA  9.7 (4261)  CGA  5.7 (2472)
CUG 43.4 (18956) CCG  7.8 (3391)  CAG 32.9 (14396) CGG 11.3 (4955)

AUU 14.8 (6453)  ACU 11.2 (4918)  AAU 14.9 (6521)  AGU  9.5 (4158)
AUC 26.2 (11471) ACC 21.7 (9481)  AAC 23.2 (10142) AGC 18.7 (8164)
AUA  6.3 (2734)  ACA 12.9 (5640)  AAA 22.1 (9663)  AGA 10.2 (4460)
AUG 22.6 (9899)  ACG  7.7 (3376)  AAG 35.9 (15699) AGG 11.1 (4867)

GUU  9.9 (4313)  GCU 17.5 (7660)  GAU 21.0 (9196)  GGU 11.2 (4880)
GUC 16.8 (7359)  GCC 31.4 (13719) GAC 29.6 (12954) GGC 25.6 (11190)
GUA  5.8 (2551)  GCA 13.3 (5797)  GAA 26.4 (11548) GGA 16.4 (7186)
GUG 32.1 (14026) GCG  8.5 (3699)  GAG 42.2 (18464) GGG 17.6 (7692)

GenBank carries 8 cow prion DNA sequences, AB001468 (6 repeats), D26151 (promoter fragment),
 D26150 (upstream exons), D10612 + D90545 (6 repeats), S55629 (6 repeats, just CDS),
 D10614 + D90547  (5 repeats, just CDS), D10613 D90546 (6 repeats, just CDS), 
 X55882 (6 repeats, just CDS)

X55882 give this codon use for the long cow prion gene (792 bp = 264 aa)
197 A    193 C    244 G    161 T
25% A  24% C    31%G    20%T      55.2% GC

TTT 3    TCT 2    TAT 5    TGT 1    
TTC 3    TCC 2    TAC 8    TGC 2    
TTA 0    TCA 0    TAA 0    TGA 0    
TTG 0    TCG 0    TAG 1    TGG 9    

CTT 2    CCT 8    CAT 8    CGT 4    
CTC 6    CCC 4    CAC 3    CGC 0    
CTA 0    CCA 6    CAA 5    CGA 4    
CTG 1    CCG 0    CAG 12   CGG 0    

ATT 2    ACT 2    AAT 0    AGT 7    
ATC 5    ACC 8    AAC 11   AGC 2    
ATA 2    ACA 2    AAA 3    AGA 1    
ATG 8    ACG 0    AAG 7    AGG 2    

GTT 0    GCT 4    GAT 1    GGT 15   
GTC 3    GCC 1    GAC 5    GGC 14   
GTA 2    GCA 4    GAA 4    GGA 15   
GTG 10   GCG 0    GAG 4    GGG 6    

Squirrels: a natural reservoir for TSEs?

15 Aug 1997 Per email
Dr. Joseph R. Berger writes that he has an epidemiological study correlating meat and game consumption in Kentucky that should be coming out as a research correspondence in Lancet in the upcoming months. As yet he has no hard data that TSE exists in squirrel populations and will look at that in the near future.

Agent causes brain illness

July 1, 1997
Huntington, West Virginia Herald Dispatch
LEXINGTON, Ky. - A University of Kentucky researcher believes he may have found new information in the study of several brain-destroying illnesses that strike humans and animals. The illnesses are apparently transmitted by a mysterious agent unlike anything else found in nature. Dr. Joseph Berger, chairman of UK's departmen of neurology has discovered a common medical link in squirrel meat consumption in Kentucky, "mad cow" disease in England and a brain-destroying illness that afflicts cannibals in New Guinea.

Berger stressed that he isn't suggesting people are putting themselves at risk by eating squirrel meat or that they should stop. "We need a lot more data.

UK College of Medicine Brings Together U.S. "Virus Hunters" to Discuss Emerging Infectious Diseases

July 7, 1997 More than 250 health care professionals including physicians, nurses, and veterinarians attended the June 27-28 "Hot Zone 1997" conference sponsored by the University of Kentucky College of Medicine on June 27 and 28 at the Hyatt Regency in Lexington. ...

UK College of Medicine infectious disease researchers were well represented. And, Dr. Joseph Berger, UK chair of neurology, presented new information "Mad Squirrel-Mad Cow-Jacob Crueutzfeldt Disease." Berger talked about discovering a common medical link in squirrel-meat consumption in Kentucky, "mad cow" disease in England and a disease that affects cannibals in New Guinea.

