Abstract
MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.
Highlights
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare early-onset leukodystrophy caused by genetic mutations in the genes encoding glial membrane proteins MLC1 and GlialCAM
An amino acid sequence alignment of six MLC1 proteins from Euteleostomi, a clade of bony vertebrates, showed that approximately 50– 90% of amino acids are identical among the species
The amino acid composition and hydrophobicity of putative TM segments are well conserved among the species, which indicates that the TM topology of MLC1 could be conserved
Summary
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare early-onset leukodystrophy caused by genetic mutations in the genes encoding glial membrane proteins MLC1 and GlialCAM. The pathological outcome of MLC disease is impaired motor coordination, cognitive function and epilepsy. Previous studies have suggested that MLC1 is crucial for astroglial interactions and water homeostasis, which might cause the pathological outcomes of MLC disease [11,12,13]. Eight TM domains were predicted from the hydropathy plot analysis [15]. None of these predictions is supported by experimental data. The only experimentally supported idea regarding the TM topology of MLC1 is that both the amino and carboxylic termini of mouse MLC1 protein reside in the cytoplasmic face [11,15], which suggests that MLC1 protein should contain an even number of (a) royalsocietypublishing.org/journal/rsob Open Biol. The only experimentally supported idea regarding the TM topology of MLC1 is that both the amino and carboxylic termini of mouse MLC1 protein reside in the cytoplasmic face [11,15], which suggests that MLC1 protein should contain an even number of (a) royalsocietypublishing.org/journal/rsob Open Biol. 11: 210103
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