Conservation of the carboxyl terminal epitope of myelin proteolipid protein in the tetrapods and lobe-finned fish.

Immunochemical analysis of the myelin proteolipid protein (PLP) has identified the carboxyl terminal amino acid phenylalanine 276 as the only PLP epitope conserved between the PLP components of rat and lungfish, species representing the phylogenetically most widely separated groups that synthesise typical CNS myelin. Immunoblotting using a rabbit antiserum raised against the carboxyl terminal sequence of rat PLP (residues 257-276) identified this epitope on the PLP components of both tetrapod (rat, chicken, lizard, and frog) and lobe-finned fish (coelacanth and lungfish) CNS myelin, including the DM-20 isoform of PLP, which is restricted to rat, chicken, and lizard CNS myelin. The conservation of the carboxyl terminus of PLP during evolution suggests this structure may play an important role in maintaining the organisation and function of PLP in the myelin membrane.

Presence of proteolipid protein in coelacanth brain myelin demonstrates tetrapod affinities and questions a chondrichthyan association.

The protein and glycoprotein compositions of CNS myelin from the living coelacanth (Latimeria chalumnae) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An unglycosylated component of 25 kilodaltons showed substantially stronger immunoblot reactivity with antibodies against mammalian proteolipid protein (PLP) than lungfish glycosylated PLP. DM-20 (intermediate protein) was not detectable in either fish. The presence of unglycosylated PLP in CNS myelin of the actinistian coelacanth contradicts an association with cartilaginous fishes but supports tetrapod affinities closer than those of lungfish.

Electrophoretic characterization and immunoblot analysis of the proteins from the myelin-like light membrane fraction of shrimp ventral nerve (Penaeus duorarum).

1. The proteins of the light membrane fraction (LMF) from the ventral nerve of the pink shrimp (Penaeus duorarum) were separated by SDS gel electrophoresis and analysed by staining and immunoblotting. 2. Shrimp LMF carried four major proteins with apparent molecular weights of Mr = 21,500, 40,000, 78,000, 85,000 and four minor components (Mr = 36,000, 41,500, 43,000, 50,000). 3. None of these proteins bound Concanavalin A. 4. The four major proteins showed no reaction with antisera against six vertebrate myelin proteins. Only the minor Mr = 50,000 component was weakly recognized by the antibodies against mammalian myelin P0 protein.

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.

Immunological evidence for the presence of myelin-related integral proteins in the CNS of hagfish and lamprey.

Antibodies against myelin proteins were utilized in the analysis of total particulate material from the brains of the agnathan hagfish and lamprey. Immunoblotting revealed in both species the presence of bands at 50,000 dalton that reacted with anti-bovine PNS-P0 antibodies. Single bands of 34,000 dalton and 51,000 dalton were immunodetected with anti-trout CNS-36K antibodies in lamprey and hagfish, respectively. Antibodies against mammalian myelin basic protein (MBP) and proteolipid protein (PLP) were not recognized. In spite of the lack of multilayered myelin in agnatha, the presence of myelin-related integral proteins suggests that agnathan glial cells have already acquired the capacity to synthesize some proteins that are similar to typical myelin proteins. This represents a crucial evolutionary step towards myelination.

Antigenic sites common to major fish myelin glycoproteins (IP) and to major tetrapod PNS myelin glycoprotein (Po) reside in the amino acid chains.

The major myelin glycoproteins in the CNS and PNS of trout (IP) were enzymatically deglycosylated with endoglycosidase F (Endo F) and examined by electro-immunoblotting. Following carbohydrate removal and loss of concanavalin A affinity each of the four IP components underwent a similar reduction in molecular size, corresponding to approximately 3,000 daltons. Immunological cross-reactivities with anti-bovine Po or anti-trout IP2 antibodies, were however fully retained by the Endo F cleavage products. This strongly implies that the antigenic sites shared by the mammalian Po protein and the various intermediate glycoproteins of trout CNS and PNS are located in the protein portion. Immunoblot analysis of the PNS myelin proteins from various species of the major vertebrate classes with anti-trout IP2 antiserum revealed striking differences in the immunological properties of the individual Po components which were not detected when anti-bovine Po antiserum was used as a probe.

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.

Myelin lipids: a phylogenetic study.