Squirrel brains and CJD

      Neurology Web-Forum
      This response submitted by Bill Alford on 8/19/96.
Dr. Eric Weisman (a behavior neurologist) and myself have recently noted at least three patients who suddenly contracted CJD, and who admittedly had consumed the brains of squirrels. The findings were so revelent, that the University of Ky has requested the brains of 100 squirrels be submitted for examination and research. I had a patient that died secondarily of CJD - she was super productive weeks prior to her diagnosis, then vegitated for less than a month before her demise. A sad situation!!!

CJD. Possible transmission to humans by consumption of wild animal brains.

 American Journal of  Medicine 1984 76 1 142-145 
 Kamin, M.; Patten, B. M. 
Although the natural mode of spread of the agent responsible for Creutzfeldt-Jakob disease is not known, several reports suggest transmission through eating contaminated food or brain. Four patients with Creutzfeldt-Jakob disease are described, who had a history of eating the brains of wild goat or squirrel. Those patients indicate the possible acquisition of Creutzfeldt-Jakob disease by ingestion of the agent from a presumptive reservoir in the central nervous system of wild animals.

Transmissible virus dementia: evaluation of a zoonotic hypothesis.

Neuroepidemiology 1986;5(4):194-206 
Davanipour Z, Alter M, Sobel E, Asher DM, Gajdusek DC
Creutzfeldt-Jakob disease (CJD) and kuru are subacute transmissible dementing encephalopathies characterized by spongiform changes in the brain. Scrapie is a similar slow viral encephalopathy which affects sheep, goats and certain other animals. Anecdotal reports suggest that Creutzfeldt-Jakob disease could be a zoonosis. To evaluate the possibility that CJD is acquired from animals, a case-control study was conducted on 26 well-documented CJD cases and 40 controls. Data were collected on exposure to animals through occupations, hobbies, sports and pets. An excess exposure to certain animals was noted among the patients compared to controls in relation to occupation (deer, monkey, squirrel; odds ratio (OR) = 8.9; p less than 0.10) and hobbies (deer, OR = 9.0; rabbit, OR = 6.0; p less than 0.05). Similarly, exposure to animal organs was significantly greater in the CJD group (OR = 20.9; p less than 0.005). Statistically significant increased exposure to sheep or goats was not found among the patients. However, since spongiform encephalopathy has a wider host range than sheep and goats, the increased exposure to certain other animals suggests that a zoonotic source for CJD should be further explored.

What kind of squirrels are we talking about?

  Rodentia Sciurognathi Sciuridae Sciurinae 
                       Sciurus aberti (tassel-eared squirrel) 
                       Sciurus carolinensis (gray squirrel) 
                       Sciurus griseus 
                       Sciurus lis 
                       Sciurus niger (fox squirrel) 
                       Sciurus stramineus 
                       Sciurus vulgaris 

Eating squirrel brain in US

15 August 97 email correspondence
"While we never ate the brains, I can remember the whole squirrel on the plate in the middle of table. Yup, pretty gross. I can remember stories about people cracking open the skull to eat the brains but no one in my family ate it. "

Gene may affect how early people develop Alzheimer's

August 20, 1997 By LEILA CORCORAN, Reuters
Researchers said on Wednesday they had found a gene that might help determine how young people develop Alzheimer's disease. The gene, HLA-A2, joins at least four other genes known to be involved in development of the disease, which causes dementia and eventual death. The HLA gene is involved in production of human leukocyte antigens, which regulate immune system response.

Haydeh Payami and colleagues at Oregon Health Sciences University found that among more than 200 Alzheimer's patients, those who had the HLA-A2 gene got the disease three years earlier than those who did not.

Reporting in the offline journal Neurology, they said 42 percent of the patients had the A2 version of the gene. The gene seemed to combine effects with another known Alzheimer's gene, APOE, they added. About 4 million Americans, including former President Ronald Reagan, suffer from the incurable disease. The researchers noted that non-steroidal anti-inflammatory drugs (NSAIDS), such as ibuprofen, may delay the onset of Alzheimer's. "Thus, Alzheimer's disease may involve an immune/inflammatory response mechanism mediated by HLA," they wrote.

"This is an interesting preliminary finding, and we're eagerly awaiting confirmation by other scientists," said Zaven Khachaturian, director of the Alzheimer's Association's Ronald and Nancy Reagan Research Institute.

