Gain-of-function Chinese hamster ovary mutants LEC18 and LEC14 each express a novel N-acetylglucosaminyltransferase activity

LEC18 and LEC14 cells are gain-of-function glycosylation mutants isolated from Chinese hamster ovary cells for resistance to pea lectin. Structural studies have shown that LEC18 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-6 position of the core GlcNAc (Raju, T. S., Ray, M. K., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302), whereas LEC14 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-2 position of the core beta-linked Man residue (Raju, T. S., and Stanley, P. (1996) J. Biol. Chem. 271, 7484-7493). Both modifications are novel and have not been reported in glycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct N-acetylglucosaminyltransferase (GlcNAc-T) activity. The LEC18 activity, termed GlcNAc-TVIII, transfers GlcNAc to GlcNAcbeta1-O-pNP and to a GlcNAc-terminating, biantennary, complex N-glycan, with or without a core fucose. By contrast, the LEC14 transferase, termed GlcNAc-TVII, does not have significant activity with simple acceptors, and transfers GlcNAc preferentially to a GlcNAc-terminating biantennary glycopeptide that contains a core fucose residue. The acceptor specificities and other biochemical properties of GlcNAc-TVII and GlcNAc-TVIII differ from previously characterized GlcNAc-transferases including GlcNAc-TIII, indicating that they represent new members of the mammalian GlcNAc-T group of transferases.     

Alpha 1,6-linked fucose affects the expression and stability of polysialic acid-carrying glycoproteins in Chinese hamster ovary cells

To determine the effect of alpha1,6-linked fucose modification of N-glycans on the expression of polysialic acids (PSAs), the expression of PSAs in a fucose-lacking mutant of Chinese hamster ovary (CHO) cells, Lec13, was compared with that in CHO K1 cells. PSA synthase activity in these cells and the antennary structures of N-glycans associated with the neural adhesion molecule (NCAM), which is a major PSA-carrying glycoprotein, did not differ between the two types of cells. Metabolic labeling of cells with [3H]glucosamine for 48 h followed by immunoprecipitation with anti-PSA monoclonal antibodies revealed that the amount of labeled PSA-carrying glycoproteins obtained from Lec13 cells was 10-times less than that from K1 cells, although the incorporation of [3H]glucosamine into total extracts and NCAM was almost the same. In contrast, when cells were pulse labeled with [35S]methionine followed by a 1 h chase, there was not such a great difference in PSA-carrying protein synthesis between K1 and Lec13 cells. However, during a prolonged chase period, PSA-carrying proteins rapidly decreased in Lec13 cells, whereas those in K1 cells did not change. The degradation of PSA-carrying glycoproteins in Lec13 cells was partly prevented when the cells were grown in fucose-containing medium. Therefore, fucose modification of core N-glycans may affect the efficient expression of PSAs through the intracellular stability of PSA-carrying glycoproteins.     

Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry

The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subjected to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and the 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.     

N-linked glycosylation of the ligand-binding domain of the human urokinase receptor contributes to the affinity for its ligand

Variations in glycosylation exist among urokinase plasminogen activator receptors (u-PARs) from different cell types. We have studied the functional role of N-linked carbohydrate within the ligand-binding domain of u-PAR. Treatment with glycosidases demonstrated that all the N-linked carbohydrates on u-PAR are complex-type oligosaccharides. Substitution of a single Asn (Asn52) to Gln by means of site-directed mutagenesis led to an active receptor mutant with a ligand-binding domain devoid of carbohydrate. The cellular distribution, the glycosyl-phosphatidylinositol anchoring, and the conformational stability after solubilization were unaffected by this single substitution. However, ligand binding analysis demonstrated a 4- 5-fold decrease in affinity as compared with the wild type receptor. Two different strategies were used in order to obtain a u-PAR type completely devoid of N-linked carbohydrates. 1) Tunicamycin treatment of wild type u-PAR-expressing cells. 2) Mutation of all glycosylation sites (Hu-PARN5-mut). In neither case, unglycosylated receptors with ligand binding activity were identified. However, immunofluorescence studies demonstrated that the Hu-PARN5-mut was retained inside the cells in the endoplasmic reticulum. The same result was found for Hu-PARN4-mut, where only the glycosylation sites outside the binding domain were mutated. These results demonstrate that some extent of glycosylation of u-PAR is necessary for cellular transport and for molecular maturation events leading to ligand binding activity. Glycosylation of the binding domain per se affects only the affinity of the receptor. The positive modulation of the Asn52 carbohydrate side chain on ligand affinity suggests that the u-PAR glycosylation variants observed in various cell types may have different functional roles.     

