Monoclonal antibodies to distinct regions of human myelin proteolipid protein simultaneously recognize central nervous system myelin and neurons of many vertebrate species

Abstract

Myelin proteolipid protein (PLP), the major protein of mammalian CNS myelin, is a member of the proteolipid gene family (pgf). It is an evolutionarily conserved polytopic integral membrane protein and a potential autoantigen in multiple sclerosis (MS). To analyze antibody recognition of PLP epitopes in situ, monoclonal antibodies (mAbs) specific for different regions of human PLP (50–69, 100–123, 139–151, 178–191, 200–219, 264–276) were generated and used to immunostain CNS tissues of representative vertebrates. mAbs to each region recognized whole human PLP on Western blots; the anti‐100–123 mAb did not recognize DM‐20, the PLP isoform that lacks residues 116–150. All of the mAbs stained fixed, permeabilized oligodendrocytes and mammalian and avian CNS tissue myelin. Most of the mAbs also stained amphibian, teleost, and elasmobranch CNS myelin despite greater diversity of their pgf myelin protein sequences. Myelin staining was observed when there was at least 40% identity of the mAb epitope and known pgf myelin proteins of the same or related species. The pgf myelin proteins of teleosts and elasmobranchs lack 116–150; the anti‐100–123 mAb did not stain their myelin. In addition to myelin, the anti‐178–191 mAb stained many neurons in all species; other mAbs stained distinct neuron subpopulations in different species. Neuronal staining was observed when there was at least approximately 30% identity of the PLP mAb epitope and known pgf neuronal proteins of the same or related species. Thus, anti‐human PLP epitope mAbs simultaneously recognize CNS myelin and neurons even without extensive sequence identity. Widespread anti‐PLP mAb recognition of neurons suggests a novel potential pathophysiologic mechanism in MS patients, i.e., that anti‐PLP antibodies associated with demyelination might simultaneously recognize pgf epitopes in neurons, thereby affecting their functions. © 2006 Wiley‐Liss, Inc.

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

“…By employing the three available anti-extracellular PLP antibodies in the OLN-PLP cell lines, we investigated the possibility that PLP could undergo such changes when sensing the presence of sulfatide at the level of the plasma membrane. Immu- nocytochemical analysis of live cells revealed that antibody 4C2, which is directed against a nonconformational epitope in the first extracellular loop (35), recognizes PLP-eGFP at the surface of virtually all OLN-PLP, OLN-PLP-G, and OLN-PLP-GS cells (Fig. 7A), confirming the biochemical analysis (cf.…”

“…Overexpression of syntaxins leads to nonfunctional SNARE complexes (43), thus mimicking a dominant negative approach. Double labeling with anti-PLP antibodies recognizing extracellular (O10) and intracellular (2D2) PLP epitopes (35,36) revealed that in OLGs that overexpress syntaxin 3, transport of PLP to myelin sheets was apparently blocked, while in cells that overexpress syntaxin 4, PLP is present at the surface ( Fig. 2A).…”

“…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). The epitope recognized by anti-PLP antibody O10 is acquired posttranslationally and is directed against a conformation-dependent epitope in the second extracellular loop (kind gift of Evi Albers-Krämer, Mainz, Germany) (36).…”

Neuronal expression of the proteolipid protein gene in the medulla of the mouse

“…Expression in glial progenitors occurs early in embryonic development and increases with postnatal maturation (LeVine et al, 1990;Spassky et al, 1998;Delaunay et al, 2008). Several lines of evidence, including our own prior work, have demonstrated expression of the Plp gene in CNS neurons (Bongarzone et al, 1999;Jacobs et al, 2003Jacobs et al, , 2004Miller et al, 2003;Greenfield et al, 2006). In oligodendrocytes, PLP is targeted by specific trafficking sequences to the membrane (Wight et al, 1993), where it is an important myelin component.…”

Autoantibody-producing plasmablasts after B cell depletion identified in muscle-specific kinase myasthenia gravis

“…Hybridomas producing monoclonal Igs to MuSK were prepared from lymph nodes of immunized mice using standard protocols (59). Mice were obtained from the Jackson Laboratory.…”