Research Article: Analysis of Myelin Basic Protein Fragmentation by Proteasome

Date Published: April , 2009

Publisher: A.I. Gordeyev

Author(s): A. V. Bacheva, A. A. Belogurov, N. A. Ponomarenko, V. D. Knorre, V. M. Govorun, M. V. Serebryakova, A. G. Gabibov.

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Abstract

The proteasome is a high molecular protein complex whose purpose is specific protein degradation in eukaryotic cells. One of the proteasome functions is to produce peptides, which will then be presented on the outer cell membrane using main histocompatibility complex (MHC) molecules of the first or second class. There are definite reasons to believe that proteasome directly takes part in the specific degradation of myelin basic protein (MBP), which make up to 30% of all proteins in the myelin sheath of neuronal axons. The details of the proteasomal degradation of MBP are still unclear. In this work, the features of specific MBP degradation by proteasome were studied.

Partial Text

Multiple sclerosis (MS) – a chronic neurodegenerative disease of autoimmune nature – is an outstanding medical–social problem, because it affects mainly the young and middle-aged. The problem of MS treatment still has no satisfactory solution, and to this day there are several medi-cines (therapies) able to suppress MS to some extent, but not to fully cure it. Neuronal degradation occurs in the brain of MS patients due to the destruction of the neuron’s myelin sheath. One biochemical characteristic which differentiates myelin from other biological membranes is the high lipid/protein ratio. Proteins comprise 25–30% of the mass of the myelin sheath dry matter. About 30% of all myelin proteins are three iso-forms of the myelin basic protein (MBP). MBP is one of the main autoantigen in MS. Earlier, we and other authors showed that catalytic anti-bodies [2–5] and some proteases [6–9] may be involved in MBP degradation. It is known that every eukaryotic cell contains a special compart-ment for targeted protein degradation (proteasome), which is a high molecular protease complex. One of the proteasome’s functions is to produce peptides, which will then be presented on the cell membrane using main histocompatibility complex (MHC) molecules of the first or second class [10]. There are definite reasons to assume that proteasome directly takes part in specific MBP degradation. The details of this process are still unclear. In this work, the specific features of MBP degradation by proteasome were studied.

Proteasome was isolated and purified using the technique described in [13] with slight modifications. At the first stage, the degradation of MBP (from bovine brains, isoform with MW 18,5 kDa) was performed by a full 26S complex and catalytic 20S subparticle isolated from mice liver. It is shown in Fig. 1 that the incubation of MBP with 20S and 26S proteasomes leads to progressive MBP degradation. The 20S proteasome completely hydrolyzed MBP in 45 min, while the 26S proteasome requires 85 min to degrade the same amount of MBP. The variation in reaction rates could be ascribed to different proteasome concentrations: in the case of 20S proteasome, the enzyme/substrate ratio was 2.7 : 1 (μg/μg of protein) or 1 : 14.5 (mol/mol); in the case of the 26S proteasome, the enzyme/substrate ratio was lower, namely 1 : 1 (μg/μg of protein) or 1 : 110 (mol/mol). The pro-teasome amount was estimated by the Lowry method, using bovine serum albumin as a standard.

In conclusion, in our work it was shown that both 20S and 26S proteasomes are able to hydrolyze the myelin basic protein, and the proteasome/MBP molar ratio was found to be 1 : 14.5 and 1 : 110, respectively; the complete hydrolysis time was 45 and 85 min, respectively. After separating hydro-lyzates by HPLC, the molecular weights of the fragments were determined by MALDI mass spectrometry. After analyzing the amino acid sequence of MBP, the proteolytic sites were identified. It was demonstrated that the nonubiquitinated myelin basic protein is a good substrate for both 20S and 26S proteasomes. This was the first work to identify the sites of MBP proteolysis using a proteasome isolated from the brains of SJL/J and Balb/C mice and to show significant differences in the degradation pattern of this autoantigen. These findings could argue for a better presentation of MBP fragments on the MHC molecules in the case of mice genetically predisposed to the development of experimental autoimmune encephalomyelitis.

This work was supported by RFBR grant 07-04-12100-ofi, 09-04-01546-a, 07-04-92168-NCNI_а, NATO SFPP 982833, and the “Fundamental science for medicine – 2008” program of the presidium of the Russian Academy of Sciences.

 

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