"This is a provocative finding because it strengthens the argument for the important role the immune system may play in the expression of Alzheimer's disease."

Soluble amyloid A{beta}1-(40) exists as a stable dimer at low concentrations

1997 272: J. Biol. Chem. 1997 Aug 22 272(34). 21037
William Garzon-Rodriguez, Marisa Sepulveda-Becerra, Saskia Milton, Charles G.  Glabe
Recent studies have implicated the amyloid A peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble A or its oligomeric state, and the existing data are often contradictory.... The A-(1-40) dimers appear to be very stable, because no subunit exchange is observed after 24İh between fluorescent homodimers. Gel filtration confirms that nanomolar concentrations of 14C-labeled A-(1-40) and fluorescein-labeled A-(1-40) elute at the same dimeric position as wild type A-(1-40), suggesting that soluble A-(1-40) is also dimeric at more physiologically plausible concentrations.

Exons 16 and 17 of the amyloid precursor protein gene in familial inclusion body myopathy

K. Sivakumar, L. Cervenakova, M.C. Dalakas, et al.
Annals of Neurology  38:2 267

Presenilins are processed by caspase-type proteases

272: J. Biol. Chem. 1997 Aug 15 272(33). 20655 1997 
Hansruedi Loetscher, Ulrich Deuschle, ..., Helmut Jacobsen 

A Novel Glycosylphosphatidylinositol-anchored Form of Ceruloplasmin Is Expressed by Mammalian Astrocytes

JBC Volume 272, Number 32, Issue of August 8, 1997 pp. 20185-20190
Bharatkumar N. Patel and Samuel David
Ceruloplasmin is a copper-binding protein, which is the major ferroxidase in plasma of hepatic origin. We now provide evidence for a novel membrane-bound form of ceruloplasmin expressed by astrocytes in the mammalian central nervous system. Using a monoclonal antibody (1A1), we show that the cell surface antigen recognized by this antibody is ceruloplasmin and that it is directly anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor.

Our peptide mapping and other immunochemical studies indicate that, except for the GPI anchor, the membrane-bound and secreted plasma forms are similar. We also show that the membrane-bound form of ceruloplasmin has oxidase activity. These studies therefore suggest that the GPI-anchored form of ceruloplasmin may play a role similar to the secreted form in oxidizing ferrous iron.

The GPI-anchored form of ceruloplasmin expressed by astrocytes is likely to be the major form of this molecule in the central nervous system because serum ceruloplasmin does not cross the blood-brain barrier. Lack of this form of ceruloplasmin in the central nervous system could lead to the generation of highly toxic free radicals, which can cause neuronal degeneration as seen in aceruloplasminemia and other neurodegenerative diseases such as Parkinson's and Alzheimer's disease. [At one time, this role below seemed like a possible function for prion protein -- and it still might be. The repeat region has 4 conserved and periodic dentate histidines that bind copper at one micromolar concentrations, possibly creating the necessary prosthetic group for oxidative catalysis. The other interesting aspect raised by this is whether prion protein too might normally be found in the plasma -- webmaster]

See also:

Fumonisin B1-induced sphingolipid depletion inhibits vitamin uptake via the glycosylphosphatidylinositol-anchored folate receptor

272: J. Biol. Chem. July 18 272(29)  18020 1997 
Victoria L. Stevens, Jianhua Tang 

A transmissible subacute spongiform encephalopathy in a visitor to the eastern highlands of New Guinea.

Brain 1976 Dec;99(4):637-658 
Grabow JD, Campbell RJ, Okazaki H, Schut L, Zollman PE, Kurland LT
A Caucasian male, clinically ill with a respiratory disease, visited the Eastern Highlands of New Guinea (endemic for kuru in the Fore people) and developed subacute spongiform encephalopathy (Jakob-Creutzfeldt disease) ten weeks later, from which he subsequently died. Brain material was inoculated intracranially into squirrel monkeys, and several of them developed a spongiform encephalopathy. Monkeys that received control material (normal brain) were normal. Electronmicroscopic features in affected brain tissue are described, and the question of a relationship between Jakob-Creutzfeldt disease and kuru is considered.

Creutzfeldt-Jakob disease after liver transplantation

Annals of Neurology  38:2 269 August 1995
A. Creange, F. Gray, P. Cesaro, et al.

