doi: https://www.frankenthalerfoundation.org
The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.
Myelin sheaths are multilamellar membrane wrappings insulating selected neuronal axons, enabling 20-100 times faster conduction of action potentials along myelinated axons. High conduction velocity is fundamental for motor, sensory and cognitive functions. Myelin is formed by specialized glial cells; oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). In the CNS, where larger than 0.2 µm diameter axons are generally myelinated, cell processes of oligodendrocytes spirally wrap around the axons to form an insulating myelin sheath.
Myelin can be divided into two distinct compartments: compact and non-compact myelin. Compact myelin, found in the internodal segments, is a tightly packed multilayered structure, in which the distance between two apposing outer membrane surfaces is only ∼2 nm and no free cytoplasm is present. The thickness of the myelin sheath generally depends on the axon diameter; mature CNS myelin may comprise up to 160 membrane turns with a total thickness of 1.7 µm. CNS myelin is lipid-rich (70-75% lipid of total dry weight), with a unique lipid profile, mainly consisting of cholesterol (CH), galactosyl ceramide, and ethanolamine plasmalogen.
Only a few specific proteins comprise the majority of CNS myelin protein; these include myelin proteolipid protein (PLP), myelin basic protein (MBP), cyclic nucleotide phosphodiesterase (CNPase), myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG). Each of these myelin-specific proteins has a particular function and localization within the myelin sheath. PLP and MBP are the major constituents of CNS myelin, forming 38% and 30% of the total protein mass, respectively. Both PLP and MBP exist in the compact compartment and interact tightly with lipid bilayers. MBP is a membrane-embedded protein, located on the cytoplasmic leaflet of the myelin membrane, within the major dense line. MBP is expressed by both oligodendrocytes and Schwann cells and is crucial for CNS myelin compaction. MBP has a major role in myelination, forming a molecular barrier to restrict redundant cytoplasmic and membrane-bound proteins from entering between the myelin lamellae in compact myelin.
PLP is a 30-kDa integral membrane protein mainly expressed by CNS oligodendrocytes. Minor expression can be observed in PNS myelin, as well as in kidney distal and proximal tubules. PLP is a tetraspan integral membrane protein with cytoplasmic N and C termini. Two alternative isoforms of PLP are expressed; the minor isoform DM20 (26 kDa) lacks 35 residues in its intracellular loop. PLP and DM20 are extremely lipophilic and predicted to contain several cysteine-linked fatty acid moieties. PLP interacts with membranes and has a high affinity towards CH-rich lipid rafts.
PLP is a target for autoantibodies associated with multiple sclerosis (MS). Copy-number variation or mutations in the PLP-encoding gene, PLP1, result in pathological conditions, such as lethal Pelizaeus-Merzbacher disease (PMD) and milder spastic paraplegia type 2 (SPG2), which both lead to CNS hypomyelination, hypotonia, ataxia, spasticity, and delayed development of motor and cognitive skills. Additionally, a complete deletion of PLP1 results in the demyelination of peripheral nerves. In transgenic mice, over-expression of PLP1 produces myelin defects similar to those observed in PMD patients, as well as the accumulation of PLP in vacuoles of the oligodendrocyte soma. Respectively, PLP-deficient mice show moderate phenotypic changes with a slightly reduced or delayed myelination of small-diameter axons, increased number of cytosolic channels in compact myelin, inadequate compaction of myelin, and axonal damage. Hence, a key function of PLP is to adhere myelin lamellae together and enhance the compaction of myelin, thus increasing the physical stability of myelin, as well as to support axon-myelin metabolism.
