Synaptic membrane phospholipids: effects of maternal ethanol consumption

The cholesterol and phospholipid content and phospholipid composition were determined in synaptic membranes from the 17- to 31-day-old offspring of rats that were pair-fed either a control or 6.6% (v/v) ethanol liquid diet on a chronic basis prior to parturition. At all ages examined, the major synaptic membrane phospholipid was phosphatidyl choline (greater than 40%). Other prominent synaptic membrane phospholipids included phosphatidyl ethanolamine (approximately 17 to 21%), ethanolamine plasmalogen (approximately 5 to 16%), and phosphatidyl serine (approximately 13%). Smaller proportions of sphingomyelin (4 to 7%), phosphatidyl inositol (approximately 1%), and phosphatidic acid (approximately 1%) were detected. Between 17 and 31 days of age, there was a significant decrease in the proportion of phosphatidyl choline and a significant increase in the proportion of ethanolamine plasmalogen. When the offspring of control and ethanol-treated rats were compared, no significant differences were found in either the yield of synaptic membrane protein, or in the concentration of synaptic membrane cholesterol and total phospholipid. However, the proportion of ethanolamine plasmalogen was significantly decreased in the 24-day-old offspring of ethanol-treated rats, suggestive of a delay in the normal development-related increase of this lipid. In addition, there was a small increase in the proportion of sphingomyelin in the 31-day-old offspring of ethanol-treated rats.     

Isolation and partial characterization of rat CNS axolemma enriched fractions

Axolemma-enriched fractions were prepared from rat brain by osmotic shock of a purified preparation of myelinated axons and subsequent separation of myelin, two axolemma-enriched fractions and myelin-free axons by density gradient centrifugation. Compared with the starting whole homogenate, the fractions were enriched in specific activity of Na+K+ ATPase, acetylcholinesterase, 5'nucleotidase as well as 2',3'-cyclic nucleotide 3'phosphohydrolase. Compared with myelin, the axolemmal fractions are greatly enriched in high molecular weight proteins. The 1.0/1.2 fraction has a predominant peak of fucose-labeled glycoprotein with a molecular weight between that of the myelin associated glycoprotein and the Wolfgram protein which is absent from the myelin glycoprotein profile. Polyacrylamide gel electrophoresis showed that the protein profile of myelin isolated by this procedure was similar to that of myelin isolated by other procedures and that the myelin specific basic and proteolipid proteins were virtually absent in the axolemma-enriched fractions. Both axolemma fractions were enriched in higher MW proteins, some of which resembled proteins in the myelin protein profile. Both axolemma-enriched fractions specifically bind between 2 and 3 pmoles of [3H]tetrodotoxin per mg protein. The axolemma-enriched fractions incorporated [3H]leucine and [14C]fucose exclusively into high molecular weight proteins and glycoproteins. In contrast myelin concomitantly isolated with the axolemma-enriched fractions had a significant amount of [3H]leucine labeled protein in myelin proteolipid and basic proteins. In addition to the myelin associated g-ycoprotein the [14C]fucose labeled a glycoprotein of slightly larger apparent molecular weight than proteolipid protein was found in the myelin fraction while the comparable labeled glycoprotein was absent in the axolemma-enriched fractions. The possible extent of contamination of these fractions by myelin or myelin subfractions and relationship of these axolemma-enriched fractions to other axolemma preparations are discussed.     