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryoni development

PNAS August 1997
Patrick J. Babin*,, Christine Thisse, ..., and Bernard Thisse 
Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library.

Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

Dwarf marmoset tiny as a mouse found in the Amazon

By CANDACE PIETTE, The Guardian  August 12, 1997 
[Prion sequenes for primates and ungulates have been intensively sequenced, leading to models of how this gene evolved. These newly discovered species allow predictions of the model to be tested in these newly discovered species. -- webmaster]

SAO PAULO, Brazil -- A Dutch biologist working in Brazil's Amazon rain forest says he has found a new species of monkey. There are only about 240 species of primate in the world.

Marc Van Roosmalen, who works for the National Institute for Amazon Research in Manaus, Brazil, says the monkey, which is related to the marmoset family and is about the size of a mouse, was captured in the middle reaches of the Madeira river. It was then brought downriver from Manicore, 300 miles to the south of Manaus, by a local caboclo, a backwoodsman of mixed white and Indian ancestry.

"The creature was inside an empty milk can punched with airholes. Its squeak resembled the chirping of a grasshopper. I saw immediately it wasn't a common pygmy marmoset," Van Roosmalen said.

"This animal had a white border of fur around its face and a black crown, both of which distinguished it. Its ears were exposed and its tail was black and unringed instead of brownish and ringed. I didn't show any excitement in case the guy might not have given it to me."

No place has been so rich in new finds of mammals and plants as the Rio Madeira region of the Amazon. Of the four great river barriers of the Amazon basin, the Madeira is one of the fiercest. With its fast white water infested with caimans, a kind of crocodile, it forms a very effective barrier for small mammals.

"The region is a hotbed of evolution," said Van Roosmalen, who has devoted close study to a number of primates spread across the 2.7 million square miles of Amazon.

"I believe that this new monkey, which we're calling the black-capped dwarf marmoset, is a missing link between the full-size Amazonian marmosets of the genus callithrix, which are only found south of the Amazon and east of the Madeira, and the pygmy marmoset of the genus cebuella, which are found west of the Madeira."

He believes ancestors of the dwarf marmoset must have crossed the river during flooding, probably in a tree which toppled into the current. Isolated between the Madeira and Aripuana rivers, in a small area, it managed to survive alongside the callithrix, which evolved.

New artiodactyl reportedly discovered in Vietnam

August 22, 1997  By GENE KRAMER, Associated Press The Associated Press 
WASHINGTON -- A deer-like animal with tiny antlers that barks like a dog has been identified as a previously unknown species by scientists from skulls collected in the dense jungles of Vietnam, the World Wildlife Fund said Thursday.

The group said an international expedition will return to Vietnam in October hoping for live sightings of what a genetic analysis of tissue samples shows to be a previously unknown species of muntjac.

Named after the rugged mountain range separating Vietnam and Laos, the Truong Son muntjac is more dog-sized than deer-sized, weighing an estimated 34 pounds and standing about 14 inches at the shoulder, the WWF said.

Vietnamese call it sam soi cacoong, meaning "the deer that lives in the deep, thick forest," and "its small size allows it to move freely through dense vegetation," the wildlife group said.

The animal is widely hunted for its meat. Sixteen adult and two fawn skulls were obtained from hunters during an expedition to Vietnam in March and April and were analyzed at the University of Copenhagen, Denmark.

"The Truong Son muntjac has a black coat and extremely short antlers about a thumbnail's length," with no lower "brow tine," said the WWF. It is about half the size and weight of the region's giant muntjac.

Besides body size and weight, the WWF said other distinctive features establishing Truong Son muntjac as a new species were its very short antlers and lack of a lower antler spike. It also noted that the canine teeth of males and females are the same size, in contrast the much smaller females' teeth in giant muntjac.

"Vietnam continues to yield some of this century's most exciting wildlife discoveries (in) ... many areas relatively untouched since prehistoric times," said Dr. Eric Wikramanayake, senior conservation scientist for the wildlife group's Indochina program. "WWF fully expects other new species, both large and small, will be discovered in these regions in the coming years."

Two other mammal species unknown to the world until identified in 1992 and 1993 in Vietnam, he said, were the soala, or Vu Quang ox, and the giant muntjac.

This year's expedition was conducted under the Indochina Subregional Biodiversity Conservation Project financed by the U.N. Development Program, with participation by the Vietnamese Agricultural and Rural Development Ministry and Danang University.

