Cleavage of the PO glycoprotein of the rat peripheral nerve myelin: Tentative identification of cleavage site and evidence for the precursor-product relationship

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37°C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24kDa protein with a concomitant decrease of PO protein. The conversion of PO into 24kDa protein was blocked by heating isolated myelin at 100°C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24kDa protein. Similarly, addition of CaCl₂ to isolated myelin did not accentuate the formation of 24kDa protein suggesting that the conversion of PO into 24kDa protein may not be due to Ca²⁺ activated protease. It is postulated that the formation of 24kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24kDa protein was purified and characterized. The N-terminal sequence of 1–17 amino acid residues of 24kDa protein was identical to PO. 24kDa protein was immunostained and immunoprecipitated with anti-PO antiserum indicating the immunological similarities between PO and 24kDa protein. Labeling of 24kDa protein with [³⁵S]methionine provided evidence that PO may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24kDa protein was phosphorylated, glycosylated and acylated like PO. Phosphorylation of 24kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24kDa protein. These results suggested that serine residue phosphorylated by protein kinase C may be located in amino acid residues 1-168. 24kDa protein was stained with periodic Schiff reagent. In addition, 24kDa protein was fucosylated and the fucosylation of 24kDa protein was inhibited (70%) by tunicamycin, providing evidence that it is N-glycosylated. Recently, it was demonstrated that both PO and 24kDa protein were fatty acylated with [³H]palmitic acid in the nerve slices and fatty acids are covalently linked to these proteins (Agrawal, H.C. and Agrawal, D. 1989, Biochem. J. 263:173–177). The time course of inhibition of acylation by cycloheximide of 24kDa protein was identical to PO. Cycloheximide inhibited acylation of PO and 24kDa protein by 61% and 58% respectively, whereas, monensin had little affect on the fatty acylation of these proteins. Less [³H]palmitic acid and¹⁴C-amino acids were incorporated into 24kDa protein when compared to PO between 5–30 min after incubation of the nerve slices. However, more radioactivity was incorporated into 24kDa protein after 60 min when compared to PO under identical conditions. These results provided evidence of a precursor-product relationship between PO and 24kDa protein. Therefore, PO may be cleaved into 24kDa protein in the myelin membrane following its acylation and glycosylation in the Schwann cells.     

Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3×106. This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350–400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39–1.40 g ml–1, suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources 001contain three common structures, namely Gal13GalNAc, GlcNAc16 (Gal13) GalNAc and Gal14 GlcNAc6 (Gal13)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.     

Myelin-Associated Glycoprotein and Related Glycoconjugates in Developing Cat Peripheral Nerve: A Correlative Biochemical and Morphometric Study

The expression and accumulation of the myelin-associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK-1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60-kilodalton (kD) glycoprotein and a group of 20-26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate-3-glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20-26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.     

Mycobacterium leprae binds to a major human peripheral nerve glycoprotein myelin P zero (P0)

We have previously shown that a major phosphorylated 25-kDa glycoprotein of the human peripheral nerve binds to Mycobacterium leprae. In the present study, we confirm that the 25-kDa glycoprotein of the human peripheral nerve is myelin P zero (P₀) by immunoprecipitation and Western blot experiments using monoclonal antibodies to myelin P₀. Immunohistochemical studies on human nerve using these antibodies to myelin P₀ exhibited a strong immunoreactivity to the myelin and Schwann cells. Myelin P₀ is a peripheral nerve specific protein; therefore it could likely be one of the key target molecules for M. leprae binding/internalization or even contact-dependent demyelination. This finding of M. leprae binding to myelin P₀ adds to the present understanding on neural predilection of M. leprae.     

High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.     

Subcellular distribution of sulfated glucuronyl glycolipids in human peripheral motor and sensory nerves

Sulfated glucuronyl glycolipids (SGGL) have been implicated as important target antigens in patients with demyelinating polyneuropathy and IgM paraproteinemia. Sulfated glucuronyl paragloboside (SGPG), a major species of SGGL, was identified in the subcellular fractions of human peripheral motor and sensory nerves using a simple and quantitative method. SGPG was found to be concentrated in the myelin-enriched fractions of both motor and sensory nerves (1.3±0.3 and 1.5±0.4 µg/mg protein, respectively), whereas its concentration was 0.9±0.2 and 1.8±0.6 µg/mg protein in the axolemma-enriched fractions of motor and sensory nerves, respectively. Our finding that SGPG is more abundant in the human sensory nerve axolemma-enriched fraction may account for the clinical and pathological observations that the lesions are more heavily concentrated in the sensory nerve than in other parts of the nerve tissues in this disorder.     