Role of asparagine-linked oligosaccharides in protein folding, membrane targeting, and thyrotropin and autoantibody binding of the human thyrotropin receptor

The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.     

LEC18, a dominant Chinese hamster ovary glycosylation mutant synthesizes N-linked carbohydrates with a novel core structure

The dominant Chinese hamster ovary cell glycosylation mutant, LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells express altered cell surface carbohydrates with markedly reduced binding to 125I-PSA and increased binding to 125I-labeled Datura stramonium agglutinin (DSA) compared with parental cells. Desialylated [3H]Glc-labeled LEC18 cellular glycopeptides that did not bind to concanavalin A-Sepharose exhibited an increased proportion of species that were bound to DSA-agarose. Most of these glycopeptides bound to ricin-agarose and were unique to LEC18 cells. This fraction was purified from approximately 10(10) cells and shown by 1H NMR spectroscopy and methylation linkage analysis to contain novel N-linked structures. Digestion of these glycopeptides with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases gave core glycopeptides that, in contrast to cores from parental cells, were mainly not bound to concanavalin A-Sepharose or to PSA-agarose. 1H NMR spectroscopy, matrix-assisted laser desorption ionization/time of flight mass spectrometry, electrospray mass spectrometry, and collision-activated dissociation mass spectrometry showed that the LEC18 core glycopeptides contained a new GlcNAc residue that substitutes the core GlcNAc residues. Methylation linkage analysis of the parent compound provided evidence that the GlcNAc is linked at O-6 to give the following novel, N-linked core structure. [formula: see text]     

Sialylated N-glycans in adult rat brain tissue--a widespread distribution of disialylated antennae in complex and hybrid structures

This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40% of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7%, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for 'brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core alpha1-6-linked and outer-arm alpha1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewis(x) and NeuAc alpha2-3 substituted Galbeta1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal beta1-3-linked galactose residues was observed. The presence of beta1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAc alpha2-3Galbeta1-3(NeuAc alpha2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one beta1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing beta1-3-linked galactose and 'polylactosamine' N-glycans with one to three terminal beta1-3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal beta1-3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.     

Core fucosylation of high-mannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec1) CHO cells

During studies on the fucosylation of endogenous proteins in parental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferase I-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observed that Lec1 cells incorporate approximately 10-fold less [3H]fucose into macromolecules than Pro5 cells. Interestingly, most of the labelled oligosaccharides from both cell types could be released from the macromolecules by digestion with peptide N-glycosidase F (PNGase F). This was unexpected for Lec1 cells because they do not synthesize complex- or hybrid-type N-glycans. Structural analyses of the fucosylated oligosaccharides from Lec1 cells showed the fucose to be in an alpha 1,6 linkage to the core GlcNAc of relatively small oligomannose N-glycans (Man4GlcNAc2 and Man5GlcNAc2, where Man is D-mannose). Comparing the sizes of oligomannose N-glycans from Pro5 and Lec1 cells demonstrated a much higher proportion of the small (Man4GlcNAc2 and Man5GlcNAc2) oligomannose species in Lec1 cells. These results suggest that the core alpha 1,6 fucosyltransferase will fucosylate small (Man4-Man5GlcNAc2), but not large (Man8-Man9GlcNAc2) oligomannose N-glycans.     

Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.     

