Tài liệu Báo cáo khoa học: Binding of N- and C-terminal anti-prion protein antibodies generates

Binding of N- and C-terminal anti-prion protein antibodies

generates distinct phenotypes of cellular prion proteins

(PrPC) obtained from human, sheep, cattle and mouse

Thorsten Kuczius1

, Jacques Grassi2

, Helge Karch1 and Martin H. Groschup3

1 Institute for Hygiene, University Hospital Muenster, Muenster, Germany

2 CEA, Service de Pharmacologie et d’Immunologie, CEA ⁄ Saclay, Gif sur Yvette, France

3 Institute for Novel and Emerging Infectious Diseases, Friedrich Loeffler-Institute, Federal Research Centre for Virus Diseases of Health,

Greifswald – Isle of Riems, Germany

Prion diseases, also known as transmissible spongi￾form encephalopathies, are a group of neurodegener￾ative disorders affecting both humans and animals.

The human forms encompass sporadic and familiar

Creutzfeldt–Jakob disease and the new variant Cre￾utzfeldt–Jakob disease (vCJD), which has been linked

to BSE, the bovine spongiform encephalopathy of

cattle [1,2]. Scrapie is the prion disease in sheep and

goats.

The main characteristic of the disease is the accumu￾lation of an abnormal prion protein (PrPSc), thought

to be the only infectious agent associated with prion

neurodegeneration [3]. The pathogenic mechanism is

assumed to involve conversion of physiological cellular

prion protein (PrPC) to a pathological isoform (PrPSc)

accompanied by a conformational change from a

largely a-helical form into a b-sheet structure [4]. In

contrast to PrPC, the infectious PrPSc protein is deter￾gent-insoluble. PrPC and PrPSc protein samples can be

differentiated by pretreatment with proteinase K (PK),

which completely hydrolyses PrPC but only removes

55–70 amino acid residues in the N-terminal region of

PrPSc resulting in a molecular reduction of 6–8 kDa.

The western blot method is a useful in vitro assay

for the characterization of PrPSc and PrPC, in which

fully glycosylated mouse PrP migrates at 33–35 kDa

Keywords

antibody; glycotyping; prion protein; PrPC;

signal intensity

Correspondence

T. Kuczius, Institute for Hygiene, University

Hospital Mu¨nster, Robert Koch Strasse 41,

48149 Mu¨nster, Germany

Fax: +49 251 9802868

Tel: +49 251 9802897

E-mail: [email protected]

Website: http://www.hygiene.uni-muenster.de

(Received 14 July 2006, revised 20 Decem￾ber 2006, accepted 12 January 2007)

doi:10.1111/j.1742-4658.2007.05691.x

Prion diseases are neurodegenerative disorders which cause Creutzfeldt–

Jakob disease in humans, scrapie in sheep and bovine spongiform

encephalopathy in cattle. The infectious agent is a protease resistant iso￾form (PrPSc) of a host encoded prion protein (PrPC). PrPSc proteins are

characterized according to size and glycoform pattern. We analyzed the

glycoform patterns of PrPC obtained from humans, sheep, cattle and mice

to find interspecies variability for distinct differentiation among species. To

obtain reliable results, the imaging technique was used for measurement of

the staining band intensities and reproducible profiles were achieved by

many repeated immunoblot analysis. With a set of antibodies, we discov￾ered two distinct patterns which were not species-dependent. One pattern is

characterized by high signal intensity for the di-glycosylated isoform using

antibodies that bind to the N-terminal region, whereas the other exhibits

high intensity for protein bands at the size of the nonglycosylated isoform

using antibodies recognizing the C-terminal region. This pattern is the

result of an overlap of the nonglycosylated full-length and the glycosylated

N-terminal truncated PrPC isoforms. Our data demonstrate the importance

of antibody selection in characterization of PrPC.

Abbreviations

BSE, bovine spongiform encephalopathy; PK, proteinase K; PrP, prion protein; SAF, scrapie-associated fibril; vCJD, variant Creutzfeldt–Jakob

disease.

1492 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS