Conservation of Topology, But Not Conformation, of the Proteolipid Proteins of the Myelin Sheath

Abstract

The proteolipid protein gene products DM-20 and PLP are adhesive intrinsic membrane proteins that make up >/=50% of the protein in myelin and serve to stabilize compact myelin sheaths at the extracellular surfaces of apposed membrane lamellae. To identify which domains of DM-20 and PLP are positioned topologically in the extracellular space to participate in adhesion, we engineered N-glycosylation consensus sites into the hydrophilic segments and determined the extent of glycosylation. In addition, we assessed the presence of two translocation stop-transfer signals and, finally, mapped the extracellular and cytoplasmic dispositions of four antibody epitopes. We find that the topologies of DM-20 and PLP are identical, with both proteins possessing four transmembrane domains and N and C termini exposed to the cytoplasm. Consistent with this notion, DM-20 and PLP contain within their N- and C-terminal halves independent stop-transfer signals for insertion into the bilayer of the rough endoplasmic reticulum during de novo synthesis. Surprisingly, the conformation (as opposed to topology) of DM-20 and PLP may differ, which has been inferred from the divergent effects that many missense mutations have on the intracellular trafficking of these two isoforms. The 35 amino acid cytoplasmic peptide in PLP, which distinguishes this protein from DM-20, imparts a sensitivity to mutations in extracellular domains. This peptide may normally function during myelinogenesis to detect conformational changes originating across the bilayer from extracellular PLP interactions in trans and trigger intracellular events such as membrane compaction in the cytoplasmic compartment.

Misalignment of PLP/DM20 Transmembrane Domains Determines Protein Misfolding in Pelizaeus–Merzbacher Disease

“…puzzling that some EC2 substitutions cause the retention of PLP but not of the corresponding DM20 mutant (Gow and Lazzarini, 1996;Gow et al, 1997;Thomson et al, 1997) ) were ER retained, whereas the corresponding DM20 mutants were cell surface expressed and accumulated in late endosomes (Fig. 5A, and data not shown).…”

The Major Myelin-Resident Protein PLP Is Transported to Myelin Membranes via a Transcytotic Mechanism: Involvement of Sulfatide

“…PLP consists of four hydrophobic ␣-helices that span the lipid bilayer with two extracytoplasmic domains and three cytoplasmic domains, while both the C and N termini face the cytoplasm (33,34). Anti-PLP antibodies 4C2, directed against a nonconformational epitope in the first extracellular loop (PLP amino acids 50 to 69), and 2D2, directed against an intracellular region absent from DM-20 (PLP amino acids 100 to 123), were kind gifts of Vijay Kuchroo (Harvard Medical School, Boston, MA) (35).…”

Disease-associated mutations cause premature oligomerization of myelin proteolipid protein in the endoplasmic reticulum

“…The amino acid sequence of PLP is highly conserved, and a large number of naturally occurring mutations are associated with disease. Most of these mutations prevent PLP from reaching the cell surface (11, 12), and evidence suggests that the mutant PLP is misfolded (13,14). Mutant forms of PLP are retained in the ER, and the resulting accumulation of mutant PLP in the ER is thought to be a direct cause of the oligodendrocyte cell death that is the primary clinical feature of PMD (12, 15).…”