Orientation of myelin proteolipid protein in the oligodendrocyte cell membrane

The orientation of proteins within a cell membrane can often be difficult to determine. A number of models have been proposed for the orientation of the myelin protein, proteolipid protein (PLP), each of which includes exposed domains on the intracellular and extracellular membrane faces. Immunolabeling experiments have localized the C-terminus and the region spanning amino acids 103–116 to the cytoplasmic face of the membrane, but no well characterized antibodies have been available that label extracellular PLP domains. In this report, we describe the generation and characterization of mouse monoclonal antibodies (mAb) against putative extramembrane domains. Three of the mAb, specific for PLP peptides 40–59, 178–191, or 215–232, immunostain live oligodendrocytes, indicating that these regions of the molecule are exposed on the external surface of the cell. In addition, we have used these mAb to study the time-course of incorporation of PLP into the oligodendrocyte membrane. These studies increase our knowledge of the orientation of PLP in the lipid bilayer and are relevant for understanding myelin function. Special issue dedicated to Dr. Marion E. Smith. Marion has filled many roles in my life (M. Lees): She has been a long time colleague, personal friend, meeting roommate, and traveling companion. Even our husbands have become good friends. Further, Marion’s scientific contributions in multiple aspects of neurochemistry have made her a role model for all scientists, and particularly for young women. It should be noted that all of the authors of this paper just happen to be women.     

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.     

Cell-cell fusion induced by the avian reovirus membrane fusion protein is regulated by protein degradation

The p10 fusion-associated small transmembrane protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the protein for degradation following p10 insertion into an early membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral membrane fusion protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell fusion.     

Cysteine-108 is an acylation site in myelin proteolipid protein.

Myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids. A large proportion of these acyl moieties are bound in thioester linkages, as demonstrated by alkylation of newly formed SH groups upon deacylation. To identify the Cys residue(s) involved in the thioester linkage(s), reduced and carboxyamidomethylated proteolipid protein was labeled with [14C]iodoacetamide upon deacylation with neutral hydroxylamine. The labeled protein was digested with trypsin or pepsin, and peptides analyzed by RP-HPLC. Identification of the isolated radioactive peptides by amino acid analysis, peptide sequencing and/or fast-atom bombardment-mass spectrometry revealed that Cys108 in the bovine PLP sequence is an acylated site. The sequence surrounding the palmitoylation site in the myelin PLP is strikingly similar to that found in rhodopsin. Furthermore, as in rhodopsin and other members of the G protein-coupled receptor family, this Cys residue is located within a hydrophilic, basic, and possibly cytoplasmic, domain.     

Minireview: Autoimmune responses to myelin proteolipid protein

The authors present a brief historical sketch of the development of our understanding of immune responses to myelin proteolipid protein (PLP) and the acceptance of PLP as a potent antigen in the induction of experimental allergic encephalomyelitis (EAE). The distinct characteristics of the PLP molecule that may contribute to complex immune responses to this protein are reviewed and these responses are compared with those to MBP, both in the pathology of EAE and at the level of the T cell. Recent evidence demonstrating differences between T cell responses to PLP and MBP is reviewed. Finally, the potential contribution of immune responses to PLP in human diseases, particularly mutiple sclerosis (MS), that have been identified to date are then summarized.For the authors to write a review on PLP and its role in EAE without Marjorie is like their sailing a ship without a captain, compass or rudder. This review is largely based on work and ideas generated in Marjorie's laboratory, but it was prepared without her input. Consequently, it lacks her meticulous reflection on the structure of each of its sentences and on the use of each word. Papers written with Marjorie are usually honed to near perfection late into the evening at her kitchen table in Newton, where food, ideas, and warmth abound, and where her very patient and accommodating husband Sidney and a demanding but lovable canine are close at hand. Writing this essay gave the authors a chance to recognize our scientific forebears, to consider where we are at this point and to contemplate our future directions in studying immune responses to PLP. We are, indeed, very grateful and indebted to Marjorie for her generous personal and scientific support, wise guidance, inspiration, strength, energy and, most importantly, friendship. Marjorie, we thank you, you are our role model, and we affectionately anticipate many more years of continued collaboration with you.Abbreviations used in this paper CNS central nervous system - EAE experimental allergic encephalomyelitis - MBP myelin basic protein - MHC major histocompatibility complex - MOG myelin oligodendrocyte cyte glycoprotein - MS multiple sclerosis - PLP myelin proteolipid protein - PNS peripheral nervous system - TcR T cell receptor Special issue dedicated to Dr. Marjorie B. Lees.     

