Marjorie B. Lees

Modification of the lowry procedure for the analysis of proteolipid protein

Abstract A modification of the Lowry procedure is described for the rapid and reliable analysis of proteolipid protein. The procedure involves the addition of sodium dodecyl sulfate to dissolve the lipid-containing protein samples or to maintain the water-soluble apoprotein in solution. All steps in the analysis are carried out at room temperature. Chymotrypsin and Versatol are more suitable standards for proteolipid protein than is bovine serum albumin. The method has been applied to the analysis of myelin proteins.

An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, we have previously shown that the disease is mediated by Th1 cells, which recognize tryptophan 144 as the primary TCR contact point. Here we describe an altered peptide ligand (APL), generated by a single amino acid substitution (tryptophan to glutamine) at position 144 (Q144), which inhibits the development of EAE induced with the native PLP 139-151 peptide (W144). We show that the APL induces T cells that are cross-reactive with the native peptide and that these cells produce Th2 (IL-4 and IL-10) and Th0 (IFN gamma and IL-10) cytokines. Adoptive transfer of T cell lines generated with the APL confer protection from EAE. These data show that changing a single amino acid in an antigenic peptide can significantly influence T cell differentiation and suggest that immune deviation may be one of the mechanisms by which APLs can inhibit an autoimmune disease.

A linear Lowry--Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins.

Abstract The protein method of Lowry, Rosebrough, Farr and Randall was modified to give a linear standard curve of absorbance versus μg of bovine serum albumin at 650 and 750 nm wavelengths (1 cm optical path) over the range up to 50 μg protein per ml and an absorbance of 1.1. This was achieved mainly by using a high concentration of Folin-phenol reagent, added rapidly in a volume that was large relative to the final volume. Sodium dodecyl sulfate (SDS) incorporated in the test did not change the albumin standard curve, and linear results were obtained with water-insoluble membrane proteins (myelin proteolipid and rhodopsin) solubilized by SDS.

Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis.

Peripheral blood lymphocytes (PBL) from active and stable multiple sclerosis (MS) patients, patients with other neurologic diseases (OND), and control subjects were tested for sensitization to two myelin antigens not previously examined in multiple sclerosis, using a [3H]thymidine incorporation assay. The antigens investigated were myelin-associated glycoprotein (MAG) and proteolipid protein (PLP). In addition, sensitization to myelin basic protein (MBP) was also tested. Lymphocyte stimulation indices in active MS patients that were greater than 2 standard deviations above controls were as follows: 9/30 for MAG, 0/17 for PLP, and 8/81 for MBP. No control subjects responded to MAG or PLP, and only 1/29 control subjects responded to MBP. Three of the patients that responded to MAG also responded to MBP. Although the mean proliferative response to MAG and to MBP was greater in the population of active MS patients than in stable MS, ONDs, or controls, the difference was not statistically significant. The OND group was the only population which proliferated to PLP (6/16). The only statistically significant differences among the groups for all myelin antigens tested were the proportion of individuals with active MS vs. controls that responded to MAG (P less than 0.05), and OND vs. controls and active MS that responded to PLP (P less than 0.025). The greatest individual responses to the three antigens tested were to MBP in active MS patients. Elimination of the T8 (cytotoxic/suppressor) subset amplified the responses to myelin antigens in some patients and ONDs studied. These studies have demonstrated reactivity to MAG but not PLP in some patients with active MS, and reactivity to PLP in some patients with other neurologic diseases.

Monoclonal Antibodies Against Myelin Proteolipid Protein: Identification and Characterization of Two Major Determinants

: This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme‐linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl‐terminal sequence when tested against a panel of synthetic peptides in a solid‐phase assay. A carboxyl‐terminal pentapeptide (residues 272–276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen‐antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209–217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202–217 and 205–221, suggesting that flanking residues may interfere with peptide‐antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM‐20 isoforms. Twenty‐three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209–217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl‐terminus‐specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.

