Estrogen induces N-linked glycoprotein expression by immature mouse uterine epithelial cells

Characterization of complex glycoconjugates and the effects of estrogen on their expression in immature mouse uterine epithelial cells are reported. The secreted fraction contained nonanionic, O-linked lactosaminoglycan (LAG)-bearing proteins of Mr 30,000-40,000 as well as anionic, O-linked, LAG-bearing glycoproteins with very high apparent molecular weight (greater than 670K). Heparan sulfate (HS) proteoglycans and HS linked to little or no protein were found in the secreted fraction as well. A very similar array of glycoconjugates was found in the nonhydrophobic fraction of cell-associated macromolecules. In addition, the hydrophobic cell-associated fraction contained nonanionic, LAG-bearing glycoproteins of approximately 250K, anionic LAG-bearing glycoproteins distributing over a wide range of molecular weights, and HS proteoglycans with median molecular weights of approximately 250K. In contrast to the glycoproteins produced by their mature counterparts, virtually all glycoproteins produced by immature cells were O-linked. Estrogen treatment of immature mice caused uterine epithelial cells to secrete anionic, high molecular weight (greater than 670K) N-linked glycoproteins as a major product. These estrogen-responsive glycoproteins did not appear to contain LAGs. Estrogen treatment also markedly decreased the proportion of all hydrophobic glycoconjugates in the cell-associated fraction. Collectively, these observations indicate that one aspect of the estrogen-induced maturation of uterine epithelial cells is the stimulation of N-linked glycoprotein synthesis and secretion. Furthermore, stimulation of N-linked glycoprotein synthesis by itself is insufficient to support N-linked LAG glycoprotein production.     

srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates

srf-3 is a mutant of C. elegans that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis and Yersinia pestis. Recently, SRF-3 was characterized as a nucleotide sugar transporter of the Golgi apparatus occurring exclusively in hypodermal seam cells, pharyngeal cells, and spermatheca. Based on the above observations, we hypothesized that srf-3 may have altered glyconjugates that may enable the mutant nematode to grow unaffected in the presence of the above pathogenic bacteria. Following analyses of N- and O-linked glycoconjugates of srf-3 and wild type nematodes using a combination of enzymatic degradation, permethylation, and mass spectrometry, we found in srf-3 a 65% reduction of acidic O-linked glycoconjugates containing glucuronic acid and galactose as well as a reduction of N-linked glycoconjugates containing galactose and fucose. These results are consistent with the specificity of SRF-3 for UDP-galactose and strongly suggest that the above glycoconjugates play an important role in allowing adhesion of M. nematophilum or Y. pseudotuberculosis biofilm to wild type C. elegans. Furthermore, because seam cells as well as pharyngeal cells secrete their glycoconjugates to the cuticle and surrounding surfaces, the results also demonstrate the critical role of these cells and their secreted glycoproteins in nematode-bacteria interactions and offer a mechanistic basis for strategies to block such recognition processes.     

Compartmentalization of cyclic AMP elevation in neurons ofAplysia californica

We have measured by radioimmunoassay the amount of total, free, and bound forms of cyclic AMP (cAMP) within the abdominal ganglion and in five identified cell bodies of neurons from Aplysia californica. In the abdominal ganglion the unbound (free) cAMP levels comprised approximately 25-30% of the total cAMP content under the unstimulated condition, i.e., bathed in high-magnesium saline. Under pharmacological conditions that blocked endogenous phosphodiesterase and activated adenylate cyclase, ganglionic free cAMP levels were elevated more than fourfold, while bound cAMP levels more than doubled. Freeze-substitution techniques were employed to facilitate isolation of individual cell bodies either before or after pharmacological manipulation of cAMP levels. The basal, free cAMP content of cells R2, LP1, R15, L11, and L2-L6 was in the range of 10-40 pmol/mg of cell protein, which accounted for approximately one-half of the total cAMP content per cell body. Determinations of individual cell volumes indicated that the basal, free cAMP concentrations ranged from 1 to 6 microM. Under the same pharmacological conditions that elevated ganglionic cAMP in levels, no changes were measured in either the free or the bound forms of cAMP in isolated cell bodies. Our results indicate that the cAMP elevation was compartmentalized within the neuropilar region of the ganglion, most likely within the processes of the nerve cells. Previous results demonstrated that cAMP injections into the same Aplysia neurons studied here induced a cAMP-activated sodium current, INa (cAMP). In this report we discuss the possibility that pharmacological elevation of cAMP within neuronal processes may reach concentrations similar to those produced by cAMP injections into somata.     