Maternal ethanol consumption and synaptic membrane glycoproteins in offspring

The present study was undertaken to assess the influences of chronic maternal ethanol consumption, prior to and during gestation, on the development of synaptic plasma membranes (SPMs) and on the synthesis of SPM glycoproteins in offspring. Comparisons were made between animals whose mothers were pair-fed a control or 6.6% (v/v) ethanol liquid diet in which protein accounted for either 18% (original) (C & E) or 21% (revised) (*C & *E) of the calories. In addition, groups of pups that were either cross-fostered (*C & *E) with chow-fed surrogate mothers or reverse cross-fostered (offspring of chow-fed mothers with *C & *E mothers) were examined. Ethanol and matched (same dietary group) control pups had comparable brain and body weights, brain protein content, and yield of SPM proteins during the 10–24 day age period examined. However, the yield of SPM proteins from ethanol and control offspring of and/or reared by the three groups of rat mothers that received the *E and *C liquid diets was greater than that of the offspring of rats that were fed the original diets. This suggests that the original diets were not nutritionally adequate for pregnant rats. Despite the fact that the content of SPM proteins was comparable in ethanol and matched control pups, the offspring of ethanol-treated rats had an abnormal distribution of [³H]-or [¹⁴C]-fucose-derived radioactivity among SPM glycoproteins. The SPM abnormalities were most severe in the non-cross-fostered offspring of E rats. No SPM glycoprotein abnormalities were found in the reverse cross-fostered group. The results of the present study demonstrate that chronic maternal ethanol consumption prior to parturition has a severe effect on the synthesis of SPM glycoproteins in developing offspring without affecting the content of SPMs per se. It also demonstrates the importance of optimizing the composition of liquid diets used to feed pregnant rats.     

Effects of maternal nutritional stress on synaptic plasma membrane proteins and glycoproteins in offspring

An earlier study in this laboratory demonstrated a 33 to 50% deficiency of synaptic plasma membrane (SPM) proteins in the brains of developing offspring of rats that were fed a protein-deficient diet during lactation. We have demonstrated that an SPM deficit persisted if the nutritional stress (in this case postnatal protein-calorie deficiency) was continued on a long-term basis. If nutritional rehabilitation was instituted on the 21st postnatal day, however, the SPM deficit was reversible. We also demonstrated abnormalities in the synthesis of fucosylated SPM glycoproteins in the developing offspring rats subjected to protein or protein-calorie deficient diets and in rehabilitated offspring. These abnormalities were detected using sensitive double-label isotopic procedures. Few abnormalities were found in the relative distribution of the dye-binding capacity of SPM proteins from developing control, undernourished, or rehabilitated rats on polyacrylamide gels that had been stained with Coomassie blue. The potential cause and importance of abnormalities in synaptic glycoproteins is discussed.     

Inhibition of reactive gliosis in vivo by exogenous axolemma and myelin fractions

Exogenous myelin- or axolemma-enriched fractions were assessed for the ability to inhibit biochemical and morphological expressions of reactive gliosis in rat optic nerve. Elvax pellets containing exogenous myelin, axolemma, whole-brain homogenate, liver, or red cell extracts or no homogenate were inserted into a dural slit in distal regions of crushed optic nerve. Biochemical and morphological expressions of reactive gliosis were assessed at 7 or 14 days postoperatively. Post-traumatic elevations in lactic dehydrogenase activity normally seen at 7 days postoperatively were prevented by placement of Elvax pellets containing myelin or axolemmal fractions into the optic nerve. Morphological analyses indicated an inhibition of post-traumatic elevations in glial cell numbers, surface area, and nuclear size at the 14-day time point. Exposure of the axolemmal fraction to heat or trypsin inactivated its ability to modulate reactive gliotic changes. Myelin fractions were trypsin-sensitive, but not heat-sensitive. In contrast, Elvax pellets containing whole-brain tissue homogenates or liver and red cell membranes had no significant effects on post-traumatic glial changes relative to preparations in which homogenate-free pellets were used.     