Expression, purification, and reconstitution of receptor for pituitary adenylate cyclase-activating polypeptide. large-scale purification of a functionally active G protein-coupled receptor produced in Sf9 insect cells

Human pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was expressed in Sf9 insect cells and Chinese hamster ovary (CHO) cells. The recombinant receptor in Sf9 cell membranes had low affinity for 125I-PACAP27 (Kd = 155.3 pM) and was insensitive to guanosine 5'-O-3-thiotriphosphate (GTPgammaS), whereas the receptor in CHO membranes had a high affinity (Kd = 44.4 pM) and was GTPgammaS sensitive. The receptor in Sf9 membranes was converted to a high affinity state (Kd = 20-40 pM) following solubilization with digitonin. A large quantity (2 mg from 8 liters of insect cells) of the purified PACAP receptors (Bmax = 23.9 nmol/mg of protein) were obtained in a digitonin-induced high affinity state (Kd = 17.3 pM) using biotinylated ligand affinity chromatography. The apparent molecular weight of the purified receptor (Mr = 48,000) was smaller than that of the receptor from CHO cells (Mr = 58,000) due to differences in asparagine-linked sugar chains. The purified receptor reverted to a low affinity state (Kd = 182.6 pM) upon reconstitution into lipid vesicles, however, the receptor reconstituted with Gs protein had a high affinity (Kd = 40.2 pM) and was GTPgammaS sensitive. [35S]GTPgammaS binding to the reconstituted Gs protein was enhanced by PACAP27 and PACAP38 (EC50 = 42.5 and 9.4 pM, respectively) but not by antagonist PACAP(6-38), indicating that the purified receptor was functionally active.     

Glycosylation of active human renin is necessary for secretion: effect of targeted modifications of Asn-5 and Asn-75

Renin is a mammalian aspartic protease that is rate-limiting in the renin-angiotensin cascade. Preprorenin is the translational product of the human renin gene and is secreted as prorenin, an inactive zymogen, primarily from the juxtaglomerular cells of the kidney. It has previously been shown that the 46-amino-acid pro domain is not necessary for the secretion of fully active or mature renin from mammalian cells. Additionally, previous reports indicated that glycosylation of Asn-5 and Asn-75, the two potential sites of N-glycosylation in renin, is not necessary for the secretion of prorenin from mammalian cells. In the present study, the role of N-glycosylation in the secretion of mature renin was examined. Asn to Ser mutations at one or both of the glycosylation sites of mature renin were made and the expression of these constructs was examined in COS, CHO, and Sf9 insect cells. In the absence of the pro sequence, N-glycoylation at Asn-75 was essential for the secretion of active renin protein from all three cell types. The mutation at Asn-75 caused a more dramatic reduction in renin secretion than the mutation at Asn-5. This is in contrast to results previously reported for prorenin.     

Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells

The complex biantennary oligosaccharide at Asn297 of IgG is essential for some effector functions. To investigate the effect of carbohydrate structure on Ab function, we have now expressed mouse-human chimeric IgG1 Abs in Chinese hamster ovary (CHO) cells with defined defects in carbohydrate biosynthesis. We had previously shown that IgG1 Abs produced in the cell line Lec 1, which attaches a high-mannose intermediate carbohydrate, were severely deficient in complement activation, showed a slightly reduced affinity for Fc gammaRI, and had a reduced in vivo half-life. We have extended these studies by producing the same dansyl-specific IgG1 in cell lines deficient in attachment of sialic acid (Lec 2) and galactose (Lec 8). IgG1-Lec 1, IgG1-Lec 2, and IgG1-Lec 8 all showed varying reactivity with a mAb specific for an epitope in the amino terminal region of C(H)2, suggesting that the conformations of these proteins were altered by the different carbohydrate structures. Functionally, IgG1-Lec 2 and IgG1-Lec 8 were comparable to wild type with respect to in vivo half-life, affinity for Fc gammaRI, and capacity for complement-mediated hemolysis. While IgG1-Lec 2 was essentially identical to wild type in its capacity to interact with individual components of the classical complement activation pathway, IgG1-Lec 8 demonstrated equivalent maximal binding at lower concentrations and was preferentially bound by mannose-binding protein. Although IgG1-Lec 1 was deficient in activation of the classical pathway, it had a superior capacity to activate the alternative pathway. These studies demonstrate that Abs bearing C(H)2-linked carbohydrate of differing structures have different functional properties.     

