Rapidly metabolized glycoproteins in a neuroplastoma cell line

The metabolism of neuroblastoma cell glycoproteins was examined using l-[³H]fucose. Incubation of monolayer cultures with [³H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [³H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h)_ of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [³H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 · 10^?4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [³H]fucose. Although the major proportion of the radioactivity remained as [³H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components. However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [⁴C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.     

The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line

When monolayer cultures of neuroblastoma N2a cells were prelabelled with [³H]fucose to steady state, and then reincubated in complete medium in the presence of unlabelled 40mm-l-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2h. After this period of time the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is known to be associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In one set of experiments, with a similar incubation procedure to that used with intact cells, three membrane components were rapidly degraded (150000, 130000 and 48000 daltons), but another surface glycoprotein (68000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analysed by dual-label experiments that used both [¹⁴C]fucose and [³H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells were found to turn over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, were also shown to turn over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [³H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents.     

The relationship between glycosylation and glycoprotein metabolism of mouse neuroblastoma N18 cells.

Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition.     

In vitro synthesis and secretion of glycoprotein by human mammary tissue

Summary Organ cultures of human surgical specimens can be used to investigate glycoprotein production in vitro under conditions in which three-dimensional tissue structures and cell-cell interactions resemble those present in vivo. In this report, an organ-culture system is used to investigate the synthesis, transport and release of glycoprotein by normal and benign hyperplastic human mammary epithelium. Autoradiography of explants pulse-labeled with individual glycoprotein precursors ([³H]glucosamine, [³H]fucose, [³H]acetylmanosamine) and maintained in organ culture for intervals up to 72hr revealed that glycoprotein is synthesized and then secreted by mammary epithelium. Incorporation of each isotope took place in the Golgi apparatus. Most of the newly synthesized glycoprotein, labeled with each of the three precursors, then was transported to apical cell surfaces and secreted into gland lumina. Observations were indistinguishable in normal and benign hyperplastic glands. Thus nonlactating human mammary epithelium exhibits a glycoprotein secretory activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [³H]glucosamine-labeled macromolecules released into the medium showed a group of glycoproteins with a molecular weight of 48,000±6,000 daltons plus high-molecular-weight glycosylated components at the top of gels. The nature of gp48 is not known, but similar molecular-weight glycoproteins also are released by surgical specimens of human mammary cancer maintained in organ culture. Z. A. T. received support from NCI Grant No. CA-14089.     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

Synthesis of glycoproteins in a single identified neuron of Aplysia californica

Incorporation of L-[³H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [³H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [³H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [³H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [³H]fucose, gel profiles of particulate fractions labeled with [¹⁴C]valine were much more complex.     

Incorporation of fucose into the carbohydrate moiety of phytohemagglutinin in developing Phaseolus vulgaris cotyledons

Incubation of developing cotyledons of P. vulgaris with [³H]fucose resulted in the incorporation of radioactivity into the cell wall, membranous organelles and soluble macromolecules. Fractionation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography, showed that phytohemagglutinin (PHA) was the major fucosylated protein synthesized in the cotyledons. Incorporation of fucose into PHA occurred in the membranous organelle fraction, and the radioactive fucose remained associated with the PHA during a 20-h chase of the radioactivity. Tunicamycin inhibited the incorporation of glucosamine and fucose into PHA to the same extent (65%), indicating the involvement of a lipid intermediate in the incorporation of fucose, or the attachment of fucose to the high-mannose oligosaccharide moiety of newly synthesized PHA. Digestion with proteinase K of [³H]fucose- or [³H]glucosamine-labeled PHA resulted in the formation of glycopeptides of similar size. These glycopeptides were partially resistant to digestion with endo-β-N-acetylglucosaminidase H, even after the removal of fucose by mild acid hydrolysis. We postulate, on the basis of these experiments, that the transport of PHA from the endoplasmic reticulum to the protein bodies is accompanied by the modification of its oligosaccharide side-chain. This modification involves inter alia the attachment of fucose, and renders the oligosaccharide side-chain resistant to digestion with endo-β-N-acetylglucosaminidase H. Analogy with animal glycoproteins indicates that this modification probably occurs in the Golgi apparatus.     