Analysis of the apparent biphasic axonal transport kinetics of fucosylated glycoproteins

Following intraocular injection of [3H]fucose, which labels many glycoproteins of retinal ganglion cells, the accumulation of transported radioactivity arriving at the superior colliculus (nerve terminals) peaks within a few hours and decays with a time course of hours. Then, over a period of several days, radioactivity again accumulates at the superior colliculus and then decays with a half-life of days. The second peak also represents fast transported material since it occurs almost simultaneously along the optic nerve and tract as well as at the nerve endings. Such data have been interpreted as evidence for both a group of rapidly released, rapidly transported glycoproteins (first peak) and a group of slowly released but rapidly transported glycoproteins (second peak). We investigated this supposition by studying in more detail the metabolism of some individual fucosylated proteins in both the retina and superior colliculus. We noted that much of the radioactivity incorporated in fucosylated glycoproteins at the retina was rapidly metabolized (with a turnover on the order of hours), while the remainder of the fucosylated moieties had a metabolic half-life on the order of days. This was also true of the metabolic behavior of several individual glycoproteins, selected for study because they are major components of the group committed to transport and accumulating in two waves at the superior colliculus. In other experiments we injected [35S]methionine intraocularly and examined the metabolism in the retina and the kinetics of transport to the superior colliculus of the peptide backbone of these same individual proteins. In contrast to the two waves of accumulation of radioactivity from [3H]fucose, accumulation of radioactivity of the peptide backbone of the same glycoproteins was monophasic. Our explanation of these data involves the presence of two types of fucose moieties on the peptides. One group of fucose moieties is labile and is lost from the peptide backbone over a period of hours. Other fucose moieties are approximately as metabolically stable as the peptide backbones to which they are attached. The actual peptide backbones of the glycoproteins are committed to rapid transport over a period of several days. Thus, the first (and most prominent) peak of transported radioactivity in [3H]fucosylated glycoproteins does not represent a discrete phase of transport but, rather, is the summation of kinetics of gradual arrival of proteins and the rapid drop in their specific radioactivity as the more labile moieties of [3H]fucose are lost.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of prenatal ethanol exposure on dendritic spines of layer V pyramidal neurons in the somatosensory cortex of the rat

The number of dendritic spines on consecutive segments from the cell body along the 400-600 micron proximal region of the apical dendrites of layer V pyramidal neurons, impregnated with the rapid Golgi method, in the somatosensory cortex was counted in ethanol-treated rats during gestation (25% ethanol in drinking water representing 30-35% of the total caloric intake) and in age-matched controls at postnatal ages 4, 15, 30 and 90 days. Although the mean values were lower in ethanol-treated rats than in controls during the first fortnight of postnatal life, significantly lower numbers of spines were observed only in the 15-day-old rat (Student's t-test, P less than 0.01-0.001). Spines with long, thin pedicles were characteristically encountered in ethanol-treated and controls aged 4 days; this sort of spine also predominated in ethanol-treated rats aged 15 days, but not in age-matched controls. The decrease in number and the abnormal morphology of spines was no longer present in ethanol-treated rats aged 30 and 90 days. These data suggest that impaired maturation of dendritic spines on cortical pyramidal cells, followed by recovery of the altered parameters at the end of the first postnatal month, occurs in the offspring of ethanol-treated rats during gestation.     

Isolation and characterization of periaxolemmal and axolemmal enriched membrane fractions from the rat central nervous system.

In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.0M sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K+ ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.0M fraction was also highly enriched in Na+, K+ ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage-dependent Na+ channel, axon-specific fodrin, and MAP-1B. Based on these characteristics we concluded that the 0.8M and 1.0M subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipin10 was unique with equally high levels in myelin and in the 0.8M and 1.0M subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GA/1. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K+ and Cl- channels, which based on channel sizes and open times, are largely distinct from one another.     

