Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1.

The capacity of the adult brain and spinal cord to repair lesions by axonal regeneration or compensatory fibre growth is extremely limited. A monoclonal antibody (IN-1) raised against NI-220/250, a myelin protein that is a potent inhibitor of neurite growth, promoted axonal regeneration and compensatory plasticity following lesions of the central nervous system (CNS) in adult rats. Here we report the cloning of nogo A, the rat complementary DNA encoding NI-220/250. The nogo gene encodes at least three major protein products (Nogo-A, -B and -C). Recombinant Nogo-A is recognized by monoclonal antibody IN-1, and it inhibits neurite outgrowth from dorsal root ganglia and spreading of 3T3 fibroblasts in an IN-1-sensitive manner. Antibodies against Nogo-A stain CNS myelin and oligodendrocytes and allow dorsal root ganglion neurites to grow on CNS myelin and into optic nerve explants. These data show that Nogo-A is a potent inhibitor of neurite growth and an IN-1 antigen produced by oligodendrocytes, and may allow the generation of new reagents to enhance CNS regeneration and plasticity.

rMAL is a glycosphingolipid-associated protein of myelin and apical membranes of epithelial cells in kidney and stomach.

rMAL, the rat myelin and lymphocyte protein, is a small hydrophobic protein of 17 kDa with four putative transmembrane domains and is expressed in oligodendrocytes and Schwann cells, the myelinating cells of the nervous system. In addition, transcript expression has been found in kidney, spleen, and intestine. Confocal microscopy and immunoelectron microscopy with an affinity-purified antibody localized rMAL to compact myelin in a pattern similar to the structural myelin proteins: myelin basic protein and proteolipid protein. In kidney and stomach epithelia, rMAL is located almost exclusively on the apical (luminal) membranes of the cells lining distal tubuli in kidney and the glandular part of the stomach. Biochemical analysis of plasma membranes isolated from spinal cord and kidney demonstrated that rMAL is a proteolipid that is present in detergent insoluble complexes typical for proteins associated with glycosphingolipids. Lipid and protein analysis showed a co-enrichment of glycosphingolipids and rMAL protein within these complexes, indicating a close association of rMAL to glycosphingolipids in myelin and in kidney in vivo. We conclude that specific rMAL-glycosphingolipid interactions may lead to the formation and maintenance of stable protein-lipid microdomains in myelin and apical epithelial membranes. They may contribute to specific properties of these highly specialized plasma membranes.

Transport of proteolipid protein to the plasma membrane does not depend on glycosphingolipid cotransport in oligodendrocyte cultures.

The possibility that transport of proteolipid protein (PLP) from its site of synthesis to the plasma membrane is dependent on cotransport with (sulfo)galacto-cerebrosides was investigated in primary cultured oligodendrocytes and Chinese hamster ovary (CHO) cells expressing PLP. Sulfation was inhibited by growing oligodendrocytes in the presence of a competitive inhibitor of this process, sodium chlorate. Under these circumstances, sulfatide synthesis was inhibited by 85%. Nevertheless, PLP was still delivered to the plasma membrane in quantitative amounts. Furthermore, when PLP was expressed in CHO cells, which normally synthesize very low amounts of galactosyl ceramide (GalCer) and no sulfatide, PLP was transported to the plasma membrane. Moreover, in CHO cells coexpressing PLP and ceramide galactosyl transferase, PLP cell surface labeling was unaltered. Noting that it has been demonstrated that proteins destined for the apical surface of epithelial cells colocalize with glycolipid-enriched microdomains, we isolated detergent-insoluble membrane complexes from cultured oligodendrocytes. We found, however, that most of the PLP is present in the detergent-soluble fraction and, furthermore, that PLP could not be chased into or out of the insoluble fraction. Taken together, these data make it very likely that in oligodendrocytes PLP transport takes place irrespective of the presence of glycosphingolipids GalCer and sulfatide.

Differential and cell development-dependent localization of myelin mRNAs in oligodendrocytes.

