Disruption of O-GlcNAc Cycling in C. elegans Perturbs Nucleotide Sugar Pools and Complex Glycans.

The carbohydrate modification of serine and threonine residues with O-linked beta- N-acetylglucosamine (O-GlcNAc) is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C. elegans lacking O-GlcNAc transferase and O-GlcNAcase activities. In ogt-1 null animals, steady-state levels of UDP-GlcNAc/UDP-GalNAc and UDP-glucose were substantially elevated. Transcripts of genes encoding for key members in the HBP (gfat-2, gna-2, C36A4.4) and trehalose metabolism (tre-1, tre-2, tps-2) were elevated in ogt-1 null animals. While there is no evidence to suggest changes in the profile of N-linked glycans in the ogt-1 and oga-1 mutants, glycans insensitive to PNGase digestion (including O-linked glycans, glycolipids, and glycopolymers) were altered in these strains. Our data support that changes in O-GlcNAcylation alters nucleotide sugar production, overall glycan composition, and transcription of genes encoding glycan processing enzymes. These data along with our previous findings that disruption in O-GlcNAc cycling alters macronutrient storage underscores the noteworthy influence this posttranslational modification plays in nutrient sensing.

Submicrometer fiber-optic Fabry-Perot interferometer formed by use of the Langmuir-Blodgett technique.

The fabrication of an optical cavity at the distal end of an optical fiber has been achieved by Langmuir-Blodgett (LB) deposition of tricosanoic acid. This technique allows nanometer-scale control over the cavity length to a total thickness of ~0.5 microm . The cavity has been shown to act interferometrically and, thus, has potential sensing applications.

Second-harmonic generation in Langmuir Blodgett waveguide overlays on single-mode optical fiber.

Second-harmonic radiation has been obtained from Langmuir-Blodgett films of E-N -octadecyl-4-[2-(4-dibutylaminophenyl)ethenyl]quinolinium octadecylsulfate, deposited as a waveguide overlay upon optical fiber that is single mode at the pump wavelength (lambda=1064 nm) . A quadratic relationship between the pump power and second-harmonic intensity was observed.

Fiber-optic chemical sensing with langmuir-blodgett overlay waveguides.

Fiber-optic chemical sensing has been demonstrated with a side-polished single-mode optical fiber, evanescently coupled to chemically sensitive Langmuir-Blodgett (LB) overlay waveguides. The sensors exhibit a channel-dropping response centered on a wavelength that is dependent on the thickness and the refractive index of the overlay waveguide. It has been shown that pH-sensitive organic dyes proved to be suitable materials for the formation of an overlay waveguide whereas LB deposition provides the required thickness control. A theoretical model of the sensor response, based on the Kramers-Kronig relations and phase matching of the guided modes within the optical fiber and overlay waveguide, shows good agreement with experimental results.

Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin.

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.

pH sensor using Langmuir-Blodgett overlays on polished optical fibers.

Evanescent coupling between a side-polished single-mode optical fiber and a single-mode, pH-sensitive Langmuir-Blodgett overlay is used to demonstrate an intrinsic fiber-optic pH sensor. The sensor shows a wavelength sensitivity of 18.8 +/- 0.8 nm/pH and a transmission sensitivity of 9.7 +/- 0.8 dB/pH when operating at 750 nm.

The major subunit of the asialoglycoprotein receptor is expressed on the hepatocellular surface in mice lacking the minor receptor subunit.

The mammalian asialoglycoprotein receptor (ASGPR) is located on the sinusoidal membrane of hepatocytes where it binds and endocytoses galactose-terminated glycoproteins (asialoglycoproteins). ASGPR is composed of two highly homologous subunits, termed hepatic lectin 1 and 2. Despite numerous studies the contribution of both subunits to biosynthesis and functional activity of ASGPR in vivo has remained controversial. Mice lacking the murine hepatic lectin (MHL)-2 subunit are viable and fertile without obvious phenotypic abnormalities. In the absence of MHL-2, knockout mice express MHL-1 protein at reduced levels. Here, we examine the intracellular fate and function of this remaining subunit. The results show that MHL-1 reaches the hepatocellular surface in knockout mice but is unable to effectively remove any one of three different radiolabeled ligands within 30 min. A small but detectable residual ligand clearance in knockout mice at 4 h is apparently not mediated by remaining MHL-1. Serum concentrations of galactose-terminating glycoproteins are not elevated in these ASGPR-deficient mice. However, competitive in vitro degradation experiments suggest that other endogenous ASGPR ligands, the nature of which remain to be determined, accumulate in serum of knockout animals.