The lipid composition of CNS and PNS myelin was studied in rat, Xenopus, trout and Torpedo. The main difference lay in the proportion of cerebrosides, which decreased in the sequence rat greater than Xenopus greater than Torpedo greater than trout. In addition Torpedo CNS and PNS myelins were extremely rich in sulfatides. In some respects, Torpedo appeared closer to tetrapods than trout. Otherwise the proportion of the different lipid classes did not reveal any clear evolutionary trends. The presence of hydroxylated galactolipids in CNS myelin was investigated in several additional species. Considerable amounts were found in Torpedo, Polypterus, Protopterus, lizard, and chicken, with the highest values in rat and anurans. Only very small amounts of hydroxylated cerebrosides were detected in trout and in axolotl, while newt had none. This parameter appears therefore of doubtful usefulness for phylogenetic studies. In contrast to myelin proteins, myelin lipids are of limited value for establishing phylogenetic relationships among vertebrates.

Changes of myelin proteins during Wallerian degeneration in situ and in millipore diffusion chambers preventing active phagocytosis.

Changes of myelin proteins in mouse sciatic nerves were studied comparing nerves degenerating in situ with nerves enclosed in millipore diffusion chambers which eliminate invasion of non-resident cells. Nerves kept in chambers showed nearly complete preservation of myelin sheaths with a very slow degradation of myelin proteins. Nerves degenerating in situ showed rapid myelin phagocytosis by macrophages with almost complete disappearance of myelin proteins after 28 days. These data elucidate the role of macrophages for removal of myelin proteins.

Expression of myelin proteins characteristic of fish and tetrapods by Polypterus revitalizes long discredited phylogenetic links.

CNS myelin constituents were used as evolutionary markers to study the controversial relationships of Polypterus with bony fishes, lungfishes and amphibians. The occurrence in Polypterus CNS of myelin proteins similar to those observed in the sterlet confirms its close relationship to Chondrostei. However, the simultaneous presence of proteolipid protein (PLP) demonstrates the existence of a long discredited relationship between Polypterus and tetrapods, and also with the lungfish Protopterus, an ally of tetrapods. As in the lungfish Protopterus, Polypterus cerebrosides and sulfatides contained alpha-hydroxy fatty acids. In contrast to Protopterus CNS myelin which carries glycosylated PLP but lacks Po-like component and the 36 kDa protein, Polypterus possesses these two bony fish myelin components. Furthermore, the presence of aglycosylated PLP in Polypterus, as in higher vertebrates, differentiates it from Protopterus. The simultaneous presence in Polypterus of myelin constituents from fish and land vertebrates indicates that Polypterids occupy an unusual intermediate phylogenetic position. The near absence of 2',3'-cyclic nucleotide 3'-phosphodiesterase activity in Polypterus. Protopterus, and in other fishes, confirmed by the lack of Wolfgram protein, establishes this myelin enzyme as the only CNS myelin constituent specifically expressed by tetrapods.

Major central nervous system myelin glycoprotein of the African lungfish (Protopterus dolloi) cross-reacts with myelin proteolipid protein antibodies, indicating a close phylogenetic relationship with amphibians.

CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti-human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A-horseradish peroxidase and bound to anti-rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti-PLP reactivity. These results agree with a concanavalin A-binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g-PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP-cross-reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.

Bony fish myelin: evidence for common major structural glycoproteins in central and peripheral myelin of trout.

Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two-dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IP0. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP.(ABSTRACT TRUNCATED AT 250 WORDS)

Central nervous system myelin of teleosts: comparative electrophoretic analysis of its proteins by staining and immunoblotting.

CNS myelin was isolated from 24 teleostean fishes and the proteins were analyzed by staining and immunoblotting. All species showed a 36 K protein, two or more glycosylated hydrophobic intermediate protein (IP) components and several myelin basic protein bands (BP). The 36 K protein was specific for teleostean fishes. The IP and BP components displayed substantial variations in their proportions as well as in molecular sizes when comparing the different teleosts. This contrasts with CNS myelin proteins which appear more stable in terrestrial vertebrates.

Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistochemical localization of 36K and IP2 proteins in trout nerve tissue.

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.

Myelin-associated glycoprotein and myelin basic protein are present in central and peripheral nerve myelin throughout phylogeny.

