title: A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins.
abstract: Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc 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 approximately 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 PrPSc. 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 PrPSc in clinical samples, in particular, urine.
authors.affiliation: Departments of Pharmacology 1, Nagasaki University Graduate School of Biomedical Sciences, Japan. hisako@net.nagasaki-u.ac.jp
authors.forename: Hisako
authors.initials: H
authors.lastname: Furukawa
authors.forename: Katsumi
authors.initials: K
authors.lastname: Doh-ura
authors.forename: Ryo
authors.initials: R
authors.lastname: Okuwaki
authors.forename: Susumu
authors.initials: S
authors.lastname: Shirabe
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Heiichiro
authors.initials: H
authors.lastname: Udono
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
authors.forename: Shigeru
authors.initials: S
authors.lastname: Katamine
authors.forename: Masami
authors.initials: M
authors.lastname: Niwa
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000652:urine*; D003429:Cross Reactions; D004755:Enterobacteriaceae / Q000378:metabolism; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods; D018620:PrPSc Proteins / Q000652:urine*; D017096:Prion Diseases / Q000175:diagnosis* / Q000652:urine; D012680:Sensitivity and Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D018620:PrPSc Proteins