Due to the high abundance of myelin PLP in brain tissue, PLP has been a target for extensive research for over 70 years. Thus far, the challenging extreme hydrophobicity of PLP has hindered its structural and functional characterization. Here, we established a recombinant production system for biologically active human PLP and its shorter isoform DM20. We used small-angle X-ray (SAXS) as well as neutron scattering (SANS), the latter with contrast matching, to reveal the low-resolution 3D structure of dimeric human PLP and DM20 in membrane-mimicking detergent complexes. We demonstrate that lipid membrane-reconstituted recombinant PLP, as well as DM20, induces formation of multilamellar, highly organized membrane assemblies with a repeat distance resembling CNS myelin. The characteristics and determinants of PLP/DM20 membrane stacking were further unraveled using X-ray diffraction, electron microscopy (EM), and atomic force microscopy (AFM). Through our experiments, we provide a novel insight into the structure of two integral membrane proteins of human CNS myelin and the membrane multilayers they assemble together with lipids.
Myelin PLP is highly conserved among vertebrates. The human and mouse proteins share 100% identity, and even birds and amphibian proteins are more than 85% identical to mammalian proteins. Regardless of the extreme lipophilicity of PLP, we managed to establish a system to produce recombinant human PLP and DM20 in large scale for structural and functional studies. We overexpressed human PLP and DM20 with a cleavable green fluorescent protein (GFP) and an octahistidine tag in the baculovirus-insect cell expression system. PLP and DM20 were solubilized from expression host cell membranes using maltose-based detergents and purified with standard purification techniques. Briefly, after solubilization, immobilized metal affinity chromatography (IMAC) followed by tag cleavage by Tobacco etch virus protease and reverse IMAC were used prior to a final size-exclusion chromatography (SEC) step to obtain pure protein. In SEC, PLP and DM20 showed similar elution patterns with one major peak and minor peaks with higher-order oligomers. Pure elution fractions were combined from the main peak for further experiments.
Fig 1 Purification of folded PLP and DM20.
A SEC-MALS shows dimeric state for PLP (upper panel) and DM20 (lower panel) in a detergent complex. B The pure PLP and DM20 SEC fractions on Coomassie stained SDS-PAGE. Molecular weight markers in kDa are shown on the left. C SRCD indicates α-helical conformation for PLP and DM20. The lipid composition and detergent tail length slightly affect the protein secondary structure content.
Since PLP and DM20 are transmembrane proteins, they were purified in a complex with a detergent that mimics the membrane bilayer and embeds the hydrophobic transmembrane regions of the protein. This complicates the analysis of the protein oligomeric state, however. To study the oligomeric status of PLP and DM20, the protein molecular weight in detergent complexes, and the amount of bound detergent, were analyzed using multi-angle light scattering (MALS). The total particle molecular weights of the main elution peaks of PLP and DM20, comprising both the protein and bound n- decyl - β-D - maltopyranoside (DM) molecules, were 103.4 kDa and 96.5 kDa, respectively. The molecular weight of the protein fraction was further determined using protein conjugate analysis, showing a molecular weight of 57.0 kDa for PLP and 54.1 kDa for DM20, which is 35 residues shorter. These values are close to the calculated dimeric molecular weights of PLP (60.0 kDa) and DM20 (52.4 kDa), indicating that each protein-detergent complex is composed of two protein molecules surrounded by a detergent belt. Both protein-detergent complexes include ∼45% of detergent, corresponding to 96 and 87 DM molecules for PLP and DM20, respectively.
To follow the folding of human PLP and DM20, we explored the secondary structure content of recombinant PLP and DM20 in complex with maltose-based detergents with either a 10- (DM) or an 11-carbon tail (n- undecyl-β-D-maltopyranoside (UDM)) using synchrotron radiation circular dichroism spectroscopy (SRCD). SRCD spectra for both PLP and DM20 indicate a predominantly α-helical conformation, indicating the proper folding of the recombinant proteins.
A clear difference between detergents was observed for PLP; UDM gave rise to more intense spectral features despite the equal protein concentration in the samples, which can be a sign of more rigid ordering of the transmembrane helices. A minor variation in the spectral shape was detected at 213-222 nm, also possibly reflective of a slightly lower helical content of PLP in DM. On the other hand, DM20 showed no intensity difference between detergents, but spectral shape was slightly altered (at 207-215 nm) when comparing DM20 spectra in DM and UDM; this could relate to minor differences in either helical content or helix packing. These results illustrate an alteration in pr