This phylogenetic study of central and peripheral nervous system myelin proteins demonstrates that important changes occur in the composition of certain myelin proteins during evolution. Only two components, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are present in all Gnathostomata representatives investigated. While MBP components varied considerably even among the representatives of a given order, the apparent molecular weight of MAG showed little variation indicating that the conservation of the molecular structure could be important for the function of MAG in glia axon interactions.

Appearance of Myelin proteins during vertebrate evolution.

Myelin, defined as an arrangement of spirally fused unit membranes, is an acquisition of vertebrates and first appeared during evolution in Gnathostomata. In all species studied PNS and CNS myelins contain the myelin-associated glycoprotein (MAG) and the myelin basic protein (MBP). Throughout phylogeny PNS myelin is characterized by the major P(0) glycoprotein which is called IP in fishes. The PNS myelin proteins did not evolve further except for the addition of P(2) protein from reptiles onward. In Elasmobranchii and Chondrostei, PNS and CNS myelin proteins are similar. CNS myelin of actinopterygian fishes possesses a 36,000 Da protein (36K) in addition to P(0)-like IP glycoproteins. In tetrapod CNS myelin, P(0) is replaced by the proteolipid protein (PLP) and the Wolfgram protein (WP). Of particular interest in a transitional phylogenetic sense are the lungfish Protopterus, carrying glycosylated PLP (g-PLP) but no P(0), 36K or WP, and the bichir Polypterus, showing simultaneous presence of P(0), 36K and PLP. These results indicate that myelin proteins could be valuable molecular markers in establishing vertebrate phylogenetic relationships and in reconstructing the fish-tetrapod transition.

Biosynthesis and insertion of Wolfgram protein into optic nerve membranes.

Antibodies against pig brain Wolfgram protein (WP) were prepared and utilized in the analysis of WP biosynthesis in membranes from optic nerves of 20 day-old rats. Newly synthesized WP appeared rapidly (less than 5 min) in myelin and in a non-myelin microsome fraction and accumulated in both thereafter. Monensin did not affect the insertion of WP in either membrane fraction. These results are consistent with biosynthesis of WP on free ribosomes.

Phylogenetic examination of vertebrate central nervous system myelin proteins by electro-immunoblotting.

Central nervous system (CNS) myelin proteins from vertebrate classes were examined by immunoblotting with antisera against mammalian CNS myelin proteins. Higher vertebrates possessed proteolipid (PLP), DM-20 and Wolfgram (WP) proteins, except that DM-20 was missing in amphibia. Fish CNS myelins contained neither PLP nor WP; instead they bound antisera to mammalian peripheral nervous system P0 protein. All classes carried myelin basic protein, but only mammals exhibited a component equivalent to rat 21.5K (21,500 dalton). These phylogenetic data are consistent with major changes in CNS myelin protein composition at the transition from fishes to higher vertebrates.

Biochemical characterization of the central nervous system myelin proteins of the rainbow trout, Salmo gairdneri.

Central nervous system myelin isolated from the rainbow trout (Salmo gairdneri) displays a very low median density on zonal gradient centrifugation, banding at approximately 0.32 M sucrose. Its proteins consist of a 36 K (36,000 mol.wt.) component, two Concanavalin A-reactive intermediate proteins IP1 (23,000 mol.wt.) and IP2 (26,200 mol.wt.), and two basic proteins BP1 and BP2, of which the latter co-migrates with rat SBP while BP1 is of slightly smaller size. The trout myelin proteins electrofocus at pH positions similar to those of their mammalian counterparts. Immunoblotting shows that antibodies against rat PNS myelin P0 glycoprotein are bound by IP1 and IP2, but not by 36K. None of the trout myelin proteins react with anti-rat CNS myelin proteolipid protein (PLP) antiserum. The basic proteins BP1 and BP2 bind strongly to antibodies directed against human myelin basic protein. In vivo injection of tritiated fucose or palmitate leads to radiolabeling of IP1 and IP2. Under autolytic in situ conditions the appearance of a glycosylated 20,000 mol.wt. component (IP0) is noted, with parallel reduction of both IP1 and IP2, indicating sequence homologies between IP1 and IP2. The 36K protein is not affected by autolysis.