Hydrocephalus following prenatal exposure to ethanol

Pregnant rats were administered a liquid diet containing 5% (w/v) ethanol between gestational days 10 and 21, and the brains of nine offspring were examined at 8 weeks of age. Two ethanol-treated offspring showed obvious hydrocephalus characterized by markedly enlarged lateral ventricles. Most of the other ethanol-treated rats only showed a slight enlargement of the lateral ventricles. An ethanol-treated offspring showed no neuropathological signs of hydrocephalus. Histological observation of the hydrocephalic brains revealed dilation of the lateral ventricles, loosely bundled corpus callosum, hypoplasia of the septum and fimbria, and thinning of the cerebral cortices. There were no differences in the shape of the third ventricle and aqueduct between hydrocephalic and non-hydrocephalic rats. Ectopic cell clusters were found on the surface of the lateral ventricle and in the interventricular foramen in ethanol-treated rats with hydrocephalus. However, leptomeningeal heterotopias were found on the cortical surface in ethanol-exposed rats independently of whether or not they showed hydrocephalus. Thus, heterotopias within the ventricles may be related to the genesis of hydrocephalus following prenatal ethanol exposure. However, whether they could be a direct cause of the hydrocephalus is uncertain as they seem to be not enough large to block the current of the cerebrospinal fluid. We also examined the expression of L1, a cell adhesion molecule suspected of involvement in the genesis of hydrocephalus after prenatal ethanol exposure, in 1-day-old rats. Western blot analysis using specific antibody against L1 showed no significant change in L1 protein expression in ethanol-exposed rats. These data suggest that L1 protein expression is not affected by ethanol.     

Properties of acetylcholinesterase in axolemma-enriched fractions isolated from bovine splenic nerve

The properties of acetylcholinesterase (AChE) in axolemma-enriched fractions (AEF) from bovine splenic nerve were investigated to see if they differed in any way from those of the AChE in diaphragm muscle. The axolemmal enzyme had a low Km for acetylthiocholine (ca. 90 microM), exhibited substrate inhibition, and had a well-defined optimum of substrate concentration of 1 mM. The rate of hydrolysis of substrate decreased with increasing acyl chain length (acetyl- greater than propionyl- greater than butyryl-). The AChE inhibitors eserine and hexamethonium were competitive inhibitors of the membrane-bound enzyme, whereas lidocaine was a noncompetitive inhibitor; these results were comparable to the effect of these inhibitors on diaphragm muscle AChE. The axolemmal enzyme was more efficiently solubilized and more stable in nonionic detergents such as Triton X-100 and Tween 20 than charged detergents such as lysolecithin and zwitterionic detergents. These results indicate that the AChE present in bovine splenic nerve AEF is identical to the previously characterized AChE from other sources.     

Entrapment in anti myelin-associated glycoprotein neuropathy

Anti-myelin associated glycoprotein (MAG) neuropathy is a chronic disorder in which IgM antibodies react with Schwann cell glycoproteins, including MAG and peripheral myelin protein 22 (PMP22). Nerve conduction studies show features of axon loss and predominantly distal slowing consistent with demyelination. Because a genetic loss of PMP22 function yields hereditary neuropathy with liability to pressure palsies (HNPP), loss of PMP22 function due to anti- MAG antibodies may result in increased sensitivity to entrapment. We investigated this by performing standardized electrophysiological studies in 16 patients with anti- MAG neuropathy and 16 disease controls with genetically confirmed HNPP. Disproportionate slowing relative to adjacent segments occurred in similar proportions of patients with anti-MAG neuropathy and HNPP, and was of the same magnitude in each group. Affected were the elbow, carpal tunnel and the wrist-hand segments of the median and ulnar nerves. However, in anti- MAG neuropathy as compared to HNPP, absolute values of distal motor latencies and conduction velocities outside entrapment sites were slower and amplitudes were lower. In conclusion, increased sensitivity for entrapment may occur in anti-MAG neuropathy and contribute to part of the nerve damage.     