Architecture of the yeast cell wall. The linkage between chitin and beta(1-->3)-glucan.

To isolate the putative linkage region between chitin and beta(1-->3)-glucan, Saccharomyces cerevisiae cell walls were digested with beta(1-->3)-endoglucanase and the reducing ends of the enzyme-resistant glucose chain stubs were labeled by reduction with borotritide. The radioactive material was further digested with exochitinase to remove the bulk of the chitin, and the liberated oligosaccharides were fractionated on a sizing column. A single peak (compound I) was found to consist of N-acetylglucosamine, glucose, and glucitol residues in the ratio 1:2:1. By digestion with beta-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing terminus, linked to laminaritriitol by a beta(1-->4) bond. Five additional oligosaccharides were recovered, two being analogs of compound I, with 1 or 3 glucose units, respectively; the remaining three were shown to be the reduced analogs of laminaribiose, laminaritriose, and laminaritetraose. The presence of N-acetylglucosamine-containing oligosaccharides arises from the activity of chitinase in cleaving 2 sugar units sequentially in those chains containing an odd number of N-acetylglucosamine residues; correspondingly, oligosaccharides containing only glucose and sorbitol derived from even-numbered chitin chains, a result implying that chitinase can hydrolyze the linkage between N-acetylglucosamine and glucose. It is concluded that the terminal reducing residue of a chitin chain is attached to the nonreducing end of a beta(1-->3)-glucan chain by a beta(1-->4) linkage. Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chitin synthetase 3. The timing and possible mechanism of formation of the chitin-glucan linkage is discussed.

alpha 2-8 Sialic acid polymers: size, structure, and compositional analysis.

A series of three variable assay procedures is described to provide overlapping information on the size, structure, and composition of the alpha 2-8 linked polysialic acid chains present on a wide variety of critical cell surface glycoproteins. Technical advances in instrumentation have permitted the development of new applications for a methodology involving the sequential use of periodate and borohydride to modify terminal sialic acid residues. The procedures described here provide a rapid and facile assay for (a) the determination of polysialic acid chain length, (b) the simultaneous identification of N-acetylneuraminic acid, N-glycolyneuraminic acid, and KDN (deaminated sialic acid) when present in a single preparation, (c) the ability to distinguish qualitatively between reducing and nonreducing polymers, and (d) the ability to determine the number of chains bound to a glycoprotein of known molecular weight.

Modulation of the asialoglycoprotein receptor in human hepatoma cells: effect of glucose.

The hepatic receptor for asialoglycoproteins was found to be modulated by the glucose concentration in the medium of the human hepatoma cell line HepG2. The surface binding of asialoorosomucoid, a well-documented ligand for this receptor, increased from 20 ng/mg of cellular protein to about 40 ng/mg as the glucose concentration was increased from 10 to 50 mg/dl. The up-modulating effect of glucose was mimicked by pyruvate, a product of glucose metabolism, and abolished by both 2-deoxyglucose, an inhibitor of glucose metabolism, and by cycloheximide, an inhibitor of protein synthesis. Scatchard plot analysis indicated a rise in the number of binding sites and a twofold increase in binding affinity. In contrast, the binding of antibody remained unchanged with respect to alterations in glucose concentration, an indication that the actual number of receptors remained constant in face of an increased number of binding sites. Specificity of the glucose effect was shown by the binding of insulin and transferrin to their respective receptors, which was unaffected by the high glucose concentration that increased asialoorosomucoid binding. The repression of receptor binding seen with cells grown in biotin-deprived medium was reversed by increasing the glucose concentration of the medium. In this case, binding was restored to a level sixfold to sevenfold higher than that of the control cells grown in dialyzed serum. The stimulatory effect of glucose was shown to be independent of and significantly greater than that of cyclic GMP, a known regulator of receptor expression of biotin-deficient HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin regulation of sialic acid expression in rat thyroid cells.