Rare baby addax keeping his species in play

Scripps Howard August 21, 1997
[The addax prion gene was recently sequenced by Schaetzl et al. SAN FRANCISCO -- Six days old, wobbly-legged and frisky, the so far nameless male addax butts his nubbin horns against a Sonoma County oak, unaware that there aren't many more of him on Earth. Partying sheiks, trigger-happy soldiers and big game hunters have used the addax, a spiral-horned antelope, for target practice. Consequently, it is thought to be extinct in its North African habitat, although about 250 may be wandering around in the stony deserts of Mali and Mauritania.

The sand colored addax calf, whose mother "Fuzz" and father "Valentino" were hand-reared at Safari West, a privately-owned wildlife park east of Santa Rosa, Calif., is one of about 2,000 of her species left -- virtually all in zoos, not including San Francisco's. Like his parents, the newborn addax is curious -- a trait that has contributed to the decimation of the species, according to Safari West naturalist Leslie Loutzenhiser. Unlike his parents' forebears, the little fellow is unlikely to be able to take care of himself in his native habitat, because of the human imprint on Mom and Dad and on himself.

Addax "don't drink in their native environment. They use succulents" to obtain the water they require, Loutzenhiser said. At Safari West, it's all the water they can drink and hay and munchies provided by a benevolent staff instead of tough desert forage. Besides, after hand-rearing at Safari West by affectionate human keepers, "they've lost their fear of predators," Loutzenhiser said. It would take a generation or two to condition zoo- or preserve-reared addaxes to return to the wild, she said. The preserve, 65 miles north of San Francisco, was established 10 years ago by Peter Lang, a real estate man and artist who claims to be the last cattle rancher in Beverly Hills.

In a way, it is a museum of the almost past: most of its animal and bird occupants are representatives of threatened or endangered species, such as Asian white necked cranes, scimitar horned and Arabian oryx and bongos. The preserve's inhabitants become semi-pets. They were, in effect, born -- or transferred -- into a sort of humanitarian, animal-loving jail.

Safari West, 400 acres of oak foothills and meadows, is one of half a dozen privately-owned wild animal reserves in the U.S. accredited by the American Zoological Association to buy, loan and trade exotic wild animals. The animal herds are periodically thinned via transfer or sale to other zoos or preserves to invigorate the genetic pool of the endangered or threatened species on the premises.

Clarification of cross-species in vitro barriers

22 Aug 1997  Q&A with Alex Bossers
Question: What are the kinetic consequences of fibril dissociating and forming new nucleation sites, ie, can you measure this?

Answer: Indeed you would expect that if more PrP-res is formed during the conversion, more seed for 'secondary' polymerization should become available. This depends however on the polymer to 'break' in smaller seeds (as expected to hapen in vivo). Whether this happens in vitro (cell-free) is still the question but probably not. Therefore the same amount of polymerizing ends will be present during the growth of the polymer and the new formed PrP-res is not able to speed up the conversion process.

However it should be possible to measure this effect:
- Start a conversion using the absolute minimal amount of PrP-res required to visualize conversion (= starting conversion activity). Use non-labeled PrP-sen and perform a conversion until most of the PrP-sen is converted into PrP-res (incl. PK-digestion).

- SONICATE the conversion sample (including new formed res) by which the PrP-res polymers, which should have become longer, break into smaller seeds again. Perform a second conversion using this PrP-res sample and compare it with the starting converting activity. You should expect the second sample to convert better than the starting sample, whether it is within the level of detection will be the question. If it is, you should also be able to perform 'strain adapting' experiments representing in vivo serial passages of TSE sources.

Question: Do you know if this experiment already has been done unsuccessfully?

Answer: Until now I am not aware of any of these experiments, however the group of Byron Caughey and collegues make a lot of progress with the conversion experiments and they're working always working in a lot of different directions to use the conversion. So, maybe we can expect to hear/see some of these results soon!?

Question:Are your new sheep alleles in GenBank and were there any changes?

Answer: Yes. AJ000681, AJ000680, AJ000679. No changes. I introduced a common nomenclature for the different sheep allelic-variants in the database.

Question:Did you find a sheep allele with a missing cysteine? The disulphide bond, if missing, could have interesting effects on the stability of the prion protein. Have you tried in vitro conversion in the presence of a reducing agent like dithiothreitol?