Acute Experimental Allergic Encephalomyelitis in SJL/J Mice Induced by a Synthetic Peptide of Myelin Proteolipid Protein

Clinical, histologic, and ultrastructural characteristics of acute experimental allergic encephalomyelitis (EAE) induced by sensitization with a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 139-151 HCLGKWLGHPDKF were studied in SJL/J mice. Groups of mice were immunized with 20, 50, or 100 nmol of the peptide and were killed from seven to 28 days after sensitization or when they were moribund. Beginning on Day 9, the mice showed signs of EAE and the disease progressed rapidly to paralysis. Central nervous system (CNS) inflammation, edema, gliosis, and demyelination were found in all mice killed between Days 10 and 28 and white matter lesion areas correlated with clinical score at the time the mice were killed. Peripheral nerve roots and the cauda equina did not have lesions. Within the range studied, the severity of clincal or histologic disease was the same regardless of the PLP peptide dose. Two often mice immunized with 100 nmol and none of 14 mice given smaller doses of a synthetic peptide of mouse myelin basic protein (MBP) showed clinical EAE. These mice had small numbers of CNS lesions that were indistinguishable from those in PLP peptide-sensitized mice. These findings demonstrate that immunization of SJL/J mice with PLP peptide 139— 151 produces a disease with the clinical and morphologic features of CNS tissue-, whole PLP-, whole MBP-, and MBP peptide-induced acute EAE. Thus, PLP is a major encephalitogen and immune reactions to epitopes of different myelin proteins may induce identical patterns of injury in the CNS.

Chronic experimental allergic encephalomyelitis induced in rabbits with bovine white matter proteolipid apoprotein

A chronic, progressive form of experimental allergic encephalomyelitis was produced by immunization of rabbits with bovine brain white matter proteolipid apoprotein. Clinical signs appeared 4 to 13 months after sensitization, and were characterized by ataxia and limb paresis which progressed to flaccid paralysis and incontinence. Light and electron microscopic observations showed both acute and chronic nonsuppurative myelitis or encephalomyelitis accompanied by primary demyelination. Myelin damage was most evident in the spinal cord but was also present in the optic nerve and brain. The neuropathology was consistent with lesions of chronic experimental allergic encephalomyelitis produced by central nervous system tissue, and resembled lesions of multiple sclerosis as well. These observations suggest that protein may be involved in the pathophysiology of demyelinating diseases. A mechanism for the chronic course of the disease is discussed.

Myelin proteolipid protein - the first 50 years

Myelin proteolipid protein (PLP), the most abundant protein of central nervous system (CNS) myelin, is a hydrophobic integral membrane protein. Because of its physical properties, which make it difficult to work with, progress towards determining the exact function(s) and disease associations of myelin PLP has been slow. However, recent molecular biology advances have given new life to investigations of PLP, and suggest that it has multiple functions within myelin and is of importance in several neurological disorders.

Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice ☆

Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in SWR/J (H-2q) and SJL/J (H-2s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In SWR/J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in SWR/J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.

Interactions of Folch-Lees proteolipid apoprotein with planar lipid bilayers.

SummaryWater-soluble Folch-Lees proteolipid apoprotein from bovine CNS white matter induces a voltage-dependent conductance in black lipid membranes. Na+ is required for the induced conductance change but the established conductance has very low ionic selectivity. The induced conductance fluctuates with a minimum amplitude of 10−11–10−10 mho. The magnitude of the conductivity change is dependent on protein concentration and on the composition of lipid bilayers. At a fixed voltage the induced conductance of a phosphatidylcholine-cholesterol membrane is proportional to the sixth power of the protein concentration and the first power of Na+ concentration. The interactions between the apoprotein and the lipids are both electrostatic and hydrophobic, but the interaction leading to the conductance increase appears to be mainly hydrophobic. Both the increase in conductance and the current fluctuations remain after extensive washing of the chambers to remove the protein. Furthermore, pronase or glutaraldehyde added to either the cis or trans side of the membrane does not affect the apoprotein-established conductance. However, if the bilayer is formed in the presence of both the apoprotein and pronase or if the apoprotein is treated with pronase prior to its addition to the chamber, no conductance change is observed. The association of the apoprotein with the membrane thus appears to render the protein inaccessible to proteolytic digestion, suggesting that the apoprotein is at least partially imbedded in the membrane interior.