N-linked glycoprotein biosynthesis in the developing mouse embryo

We have developed microenzymic assays that have, for the first time, enabled analysis of several enzymes in the pathway for N-linked glycoprotein biosynthesis in pre- and peri-implantation mouse embryos. The in vitro activities of the glycosyl transferases responsible for the formation of N-acetylglucosaminylpyrophosphoryldolichol,N, N'-diacetyl-chitobiosylpyrophosphoryldolichol, mannosylphosphoryldolichol, and glucosylphosphoryldolichol were found to decrease after fertilization before increasing significantly at the blastocyst stage, a stage that was also found to be highly sensitive to the glycosylation inhibitor, tunicamycin. The observed elevation in the activities of these enzymes in blastocysts still occurred when ebbryos were cultured in alpha-amanitin, indicating that de novo mRNA synthesis is unnecessary for the observed increase in their activities. Thus, an elevated capacity for N-glycosylation exists at the blastocyst stage, a time when dramatic increases in cell-cell interactions are known to occur.     

A single enzyme catalyzes the synthesis of the mannosylphosphoryl derivative of dolichol and retinol in rat liver and Chinese hamster ovary cells

It is well established that mannosylphosphoryldolichol participates in the synthesis of N-linked glycoproteins by donating mannosyl residues to oligosaccharide-lipid intermediates. It has been suggested that mannosylphosphorylretinol also is involved in glycoprotein biosynthesis. We conclude that one synthase catalyzes the synthesis of both mannosylphosphoryldolichol and mannosylphosphorylretinol in rat liver tissue and Chinese hamster ovary cells, based on the following results. 1) The enzyme in rat liver microsomes that synthesizes mannosylphosphoryldolichol and mannosylphosphorylretinol is inactivated at the same rate at 55 degrees C. 2) In membranes of both rat liver and Chinese hamster ovary cells, exogenous dolichyl phosphate and retinyl phosphate compete with each other for mannosyl-lipid synthesis. However, in both systems adding exogenous retinyl phosphate has no effect on the synthesis of mannosylphosphoryldolichol from endogenous dolichyl phosphate in the membranes. 3) Membranes prepared from a mutant of Chinese hamster ovary cells which is devoid of mannosylphosphoryldolichol synthase lack the ability to synthesize mannosylphosphorylretinol.     

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

Glycoprotein synthesis and embryonic development

One of the most striking morphogenetic events during embryonic development is gastrulation, a process that leads to formation of the primitive gut. Using sea urchin embryos, we have studied the synthesis and function of glycoproteins during gastrulation. These studies have revealed that at least three processes are induced prior to gastrulation: de novo synthesis of dolichol; phosphorylation of dolichol by dolichol kinase, which may catalyze the final step in the de novo pathway; and initiation of the synthesis of N-linked glycoproteins. Whether or not activation of the glycosylation process results merely because of the production of dolichyl monophosphate or because, in addition, proteins containing glycosylatable-Asn-X-Ser/Thr-sequences are first translated just prior to gastrulation, is currently being investigated.     

Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell-Specific Antigen and Its Biological Roles

Primary mesenchyme cell (PMC)-specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al. , 1987) P4 reacted with the N-linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius , P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate-deficient sea water were immuno-reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4-reactive glycoproteins may play important roles in cell-substrate interaction and spicule formation.     

Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed.     

Structure of a major yolk glycoprotein and its processing pathway by limited proteolysis are conserved in echinoids

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, Strongylocentrotus purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein found in the embryo was prepared. Immunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa yolk glycoprotein of the egg during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified from S. purpuratus and analyzed by limited proteolysis and peptide mapping. This analysis revealed that these glycoproteins were cleavage products derived from the major yolk glycoprotein. The antibody to the 90-kDa glycoprotein in S. purpuratus embryos was used to identify a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: Arbacia punctulata, Lytechinus pictus, and Dendraster excentricus. However, eggs from other echinoderm classes and from Xenopus laevis, Drosophila melanogaster, and the chicken did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed. Comparison of the physical and chemical properties of these glycoproteins revealed striking similarities in pI and in amino acid and monosaccharide composition. The results of peptide mapping also supported the conclusion that the 160- to 170-kDa glycoproteins from the four echinoids are structurally homologous glycoproteins containing N-linked polymannose chains. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout development, and that externalization of the 160-kDa glycoprotein or its cleavage products was not detectable.     

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.     

Galactosyl- and fucosyltransferases in etiolated pea epicotyls: product identification and sub-cellular localisation

Particulate membrane preparations from etiolated pea epicotyls were found to contain fucosyltransferases, which transferred fucose from GDP-fucose onto xyloglucan and N-linked glycoprotein, and galactosyltransferases, which transferred galactose from UDP-galactose onto galactan, xyloglucan, and N-linked glycoprotein. The products were characterised by specific enzyme degradation and by acid and alkaline hydrolysis. All the enzymes were found to be concentrated in the Golgi apparatus. The Golgi apparatus was further fractionated into membranes of low, medium and high-density. The glycoprotein fucosyltransferase activity was present in highest amounts in the medium-density Golgi membranes, while the majority of the xyloglucan fucosyltransferase was present in the low-density Golgi membranes. The majority of the galactan galactosyltransferase (galactan synthase) was found in the low-density membranes, while the glycoprotein galactosyltransferase was equally distributed in all three subfractions.     

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.     

Glycoprotein expression in mouse cerebellum: effects of inhibitors of N-linked glycosylation.