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid axonal transport in focally demyelinated sciatic nerve

Focal demyelination was produced in rat sciatic nerve by unilateral intraneural injection of anti-galactocerebroside serum. A functional lesion was confirmed by the presence of nerve conduction block. Histologically, this corresponded to demyelination of 50-70% of the fibers in nerve cross sections; axonal structures appeared intact. At the time of maximal demyelination (7 d), 35S-methionine or 3H-fucose was injected bilaterally into the spinal cord ventral horn. At later times (5 hr-7 d), the sciatic nerve was removed and radioactivity in successive nerve segments was quantitated. The transport rates (approximately 260 mm/d) and the composition of transported proteins and glycoproteins (separated on 7-15% polyacrylamide gradient gels) were not altered in lesioned nerves relative to contralateral control nerves. Light microscopic autoradiographic analysis revealed a similar localization of axonally transported and deposited glycoproteins in demyelinated and control fibers. Initially (8 hr), the majority of label was over axons. Labeled glycoproteins remaining in the nerve after 1 week were retained mainly in axolemmal regions. We conclude that acute focal primary demyelination does not lead to major alterations in the transport or deposition of newly synthesized macromolecules.     

Adhesion of axolemmal fragments to Schwann cells: a signal- and target-specific process closely linked to axolemmal induction of Schwann cell mitosis

Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125I-axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more.     

Distribution of adrenocorticoid receptors in the rat CNS measured by competitive PCR and cytosolic binding

A growing number of glycoproteins have been identified and characterized in myelin and myelin-forming cells. In addition to the major P0 glycoprotein of compact PNS myelin and the myelin-associated glycoprotein (MAG) in the periaxonal membranes of myelin-forming oligodendrocytes and Schwann cells, the list now includes peripheral myelin protein-22 (PMP-22), a 170 kDa glycoprotein associated with PNS myelin and Schwann cells (P170k/SAG), Schwann cell myelin protein (SMP), myelin/oligodendrocyte glycoprotein (MOG), and oligodendrocytemyelin glycoprotein (OMgp). Many of these glycoproteins are members of the immunoglobulin superfamily and express the adhesion-related HNK-1 carbohydrate epitope. This review summarizes recent findings concerning the structure and function of these glycoproteins of myelin sheaths with emphasis on the physiological roles of oligosaccharide moieties.     

Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chicken ciliary ganglion after intracerebral injection of (3H)fucose and (3H)-glucosamine

The axonal transport of glycoproteins to nerve terminals has been studied in chicken ciliary ganglion after intracerebral injection of [³H]fucose. Light microscopic radioautographs of ciliary ganglion examined at several time intervals showed from 1.5 to 12 h a slow rise of the radioactivity in preganglionic axons and a tremendous increase of the amount of label in their large caliciform nerve terminals. The concentration of the tracer in presynaptic calices reached a maximal value between 12 and 48 h, and then decreased progressively at a rate indicating a turnover time of 10 days. At any time interval, the radioactivity in the preganglionic axons was lower than in their preterminal segment and in their presynaptic calices. No significant reaction was seen over postsynaptic perikarya. Electron microscopic radioautographs of preganglionic axons showed a slight reaction mainly located over the axolemma; in nerve terminals, the reaction predominated over regions occupied by synaptic vesicles and plasma membranes. A similar distribution of the label was observed after intracerebral injection of [³H]glucosamine. In conclusion, glycoproteins manufactured from labeled sugars in preganglionic nerve cell bodies are transported rapidly (220–290 mm/day) along the axons to nerve endings. One fraction accumulates mainly as components of synaptic vesicles and presynaptic plasma membranes; another one participates in the renewal of axolemmal glycoprotein.     

EFFECTS OF PRENATAL ETHANOL EXPOSURE ON PHYSICAL GROWTH, SENSORY REFLEX MATURATION AND BRAIN DEVELOPMENT IN THE RAT

In the offspring of ethanol-treated rats during gestation (25% ethanol in drinking water) decreased litter size, increased postnatal mortality rate, reduced body weight and body size, delayed ear opening, eyelid opening and teeth eruption, retarded air righting reflex acquisition, impaired brain growth, reduced cortical thickness and delayed maturation of layer Vth's pyramidal neurons: reduced basilar dendritic arborization and decreased number of spines in the apical dendrite, were observed when compared with age-matched controls fed with a standard diet. Minimal effects were found in the offspring of fibre-treated rats during gestation (standard diet mixed with cellulose) in which the body weight was similar to that of controls, although both the calorific intake from food and the mother's weight gain during pregnancy were similar to those of the ethanol-treated group. All these abnormal parameters became normal at the end of the first month of postnatal life, indicating recovery of these developmental defects produced by prenatal ethanol consumption.     