In oligodendrocytes (OLG), the mRNAs for the various myelin proteins localize to different intracellular sites. Whereas the confinement of myelin basic protein (MBP) mRNA to the processes of the cell has been well established, we demonstrate that most other myelin mRNA species are mainly present in the perinuclear region. Using in situ hybridization of cultured rat OLG we found that mRNAs are localized to at least three different locations: 1) to the perinuclear region [myelin-associated glycoprotein (MAG) mRNA]; 2) mainly to the processes (the mRNA for the 14-kDa isoform of MBP); and 3) to both the perinuclear region and the primary processes [2',3'-cyclic nucleotide phosphodiesterase (CNPase) and proteolipid protein (PLP) mRNAs]. Thus, depending on their primary structure, the mRNA species in OLG either remain near the nucleus or localize to primary or secondary processes before their translation. The myelin mRNA localization correlates well with that of the proteins encoded in them, as demonstrated by immunocytochemistry. Since different isoforms of MBP have different locations in transfected HeLa cells (Staugaitis et al.: J Cell Biol 110:1719-1727, 1990), we also have investigated the localization of the various mRNAs in OLG, using exon 2-minus and exon 2-specific probes in situ hybridization. The exon 2-minus MBP mRNAs are transported far into the processes, whereas exon 2-specific mRNA was only detected in the cell body. This suggests that sorting and trafficking of MBP mRNA are regulated by the presence or absence of the exon 2 sequence. Furthermore, during maturation of OLG, exon 2-plus mRNAs disappear, whereas exon 2-minus mRNAs increase. The developmentally regulated expression of exon 2-plus transcripts suggest a role of their protein products in differentiation rather than in myelination.

Transport of biosynthetic sphingolipids from Golgi to plasma membrane in HT29 cells: involvement of different carrier vesicle populations.

Intracellular transport of the sphingolipids glucosylceramide (GlcCer) and sphingomyelin (SM), was examined in HT29 human colon adenocarcinoma cells. After synthesis from a fluorescent precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-Cer), transfer of SM from the Golgi complex to the plasma membrane can occur independently of that of GlcCer, as revealed by temperature-dependent experiments. Thus, at 20 degrees C, SM trafficking to the cell surface is essentially unaffected, whereas GlcCer transport to the plasma membrane is inhibited by approximately 75%, when compared to the transfer of both lipids at 37 degrees C. The mechanism by which SM and GlcCer are transported to the cell surface involves at least in part a vesicular mechanism. Transport vesicles, containing both lipids at their luminal surface, as revealed by the inaccessibility of the NBD fluorescence to the quencher sodium dithionite, have been isolated from cells, permeabilized by filter stripping. As evidenced by electron microscopic and biochemical criteria, no vesicles or lipids were released when cell permeabilization had been carried out with streptolysin. Density gradient analysis indicates the potential existence of several vesicle populations, distinctly enriched in either lipid, involved in transport of sphingolipids to the plasma membrane in HT29 cells.

Outstations of the Golgi complex are present in the processes of cultured rat oligodendrocytes.

Primary cultures of rat oligodendrocytes were incubated with a fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoylceramide+ ++ (C6-NBD-ceramide). This compound is known to stain the Golgi complex specifically. Within 30 min of incubation at 37 degrees C most of the C6-NBD-ceramide was incorporated into the perinuclear Golgi system, as revealed by conventional and confocal laser fluorescence microscopy. Interestingly, C6-NBD-ceramide was found to accumulate also in smaller, oval-shaped structures in many of the processes, at distances up to 30 microns from the nucleus. This implies the possibility that these structures are Golgi (-derived) complexes. Indeed, after incubation of oligodendrocytes with C6-NBD-ceramide and rhodamine-labeled transferrin both fluorescent labels colocalized in the Golgi system of the cell body as well as in the structures in the processes. Additional support for the Golgi character of these structures was obtained by transmission electron microscopy. Particularly in oligodendrocytes cocultured with neurons, many Golgi structures were present all over the processes. The results lead us to conclude that, in the oligodendrocyte, the Golgi complex does not only reside in the perikaryon, but also in the processes. One can speculate that a polarized biosynthetic activity, involving the presence of the Golgi near the site of myelin synthesis, may be advantageous to the oligodendrocyte for assembly and/or repair of the myelin membrane at the distal end of the processes.

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product.

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. sterothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8 degrees C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostability was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B. caldolyticus.