The present study, utilizing the hormone-responsive rat thyroid cell line FRTL-5, presents evidence establishing the regulatory role of thyrotropin in modulating mRNA for beta-galactoside alpha 2,6-sialytransferase, the enzyme responsible for the expression of alpha 2,6-linked sialic acid. Both the cell surface membrane and the thyroglobulin secreted by cells grown in the presence of this hormone exhibit a marked decrease in the level of alpha 2,6-bound sialic acid with little or no change in the number of alpha 2,3-sialic acid residues. An additional, and unexpected, sequel is the finding of a coordinated decrease in all of the core monosaccharide constituents of the secreted thyroglobulin. Both of the above phenomenological changes appear to be at some variance with previously described systems wherein thyrotropin was deemed to increase glycosylation. It is anticipated that further resolution of this apparent difference may provide a clearer definition for the role of the carbohydrate moiety in affecting the biological function of thyroglobulin.

Tritiated borohydride reduction of carbohydrates: reduction of individual monosaccharides, alone or in groups, results in highly divergent specific activities.

Reduction of carbohydrates by tritiated borohydride resulted in the production of alditols or glycosides with characteristically divergent specific radioactivities. Simultaneous reduction of individual sugars in the presence of a reference standard, talose, permitted the assignment of a unique specific radioactivity with respect to talitol as 100%. A variety of structures was examined, including neutral hexoses, free and acetylated aminosugars, ketohexoses and glycosides containing a fixed pyranose ring adjacent to a carbonyl group. In the latter case, the resulting steric hindrance severely restricted the incorporation of tritium. In both of the ketohexoses tested, the minor product of the two epimeric alditols exhibited the higher specific radioactivity. In all cases, reduction produced a characteristic and reproducible specific activity in which the values varied from 51 to 182% of that found for talose. These results are interpreted on the basis of generalizations concerning mechanism and predictive value.

Hyposialylated thyroglobulin in a patient with congenital goiter and hypothyroidism.

A large family (14 children) with congenital goiter whose parents are first cousins was studied. Thyroid tissue was obtained, after 125I in vivo labeling, from one of the siblings (JBM). Gel filtration of thyroid proteins indicated that thyroglobulin (Tg) eluted as a single symmetrical peak in the same position as authentic 19S Tg. Gel electrophoresis in a 7.5% sodium dodecyl sulfate-polyacrylamide gel revealed a major band with the same mobility and immunoreactivity as normal 19S Tg. Hydrolysis of the patient's Tg indicated that most of the radioactivity was mono- and diiodotyrosines. The yield of T4 from JBM Tg (26 pmol/mg protein) was 5-fold less than normal thyroid tissue (140 pmol/mg protein) and approximately half of that in thyroid tissue from endemic goiter (51 pmol/mg). Total T3 released from JBM Tg was similar to the other two tissues. When the carbohydrate content of normal and patient Tg was analyzed, there was no differences in glucosamine, galactose or mannose content. However, unlike normal and endemic-goiter Tg, that had a mean sialic acid content of 7.3 and 5.6 micrograms/mg protein, respectively, the sialic acid concentration of the patients Tg was only 0.3 microgram/mg. Sialyltransferase activity was readily demonstrated in homogenate from normal thyroid or endemic goiter, but no sialyltransferase activity was detectable in a homogenate of JBM-thyroid tissue. We conclude that the finding of severely hyposialylated Tg is linked to a defect in iodotyrosine coupling seen in this patient with a possibly abnormal migration of Tg into the follicular lumen.

Sialic acid metabolism in sialuria fibroblasts.

Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid (N-acetylneuraminic acid, NeuAc). Fibroblasts cultured from the three known cases of sialuria contained 70-200-fold increases in soluble sialic acid, but normal concentrations of bound sialic acid. The sialic acid appeared in the cytosolic fraction of the cells on differential centrifugation, and was susceptible to borohydride reduction, suggesting that accumulated sialic acid was in the form of NeuAc and not CMP-NeuAc. In biochemical studies, CMP-NeuAc (50 microM) inhibited the UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase of normal fibroblasts by 84-100%, but inhibited the epimerase from sialuria cells by only 19-31%. Feeding sialuria cells up to 5 mM D-glucosamine for 72 h increased free sialic acid content 20-60%, but normal cells were unaffected by this treatment. Cytidine feeding (5 mM, 72 h) reduced the NeuAc content of sialuria cells, initially 112, 104, and 266 nmol/mg protein, by 63-71 nmol/mg protein; CMP-NeuAc concentrations, initially 4, 2, and 5 nmol/mg protein, increased by 14-33 nmol/mg protein. Consequently, the total cellular content of soluble sialic acid (NeuAc + CMP-NeuAc) was lowered 14-46% by cytidine feeding. The inheritance pattern of sialuria has not been determined. However, cells from both parents of one sialuria patient contained normal concentrations of free sialic acid, and the parental epimerase activity also responded normally to CMP-NeuAc. We conclude that the basic biochemical defect in all known cases of sialuria is a failure of CMP-NeuAc to feedback-inhibit UDP-GlcNAc 2-epimerase and cytidine feeding can lower the intracellular soluble sialic acid concentration of sialuria cells.