Answer: No. This is one of many interesting variations that could be studied by site-specific mutagenesis. But be careful of just knocking out one cysteine --look at Muramoto et al in PNAS (1996) 93:p15457-15462 -- a C178A engineered allele prevented conversion to PrP-res, which I think also could be caused not only by preventing the disulfide bridge but also by a possible homodimerizations. Question:Have you looked at conversion of human met 129 val heterozygotes yet?

Answer: Indeed Raymond et al. only did conversions using the 129 homozygotes. Since doing these experiments in such hugh amounts (you have to process each set of conversions you want to compare at the same time, preferable on the same gel, all quantitate etc....) is very very hard work. And using only the homozygotes finally adresses the question after all. Of course a logical follow-up is to do conversions using the heterozygotes and maybe even the other CJD associated allelic variants to construct a paper like our latest PNAS paper.

Note that the Nature paper used the most common variant of bovine-PrP (the 6 octa-repeat variant). Since the 5-octa-repeat variant of bovine PrP is more homologous with sheep, hamster, mouse AND human PrP you could expect different conversion efficiencies form using a 5 instead of a 6 octa-repeat variant. If it would make the final conclusion different is probably another question since sheep and human differ both equally from bovine in the octarepeat section. This question is another follow-up.

Recombinant scrapie-like prion protein of 106İamino acids isİsoluble

 PNAS
Vol. 93, pp. 15457-15462, December 1996 free fulltext online! Tamaki Muramoto, Michael Scott, FredİE. Cohen, and StanleyİB. Prusiner
The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. We previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation.

Removal of a 36-residue loop between helices 2İand 3İdid not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106İresidues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K.İThese results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3İand 4.İThe discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.

We produced synthetic peptides corresponding to each of the four regions designated H1, H2, H3, and H4; unexpectedly, in aqueous buffers H1, H3, and H4 adopted beta-sheet structures ....

Familial spongiform encephalopathy associated with a novel prion protein gene mutation.

Ann Neurol 1997 Aug;42(2):138-146 
Nitrini R, Rosemberg S, ... Albrecht S, Zatz M, LeBlanc A
... We studied a Brazilian family with an autosomal dominant form of dementia. Nine members of the family were affected by a dementia with frontotemporal clinical features, with a mean age at onset of 45 years and a mean duration of symptoms of 4.2 years. Neuropathological examination of 3 patients showed severe spongiform change and neuronal loss in the deep cortical layers and in the putamen, but minimal gliosis in the most severely affected areas. The putamen and cerebellum, but not other areas of the affected brain, displayed prion protein immunoreactivity.

A novel prion protein gene mutation causing a nonconservative substitution T183A was identified in 2 neuropathologically confirmed affected individuals (mother and son). The mutation was transmitted in a mendelian fashion to 12 members of the family. This is a novel prion disease variant characterized by an early onset and long duration of the symptoms, severe spongiform change with minimal gliosis, associated with a prion protein gene mutation at codon 183.

Commentary: T183A disrupts, or should disrupt, the glycosylation site at 181, which wants NXT. So this is an interesting mutation in the context of Collinge's method of strain-typing, which depended on the glycosylation ration of 181/198. Again, the context is 179 CVNITIKQHTVT TTTKGEN 198 in humans. The prion protein might not mature and accumulate inside the cell. This situation was looked at earlier in 1990 hamster transfection work , where it accumulated in ER and cis-Golgi. They didn't get into this nor strain-typing in the present work. Lehmann and Harris find in a very recent JBC that mouse prion at "198" can still be detected at the cell surface, but not so for 181.

The family was tracked for 5 generations,100 or so individuals, with the mutant allele tracked back to Malaga, Spain before 1912. It is inherited as a clear-cut autosomal dominant with ordinary mendalian genetics. Age of onset about 47 but progression is slow. Not much by way of gliosis or periodic EEG, small plaques maybe in putamen, no proteinase K testing, negative on Congo red. The disease did not fit into the GSS, CJD, FFI nomenclature scheme (which needs to be abandoned in favor of CJD T183A etc.)

Some of the affected were met/met, others were met-ala/val-thr, with a more rapid downward spiral in the homozygous case but similar age of onset. Note the met is on the same allele as the mutation; they didn't have any cases so far with met-thr/val-ala. They call this a non-conservative substitution which is has some validity in terms of hydrxyl vs aliphatic but not sterically; also Dayhoff's matrix shows 59 thr to ala PAMs versus 39 for randomly expected, so it is a fairly common accepted change.