ELECTROPHORETIC CHARACTERIZATION OF LEUCINE-, GLUCOSAMINE- AND FUCOSE-LABELLED PROTEINS RAPIDLY TRANSPORTED IN FROG SCIATIC NERVE

—[³H]Leucine, [³H]glucosamine and [³H]fucose were incorporated in vitro into proteins in frog sciatic ganglia and subsequently transported at a rapid rate along the sciatic nerve towards a ligature, in front of which they accumulated. The synthesis of transported fucose-labelled proteins is closely linked to protein synthesis but is not dependent on RNA synthesis, as judged by effects after incubation for 17 h in the presence of cycloheximide and actinomycin D. Labelled ganglionic as well as transported material were solubilized in sodium dodecyl sulphate and characterized by polyacrylamide gel electrophoresis. The bulk of ganglionic proteins, labelled with any of the precursors used, had molecular weights exceeding 40,000. The radioactivity patterns of leucine- and glucosamine-labelled ganglionic proteins showed similarities with dominant peaks corresponding to molecular weights of about 75,000 and 50,000. The last peak was almost lacking in fucose-labelled ganglionic components. Leucine- and glucosamine labelled-transported proteins exhibited characteristic and similar electrophoretic distributions in contrast to the pattern of fucose-labelled nerve proteins, which was more polydisperse. The most conspicious nerve proteins corresponded to molecular weights of about 75,000 and 18,000. There was a remarkable agreement in the profile of leucine-labelled transported nerve proteins and fucose-labelled ganglionic proteins. In the light of these observations the possibility that glycoproteins constitute a large part of rapidly transported proteins will be discussed.     

Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices.

1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000--150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.     

Axonal transport of newly synthesized glycoproteins in a single identified neuron of Aplysia californica

Increasing amounts of glycoprotein synthesized from L-[³H]fucose injected into the cell body of R2, an identified Aplysia neuron, were found in the right pleuro-abdominal connective. Autoradiography revealed that the glycoproteins were localized in the axon of R2. Glycoproteins appearing in the axon presumably were synthesized in the cell body, since no significant incorporation was observed when [³H]fucose was injected directly into the axon. [³H]glycoproteins were detected in the connective after a delay of 1 h after intrasomatic injection. Thereafter, transport from the cell body was rapid, and by 10 h after injection, 45% of the total neuronal [³H]glycoprotein had appeared in the axon. By analysing the radioactivity in cell body and connective 4, 10, and 15 h after injection, we found that [³H]glycoproteins were transported selectively compared to nonmacromolecular material. Sequential sectioning of the connective revealed that [³H]glycoproteins were transported in discrete waves. The population of membrane-associated [³H]glycoproteins in the axon differed from that in the cell body. Two of the five somatic components appeared to be transported preferentially. In addition a new component appeared in the axon 10 h after injection.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Synthesis and secretion of sulphated glycoproteins by rabbit oviduct explants in vitro

The ability of rabbit oviduct explants to incorporate radiolabelled precursors into specific secretory products was investigated. Ampullary and isthmic oviduct segments were cultured in the presence of [3H]glucosamine or [35S]sodium sulphate. Medium samples were analysed for the presence of secreted, labelled macromolecules. Explants incorporated the [3H]glucosamine and secreted labelled glycoproteins in vitro. SDS gel electrophoresis and subsequent fluorographic analysis of culture medium demonstrated a differential secretion of glycoproteins between the ampulla and the isthmus. Although ampullary tissue secreted a greater amount of labelled glycoproteins during the sampling period, the major secretory constituent of Mr approximately 66,000 was common to both oviduct segments. Tissue incubated with [35S]sodium sulphate also secreted a labelled glycoprotein or subunit of Mr approximately 66,000. The results indicate that rabbit oviduct explants are capable of synthesis and secretion of specific sulphated glycoproteins in vitro and that there is a difference in the type and amount of secretion produced between the two oviduct segments.     

Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Evidence for two catabolic endoglycosidase activities in β-mannosidase-deficient goat fibroblasts

Cultured skin fibroblasts derived from Nubian goats deficient in lysosomal β-mannosidase, which had previously been shown to accumulate storage oligosaccharides with the structures Manβ4GlcNAcβ4GlcNAc and Manβ4GlcNAc (in the ratio of 2.7:1) were evaluated for their ability to catabolize exogenous [³H]GlcN-labelled glycoproteins isolated from the secretions of cultured goat or human fibroblasts. Regardless of the source of exogenous labelled glycoprotein, affected goat fibroblasts took up the labelled glycoprotein from the culture medium and subsequently accumulated the same major labelled oligosaccharide, identified as Manβ4GlcNAcβ4GlcNAc; no such oligosaccharide accumulated in normal goat fibroblasts under the same conditions. Tunicamycin-treated affected fibroblasts also took up labelled exogenous glycoprotein and accumulated labelled storage trisaccharide, further suggesting the direct accumulation of storage trisaccharide from impaired glycoprotein-associated oligosaccharide catabolism. Treatment of metabolically labelled affected fibroblasts with leupeptin, an inhibitor of lysosomal cathepsins, resulted in the 2- to 6-fold inhibition of trisaccharide accumulation, while having little effect on the uptake of [³H]GlcN or the accumulation of labelled disaccharide. The results are most consistent with the presence of two endoglycosidases, an endo-β-N-acetylglucosaminidase and an endo-aspartylglucosaminidase, in goat fibroblasts. These two activities, rather than heterogeneous core oligosaccharide structures, are responsible for the ultimate accumulation of storage oligosaccharides with one and two GlcNAc residues at their reducing terminus.     

