Biochem. J. (2007) 403
(343–351) (Printed in Great Britain)
Aggregation of prion protein with insertion mutations is proportional to the number of inserts
Shuiliang YU*, Shaoman YIN*, Chaoyang LI*, Poki WONG*, Binggong CHANG*, Fan XIAO*, Shin-Chung KANG*, Huimin YAN*†, Gengfu XIAO†, Po TIEN†‡ and Man-Sun SY*1
*Department of Pathology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44120, U.S.A., †Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, People's Republic of China, and ‡Institute of Microbiology, Chinese Academy of Science, Beijing 100080, People's Republic of China
Mutation in the prion gene, PRNP, accounts for approx. 10–15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrPC (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP8OR, and the other with five more octapeptide repeats, rPrP10OR. We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and a-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrPC. Accordingly, rPrP10OR is also more efficient in promoting the aggregation of rPrPC than rPrP8OR. These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.
Key words: aggregation, antibody blocking, insertion mutation, recombinant wild-type prion protein (rPrPC).
Abbreviations used: AS-ELISA, aggregation-specific ELISA; GAG, glycosaminoglycans; GdmCl, guanidinium chloride; HRP, horseradish peroxidase; mAb, monoclonal antibody; PBS-T, PBS containing 0.05% Tween 20; PK, proteinase K; PrP, prion protein; PrPC, normal cellular PrP; PrPSc, scrapie PrP; rPrPC, recombinant wild-type PrPC; rPrP8OR, rPrPC with eight octapeptide repeats; rPrP10OR, rPrPC with ten octapeptide repeats; ThT, thioflavin T.
1To whom correspondence should be addressed (email man-sun.sy@case.edu).
Received 20 October 2006/6 December 2006; accepted 22 December 2006
Published as BJ Immediate Publication 22 December 2006, doi:10.1042/BJ20061592
The Biochemical Society, London ©2007
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