You don't need to run 181A through a neural net to see if it would buy into NIA at all, that's for O-glycosylation. Here the rules are N-{P}-ST-{P}, ie no prolines but ser or thr okay. NXTX or NXSX or rarely NXCX ; however X must NOT be Pro; not all sites with these consensus sequences are actually utilized (N-glycosylated). These asparagine codons are reminiscent of some on-going work on making and breaking the disulphide bridge, to see what effect that has on rogue conformer formation. An existing parallel is M232R within the GPI anchor, missing in mature protein, that is in some sense analagous to gylcosylation errors in that non-native prion protein might accumulate intra-cellularly if export processing is blocked. This may simply be tantamount to overproduction (and difficult to sort out from conformational effects), especially if a feedback loop keeps calling from the cell surface for more mature prion.

The other issues raised by T183A are, why hasn't this been seen before, what other mutations are we 'missing,' and are the sites that we have all that special?

The issues here are mis-diagnosis, residue function, and hot spots. That is, since the symptoms did not match earlier forms of CJD, the allele could be more common and simply have been missed. Hot spot mutations such as P102L and D178N turn up quite frequently, even discounting for remote ancestral connections. The latter has been explained by possible spontaneous deamidation of methlylated cytosine or neutral drift (M129V).

In other words, the mutation set does not necessarily reflect the importance of the residue at hand to prion structure and function, it represents a screen for neurological disease, hot spots and allele drift. We cannot conclude that glycosylation at asn 181 is more important than glycosylation at 198 just because only the183 mutation has been detected. T183A might cause disease through disruption of helix H2 and not be attributable to a lack of glycosylation.

Still, with many thousands of individual genes sequenced, why haven't we seen CJD cases arise from mutations at the disulphide cysteines, the asparagines themselves, or the second threonine NFT 200? And surely many other changes would disrupt helices or internal packing etc. And at sites like 178 there are 18 other choices besides asn that could be disruptive (though asn often promotes beta structure). There are only 14 out of 256 codons affected by point mutation with no multiple variants at any given position.

Part of the problem could be technical, mutations are missed in sporadic CJD and neurological patients simply checked for gain or loss of restriction sites and not sequenced or studied by SSCP. Mutations at some sites could be lethal in utero and be lost, though T145s doesn't support this. Note that Y145s lacks both asparagine recognition sites, the disulphide, and the GPI anchor: is it exported or does it pile up at the endoplasmic reticulum? Again, the problem could be partly over-production: only half the mRNA transcripts produce cell-surface product, causing 2x production of T245s.

A disease screen, in this case, would not necssarily pick up mutations that merely affected mature protein function, that is maybe export-blocking and consequent over-production are key. CJD is not a disease of missing function; it is a post-production conformational disease of a degradation byproduct. T183A is autosomal dominant but some mutant merely knocking out function might be recessive. Even double knock-outs are barely a problem in mice -- either the normal function is inessential or the cell has compensatory mechanisms. Of course, many changes would be nearly neutral and certain of these may be confidently predicted. Perhaps mutations are rare because they must cause both overproduction and the subtle conformational change needed for rogue seed establishment and recruitment, or if not seeming overproducers (codon P102L, V180I, V210I) then cause an extra subtle conformational change

One peculiar aspect of CJD is that the disease screen does not necessarily pick up mutations that knock out mature protein function. We are yet to see a case of CJD attributable to loss of normal function, like Tay-Sachs or Lesch-Nyan or phenylketonuria. (Of course, such a disease could be out there lurking under some other name.) CJD is not a disease of missing function; it is a post-production conformational disease of a degradation byproduct, where perhaps export-blocking, consequent over-production, plus subtle conformational change needed for rogue seed establishment and recruitment give hard-to-meet double selection criteria. Mutations that are implausible as over-producers (eg, codon P102L, V180I, V210I) are, in this scenario, extra-subtle extra-efficient conformational changes.

Sure, T183A is a garden variety autosomal dominant, but some mutant merely knocking out normal function might be recessive. In fact, double knock-outs are barely a problem in mice, so why would single knockouts be detectable -- either the normal function is inessential or the cell has adequate compensatory mechanisms (very common). But what about the 'homozygoous' double mutations in humans --would we even recognize them? You can only go so far with neurological symptoms in mice -- their nervous system diverged from ours 85 million years ago. There is no particular reason why prion function mutations should additionally cause CJD.

