John J. Lucas

Distribution of transferrin binding protein immunoreactivity in the chicken central and peripheral nervous systems

Transferrin binding protein (TfBP) is a glycoprotein originally purified from chicken oviduct that exhibits transferrin binding activity. Recent work has shown that TfBP is a post‐translationally modified form of the heat shock protein (HSP108), the avian homologue of a glucose regulated protein, GRP94. The function of this protein, however, has not yet been clearly defined. Antiserum to TfBP was found to selectively stain oligodendrocytes of the avian brain. In this study, we further describe these oligodendrocytes and other cell types positive to anti‐TfBP in the chick nervous system. In accordance with previous studies, the most prominent cell type that labels with antiserum to TfBP is the oligodendrocyte. At the electron microscopic level, the immunoreactive product is confined to the perinuclear cytoplasm and fine processes of the oligodendrocytes, whereas myelin and axoplasm are devoid of staining. The immunoreactive product is found both in the cytoplasmic matrix and bound to the endoplasmic reticulum and plasma membrane, suggesting that TfBP may have properties of both a soluble and an integral membrane protein. There is great variability in the number of TfBP‐oligodendrocytes in different areas of the central nervous system (CNS); large numbers of cells are associated with the white matter regions and are found in the myelinated tracts, whereas few cells are present in the gray matter regions. In the retina, TfBP is localized specifically in the cells that are morphologically oligodendrocytic and is present in the optic nerve fiber layer and the ganglion cell layer. Obvious staining is also seen in the Bergmann glial cells of the cerebellum and in the Schwann cells of the sciatic nerve. Furthermore, the choroid plexus cells similarly exhibit a strong reaction. The association of TfBP in these specific cell types responsible for myelination and sequestering iron and transferrin implies that TfBP may be involved in myelination and iron metabolism of the chick nervous system, perhaps through a role in transferrin concentration in these cells. A putative role of TfBP, as HSP108, is considered. J. Comp. Neurol. 260‐271, 1997.

Evidence for a compound with the properties of 2,3-dehydrodolichyl pyrophosphate

The dolichols are a family of long chain polyisoprenols, usually consisting of 16-21 isoprene units linked in a linear, head-to-tail sequence. All but 4 or fewer of the double bonds are in the cis configuration, and the ar-isoprene unit is saturated [ 11. The monophosphate ester of dolichol functions as a carrier of glycosyl units in,

Immunocytochemical study with an anti-transferrin binding protein serum : a marker for avian oligodendrocytes

eir transfer from sugar nucleotide donors to protein acceptors [2-4]. In spite of the physiologic importance of dolichyl phosphate, little is known concerning its biosynthesis, although a pathway has been postulated [5]. Evidence has been reported for the biosynthesis of dolichyl monophosphate, utilizing isopentenyl pyrophosphate as substrate and a 25 000 X g supernatant fraction from pea epicotyl as enzyme source [6]. Subsequently, different membrane fractions from rat or chicken liver were reported to form dolichyl monophosphate, and the results suggested that mitochondrial and Golgi membranes are the primary sites of synthesis [7]. Similar findings have been reported in a study of algae membrane fractions [8]. Apparently these enzyme preparations contain the enzymes required to produce the longchain polyprenyl phosphate, including the enzyme(s) responsible for selective reduction of the ar-isoprene unit. Evidence has been reported for the biosynthesis of 2,3dehydrodolichyl monophosphate, utihzing isopentenyl pyrophosphate as substrate and a 1000 X g particulate fraction from hen oviduct as

Transferrin binding protein is expressed by oligodendrocytes in the avian retina

We have investigated immunocytochemically the localization of a transferrin binding protein (TfBP) in adult CNS of avian and mammalian species using a polyclonal antibody raised against the protein purified from hen oviduct membranes (alpha OV-TfBP). TfBP has recently been shown to be HSP108. An overall strong immunoreactivity was revealed in most parts of the avian brains, especially in the white matter. The main immunoreactivity originated in small, intensely reacting cells interpreted as oligodendrocytes. The density of TfBP-labeled oligodendrocytes of the avian brains was generally proportional to the degree of myelination. There were no marked differences in TfBP-immunostaining pattern between avian species (chick, pigeon and lovebird). On the other hand, in rat, rabbit and cat brains we could not find any TfBP-immunoreactivity. Immunoelectron microscopy has further revealed that TfBP is present in the light and medium types of oligodendrocytes which are known to have high metabolic activities. TfBP reaction product was homogeneously dispersed throughout the perinuclear cytoplasm and fine processes of oligodendrocytes. The intracytoplasmic organelles such as mitochondria and Golgi apparatus were devoid of reaction product. The presence of TfBP in oligodendrocytes implies that this protein may play an important role in transferrin-mediated iron metabolism in the CNS. The complete lack of cross-reactivity between alpha OV-TfBP and mammalian tissues suggests that there is species variability in TfBP structure. We conclude that this chick TfBP antiserum will prove useful in studies of oligodendrocytes and myelination in the avian CNS.

Loss of mannosyl phosphoryl polyisoprenol synthesis upon conversion of reticulocytes to erythrocytes

It has been documented that some axons of ganglion cells in the nerve fiber layer of avian retina are wrapped in a myelin sheath. However, the identity of myelin-forming cells has not been established. In this study we demonstrated immunohistochemical evidence for the existence of a large population of oligodendrocytes in avian retina, using an antiserum against transferrin binding protein (TfBP), the avian homologue of the mammalian GRP 94 family of stress-regulated proteins. TfBP+ cells were mostly confined to the ganglion cell and optic nerve fiber layers of the retina, in which they were closely associated with the soma and axons of ganglion cells. The double-labeling experiments clearly show that TfBP is specific to oligodendrocytes. The morphology, distribution, and antigenic properties indicated by our findings suggest that TfBP+ cells are retinal oligodendrocytes that may be responsible for the myelination of ganglion cell axons in avian retina. A putative tropic role of TfBP+ oligodendrocytes to the ganglion cells is also discussed in conjunction with the physical properties of TfBP and avascular retinae of birds.

