The detection of corrupt prions (2)
Continued from yesterday
THIS is reflected by its maximum absorbance in the infrared at 1656cm-1 wavelength while the normal diseased prion protein (PrP-res) has a maximum absorbance at 1628cm-1 due to its higher beta sheet content. In fact infectious prion protein (Pr P-res) contains 43-61% beta sheet content compared to only 4% for the normal prion protein. Thus the infectious and normal prion protein (PrP-res) differ mainly in conformational changes due to the secondary structure of otherwise the same protein, with the same covalent structure and amino acid sequence.
One of the most intriguing problems of molecular biology and biochemistry is the propagation of pathology or to show how normal prion protein (Pr P-sen) is converted by the infectious and abnormal prion protein (PrP-res) into an abnormal newly infectious prion protein (PrP-res).
While conversion mechanistically involved both contact and conformational changes by the normal and pathological prion protein, invitro experiments with radioactive sulfur tagged normal prion protein 35S –PrP-sen incubated with infectious prion (PrP-res) and treated with proteinase K gave the product of 35S – Pr P-res, as an intermediate product not identical to the initial pathological protein which indicated the conversion of normal prion protein (PrP-sen) to its infectious isoform, PrP-res (Kocisko et al, 1994).
Furthermore, continuous amplification of infectivity when crude brain homogenate is used as a source of infectious prions (PrP-res) has also been observed (Saborio et al, 1999; 2001). Also, the structure of the various domains of Pr P-sen upon conversion to PrP-res (infectious) oligomers have been assessed and it can be seen that the octarepeat region which normally binds four copper atoms remain exposed to proteases and easily undergoes alteration. Independent deletion mutagenesis studies have shown that the N-terminus of PrP facilitates prion conversion to its pathological PrPres form (Supattapone et al, 2001; Flechsig et al 2000). However, residues 90 to 231 which include the helix-1 salt bridges are unaltered and stabilized against conversion. This region is also known to form strain dependent beta sheet identified by various infra-red bands to reflect strain specific conformations.
In general, the experience of most researchers about normal prion (PrP-sen) conversion to its pathological form (PrP-res) show that it is stimulated by sulfated glycans, chaperone proteins, partially unfolding detergents and an increase in temperature to 65oC (Wong et al, 2000). It is inhibited by disulfide bond reduction and requires PrP-res (the pathological form) as multimers for seeding the process. Additionally, there appears to be specific bonding of the hosts normal PrP-sen to the diseased prion (PrP-res) polymer (DeBurman et al; 1997). This specificity is thought to be responsible for the strain specificity observed in infected animals. For instance, the pathological form of the prion protein from humans shows greater degree of virulence to humans than to other animals.
Several other factors have been observed to influence the rate of conversion of PrP-sen (normal) to Pr P-res (pathological). (Korth et al, 2000, Saborio et al; 2001). Correlations between PrP sequences show that the closer the sequences between PrPsen and PrPres, the higher the conversion rate. Thus, the more the similarities in structure between species, the higher the rate of conversion and inter-species transmissibilities of prion diseases. It is, therefore, reasonable that the highest rate of conversion is obtainable within the same species (Prusiner et al; 1990).
What is peculiarly striking about the mode of prion infection is that it is characteristically different from all our previous knowledge on how infections occur, with the examples of bacteria, plasmodium in malaria, other micro-organisms and viruses. Our past accumulated knowledge show that infections can only occur through the invasion of an infectious agent into a host cell and the subsequent replication of its nucleic acid. However, prion is a simple inanimate protein without any known nucleic acid for self-replication, yet its abnormal isoform is capable of transforming the normal prion protein to an infectious form.
Based on the capacity of the infected prion protein (PrP-res) to induce the conversion of the normal prion protein to its infectious isoform, we have developed an ultra-sensitive means for the detection and diagnosis of infectious prions and its resulting diseases. In this work, we obtained a quick and steady supply of normal recombinant forms of prion protein (PrP-sen) from bacteria (E. coli) for various species of sheep, human, cattle, hamsters, mouse, etc. Normal prion proteins obtained are labeled as rec – Hu PrP, rec-Ha-PrP, rec-Mo-PrP, rec-Sh-PrP, respectively for human, hamster, mouse, and sheep.
Of course, obvious concerns arise which include the dangers of obtaining test samples of cerebrospinal fluid or brain homogenates from animals in the field without subjecting them to trauma, while applying this technique to test for prion diseases. Many animals which include humans and livestock can go into shock, leading to their death in an attempt to obtain test samples from them. Fortunately, on-going experiments at the Laboratory of Persistent Viral Diseases in Hamilton Montana, a branch of the National Institute for Allergy and Infection Diseases (NIAID) a sub-sector of the United States National Institutes of Health (NIH), has since shown results that nose swaps and saliva or other body fluids could serve as harmless substitutes in the application of our ultrasensitive method for the diagnosis of prion disease, thus putting these results beyond dispute as a significant contribution to the advancement of science in the battle against the present resurgence of infectious diseases.
•Onwubiko is Professor of Biochemistry. This an abridged version of his Inaugural Lecture titled “Ultrasensitive Detection of Corrupt Prions and Diagnosis of Their Neurodegenerative Infectious Diseases: Mad Cow Disease in Cattle, Scrapie In Sheep And CJD in Humans” delivered at the University of Nigeria on Thursday 9th July, 2015
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