Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.     

Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.     

High-mannose glycans are elevated during breast cancer progression

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.     

Inhibition of Glycosylation of the Influenza Virus Hemagglutinin

d-Glucosamine and 2-deoxy-d-glucose interfere with the biosynthesis of the hemagglutinin glycoproteins. With increasing inhibitor concentrations a progressive decrease in size of the precursor HA and the cleavage products, HA(1) and HA(2) can be observed. The shift in molecular weight is paralleled by a decrease of the carbohydrate content. This was shown by labeling studies with radioactive sugars which revealed that the inhibitors block the incorporation into glycoproteins, whereas they have no or only slight effects on the uptake and activation of sugars. Under conditions of maximal inhibition, the hemagglutinin proteins lack all or most of their carbohydrates. These findings indicate that the inhibitory effect of d-glucosamine and 2-deoxy-d-glucose is due to an impairment of glycosylation. When glycosylation is inhibited, the precursor polypeptide is synthesized at normal rates. Its cleavage products, however, are very heterogeneous. This suggests that carbohydrate protects the hemagglutinin from proteolytic degradation.     

Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.

A high molecular weight glycoprotein consisting of three disulfide-linked 142,000 molecular weight chains has been isolated from human blood platelets. The glycoprotein, designated thrombospondin, is released by platelets in response to thrombin treatment and is proteolyzed when left in the presence of platelets after liberation. It is relatively insensitive to degradation by thrombin. Thrombospondin is a filamentous protein of dimensions approximately 7 X 70 nm and contains 1.9% neutral sugars, 1.4% amino sugars, 0.7% sialic acid, and no hexuronic acid. Amino acid analysis reveals that the level of cysteine is approximately 260 residues per molecule. Thrombospondin binds to immobilized heparin but is released by 0.45 M sodium chloride. A single band is obtained by isoelectric focusing, indicating a pI of 4.7 as well as a relatively high degree of purity. Degradation of the intact molecule with trypsin yields a stable core particle of molecular weight 210,000 comprised of three 70,000 chains.     

Effects of tunicamycin on protein glycosylation and development inVolvox carteri

Summary The involvement of protein glycosylation in regulation of the development of the multicellular green alga,Volvox carteri, was studied using the antibiotic, tunicamycin. Three specific developmental processes were found to be affected by the antibiotic: reproductive cell maturation; establishment of polar cellular organization during embryogenesis and release of progeny spheroids from the parental spheroids. Tunicamycin inhibited the transfer of GlcNAc-1-phosphate to dolichyl phosphate which is catalyzed byVolvox membrane preparations. Changes in the glycosylation of several secreted and cellular glycoproteins were observed when proteins were labelled with radioactive amino acids and sugars in the absence and presence of tunicamycin and then electrophoresed on sodium dodecylsulfate-polyacrylamide slab gels. The levels of a few secreted proteins were reduced in tunicamycin treated cultures and one protein band appeared exclusively in the treated cells. Tunicamycin treatment also altered the electrophoretic mobility of radio-iodinated surface macromolecules. Binding of concanavalin A by tunicamycin treatedVolvox spheroids was drastically reduced. It is there-fore likely that the aberrant development results from inhibition of protein glycosylation and the consequent changes in the structure of the cellular, secreted and surface glycoproteins.     

Purification and identification of the lipoprotein-binding proteins from human blood platelet membrane

As reported previously, homologous plasma lipoproteins specifically bind to the plasma membrane of human blood platelets. The two major lipoprotein-binding membrane glycoproteins were purified to apparent homogeneity and identified by their mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both in the nonreduced and reduced state, by specific antibodies against glycoproteins IIb (GPIIb) and IIIa (GPIIIa), respectively, including the alloantibody anti-PlA1 and monoclonal antibodies. Furthermore, lipoprotein binding to intact platelets is also inhibited in a dose-dependent fashion by preincubation of the platelets with antibodies against these glycoproteins. From these experiments it can be concluded that lipoproteins bind to both components of the glycoprotein IIb-IIIa complex in isolated membranes and intact platelets. High density lipoprotein and low density lipoprotein bind to GPIIIa blotted to nitrocellulose in a way that binding of one species interferes with the binding of the other. Addition of fibrinogen significantly inhibits this binding. The specific binding of fibrinogen to GPIIIa is strongly inhibited in the presence of either of the two lipoproteins. LDL and HDL are specifically bound by isolated GPIIb, too. In our blotting experiments fibrinogen shows no binding to this membrane glycoprotein. On the other hand, fibrinogen significantly interferes with the interaction between GPIIb and the lipoproteins.     

Glycolipids modulate glycosyl transfer to endogenous protein acceptors

Regulation by gangliosides of glycosylation of endogenous membrane glycoproteins is indicated from in vitro studies in which incorporation of radioactive sugars into endogenous protein acceptors was measured and from in vitro studies where transferase activities of membranes were correlated with ganglioside content during hepatic tumorigenesis. Galactosyl transfer from UDP galactose exhibited a complex response pattern and was stimulated by lactosyl ceramide and the ganglioside N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM2) but was inhibited by higher gangliosides. Except for N-acetylneuraminylgalactosylglucosylceramide (GM3), which had no effect, inhibition was proportional to ganglioside complexity. Inhibition of glycosylation of the exogenous acceptor, ovomucoid, by ganglioside was slight by comparison. While marked structure-linked latency was observed with the high molecular weight exogenous acceptor, no latency was observed for incorporation into endogenous acceptors suggesting that the membranes were permeable to sugar nucleotides. Membrane disruption with detergents lessened rather than enhanced inhibition by gangliosides. Sialyl transfer from CMPsialic acid, on the other hand, was unaffected or stimulated by gangliosides. Stimulation by galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM1) was proportional to concentration and reached 2-fold at 240 micrograms/mg protein. The results suggest that the ganglioside content of membrane may affect glycosylation of membrane glycoproteins.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials.

