Omanand Koul

Modulation of rat brain cytosolic phosphatidate phosphohydrolase: Effect of cationic amphiphilic drugs and divalent cations

The effects of three cationic amphiphilic drugs on rat brain cytosolic phosphatidate phosphohydrolase and their mechanisms of action were studied utilizing membrane-bound, emulsified, and emulsified sonicated phosphatidate as substrates. With the membrane-bound substrate, chlorpromazine, desmethylimipramine, and propranolol inhibited the activity in a dose-dependent fashion with an IC50 of 30-50 microM. In the presence of the emulsified substrate, chlorpromazine was a more potent inhibitor than desmethylimipramine or propranolol but 200 microM was needed for 50% inhibition of activity. Addition of heat-inactivated microsomes to the emulsified substrate, to simulate the conditions with the membrane-bound substrate, did not alter this value. Both Mg2+ and Ca2+ stimulated the enzyme activity but only Ca2+ counteracted the effect of chlorpromazine. Kinetic studies indicate that chlorpromazine acts as a noncompetitive inhibitor of the enzyme. Emulsified sonicated phosphatidate was a good substrate at low (less than 10 microM) concentrations. It was a poor substrate at 1 mM, but at this concentration chlorpromazine stimulated the activity instead of inhibiting. This drug altered the integrity of phosphatidate vesicle membranes as visualized by electron microscopy. The different results obtained with the three types of substrate indicate the importance of the configuration of phosphatidate for the expression of enzyme activity and for its susceptibility to the action of cationic amphiphilic drugs.

Developmental Expression of HNK‐1‐Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Abstract: Monoclonal antibody HNK‐1‐reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK‐1‐reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto‐N‐neotetraosylceramide (SGGL‐1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto‐N‐norhexaosyl ceramide (SGGL‐2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK‐1‐reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK‐1‐reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK‐1‐reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N‐CAM‐180. A diffuse HNK‐1‐reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK‐1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK‐1‐reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei. In order to confirm this localization, SGGLs were analyzed by HPTLC‐immunoverlay method in micro‐dissected cerebellar layers from freeze‐dried cryocut sections: they were found primarily in the molecular layer and deep cerebellar nuclei and were undetectable in the granule cell layer and white matter. These results, along with the lack of SGGLs and disialosyl lacto‐N‐neotetraosylceramide (LD1) in several Purkinje cell‐deficient murine mutants reported previously, indicate that these glycolipids are associated specifically with Purkinje cell dendrites in the molecular layer and Purkinje cell axon synapses in deep cerebellar nuclei.

Monoclonal antibodies to distinct regions of human myelin proteolipid protein simultaneously recognize central nervous system myelin and neurons of many vertebrate species

Myelin proteolipid protein (PLP), the major protein of mammalian CNS myelin, is a member of the proteolipid gene family (pgf). It is an evolutionarily conserved polytopic integral membrane protein and a potential autoantigen in multiple sclerosis (MS). To analyze antibody recognition of PLP epitopes in situ, monoclonal antibodies (mAbs) specific for different regions of human PLP (50–69, 100–123, 139–151, 178–191, 200–219, 264–276) were generated and used to immunostain CNS tissues of representative vertebrates. mAbs to each region recognized whole human PLP on Western blots; the anti‐100–123 mAb did not recognize DM‐20, the PLP isoform that lacks residues 116–150. All of the mAbs stained fixed, permeabilized oligodendrocytes and mammalian and avian CNS tissue myelin. Most of the mAbs also stained amphibian, teleost, and elasmobranch CNS myelin despite greater diversity of their pgf myelin protein sequences. Myelin staining was observed when there was at least 40% identity of the mAb epitope and known pgf myelin proteins of the same or related species. The pgf myelin proteins of teleosts and elasmobranchs lack 116–150; the anti‐100–123 mAb did not stain their myelin. In addition to myelin, the anti‐178–191 mAb stained many neurons in all species; other mAbs stained distinct neuron subpopulations in different species. Neuronal staining was observed when there was at least approximately 30% identity of the PLP mAb epitope and known pgf neuronal proteins of the same or related species. Thus, anti‐human PLP epitope mAbs simultaneously recognize CNS myelin and neurons even without extensive sequence identity. Widespread anti‐PLP mAb recognition of neurons suggests a novel potential pathophysiologic mechanism in MS patients, i.e., that anti‐PLP antibodies associated with demyelination might simultaneously recognize pgf epitopes in neurons, thereby affecting their functions.

