Characterization of myelin basic protein charge microheterogeneity in developing mouse brain and in the transgenic shiverer mutant.
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.
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The Hospital for Sick Children, and Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
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Fredric
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D000818:Animals; D000831:Animals, Newborn / Q000254:growth & development / Q000378:metabolism*; D015153:Blotting, Western; D002845:Chromatography; D004563:Electrochemistry; D007536:Isomerism; D013058:Mass Spectrometry; D051379:Mice / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008818:Mice, Neurologic Mutants / Q000378:metabolism*; D008822:Mice, Transgenic / Q000378:metabolism*; D004676:Myelin Basic Protein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D004676:Myelin Basic Protein