Role of the peripheral myelin protein 22 N-linked glycan in oligomer stability

Peripheral myelin protein 22 (PMP22) is a 22-kDa glycoprotein containing a single N-linked carbohydrate moiety. This posttranslational modification is conserved in PMP22 across species and within members of the PMP22 gene family; however, the function of the oligosaccharide is not known. To study the role of the PMP22 carbohydrate, site-directed mutagenesis was used to alter the glycosylation consensus sequence and produce a glycosylation-deficient mutant protein. This modified PMP22 was expressed in primary Schwann cells (SCs), and the effect of the N-glycan on the turnover rate, oligomerization, and intracellular trafficking of PMP22 was determined. Our data show a slight decrease in turnover rate from a half-life of approximately 70 min for the wild-type (wt) protein to 100 min for the glycosylation mutant. Although the presence of glycosylation-deficient PMP22 oligomers could be detected in SCs, we observed a decrease in oligomer stability compared with the wt oligomers. Both wt and mutant proteins showed similar localization in the endoplasmic reticulum and Golgi compartments and were transported to the SC surface. These results suggest that the N-glycan of PMP22 facilitates, in part, the stability of the PMP22 oligomer; however, the implications of PMP22 oligomerization remain unknown.     

Use of large-scale hydrazinolysis in the preparation ofN-linked oligosaccharide libraries: application to brain tissue

In this report, we describe the preparation of a library ofN-linked glycans from whole murine brain obtained by the large-scale hydrazinolysis of an acetone powder of the tissue followed by chromatographic procedures. 84% of the characterized oligosaccharides were found to be anionic, the remainder neutral. The anionic species were successively neutralized by neuraminidase (29%), aq. hydrofluoric acid (30%), and methanolysis (26%), indicating that approximately equal portions were sensitive to desialylation, dephosphorylation and desulfation, respectively. The presence of the sulfated fraction was confirmed by direct³⁵SO₄ metabolic labelling. A residual partially characterized fraction was found to be anionic through possession of carboxylic acid groups, unrelated to sialic acid. The purified oligosaccharides, in the absence of their original protein conjugates, were shown to retain those immunological characteristics essential for recognition by a specific monoclonal antibody, LS (412), that is known to recognize a carbohydrate epitope present on a number of neural adhesion molecules and functional in neural cell adhesion. These properties confirm the viability of scaling up the size of the hydrazinolysis procedure and adapting it to whole tissue for the production of glycan libraries and for the probing of structures of interest.Abbreviations ConA concanavalin A - ELISA enzyme-linked immunosorbent assay - Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - g.u. glucose units - HRP horseradish peroxidase - HVE high voltage electrophoresis - Man mannose - MS mass spectrometry - N-CAM neural cell adhesion molecule     

High resolution neurochemical gold staining method for myelin in peripheral and central nervous system at the light- and electron-microscopic level

Myelin is a multilamellar membrane structure primarily composed of lipids and myelin proteins essential for proper neuronal function. Since myelin is a target structure involved in many pathophysiological conditions such as metabolic, viral, and autoimmune diseases and genetic myelin disorders, a reliable myelin detection technique is required that is equally suitable for light- and electron-microscopic analysis. Here, we report that single myelinated fibers are specifically stained by the gold phosphate complex, Black gold, which stains myelin in the brain, spinal cord, and peripheral nerve fibers in a reliable manner. Electron-microscopic and morphometric analyses have revealed that gold particles are equally distributed in the inner, compact, and outer myelin layers. In contrast to Luxol fast blue, the gold dye stains proteinase-sensitive myelin structures, indicating its selective labeling of myelin-specific proteins. Aiming at defining the target of gold staining, we performed staining in several mouse myelin mutants. Gold complex distribution and myelin staining in MBP^−/−/shiverer mouse mutants was comparable with that seen in wild-type mice but revealed a more clustered Black gold distribution. This gold staining method thus provides a sensitive and specific high-resolution marker for both central and peripheral myelin sheaths; it also allows the quantitative analysis of myelinated fibers at the light- and electron-microscopic level suitable for investigations of myelin and axonal disorders. This study was supported by grants from the International Human Frontier Science Program Organization (HFSPO, to N.E.S.) and the Danone Institute (to N.E.S. and I.Y.E.).     