Perhaps it is not enough to look at the neurology ward -- after all, why should the symptoms bear any resemblance to CJD? To get a rough idea of incidence, expectations could be compared to functional double knockouts in the blue opsin gene. Unlike red/green, this is autosomal, plus quite likely to be detected for free. The bottom line is that blue opsin double recessives, while uncommon, are not all that rare.

We don't even have a name for loss-of-normal-prion-function disease (I propose 'blankopathy' to distinguish it from CJD, with the blank to be filled in later when we know more what this protein does) but without a screen it will be a miracle if we ever detect or recognise it. We know from cross-gene comparative phylogeny that the gene is experiencing extremely strong selective pressure, implying important function. So there are really two different diseases out there, one of them is lurking.

[Note that Y145s lacks both asparagine recognition sites, the disulphide, and the GPI anchor: is it exported or does it pile up at the endoplasmic reticulum? Again, the problem could be partly over-production: only half the mRNA transcripts produce cell-surface product, causing 2x production of T245s. In this scenario, Y145s should be classed with T183A and M232R. Over-production has also been suggested as a consequence of long insertion mutations.]

Codon 129 may be over-rated in terms of fundamental significance. While highly invariant phylogenetically, there is no evidence that valine or leucine (elk) affects normal function and no evidence that it is especially central to the species barrier hetero-oligomer issue (except as a pragmatic matter) -- it is simply the only variant in humans that is convenient to test statistically. Methionine is an interesting amino acid that has never been implicated in an active site in any gene: Stadtman has argued that it is primarily an anti-oxidant positioned to guard active sites.

The bottom line is that we are swamped by hot spot mutations; these are unrepresentative and poorly sample sequence space. On the other hand, such mutations are precious because they occur 'in vivo' and cannot be exactly mimicked in transgenic animals and humans themselves are off-limits to experiment. The tragedy has been a focus on symptoms and gross pathology with a lack of follow-through on biochemical characterization. The pressing issues are (1) actual N- and C-termini of fibril and amyloid after in vivo proteolytic clipping (a whollly different matter fromproteinase K), (2) how the mutation is positioned relative to met/val (resolving allele ambiguity), (3) overproduction (mRNA levels) )4) establishing and sitributing cell lines expressing mutant prion on the cell surface, and (5), which alleles comprise fibril and amyloid (eg, can the mutant recruit the normal allele?). For some reason, it does not seem that in vitro cell lines are established from these patients; these need not be neural to be of use.

Blockade of Glycosylation Promotes Acquistion of Scrapie-like Properties by the Prion Protein in Cultured Cells.

J Biol Chem 1997 Aug 22;272(34):21479-21487
Lehmann S, Harris DA
The conformational conversion of the prion protein, a sialoglycoprotein containing two N-linked oligosaccharide chains, from its normal form (PrPC) to a pathogenic form (PrPSc) is the central causative event in prion diseases. Although PrPSc can be generated in the absence of glycosylation, there is evidence that oligosaccharide chains may modulate the efficiency of the conversion process, and may also serve as molecular markers of diverse prion strains. In addition, mutational inactivation of one of the N-glycosylation sites has recently been associated with a familial spongiform encephalopathy. To investigate the role of N-glycans in determining the properties of PrP, we have expressed in transfected Chinese hamster ovary cells mouse PrP molecules in which N-glycosylation has been blocked either by treatment with the drug tunicamycin, or by substitution of alanine for threonine at one or both of the N-X-T consensus sites.

We report that PrP molecules mutated at Thr182 alone or at both Thr182 and Thr196 fail to reach the cell surface after synthesis, but that those mutated at Thr196 or synthesized in the presence of tunicamycin can be detected on the plasma membrane. We also find that all three mutant PrPs, and to a limited extent wild-type PrP synthesized in the presence of inhibitor, acquire biochemical attributes reminiscent of PrPSc. We suggest that the PrP molecule has an intrinsic tendency to acquire some PrPSc-like properties, and that N-glycan chains protect against this change. However, pathogenic mutations, or presumably contact with exogenous prions, are necessary to fully convert the protein to a PrPSc state.

Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites.

Glycobiology 1990 Sep;1(1):101-109
Rogers M, Taraboulos A, Scott M, Groth D, Prusiner SB
The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor. The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr).

We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene. The T7 RNA polymerase was supplied by infection with a recombinant vaccinia virus. The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP. Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199.

Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC. Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly. The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H. Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.

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