Proteolytic processing of a peptide precursor in Aplysia neuron R14

The large neurons of the mollusc Aplysia are useful for studying the biogenesis of neuropeptides in single cells. Neuron R14 in the abdominal ganglion synthesizes large quantities of a 10-kDa neuropeptide precursor. The amino acid sequence of this precursor has been defined by analysis of the nucleotide sequence of a cDNA clone. We labeled proteins in vivo by microinjection of radioactive amino acids into individual R14 neurons. The labeled peptides were fractionated by high performance liquid chromatography and subjected to Edman degradation, thus enabling us to determine post-translational processing sites. Cleavage of the signal sequence was observed and at two internal sites. Cleavage at these internal sites occurs at basic amino acids and results in three products, a 2.9-, a 4.9-, and a 1.4-kDa peptide. These studies of protein processing serve as a basis for further investigations of the biogenesis and physiological activities of the neuropeptides.     

Development and modulation of endogenous bursting in identified neuron R15 of juvenile Aplysia

Evidence from a variety of both vertebrate and invertebrate preparations has demonstrated that modulation of the intrinsic firing patterns of individual neurons can have a dramatic effect on the functional output of a neural circuit. Although the mechanisms underlying the production and modulation of intrinsic firing patterns have been extensively studied in adult nervous systems, relatively little is known about how these two features of intrinsically active neurons develop. To address these issues, we have examined the development of endogenous bursting and its modulation by neuropeptides in the identified cell R15 of juvenile Aplysia. Confirming Ohmori (1981), we found that the mature parabolic bursting pattern of R15 is absent in early juvenile stages and develops only gradually over the last stage of juvenile development. We have then analyzed the modulatory effects of extracts made from the neurosecretory bag cells of Aplysia on the immature firing pattern of juvenile R15 cells. In the adult, neuroactive peptides released from the bag cells are known to intensify bursting. In juveniles, we have found that bag cell extract (BCE) can induce bursting prematurely as well as intensify immature bursts, whereas control extracts have no effect on the firing pattern of R15. These results show that the ionic currents necessary for the generation of endogenous bursting in R15 are present and can be modulated before the normal developmental expression of the burst pattern.     

Identification of protein-bound oligosaccharides on the surface of growth cones that bind to muscle cells

In the accompanying paper (Gabel, Den, and Ambron, in press) it was shown that eight populations of glycopeptides are synthesized by single neurons of Aplysia californica. To see which glycopeptides might mediate interactions with target cells, we first identified glycopeptides that are transported selectively to synapses and growth cones. The giant neuron R2 was injected intrasomatically with 3H-glucosamine. Twenty-four hours later, 3H-glycopeptides in the axon and cell body were isolated and resolved by serial lectin affinity chromatography. Of the eight populations, the biantennary-type glycopeptides (GPbi) and those that bind to WGA (GPwga) were preferentially associated with rapidly transported glycoproteins. In contrast, the glycopeptide that consists of N-acetylglucosamine O-linked to ser/thr was mostly retained in the cell body. GPbi and GPwga were also preferentially transported to growth cones. Analyses of RUQ cells, exposed to 3H-glucosamine in vitro for 36 h showed an enrichment of GPbi and GPwga at the growth cone relative to the cell body. The disposition of the various glycopeptides in growing neurons was also examined using FITC lectins. FITC-coupled WGA, Vicia vellosa, and lentil lectin showed extensive staining of the cell body, but only WGA stained the growth cones. To investigate if GPwga interacts specifically with target cells, these glycopeptides were isolated from the neurons of 180 abdominal ganglia. GPwga, other Aplysia glycopeptides, and glycopeptides prepared from ovalbumin were coupled separately to fluorescent spheres. The spheres were then added to muscle cells isolated from the auricle of the heart, which is innervated by many neurons from the ganglion. While spheres coupled to GPwga bound to the muscle cell surface, the other glycopeptides did not. These results indicate that glycopeptides class GPwga, found among rapidly transported glycoproteins and on the growth cone surface, is able to bind to muscle cells and may therefore play some role in neuron-target interactions.     

Determination of Glutathione and ATP in Ganglia and Individual Neurons of Aplysia californica

The levels of two gamma-glutamyl cycle substrates, glutathione and ATP, were determined in single identified nerve cell bodies from the CNS of Aplysia californica. The glutathione content of single cells averaged 30 +/- 4.9 mumol/g protein. Glutathione levels were similar in identified cholinergic, serotonergic, and histaminergic cells, as well as in neurons whose transmitters are not yet identified. The abdominal rostral white cells, which are enriched in glycine, a component amino acid of glutathione, did not possess distinctively higher glutathione concentrations. The ATP content of single Aplysia nerve cell bodies averaged 15.0 +/- 1.5 mumol/g protein. Despite the vast chemical, anatomical, and functional heterogeneity between Aplysia central neurons, no cells were found that contained unusually high or low ATP levels.     