Clinical course of myelin oligodendrocyte glycoprotein 35-55 induced experimental autoimmune encephalomyelitis is aggravated by glia maturation factor

The role of glia maturation factor (GMF) in myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) was investigated using GMF-deficient (GMF-KO) mice. We demonstrate that GMF-KO mice were resistant to the MOG 35-55 peptide-induced EAE as compared to wild type (Wt) mice (two in eight versus 10 in 10). Next, we examined the effect of administration of recombinant human GMF (rGMF) on MOG 35-55 peptide-induced EAE in mice. Daily administration of rGMF, staring days 1-14, resulted in significant exacerbation of clinical symptoms. Following rGMF injections, both GMF-KO (six in eight) and Wt mice (eight in eight) developed severe EAE (maximal clinical score of 3.5-4.0) with high frequency. The histological examination revealed severe infiltration of inflammatory cells in the spinal cord of MOG-immunized Wt mice while the resistance to EAE in GMF-KO mice was characterized by the absence of inflammatory cells. Administration of rGMF in Wt mice and GMF-KO mice resulted in a significant increase in infiltrating cells in the spinal cord following MOG-immunizations. We also evaluated cytokines and chemokines production as parameters of severity of inflammation in the spinal cord of Wt versus GMF-KO mice with and without GMF-reconstitution following MOG-immunizations. Cytokines (TNF-α, IFN-γ, IL-1β, IL-6) and chemokines (CCL2, CCL3, CXCL10, GM-CSF) production were significantly greater in Wt mice than in GMF-KO mice following MOG-immunization. Furthermore, the reconstitution experiment with rGMF showed that the administration of rGMF in both, Wt mice and GMF-KO mice produced significant increase in the GMF-mediated cytokine/chemokine production.     

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex

Senescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.     

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.     

SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

A novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa).     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.     

Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2)

Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg²⁺ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn⁹¹-Ile-Val-Thr⁹⁴-Pro-Arg-Thr⁹⁷-Pro-Pro-Pro-Ser¹⁰¹. MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91–104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position –2 or –3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.     

Evidence for the presence of major peripheral myelin glycoprotein P0 in mammalian spinal cord and a change of its glycosylation state during aging.

Glycoproteins, which react with Lens culinaris agglutinin, in the membrane preparation of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis. In contrast to the samples from brain, which showed similar staining patterns in the two different age groups, the glycoprotein patterns of spinal cords showed marked differences by the age of donors. The most prominent evidence is that a glycoprotein with an apparent molecular weight of 30 kDa (gp30) was detected in the aged rats, but not in the young adult rats. Based on the amino acid sequence data around the glycosylation site, the gp30 was identified as P0, which is a member of immunoglobulin superfamily and a major structural component of mammalian peripheral nerve myelin. This is the first report indicating that P0, which has been considered as a peripheral nerve-specific glycoprotein, occurs also in the spinal cord of mammals. In addition, nonglycosylated P0 molecule could be detected in the spinal cord of young adult rats by anti-P0 polyclonal antibody. These results indicate that the glycosylation state of the P0 molecule in the spinal cord changes during aging.     

Glutamate levels control HT22 murine hippocampal cell death by regulating biphasic patterns of Erk1/2 activation: role of metabolic glutamate receptor 5

Extracellular glutamate concentration is a critical determinant of neuronal cell fate. We recently demonstrated that HT22 murine hippocampal cell viability was reduced by exposure to high concentrations of glutamate, whereas low concentrations promoted cell survival. Extracellular signal-regulated kinase (Erk)1/2 activation by glutamate is important for both glutamate-induced cell death and survival. In this study, we investigated the role of glutamate-induced or hydrogen peroxide (H₂O₂)-induced Erk1/2 activation in HT22 cell fate determination. Glutamate and H₂O₂ treatment similarly induced early (<1 h) Erk1/2 phosphorylation regardless of concentration. On the other hand, persistent Erk1/2 phosphorylation (16–24 h) was observed only in the presence of excess glutamate. Only the latter contributed to glutamate-induced cell death, which involved metabolic glutamate receptor 5. Our findings suggest that glutamate concentration modulates two distinct phases of Erk1/2 activation, which can explain the glutamate concentration-dependent determination of HT22 cell fate.     

Identification of a peptide mimic of the L2/HNK-1 carbohydrate epitope

The L2/HNK-1 carbohydrate is carried by many neural recognition molecules and is involved in neural cell interactions during development, regeneration in the peripheral nervous system, synaptic plasticity, and autoimmune-based neuropathies. Its key structure consists of a sulfated glucuronic acid linked to lactosaminyl residues. Because of its biological importance but limited availability, the phage display method was used to isolate a collection of peptide mimics that bind specifically to an L2/HNK-1 antibody. The phages isolated from a 15-mer peptide library by adsorption to this antibody share a consensus sequence of amino acids. The peptide mimicked several important functions of the L2/HNK-1 carbohydrate, such as binding to motor neurons in vitro, and preferential promotion of in vitro neurite outgrowth from motor axons compared with sensory neurons. A scrambled version of the peptide had no activity. The combined observations indicate that we have isolated a mimic of the L2/HNK-1 carbohydrate that is able to act as its functional substitute.     

Protein phosphorylation in human peripheral nerve: Altered phosphorylation of a 25-kDa glycoprotein in leprosy

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg²⁺ or Mn²⁺ was essential for maximum phosphorylation although Zn²⁺, Co²⁺, and Ca²⁺, could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.