Effects of prostaglandin E1 on the metabolism in rat parathyroid gland in vitro

We investigated some effects of prostaglandin E₁ on the metabolism of rat parathyroid glands using a culture system containing basal Eagle's medium supplemented with 5–10% heat-inactivated rat serum. Rat parathyroid glands incorporate [³H]fucose and ¹⁴C-labeled amino acids into cellular glycoproteins and secrete some of these into the culture medium. Gel filtration chromatography separates these glycoproteins into three classes, the smallest of which (peak 3) is secreted with immunoreactive parathyroid hormone. In cultures of 48 h, prostaglandin E₁ (1 μg/ml) specifically inhibits the secretion of peak 3 and of parathyroid hormone but has no effect on the incorporation of [³H]-fucose, ¹⁴C-labeled amino acids, or [³H]uridine into parathyroid glands. Cytochalasin B inhibits the secretion of parathyroid hormone and the incroporation of isotopic fucose and amino acids. Cortisol stimulates incorporation of [³H]fucose and the secretion of parathyroid hormone even in the presence of inhibitory doses of prostaglandin E₁. It is concluded that, in organ culture, prostaglandin E₁ inhibits the secretion of parathyroid hormone and of a specific glycoprotein the function of which may be related to the secretion of the hormone.     

Surface-membrane biogenesis in rat intestinal epithelial cells at different stages of maturation.

The biosynthesis of membrane proteins and glycoproteins has been studied in rat intestinal crypt and villus cells by measuring the incorporation of L-[5,6-3H] fucose, D-[2-3H] mannose and L-[3,4,5-3H] leucine, given intraperitoneally, into Golgi, lateral-basal and luminal membranes. Incorporation of leucine and mannose was approximately equal in crypt and villus cells, whereas fucose incorporation was markedly higher (3-4 times) in the differentiated villus cells. As previously reported [Quaroni, Kirsch & Weiser (1979) Biochem J. 182. 203-212] most of the fucosylated glyco-proteins synthesized in the villus cells and initially present in the Golgi and lateral-basal membranes were found re-distributed, within 3-4h of label administration, in the luminal membrane. A similar process appeared to occur in the crypt cells, where, however, only few fucose-labelled glycoproteins were identified. In contrast, most of the leucine-labelled and many mannose-labelled membrane components found in the lateral-basal membrane of both crypt and villus cells did not seen to undergo a similar re-distribution process. The fucosylated glycoproteins of the intestinal epithelial cells represent, therefore, a special class of membrane components, most of which appear with differentiation, that are selectively localized in the luminal portion of the plasmalemma. In contrast with the marked differences in protein and glycoprotein patterns between the luminal membrane of villus and crypt cells, only minor differences were found between their lateral-basal membrane components: their protein patterns on sodium dodecyl sulphate/polyacrylamide slab gels, and the patterns of fucose-, mannose- and leucine-labelled components (analysed 3-4h after label administration) were very similar. Although the minor differences detected may be of importance, it appears that most of the surface-membrane changes accompanying cell differentiation in the intestinal epithelial cells are localized in the luminal portion of their surface membrane.     

[3 5S]Sulfate incorporation into myelin glycoproteins I. Central nervous system

The in vivo incorporation of [^{3 5}S]sulfate and [³H]fucose into rat brain myelin was investigated. Most of the ^{3 5}S in the myelin was in sulfatide, but about 4% was associated with the residual proteins after chloroform/methanol extraction. Polyacrylamide gel electrophoresis of these proteins indicated that the major ^{3 5}S-labeled component corresponded to the major fucose-labeled glycoprotein. The labeling of this predominant glycoprotein with sulfate was more selective than with fucose, since there was relatively little incorporation of sulfate into some of the minor fucose-labeled glycoproteins. There was little or no ^{3 5}S associated with proteolipid or basic protein on polyacrylamide gels. The fucose-labeled glycoproteins were converted to glycopeptides by pronase digestion and separated into two major classes by gel filtration on Sephadex-G50. Only the higher molecular weight class contained significant amounts of ^{3 5}S. The association of ^{3 5}S with the glycopeptides was not due to binding of sulfatide or free inorganic sulfate. The results indicate that the predominant myelin-associated glycoprotein in rat brain is sulfated.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.