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J. Biol. Chem., Vol. 279, Issue 22, 23661-23667, May 28, 2004

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A Pitfall in Diagnosis of Human Prion Diseases Using Detection of Protease-resistant Prion Protein in Urine

CONTAMINATION WITH BACTERIAL OUTER MEMBRANE PROTEINS^*

Hisako Furukawa, Katsumi Doh-ura¶, Ryo Okuwaki||, Susumu Shirabe**, Kazuo Yamamoto||, Heiichiro Udono, Takashi Ito, Shigeru Katamine||, and Masami Niwa

From the Departments of Pharmacology 1, ^||Molecular Microbiology and Immunology, the ^**First Department of Internal Medicine, Department of Biochemistry, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan, the ^¶Department of Prion Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Sendai 980-8575, Japan, and the Laboratory for Immunochaperones, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Tsurumi, Yokohama 230-0045, Japan

Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrP^Sc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrP^Sc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrP^Sc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrP^Sc in clinical samples, in particular, urine.

Received for publication, January 8, 2004 , and in revised form, March 12, 2004.

^* This work was supported by grants from the Ministry of Health, Labor and Welfare, Japan and the Kurozumi Medical Foundation, Japan (to H. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology 1, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel.: 81-95-849-7043; Fax: 81-95-849-7044; E-mail: hisako{at}net.nagasaki-u.ac.jp.

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