Autoacylation of myelin proteolipid protein with acyl coenzyme A

Rat brain myelin proteolipid protein (PLP) is known to contain long chain, covalently bound fatty acids. In the course of characterizing the mechanism of acylation, we found that the isolated PLP, in the absence of any membrane fraction, was esterified after incubation with [3H]palmitoyl coenzyme A (CoA). This observation demonstrated that the protein acts as both an acylating enzyme and an acceptor. Thus, acylation occurs by an autocatalytic process. The possibility of a separate acyltransferase that copurifies with PLP was essentially excluded by adding brain subcellular fractions to the reaction mixtures and by changing the isolation procedure. After deacylation, the protein was acylated at a 4-fold greater rate, suggesting that the original sites were reacylated. The palmitoyl-CoA concentration followed Michaelis kinetics, confirming that spontaneous acylation was not occurring. Pulse-chase experiments indicated that the reaction entails net addition of acyl groups. Although fatty acids are bound via an O-ester linkage, free SH groups are required in the reaction. Denaturation of the protein by sodium dodecyl sulfate or heat inhibits the reaction, whereas cerulenin has little or no effect. PO, the major protein in peripheral nerve myelin, is also an acylated protein, but it was not labeled upon incubation of either peripheral myelin or the isolated protein with [3H]palmitoyl-CoA, demonstrating that it is acylated by a different route. Several synthetic peptides derived from PLP sequences with sites known to be acylated in vivo as well as a series of deacylated PLP tryptic peptides were not labeled, indicating that integrity of the protein is required for acylation. Limited proteolysis and peptide mapping showed that the same sites are acylated in vitro or in vivo, suggesting that the autocatalytic acylation reaction is physiological.     

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.     

A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock.

We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein.     

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

Summary We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5 flanking sequence and the total 5 untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase 11 and -glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.     

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.     

A glycosylated proteolipid protein is common to CNS myelin of recent lungfish (Ceratodidae, Lepidosirenidae)

1. Myelin proteins from the CNS of recent lungfish (Lepidosiren paradoxa, Protopterus dolloi, Neoceratodus forsteri) were separated and analysed by staining and immunoblotting. 2. All species showed a glycosylated component (g-PLP) that cross-reacted with antibodies against tetrapod proteolipid protein (PLP), indicating phylogenetic relationships with amphibia. 3. Actinopterygian IP or teleostean 36k components were not detectable in lungfish CNS myelin. 4. The identical size of g-PLPs from Lepidosiren and Protopterus (Mr = 29,000) underlines the close relationship of the Lepidosirenidae. The smaller size of g-PLP from the ceratodidan Neoceratodus forsteri (Mr = 27,500) pointed to an earlier diversion.     

Topology of proteolipid protein in the myelin membrane of central nervous system. A study using antipeptide antibodies

Peptides according to amino-acid sequences of the N- and C-terminus of lipophilin (proteolipid protein, PLP) (Gly1-Phe15 = 1; Thr261-Phe276 = 6) and of the other four hydrophilic domains (Glu37-Leu60 = 2; Arg97-Leu112 = 3; Gly119-Gly127 = 3A; Trp144-Tyr156 = 3B; Lys191-Ala203 = 4; Asn222-Phe232 = 5) have been synthesized by the solid-phase Fmoc method, linked covalently to keyhole limpet hemocyanin (KLH) and used as antigens. Monospecific antibodies against these antigens were isolated by affinity chromatography. Each antibody recognized its epitope in isolated partially delipidated PLP with the ELISA technique, western blot, thin sections of paraffin embedded rat brains and in the plasma membrane of appropriately fixed/permeabilized rat oligodendrocytes in culture. After fixation with formaldehyde antipeptide 3A antibody stained intact non-permeabilized cells. Therefore the epitope 3A must be located on the extracellular surface of the membrane. This is in full support of our previous biochemical results on the orientation of lipophilin in the myelin membrane.     

Hemopexin is a developmentally regulated, acute-phase plasma protein in the chicken

Identity has been established between chicken hemopexin and alpha 1-globulin "M," a plasma known for the hormone responsiveness of its synthesis in monolayer cultures of embryonic chicken hepatocytes (Grieninger, G., Plant, P. W., Liang, T. J., Kalb, R. G., Amrani, D., Mosesson, M. W., Hertzberg, K. M., and Pindyk, J. (1983) Ann. N. Y. Acad. Sci. 408, 469-489). Identification was based on immunological cross-reactivity, electrophoretic behavior on sodium dodecyl sulfate-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes. Electroimmunoassays were used to investigate plasma protein levels, particularly those of hemopexin, in the acute-phase response and embryonic development. Acute-phase plasma protein production, elicited by injection of chickens with turpentine, bore many similarities to the pattern of hepatocellular plasma protein synthesis produced in response to the addition of specific hormones in culture. The response of the stressed chickens included elevated levels of hemopexin and fibrinogen (5- and 2-fold, respectively) accompanied by a 50% drop in albumin. Hemopexin levels of developing chick embryos were measured for several days before and after hatching. Onset of hemopexin production occurred around the time of hatching, and was followed by a steep increase (more than 1000-fold over 4 days). Similarly, it was not until the 12th h of culture that hepatocytes isolated from both early and late stage chicken embryos began to produce hemopexin, although, from their initiation in culture, they secreted a number of other plasma proteins in quantity. After 12 h, hepatocellular output of hemopexin rapidly accelerated. This precocious induction ex vivo required no hormonal or macromolecular medium supplements. These observations indicate that the embryonic chicken hepatocyte culture system will provide a useful model for studying the regulation of hemopexin biosynthesis in hepatic development and the acute-phase response.