A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine - Contamination with bacterial outer membrane proteins
A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine - Contamination with bacterial outer membrane proteins
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DOI：
10.1074/jbc.m400187200
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发表时间：
2004-05-28
影响因子：
4.8
通讯作者：
Niwa, M
中科院分区：
文献类型：
--
作者：
Furukawa, H;Doh-ura, K;Niwa, M
由于对病毒病的明确诊断有赖于对病毒蛋白异构体(PrPSc)的检测，因此迫切需要建立一种非侵入性的检测方法来检测人类PrPSc。为了评价检测尿中抗蛋白酶的普恩蛋白的诊断价值和可靠性，我们通过Western印迹、液-质联用分析和N-末端序列分析，广泛地分析了普恩疾病患者和对照组中的抗蛋白酶K(PK)蛋白。Shaked等人以前报道过PK抗性信号在32 kDa左右迁移。(Shaked，G.M.，Shaked，Y.，Kariv-Inbal，Z.，Halimi，M.，Avraham，I.和Gabizon，R.(2001)J.Biol.化学。276,31479-31482)未观察到。相反，在许多Pron病患者和两名患病对照组的尿液中发现了表观分子质量类似于37 kDa的离散蛋白条带。虽然这些蛋白也在以多种抗PrP抗体为一抗的Western印迹中发出强烈的信号，但我们发现，仅通过二次抗体的孵育，即在没有一次抗PrP抗体的情况下，这些信号仍然可以被检测到。质谱仪和N末端蛋白序列分析表明，患者尿液中耐PK的37 kDa蛋白主要为肠杆菌属的外膜蛋白(OMP)。从这些细菌分离的OMP对PK具有抗性，从肠道细菌分离的OMP在SDS-PAGE上迁移了约37 kDa。此外，OMPS与抗体的非特异性结合可能被误认为PrPSc。这些发现警告说，细菌污染可能会影响对Pron蛋白的免疫学检测。因此，临床标本，尤其是尿液中PrPSc的免疫学检测应排除肠道细菌的存在。
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 similar to 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.