O-Linked Oligosaccharide on the 75-kDa Neurotrophin Receptor

Abstract: Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75-kDa neurotrophin receptor (NTR). An O -glycosylated peptide connects the ligand-binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O -glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O-linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of desialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O-linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O-linked glycans synthesized by cultured cells had the structure (NeuNAc)_1–2-Galβ1-3GalNAc. Modification of the O-linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling.     

Endogenous neurotrophic factors enhance neurite growth by bag cell neurons of Aplysia

Mechanisms that regulate neurite outgrowth are phylogenetically conserved, including the signaling molecules involved. Here, we describe neurotrophic effects on isolated bag cell neurons (BCNs) of substrate-bound growth factors endogenous to the sea slug Aplysia californica. Sheath cells dissociated from the pleural-visceral connectives of the Aplysia CNS and arterial cells dissociated from the anterior aorta enhance neurite outgrowth when compared to controls, i.e., BCNs grown in defined medium alone. In addition, the substrate remaining after sheath cells or arterial cells are killed significantly enhances growth, relative to all other conditions tested. For instance, primary neurites are more numerous and greater in length for BCNs cultured on substrate produced by arterial cells. These results suggest that sheath and arterial cells produce growth-promoting factors, some of which are found in the substrates produced by these cell types. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we found that Aplysia collagen-like peptides are produced by dissociated arterial cells, and therefore likely contribute to the observed growth effects. Collagen-like peptides and other factors produced by sheath and arterial cells likely influence neurite growth in the Aplysia CNS during development, learning and memory, and regeneration after injury.     

Abundant expression of ras proteins in Aplysia neurons

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.     

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.     

Activity-related changes in protein phosphorylation in an identifiedAplysia neuron

The relationship between long-term electrical activity and protein phosphorylation was investigated in single, identifiable neurons in the abdominal ganglion of Aplysia californica by the intracellular injection of radiolabeled ATP followed by sodium dodecyl sulfate (SDS) gel electrophoresis. Natural and pharmacological treatments that alter the impulse activity of neurons L6 and R15 for prolonged periods did not appear to affect the phosphorylation of most of the 15 major phosphoproteins examined in these cells. Long-term excitation of L6 induced by the phosphodiesterase inhibitor IBMX correlated with phosphorylation of a 29,000-dalton protein. Long-term inhibition of L6 induced by afterdischarge of peptidergic bag-cell neurons appeared to cause dephosphorylation of a 29,000-dalton protein. Burst augmentation of R15 induced by bag-cell afterdischarge did not cause detectable changes in the phosphorylation of the major proteins we examined. These data are consistent with other studies of neural and nonneural tissues which have found a correlation between activity and the level of phosphorylation of a 29,000-dalton protein.     

Heterogeneous expression of glycoconjugates among individual glomeruli of the hamster main olfactory bulb

Glomeruli within the main olfactory bulb (MOB) are known as areas of synapse formation between axon terminals of olfactory neurons in the olfactory epithelium and dendrites of the first relay neurons (mitral and tufted cells) in the MOB, so that they serve as functional units in olfaction. We examined expression patterns of glycoconjugates in the glomeruli of the hamster MOB by lectin histochemistry using 21 biotinylated lectins. Thirteen lectins, WGA, s-WGA, DSL, DBA, SBA, WA, SJA, RCA-I, PNA, ECL, UEA-I, PSA and LCA, showed differential binding patterns among the glomeruli. To evaluate these differential binding patterns of lectins, we analysed staining intensity of each of the 13 lectins on the level of individual glomeruli by image analysis, and classified staining intensity into five grades (negative, 1+, 2+, 3+, 4+) on the basis of results obtained. This classification enables us to make detailed comparison among individual glomeruli. We further analysed the grade of staining intensity of each of the 13 lectins in the same glomerulus in adjacent serial sections by image analysis, and found that individual glomeruli varied in combination of grades of staining intensity and kinds of lectins. These results indicate that glycoconjugates are expressed heterogeneously in individual glomeruli, and that heterogeneous expression may contribute to the topographic organization of the primary olfactory pathway.     