N-Glycosylation site occupancy of rat α-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity

All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated. When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme. Results obtained with singly mutated forms of rFuc-TIV indicate that both sites are occupied in mammalian cells. Lack of glycosylation at sites 117-119, 218-220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme. These results show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218-220 playing the major role. However, N-glycosylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of staining for the unglycosylated mutants as is seen for the wild-type rFuc-TIV in transfected cells.

α1,3 Fucosyltransferase, α-L-fucosidase, α-D-galactosidase, β-D-galactosidase, and Lex glycoconjugates in developing rat brain

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Lex) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15–P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10–P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an α1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5–10) with different types of acceptors, and the differential expression of Lex containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, α-L-fucosidase, β-D-galactosidase and α-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases.

Regional Developmental and Fractional Studies on Myelin and Other Carbonic Anhydrases in Rat CNS

Myelin carbonic anhydrase (CA) was studied with respect to its development in various brain regions and light and heavy myelin (LM and HM). The data indicate that the specific activity of myelin CA has a clear neuraxial distribution, increasing rostrally. The absolute activities and relative distribution are invariant with age; this suggests the CA activity in myelin is independent of stage and degree of myelination. The studies on HM and LM illustrate that HM, like total myelin, has a constant CA activity during development. In contrast, LM although equal to HM at 14 days, progressively decays to an adult level which is one-fourth that of HM. The distribution of CA in myelin was further investigated by comparing the activity in myelin with that present in the SN4 fraction. The activity in this latter fraction, which is derived from heavy myelin, was found to be 2.2 times higher than that in the myelin fraction. Thus, in the adult there exists an almost ten-fold range of activities among the various myelin fractions, SN4 greater than HM greater than LM. This may indicate a segregation of activity towards the outer lamellae. This segregation may have physiological importance in that it is this region of the sheath which should be integrally involved in control of myelin edema. Evidence indicates that there is an interaction of chloride with the enzyme, and maybe the primary ion moved by CA in order to initiate an osmotic flux out of the sheath. The interaction of chloride with the enzyme is dependent on the CA complex with the membrane in that solubilization and partial (60-fold) purification results in a preparation which is refractory to anions.

Synthesis and Transport of Cerebrosides and Sulfatides in Rat Brain During Development

Synthesis and transport of nonhydroxy fatty acid (NFA)‐ and hydroxy fatty acid (HFA)‐containing ceramides, cerebrosides, and sulfatides were studied in vivo in rat brain during development. After an intracerebral injection of [3H]serine, incorporation into these lipids of microsomal and myelin membranes was analyzed after HPLC. Distribution of amounts and incorporation of radioactivity were also determined in individual molecular species of these lipids. The results showed that HFA‐ceramides and long‐chain NFA‐ceramides have small pool sizes and rapid turnover rates in the microsomal membranes and are preferentially utilized for the synthesis of long‐chain (≥20:0) HFA‐and NFA‐galactocerebrosides of both microsomal and myelin membranes. Glucocerebrosides are not expressed in myelin and their synthesis in microsomal membranes is predominant before the onset of myelination. With development, synthesis and accumulation of HFA‐cerebrosides increase over NFA‐cerebrosides in both microsomal and myelin membranes. In myelin, incorporation of radioactivity into HFA‐cerebrosides is even higher than that expected by transport alone from microsomal membranes and it is possible that part of the HFA‐cerebrosides in myelin could be due to de novo synthesis by myelin itself. The amount of NFA‐ and HFA‐sulfatides is about equal, both in myelin and microsomal membranes, and this relative proportion does not change with development. Similar relative rates of incorporation of radioactivity into sulfatides of microsomal and myelin membranes are consistent with the notion that both NFA and HFA sulfatides are synthesized in the microsomal (Golgi) membranes and are transported to myelin. In vitro kinetic analysis of both myelin and microsomal galactosyltransferase with different HFA‐ceramide species showed an apparent Km of about 0.12 mM, whereas the Vmax was highest for h24:1 ceramide followed by that for h18:0 and h24:0 ceramides.