Overview of N- and O-linked oligosaccharide structures found in various yeast species

Yeast and most higher eukaryotes utilize an evolutionarily conserved N-linked oligosaccharide biosynthetic pathway that involves the formation of a Glc3Man9GlcNAc2-PP-dolichol lipid-linked precursor, the glycan portion of which is co-translationally transferred in the endoplasmic reticulum (ER) to suitable Asn residues on nascent polypeptides. Subsequently, ER processing glycohydrolases remove the three glucoses and, with the exception of Schizosaccharomyces pombe, a single, specific mannose residue. Processing sugar transferases in the Golgi lead to the formation of core-sized structures (Hex<15GlcNac2) as well as cores with an extended poly-alpha1,6-Man 'backbone' that is derivatized with various carbohydrate side chains in a species-specific manner (Hex50-200GlnNAc2). In some cases these are short alpha1,2-linked Man chains with (Saccharomyces cerevisiae) or without (Pichia pastoris) alpha1,3-Man caps, while in other yeast (S. pombe), the side chains are alpha1,2-linked Gal, some of which are capped with beta-1,3-linked pyruvylated Gal residues. Charged groups are also found in S. cerevisiae and P. pastoris N-glycans in the form of mannose phosphate diesters. Some pathogenic yeast (Candida albicans) add poly-beta1,2-Man extension through a phosphate diester to their N-glycans, which appears involved in virulence. O-Linked glycan synthesis in yeast, unlike in animal cells where it is initiated in the Golgi using nucleotide sugars, begins in the ER by addition of a single mannose from Man-P-dolichol to selected Ser/Thr residues in newly made proteins. Once transported to the Golgi, sugar transferases add one (C. albicans) or more (P. pastoris) alpha1,2-linked mannose that may be capped with one or two alpha1,3-linked mannoses (S. cerevisiae). S. pombe is somewhat unique in that it synthesizes a family of mixed O-glycans with additional alpha1,2-linked Man and alpha1,2- and 1, 3-linked Gal residues.     

Effect of changes in the glycosylation of the human immunodeficiency virus type 1 envelope on the immunoreactivity and sensitivity to thrombin of its third variable domain

The influence of HIV Env glycosylation on the conformation of the third variable domain (V3) of Env was studied by both deglycosylation of mature Env and the use of Env produced by recombinant systems in which alpha-glucosidase activity was inhibited by either deoxynojirimycin (DNM) or mutation. Selective deglycosylation affected anti-V3 antibody binding. The immunoreactivity and sensitivity to thrombin cleavage of V3 presented on Env produced in baby hamster kidney cells were changed by DNM treatment. In contrast, Env expressed in alpha-glucosidase I-deficient Chinese hamster ovary cells or in their parental cells treated by DNM fully retained these V3 properties. These results are discussed in relation to the inconsistent data obtained on V3 property changes resulting from Env glycosylation changes.     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.     

Asparagine-linked oligosaccharides are localized to a luminal hydrophilic loop in human glucose-6-phosphatase

Deficiency of glucose-6-phosphatase (G6Pase), an endoplasmic reticulum transmembrane glycoprotein, causes glycogen storage disease type 1a. We have recently shown that human G6Pase contains an odd number of transmembrane segments, supporting a nine-transmembrane helical model for this enzyme. Sequence analysis predicts the presence of three potential asparagine (N)-linked glycosylation sites, N96TS, N203AS, and N276SS, conserved among mammalian G6Pases. According to this model, Asn96, located in a 37-residue luminal loop, is a potential acceptor for oligosaccharides, whereas Asn203 and Asn276, located in a 12-residue cytoplasmic loop and helix 7, respectively, would not be utilized for this purpose. We therefore characterized mutant G6Pases lacking one, two, or all three potential N-linked glycosylation sites. Western blot and in vitro translation studies showed that G6Pase is glycosylated only at Asn96, further validating the nine-transmembrane topology model. Substituting Asn96 with an Ala (N96A) moderately reduced enzymatic activity and had no effect on G6Pase synthesis or degradation, suggesting that oligosaccharide chains do not play a major role in protecting the enzyme from proteolytic degradation. In contrast, mutation of Asn276 to an Ala (N276A) destabilized the enzyme and markedly reduced enzymatic activity. We present additional evidence suggesting that the integrity of transmembrane helices is essential for G6Pase stability and catalytic activity.     

Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance

The role of the N-terminal sequence of myeloperoxidase in the intracellular targeting was examined by using glycosylated lysozyme as a reporter. A fusion protein was constructed in which the presequence residues-18 through -6 of the lysozyme moiety had been replaced by residues 1-158 of prepromyeloperoxidase. Expression of the fusion protein in Chinese hamster ovary cells demonstrated its partial secretion and partial intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glycosylated lysozyme that had been expressed in cells transfected with cDNA of glycosylated lysozyme. The retention was insensitive to NH4Cl. In the secreted protein, lysozyme contained predominantly complex oligosaccharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-beta-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein contained predominantly mannose-rich oligosaccharides. In baby hamster kidney cells, the trimming of the oligosaccharides in the lysozyme fragment was less vigorous, and a selective targeting of molecules bearing mannose-rich oligosaccharides to lysosomes was more apparent than in Chinese hamster ovary cells. In the presence of monensin, the formation of complex oligosaccharides in the fusion protein and its secretion were strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in the intracellular routing of the precursor and that this routing operates on precursors bearing mannose-rich rather than terminally glycosylated oligosaccharides and diverts them from the secretory pathway at a site proximal to the monensin-sensitive compartment of the Golgi apparatus.     

Investigation of human cyclooxygenase-2 glycosylation heterogeneity and protein expression in insect and mammalian cell expression systems

Human cyclooxygenase-2 (hCox-2) is a key enzyme in the biosynthesis of prostaglandins and the target of nonsteroidal anti-inflammatory drugs. Recombinant hCox-2 overexpressed in a vaccinia virus (VV)-COS-7 system comprises two glycoforms. Removal of the N-glycosylation consensus sequence at Asn580 (N580Q and S582A mutants) resulted in the expression of protein comprising a single glycoform, consistent with the partial N-glycosylation at this site in the wild-type (WT) enzyme. The specific cyclooxygenase activities of the purified WT and N580Q mutant were equivalent (40 +/- 3 mumol O2/min/mg) and titrations with diclofenac showed no difference in inhibitor sensitivities of WT and both mutants. Results of the expression of WT and N580Q hCox-2 in a Drosophila S2 cell system were also consistent with the N-glycosylation at this site, but low levels of activity were obtained. High levels of N-glycosylation heterogeneity are observed in hCox-2 expressed using recombinant baculovirus (BV) in Sf9 cells. Expression of a double N-glycosylation site mutant in Sf9 cells, N580Q/N592Q, resulted in a decrease in glycosylation but no clear decrease in heterogeneity, indicating that the high degree of N-glycosylation heterogeneity observed with the BV-Sf9 system is not due to partial glycosylation of both Asn580 and Asn592. N-linked oligosaccharide profiling of purified VV and BV WT and S582A mutant hCox-2 showed the presence of high mannose structures, (Man)n (GlcNAc)2, n = 9, 8, 7, 6. The S582A mutant was the most homogeneous with (Man)9(GlcNAc)2 comprising greater than 50% of oligosaccharides present. Analysis of purified VV WT and S582A mutant hCox-2 by liquid chromatography-electrospray ionization-mass spectrometry showed an envelope of peaks separated by approximately 160 Da, corresponding to differences of a single monosaccharide. The difference between the highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide.     

Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities

Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha- and beta-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the alpha-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the beta-subunit, two mutant eCGs, in which Asn 56 of the alpha-subunit was replaced with Gln (eCG alpha 56/beta) or CTP was deleted (eCG alpha/ beta-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The wild type eCG showed similar LH- and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCG alpha 56/beta was greatly reduced, whereas that of eCG alpha/beta-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the alpha-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCG alpha 56/beta was increased markedly in comparison with the wild type, and that of eCG alpha/beta-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH- and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the alpha-subunit Asn 56 or CTP-associated O-linked oligosaccharides.     

The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.