Growth cones isolated from identified Aplysia neurons in vitro: biochemical and morphological characterization

The right upper quadrant (RUQ) cells (R3-R13) of Aplysia regenerating in dissociated cell culture form unusually large growth cones. The movement of these growth cones was observed by time-lapse phase microscopy and their ultrastructure was examined by transmission electron microscopy. Their behavior and ultrastructure have features that are typical of growth cones in vitro. Additionally, they contain neurosecretory granules similar to those found in these cells in vivo. Because RUQ growth cones are large, they can be isolated by manual dissection. RUQ cells were grown in the presence of [35S]methionine and the labeled proteins transported to the growth cones were analyzed by SDS-PAGE. These proteins were compared to those in RUQ cell bodies, RUQ neurites, and to those in the neurites and cell bodies of other identified neurons grown in vitro. Most proteins synthesized by RUQ cells in vitro are transported to their growth cones, including several glycoproteins and the precursor to the R3-R14 neuropeptide. Neuropeptides are also synthesized by a number of other Aplysia neurons growing in vitro. We examined R2, LPL1, R15, and left upper quadrant neurons and found that their precursor peptides, like those of R3-R14, are readily recognized as major cell-specific radiolabeled bands on SDS gels. The presence in regenerating growth cones of neuropeptides, neurosecretory granules, and glycoproteins known to be rapidly transported toward synapses in vivo supports the emerging view that the growth cone in vitro contains not only a motility apparatus but also a macromolecular assembly capable of forming an active synapse immediately upon or shortly after contacting targets.     

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans

The prestalk and prespore cells from the Dictyostelium discoideummulticellular slug stage of development differ in assembly ofglycoconjugates. Prespore cells are 2- to 3-fold more activethan prestalk cells in the assembly of N-linked glycans and20-fold more active in their fucosylation. Only prespore cellssynthesize an O-linked glycan consisting in part of Fuc -linkedto N-acetylglucosamine. Incorporation of fucose, glucosamine,mannose and galactose into large pronase-resistant glycoconjugateswas almost exclusively into prespore cells. Such glucosamine-labelledglycoconjugates resist fragmentation by ß-eliminationand include a glycoantigen dependent on the modB genetic locus.In contrast, large fucose-labelled glycoconjugates consistedof multiple, small, O-linked oligosaccharides on carrier peptides.The spore coat protein SP96 has several fucosylated O-linkedoligosaccharides, one of which correlates with a fucose epitopepreviously shown to localize in prespore vesicles and the outerlayer of the spore coat. Dictyostelium discoideum glycoconjugates glycoproteins prespore prestalk     

Molecular cloning and characterization of chick SPACRCAN

MY-174, a monoclonal antibody that reacts with specific sialylated O-linked glycoconjugates of chick SPACR (sialoprotein associated with cones and rods), also recognizes another molecule of 300 kDa. Here, we verified that this 300-kDa molecule is chick SPACRCAN (sialoproteoglycan associated with cones and rods), another member of a novel interphotoreceptor matrix molecule family. Screening for chick SPACRCAN was carried out by plaque hybridization using a probe for chick SPACR. Specific polyclonal antibodies raised against chick SPACRCAN were used for the following experiments. To determine whether the 300-kDa molecule detected by MY-174 was identical to 300-kDa chick SPACRCAN, the migrations of these bands were examined after various glycosidase digestions. Furthermore, the expression levels were measured during retinal development and compared with those of chick SPACR. The results demonstrated that the 300-kDa molecule recognized by MY-174 was chick SPACRCAN, and we further identified it as a proteoglycan with chondroitin sulfate chains. SPACRCAN had heavily sialylated N- and O-linked glycoconjugates, and its MY-174 antigenicity was abolished by O-glycanase treatment after neuraminidase treatment, as observed for chick SPACR. During retinal development, the mRNA and core protein expression levels, MY-174 antigenicity, and hyaluronan binding ability of SPACRCAN peaked around embryonic day 17 and then gradually decreased, whereas the corresponding expression levels of SPACR simply increased, but not its hyaluronan binding ability. The MY-174 reactivity of SPACRCAN in the adult retina was decreased compared with that in the newborn retina, whereas that of SPACR was increased. The decreased hyaluronan binding of SPACR was induced by an inhibitory effect of the excess of sialic acids in the adult stage. Thus, with similar core protein structures and specific sialylated glycoconjugates but distinct chondroitin sulfate chains, SPACRCAN and SPACR may have separate roles in the retina due to their differing expression profiles during development.     

Anti-pig antibody adsorption efficacy of {alpha}-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 {beta}1, 6-N-acetylglucosaminyltransferase expression

We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha1,3 galactosyl- and core 2 beta1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.     

Histochemical study of glycoconjugates in the epididymis of the hamster (Mesocricetus auratus)

Summary The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput epididymis, with particular histochemical characteristics, has been differentiated. β-Elimination in combination with lectins was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely positive with HPA, PNA and SBA lectins. β-limination indicated that these cells contained abundant O-linked glycoconjugates. Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the lectins (especially in the acrosome), except by those with an affinity for α-l-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity was negative only in the zone between the caput and the corpus.