Overexpression and biosynthesis of CD4 in Chinese hamster ovary cells: coamplification using the multiple drug resistance gene.

CD4 is a T-cell surface glycoprotein and serves as the receptor for the human immunodeficiency virus. Glycosylation of CD4 has been shown to be necessary for proper surface expression. To study the biosynthesis and assembly of CD4, wild-type and glycosylation-deficient mutant Chinese hamster ovary (CHO) cells were cotransfected with a cDNA encoding CD4 and a cDNA for the human multiple drug resistance gene, which allowed the amplification of the transfected CD4 cDNA sequences. Clones were isolated that exhibited high-level expression of CD4 resulting from the integration of several copies of CD4 cDNA. CD4 synthesized by these cells acquired resistance to endoglycosidase H after 20-30 min of chase, suggesting a rapid translocation of the glycoprotein from the rough endoplasmic reticulum to the medial Golgi apparatus. The sensitivity of CD4 to glycosidases suggested the presence of biantennary unsialylated complex-type oligosaccharides. Consistent with this, CD4 synthesized by the Lec2 mutant, which does not add sialic acid to oligosaccharides, was identical to the glycoprotein produced by wild-type CHO cells. The amplification strategy used to express CD4 at high levels in wild-type and mutant CHO cells will have general utility.

Identification of the metabolic defect in sialuria.

Sialuria is a rare inborn error of metabolism, the hallmarks of which are moderate developmental retardation, coarse facial features, and an enormous amount of free N-acetylneuraminic acid (sialic acid) in the urine. Until now, the basic biochemical defect in this disorder has remained uncertain. In this report, the activity of the rate-limiting enzyme in the biosynthesis of sialic acid has been measured directly in whole cell lysates by a highly sensitive assay. With this technique, the basic defect in sialuria has been identified unequivocally as the loss of feedback control of uridine diphosphate N-acetylglucosamine 2-epimerase by cytidine monophosphate N-acetylneuraminic acid with resultant overproduction of sialic acid.

Ligand-induced modulation of the hepatic receptor for asialoglycoproteins. Evidence for the role of cell surface hyposialylation.

In a previous paper, we reported a ligand-induced modulation of the asialoglycoprotein receptors on HepG2 cells whereby these receptors were rendered functionally inert while remaining immunologically intact on the plasma membrane. At that time, it was speculated that the loss in receptor-binding ability was a direct result of a concomitant decrease in cell surface sialic acid residues. Experiments designed to test that hypothesis are presented. The results revealed: 1) an identical response in binding activity and sialic acid content in cells subjected to minimal exposure to neuraminidase; 2) a parallel and synchronous recovery of both parameters following modulation; 3) an invariant binding of high affinity ligands, and 4) the ability of galactose oxidase to restore, at least partially, the cell's ability to bind asialoglycoprotein. These results indicate that ligand-induced surface hyposialylation directly diminishes expression of the asialoglycoprotein receptor.

Multiple post-transcriptional regulatory mechanisms in ferritin gene expression.

We have investigated the mechanisms involved in the regulation of ferritin biosynthesis in K562 human erythroleukemia cells during prolonged exposure to iron. We show that, upon addition of hemin (an efficient iron donor) to the cell culture, the rate of ferritin biosynthesis reaches a maximum after a few hours and then decreases. During a 24-hr incubation with the iron donor the concentrations of total ferritin heavy (H) and light (L) subunit mRNAs rise 2- to 5-fold and 2- to 3-fold, respectively, over the control values, while the amount of the protein increases 10- to 30-fold. The hemin-induced increment in ferritin subunit mRNA is not prevented by deferoxamine, suggesting that it is not directly mediated by chelatable iron. In vitro nuclear transcription analyses performed on nuclei isolated from control cells and cells grown in the presence of hemin indicate that the rates of synthesis of H- and L-subunit mRNAs remain constant. We conclude that iron-induced ferritin biosynthesis is governed by multiple post-transcriptional regulatory mechanisms. We propose that exposure of cells to iron leads to stabilization of ferritin mRNAs, in addition to activation and translation of stored H- and L-subunit mRNAs.