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Identification of glycosylation sites of tau proteins

Projektdetails:

Thematik: Ursachenforschung
Förderstatus:abgeschlossen
Art der Förderung:Research
Institution:Heinrich-Heine-Universität Düsseldorf, Biologisch-Medizinisches Forschungszentrum (BMFZ)
Projektleitung:Prof. Dr. Ralf Hoffmann
Laufzeit:01. November 1999 - 31. Oktober 2001
Fördersumme:63.819,00 Euro

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Projektbeschreibung

Tau protein is the main constituent of the neurofibrillary tangles that are found inside the nerve cells of AD patients. It is not known whether tangles actually destroy neurons or if they are simply the byproducts of Alzheimer's ruinous effects. So far Dr. Hoffmann has been able to identify and characterize three AD specific modification sites on the tau protein, where phosphate groups can be bound. These are called phosphorylation sites. These phosphorylation sites are the first identified biochemical markers of sporadic AD and may be useful for early diagnosis by a simple blood test. Furthermore, Dr. Hoffmann engineered synthetic mini-proteins as models of tau to study these modifications and to develop simple clinical tests.

In Dr. Hoffmann’s opinion the AD specific phosphorylation pattern of tau is only a secondary effect observed in the brain of patients after several years, despite its value for AD diagnosis. His hypothesis is that the early events in AD development are connected to a deregulated glycosylation machinery, i.e., reversible incorporation of sugars in the tau protein catalyzed by enzymes. Recent work already suggests differences in the glycosylation patterns of tau from AD patients and from healthy age-matched controls. The exact nature of the sugars and, most important, the precise positions of tau specific glycosylation sites have not been identified so far. Moreover, there are several reports from other fields of AD research, most notably the amyloid precursor protein (APP) and the presenilins, which also indicate deregulated glycosylation as an early event in AD. Interestingly, mutations identified in familial AD appear to have similar effects as deregulated glycosylation.

In this project Dr. Hoffmann wants to elucidate the glycosylation process. He also wants to study the influence of fragments that result from this process on the functioning of tau protein. The resulting fundamental biochemical understanding would not only allow early diagnosis and prognosis but could also build the basis for new drugs. The produced antibodies may already be useful for early diagnosis of Alzheimer's disease, similar to the phosphate-specific antibodies that he produced earlier.

Zwischenbericht

Alzheimer's disease is a degenerative neurological disease that mainly affects elderly people and is characterized by nerve cells in the brain progressively dying. People begin to lose nerve cells when they get older and so a definitive diagnosis can only be made post mortem by an autopsy which can identify the two main neuropathological markers of the disease: ß-amyloid plaques and neurofibrillary tangles. ß-amyloid plaques are formed from insoluble clumps of the ß-amyloid protein, whereas the neurofibrillary tangles (NFT) are formed inside nerve cells by modified tau protein. Our project targets the glycosylated tau protein, that is, the tau protein carrying several carbohydrates. The aim of our work is to identifu the exact position of each carbohydrate in the protein. Thus, we isolated tau protein from bovine brains and Alzheimer's brains.

In our studies we had to separate the tau proteins from the total amount of other brain proteins to obtain the highest possible purity grade. Successful isolation of tau protein involved the homogenisation of whole brains to dissolve all brain proteins. We prepared tau by two different methods in which one preparation (the twice cycled preparation) yielded a highly purified tau protein that unfortunately had lost a significant amount of its glycosylation. Our second preparation (total tau preparation) showed a high degree of contamination with other brain proteins but no glycosylated tau was lost. So we focused on purification of the total tau preparation to isolate tau without any loss of glycosylation. The first purification step applied chromatographic separation according to the protein size (size exclusion chromatography) and several centrifugation and precipitation steps. Here we were able to separate the medium-sized tau proteins from smaller and larger proteins. This partially purified tau protein was further purified according to protein interactions based on their polarity. Thus it was possible to separate tau from most other proteins.

All obtained fractions were characterized in respect to their tau content and to tau modified with carbohydrates. This was done by gel electrophoresis in which the proteins were separated according to their size in an electric field. The presence of tau in each fraction was verified with a specific antibody directed against tau whereas the carbohydrates were detected by a specific carbohydrate-binding lectin to identify glycosylated tau. As it is not possible to do this in a single test we analyzed each fraction twice, once with the antibody to identify tau and in a second aliquot of the fraction with lectin. To determine the glycosylation status of tau both results were compared with each other. Fractions that were detected from both antibody and lectin showed that we had purified the tau protein with bound carbohydrates. These verification reactions had to be done before and after each purification step to be sure that we did not lose significant amounts of tau and glycosylated tau. We could show that neither tau nor glycosylated tau was lost during this purification protocol and that at the end tau was obtained at high purity with its supposedly native glycosylation status.

Most contaminating proteins were removed. Since the outlined purification strategy resulted only in a small protein amount we had to repeat the total tau preparation several times to accumulate tau including the described analysis of all intermediate steps and fractions of the complete preparation and purification protocol. Additionally, we established two-dimensional gel electrophoresis; a method that allows separation and characterization of proteins according to their charge in the first direction and their size in the second direction. This resulted in tau protein spots representing the different tau variants with their inherent modification grade. This analytical technique with its high resolution allows us to characterize each tau variant and each fraction (if necessary) by its inherent charge and size distribution.

Our next goal is to identify the exact positions of the carbohydrates bound to tau. Therefore, it is necessary to cleave the tau protein into small pieces (peptides) and sequence them on a mass spectrometer operated in our laboratory. The different carbohydrate patterns between Alzheimer and non-Alzheimer brains will provide information about carbohydrate positions on the tau protein with the promise of new insights into the development of Alzheimer's disease and new hints towards the molecular cause of Alzheimer's disease.


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