1. Explants of cerebellum from foetal mouse were cultured in vitro for 10 days in the presence of one of 4 inhibitors of N-linked glycosylation (castanospermine, deoxymannojirimycin, swainsonine, and tunicamycin). 2. The effects of the inhibitors were compared with respect to: (a) the activity of enzymes involved in glycoprotein biosynthesis and degradation; (b) the expression of N-linked glycoproteins; (c) the morphology and ultrastructure of the treated cerebellar explants.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

Biosynthesis of N-glycosidically linked glycoproteins during gastrulation of sea urchin embryos.

Embryos of the sea urchin, Stronglyocentrotus purpuratus, synthesize several classes of sulfated and non-sulfated glycoproteins during gastrulation. The antibiotic tunicamycin, which is a specific inhibitor of the N-glycosylation of proteins, inhibits the synthesis of lipid-linked oligosaccharides in these embryos at concentrations which have little effect on the biosynthesis of other classes of glycolipids or on protein synthesis. As a consequence of this inhibition, glycoproteins with oligosaccharide side chains of the general type (Man)5-7-(GlcNAc)2 are not synthesized. In addition, the biosynthesis of a novel class of sulfated glycoproteins is inhibited. In contrast, no effect upon the synthesis of sulfated glycosaminoglycans is seen. The morphogenetic consequence of tunicamycin treatment is that development of embryos from the mesenchyme blastula to the gastrula stage is arrested. The results provide evidence that during development glycoproteins containing both unsulfated and sulfated N-glycosidically linked oligosaccharide chains are synthesized via the lipid-linked pathway. The biosynthesis of these molecules appears to be a prerequisite to the differentiation and morphogenesis that occurs during gastrulation.     

beta-Adrenergic activation of glycosyltransferases in the dolichylmonophosphate-linked pathway of protein N-glycosylation

beta-Adrenoreceptor stimulation of rat parotid acinar cells increases the activity of several microsomal membrane associated, dolichylmonophosphate (Dol-P) linked glycosyltransferases. The activities of Man-P-Dol synthase and Glc-P-Dol synthase are increased by approximately 50%, and the activity of N-acetylglucosaminyl 1-phosphate transferase plus N-acetylglucosaminyl transferase increased by approximately 60%, after agonist treatment. Increases in enzyme activity are (i) independent of endogenous Dol-P levels and (ii) observed under conditions in which the specific activities of donor sugar nucleotides are kept constant. Activation of these enzymes is specific since comparable levels of NADPH-cytochrome c reductase are found in control and agonist-treated membranes. The data thus provide the initial demonstration of neurotransmitter modulation of enzymes in the dolichol-linked pathway of protein N-glycosylation.     

Enhancement of therapeutic protein in vivo activities through glycoengineering

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).     

The N-X-S/T consensus sequence is required but not sufficient for bacterial N-linked protein glycosylation

In the Gram-negative bacterium Campylobacter jejuni there is a pgl (protein glycosylation) locus-dependent general N-glycosylation system of proteins. One of the proteins encoded by pgl locus, PglB, a homolog of the eukaryotic oligosaccharyltransferase component Stt3p, is proposed to function as an oligosaccharyltransferase in this prokaryotic system. The sequence requirements of the acceptor polypeptide for N-glycosylation were analyzed by reverse genetics using the reconstituted glycosylation of the model protein AcrA in Escherichia coli. As in eukaryotes, the N-X-S/T sequon is an essential but not a sufficient determinant for N-linked protein glycosylation. This conclusion was supported by the analysis of a novel C. jejuni glycoprotein, HisJ. Export of the polypeptide to the periplasm was required for glycosylation. Our data support the hypothesis that eukaryotic and bacterial N-linked protein glycosylation are homologous processes.     

Gastrulation in the sea urchin Strongylocentrotus purpuratus is blocked by the fluorescein dye erythrosin B

Erythrosin B, a food, drug, and cosmetic dye, arrested gastrulation in embryos of the sea urchin Strongylocentrotus purpuratus. A 30 min pulse of 5 microM erythrosin B added at 18 hr postinsemination blocked gastrulation scored at 50 hr postinsemination when control embryos had completed gastrulation. Dye addition at later times had no detectable effects on development through 50 hr postinsemination. The dye may block primary invagination via its known effects on plasma membrane permeability and fluidity.     

Glycoprotein synthesis in Drosophila Kc cells. Biosynthesis of dolichol-linked saccharides

The biosynthesis of dolichol and dolichol-linked saccharide intermediates in glycoprotein synthesis was studied in an embryonic Drosophila cell line (Kc) that lacks the squalene-cholesterol branch of the polyisoprenoid biosynthetic pathway. Kc cells were labeled with [5-3H]mevalonic acid and the radioactive lipids formed were analyzed. Although the major labeled product was coenzyme Q, dolichol and a variety of dolichol derivatives could be readily detected. On the basis of their chromatographic and chemical properties, these derivatives were identified as dolichyl phosphate, glucosylphosphoryldolichol, mannosylphosphoryldolichol, and oligosaccharylpyrophosphoryldolichol. Both short term (4-h) and steady state (4-day) labeling experiments with mevalonate, rather than sugars as previously used, were performed to assess the level of these intermediates. The results of these studies, using a precursor common to all the intermediates, reveal that the early intermediates, N-acetylglucosaminylpyrophosphoryldolichol and N,N'-diacetylchitobiosylpyrophosphoryldolichol, are present at very low levels (less than 5%) relative to the other intermediates on the pathway to oligosaccharylpyrophosphoryldolichol. The total amount of dolichol intermediates remained essentially constant during the chase phase of pulse-chase experiments, indicating the absence of a major catabolic pathway for the polyisoprenoid backbone. As expected, however, the sugar moiety, studied with mannosylphosphoryldolichol, underwent rapid turnover. These results are discussed in the context of our current understanding of the pathway whereby dolichol derivatives participate in glycoprotein synthesis.