Regulation of Cerebroside and Sulfatide Metabolism in Glia Cells

Abstract: Mouse oligodendroglioma cells, G‐26 clone 20 and 24, contain galactosylceramide (cerebroside) and sul‐fogalactosylceramide (sulfatide) as determined by an HPLC technique. The synthesis of both these lipids was stimulated by 10−‐6M hydrocortisone (cortisol) and also by the removal of serum from the culture medium. Forty‐eight hours after the addition of cortisol the incorporation of H235SO4 into sulfatide, the level of sulfatide and the specific activity of the enzyme 3′‐phosphoadenosine 5′‐phosphosulfate:galactosylceramide sulfotransferase in the cells increased three‐ to fourfold. The level of cerebroside and the specific activity of UDP‐galactose: hydroxyacyl sphingosine galactosyltransferase also increased threefold in the cells on treatment with cortisol. The effect of the hormone on the synthesis of cerebroside preceded the increase in sulfatide synthesis. Experiments with cycloheximide and actinomycin D showed that the effect of the hormone on glycolipid synthesis in these cells was mediated through de novo messenger RNA and protein synthesis. Removal of serum from the culture medium resulted in an approximately twofold enhancement of H235SO4 incorporation into sulfatide within 24 h. The levels of sulfatide and cerebroside and the specific activity of the galactosyltransferase and sulfotransferase also increased significantly after serum removal. However, in contrast to the effect of the steroid, the sulfotransferase activity and the level of sulfatide increased prior to elevations in galactosyltransferase and cerebroside. The effect of serum removal was also found to be mediated by de novo RNA and protein synthesis. The effects of cortisol and serum removal on the synthesis of cerebroside and sulfatide were strictly additive. This observation plus the different temporal patterns of induction suggest that two separate regulatory mechanisms at the nuclear level exist in oligodendroglioma cells for the biosynthesis of these glycolipids.

The rat alpha1, 3-fucosyltransferase (rFucT-IV) gene encodes both long and short forms of the enzyme which share the same intracellular location.

Fucosyltransferase (FucT) activity has been detected on the surface of mouse germ cells and rat Sertoli cells, and has been postulated to play a role in cell-cell interactions. A recently cloned rat FucT (rFucT-IV) is expressed in the testes, and thus is a candidate for encoding the cell-surface FucT activity. This study maps the 5′-ends of several rFuc-T-IV mRNAs, and these results suggest that initiation of transcription may occur both upstream of the first ATG, as well as between the first two closely spaced, in-frame ATGs. Thus, in certain tissues, notably spleen, significant amounts of both a long and a short form of rFucT-IV would be predicted. This study also determines some basic properties of both the long and short forms of rFucT-IV, and investigates whether the use of alternative ATGs would allow FucT activity to be expressed both on the cell surface and in the Golgi. Plasmids that encode FLAG-epitope-labeled rFucT-IVs that initiate from either of the two ATGs were constructed, and rFucT-IV was expressed either in vitro using cell-free rabbit reticulocyte lysate, or after transfection in tissue culture. The results from these studies demonstrate that rFucT-IV is a glycosylated, transmembrane protein with a short cytoplasmic tail, and that either of the two ATGs in the 5′ region of the rFucT-IV gene are capable of acting as functional initiators of translation in vitro, to produce enzymatically active glycoproteins. However, no difference in the intracellular localization between the transferase containing a 48 amino acid or a 15 amino acid cytoplasmic tail was detected by immunocytochemistry, as both show the same pattern of Golgi-like staining in several different cell types, with no indication of surface expression. Thus, the additional amino-terminal 33 amino acids of the long form of rFucT-IV do not appear to influence its intracellular location in the cell types investigated.

UDP-galactose: ceramide galactosyltransferase of rat central nervous system myelin during development.

The activity of UDP-galactose: hydroxy fatty acid containing ceramide galactosyltransferase was studied in the myelin and microsomal fractions of rat cerebral hemispheres, cerebellum and spinal cord during development. In all three regions, the specific activity of the enzyme reached a maximum in myelin prior to that in the microsomal membranes. This temporal relationship between myelin and microsomal fraction was similar in all the three regions, although the overall timing was shifted corresponding to known differential timing of myelin deposition in these regions. The activity of the enzyme from both the membranes, during development, increased in parallel with temperature up to 45°C. Specific localization of galactosyltransferase in early myelin may suggest specific role of the enzyme in the myelination process.