The chicken oviduct and embryonic red blood cell transferrin receptors are distinct molecules

Abstract Reticulocyte membranes transfer [ 14 C]mannose from GDP[ 14 C]mannose to an endogenous lipid forming a compound that has the characteristics of β-mannosylphosphoryldolichol. The lipid co-chromatographs with hen oviduct mannosylphosphoryldolichol, is stable to mild alkali and labile to mild acid, and strong base releases mannose phosphate. Formation of the [ 14 C]mannosyl lipid by reticulocyte membranes is stimulated by the addition of exogenous dolichylphosphate. The transfer of mannose from GDPmannose to the lipid fraction is reversible upon the addition of GDP, but not GMP. The apparent K m for GDP-mannose, in the formation of the mannosyl lipid, is 0.56 μM. Erythrocyte membranes transfer less than 5% as much mannose from GDP-mannose to endogenous acceptors as do assays of reticulocyte membranes. However, assays in the presence of exogenous dolichylphosphate reveal the presence of a mannosyltransferase capable of forming a [ 14 C]mannosyl lipid that co-chromatographs in two solvent systems, on SG-81 paper, with hen oviduct and reticulocyte mannosylphosphoryldolichol. The mannosyltransferase activities from reticulocyte and erythrocyte membranes exhibit similar behavior toward divalent cations, Ammonyx detergent and dolichylphosphate. Thus, the enzymes may be identical, and the apparent absence of mannosyl phosphoryl polyisoprenol synthesis in erythrocyte membranes may be accounted for by the absence of endogenous acceptor molecules.

Effect of progesterone, estrogen withdrawal and secondary estrogen treatment of mannosylphosphoryldolichol synthesis in chick oviduct membranes

We recently described an estrogen-inducible transferrin receptor from the chicken oviduct. We now report on the comparison of the oviduct transferrin receptor with the transferrin receptor obtained from chick embryo red blood cells. Western blot analysis reveals that rabbit polyclonal antibodies raised against one receptor do not cross react with the heterologous receptor. Furthermore, peptide map analyses of either affinity purified, native [125I]-labelled transferrin receptors (dimers) or dissociated, and repurified monomers obtained from oviducts and embryonic red blood cells yield distinct patterns. Therefore, the estrogen-modulated oviduct transferrin receptor appears to be structurally distinct from the iron-modulated red cell transferrin receptor.

Effect of hormone treatments on chick oviduct β-N-acetylglucosaminidase isozymes and other acid hydrolases

Oviduct membranes from chicks treated with diethylstilbestrol have a fully induced level of an enzyme that transfers mannose from GDP-Man to form mannosylphosphoryldolichol (Lucas, J.J. and Levin, E. (1977) J. Biol. Chem.252, 4330--4336). Withdrawal of diethylstilbestrol for 5 days causes a decrease in oviduct weight, lysozyme, and 60% of the mannosyltransferase activity. Chicks withdrawn from treatment for 10 days followed by secondary stimulation with diethylstilbestrol exhibit a more rapid increase in the mannosyltransferase activity than chicks that have not been previously treated with diethylstilbestrol. Further experiments indicate that the decrease in mannosylphosphoryldolichol synthesis after hormonal withdrawal may be the result of decreased levels of endogenous dolichyphosphate in the membrane preparations.

Lack of evidence for covalently-bound carbohydrates in energy-transducing ATPases from mitochondria, bacteria, and chloroplasts

Abstract The effects of several hormone treatments on chick oviduct acid hydrolases were studied; including the effect of those treatments on the isozymes of β- N -acetylglucosaminidase. Chicks were treated for 10 days with diethylstilbestrol after which they were treated with progesterone alone, diethylstilbestrol alone, progesterone and diethylstilbestrol, or withdrawn from all hormone treatment. Protease and acid phosphatase were increased four- to fivefold upon hormone withdrawal, but they were not increased by any of the other treatments. β- N -Acetylglucosaminidase, however, increased fourfold upon hormone withdrawal and progesterone alone or progesterone and diethylstilbestrol treatment. In addition to the increase in β- N -acetylglucosaminidase activity, isozyme II increased from 20 to 35% of the total activity upon withdrawal (but not the other treatments). Isozyme I is the only form of the enzyme found in egg white.

Localization of the lipid intermediate pathway of protein glycosylation in oviduct cell types

Recently Muiioz and co-workers proposed that the energy-transducing ATPases from Mz’c~ococcus lysodeikticus (BF1 ) and spinach chloroplasts (CF, ) are glycoproteins [ 1,2]. Their evidence includes periodic acid-Schiff (PAS) staining of the enzymes’ a and /3 subunits on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE gels), PAS staining of protein bands of the appropriate molecular weight on non-denaturing PAGE gels and sugar analysis of enzyme preparations. In addition, preliminary experiments indicate that the mitochondrial ATPase (Fi) may also contain sugar moieties [3,4]. This would be consistent with a report that mitochondrial Fi may contain up to 8% non-protein material by weight [5]. Despite the potential appeal of these suggestions, the experiments reported in this communication fail to confirm the presence of covalently-bound carbohydrates in beef-heart mitochondrial F, , spinach chloroplast CF, , or Escherichia coli BFr . When SDS is carefully removed from SDS-PAGE gels prior to