Related Experiment Videos
Characterization of myelin basic protein charge microheterogeneity in developing mouse brain and in the transgenic
1The Hospital for Sick Children, and Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
Abstract:
Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 +/- 0.79. C-2 consisted of two 14-kDa species, 14,136.37 +/- 0.74 and 14,204.45 +/- 0.70. Two variants, 14,215.57 +/- 0.94 and 18,413.57 +/- 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the transgenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.
Insights
Researchers characterized mouse myelin basic protein (MBP) isoforms using adapted chromatography and gel electrophoresis. The 18.5-kDa isoform appears early in myelinogenesis, while the 14-kDa isoform emerges later and is crucial for completing the process.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Myelin basic protein (MBP) is crucial for central nervous system myelination.
- Understanding murine MBP is vital due to the use of transgenic and knockout mouse models.
- MBP exhibits heterogeneity due to alternative splicing and posttranslational modifications.
Purpose of the Study:
- To isolate and characterize the distinct MBP species in the murine brain.
- To investigate the role of different MBP isoforms during myelinogenesis.
- To analyze MBP composition in the transgenic shiverer mutant mouse.
Main Methods:
- Adaptation of scaled-down CM-52 chromatography for MBP charge isomer isolation.
- Utilized alkaline-urea slab gel electrophoresis for efficient MBP separation and Western blotting.
- Mass spectrometry was employed for precise molecular weight determination of MBP components.
Main Results:
- Murine MBP resolved into two main populations of charge isomers: 18.5-kDa and 14-kDa isoforms.
- The 18.5-kDa isoform was detected early in myelinogenesis (day 4), while the 14-kDa isoform appeared later (day 16) and became dominant.
- The shiverer mutant primarily synthesized the 18.5-kDa isoform, lacking the 14-kDa isoform, similar to early developmental stages.
Conclusions:
- The 18.5-kDa MBP isoform initiates myelinogenesis, but the 14-kDa isoform is essential for its completion.
- The shiverer mutation's inability to complete myelination is linked to the absence of the 14-kDa MBP isoform.
- This study provides critical insights into murine MBP heterogeneity and its developmental significance.