Prenatal ethanol exposure alters the effects of gonadectomy on hypothalamic-pituitary-adrenal activity in male rats

Prenatal ethanol exposure has marked effects on development of the hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes. In adulthood, ethanol-treated rats show altered gonadal hormone responses and reproductive function, and increased HPA responsiveness to stressors. Importantly, prenatal ethanol differentially alters stress responsiveness in adult males and females, raising the possibility that the gonadal hormones play a role in mediating prenatal ethanol effects on HPA function. To examine a possible testicular influence on HPA activity in males, we compared the effects of gonadectomy on HPA stress responses of adult male offspring from ethanol, pair-fed (PF) and ad libitum-fed control dams. Intact ethanol-treated rats showed increased adrenocorticotrophic hormone (ACTH) but blunted testosterone and luteinising hormone (LH) responses to restraint stress, and no stress-induced elevation in arginine vasopressin (AVP) mRNA levels compared to those observed in PF and/or control rats. Gonadectomy: (i) significantly increased ACTH responses to stress in control but not ethanol-treated and PF males; (ii) eliminated differences among groups in plasma ACTH and AVP mRNA levels; and (iii) altered LH and gonadotrophin-releasing hormone responses in ethanol-treated males. Taken together, these findings suggest that central regulation of both the HPA and HPG axes are altered by prenatal ethanol exposure, with normal testicular influences on HPA function markedly reduced in ethanol-treated animals. A decreased sensitivity to inhibitory effects of androgens could contribute to the HPA hyperresponsiveness typically observed in ethanol-treated males.     

Myelin-associated glycoprotein (MAG) and rat brain-specific 1B236 protein: Mapping of epitopes and demonstration of immunological identity

The myelin-associated glycoprotein (MAG) and the brain 1B236 protein are 100-kDa glycoproteins containing 30% carbohydrate that exist in two developmentally regulated forms and are specific to the nervous system. Recent cDNA cloning experiments in several laboratories using primarily immunological means of identification have determined the complete primary sequence of a rat brain glycoprotein that seems to correspond to both MAG and 1B236, suggesting that these proteins are identical. However, MAG was previously considered to be an oligodendrocyte/myelin specific component in the CNS at all ages, whereas 1B236 was thought to be primarily a neuronal component in adult rats but synthesized by oligodendrocytes at the time of active myelination. The composite term 1B236/MAG was proposed to describe the molecule identified by the cDNAs. In order to explore further the relationship between MAG and 1B236, as well as their developmentally regulated forms, experiments were carried out on rat samples utilizing synthetic peptides corresponding to sequences throughout the 1B236 molecule, antisera raised to synthetic peptides in the C-terminus of 1B236 that distinguish between the two developmentally regulated forms, and well-characterized polyclonal and monoclonal antibodies raised to purified MAG. Epitope mapping demonstrated that reactive sites were distributed throughout the extracellular and intracellular domains of 1B236/MAG. Only antibodies reacting with the smaller of the two forms of 1B236/MAG detected the glycoprotein in the peripheral nervous system. Both anti-MAG and anti-1B236 antibodies revealed a drastic reduction of the level of 1B236/MAG in 25-day-old myelin-deficient rats and in adult quaking mice, and both types of antibodies revealed a slight shift of 1B236/MAG toward higher apparent Mr in quaking mice as had previously been reported for MAG. The results indicate that MAG and 1B236 are almost certainly identical since they cannot be distinguished immunologically by the reagents available and that quantitatively most of the glycoprotein is associated with oligodendrocytes and myelin rather than neurons at all ages.