Changes in the glycosylation of asparagine-linked oligosaccharides have been shown in various tumor cells, including human colon cancer. Attempts were made to elucidate the difference in Asn-linked oligo-saccharides attached to lysosomal membrane glycoproteins isolated from sublines of human colon carcinoma exhibiting high and low metastatic potentials in nude mice. Lysosomal membrane glycoproteins (lamp) 1 and 2 were immunoprecipitated from the cells after labeling with radioactive sugars, and the glycopeptides prepared were fractionated by serial lectin affinity chromatography employing immobilized concanavalin A, Datura stramonium agglutinin, and tomato lectin. Comparison of Asn-linked oligosaccharides from the different colonic carcinoma cells revealed the following features. First, the highly metastatic carcinoma cells express more poly-N-acetyllactosaminyl side chains with branched galactose residues than cells with low metastatic potential. Second, sialylation is more significant in the highly metastatic carcinoma cells than in the poorly metastatic ones. Conversely, N-acetyllactosamine units are less fucosylated in the highly metastatic cells than in poorly metastatic cells. These structural changes were apparently caused by the increase in sialyltransferase and the decrease in alpha 1----3 fucosyltransferase in the highly metastatic cells. The results also suggest that highly metastatic carcinoma cells express more sialyl Lex structures at the termini of poly-N-acetyllactosaminyl side chains than poorly metastatic carcinoma cells. Further, highly metastatic cells were found to express more lamp-1 and lamp-2 on the cell surface. These results were found to be correlated to the increased expression of sialyl Lex structures with high affinity binding of anti-sialyl Lex antibody on highly metastatic cells. Increased expression of sialyl Lex in the poly-N-acetyllactosamines of the cell surface may contribute to the metastatic behavior of the cells, assuming that this structure can serve as a better ligand for selectins present on endothelial cells and platelets.     

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60, whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5). The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins.     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2

ABSTRACT: BACKGROUND: Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. RESULTS: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi alpha-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the alpha-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained alpha-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. CONCLUSIONS: We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.     

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.     

Structural changes induced in the sugar chains of glycoproteins by malignant transformation of producing cells and their clinical application

Altered glycosylation is widely observed in glycoproteins produced by tumors. One of the most consistently observed alterations is the increase of larger asparagine-linked sugar chains in the plasma membrane glycoproteins. This phenomenon is brought about by the increase of N-acetylglucosaminyltransferase V, which is responsible for the formation of the GlcNAc beta 1----6Man alpha-1----6 group. The enrichment of the complex-type sugar chains containing the -GlcNAc beta 1----6(-GlcNAc beta 1----2)Man alpha 1----6 group is correlated with tumorigenicity and metastasic potential of tumor cells. Comparative study of the sugar chains of human chorionic gonadotropin isolated from the urine of pregnant women and of patients with trophoblastic diseases including choriocarcinoma revealed that many new oligosaccharides are included in the tumor hCG. The altered glycosylation of hCG is brought about by the ectopic expression of N-acetylglucosaminyltransferase IV. With use of this altered glycosylation, a novel method useful for the diagnosis of choriocarcinoma was established.     

A re-evaluation of the surface complexity of the intact erythrocyte

Surface proteins and glycoproteins of intact human red blood cells were labelled with ¹²⁵I by the lactoperoxidase method. The radioactive proteins were then separated in each of the Fairbanks and Laemmli one-dimensional polyacrylamide gel electrophoresis systems. The radioactive polypeptides had different mobilities in the two systems, largely due to the anomalous migration of glycoproteins in polyacrylamide gels. A two-dimensional system was therefore developed using the Fairbanks and Laemmli buffer systems to exploit these anomalies. This procedure clearly resolved radioactive glycoproteins and proteins and enabled the identification of many more surface components than had previously proved possible.     

Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2-PP-dolichol in intestinal epithelial cells in culture

The lipid-linked oligosaccharides synthesized in the presence of the alpha-glucosidase inhibitors, 1-deoxynojirimycin (DJN) and N-methyl-1-deoxynojirimycin (MDJN), were compared in IEC-6 intestinal epithelial cells in culture. HPLC analysis of the oligosaccharides obtained before and after exhaustive jack bean alpha-mannosidase digestion indicates that control and MDJN-treated cells synthesize similar amounts of Glc3Man9GlcNAc2-PP-dolichol. In contrast, the formation of this compound is greatly reduced in DJN-treated cells, the major lipid-linked oligosaccharide found being Man9GlcNAc2-PP-dolichol. The decreased availability of the preferred donor for protein glycosylation may account for the impaired glycosylation and secretion of certain glycoproteins in the presence of DJN.     

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin

Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn(354) are inaccessible because the complex glycans at Asn(445) block access to the site. On IgE, the additional C(H)2 hinge domain blocks access to the oligomannose glycans at Asn(394) on one H chain by adopting an asymmetrically bent conformation. IgE contains 8.3% Man(5)GlcNAc(2) glycans, which are the trimmed products of the Glc(3)Man(9)GlcNAc(2) oligomannose precursor. The presence of these structures suggests that the C(H)2 domain flips between two bent quaternary conformations so that the oligomannose glycans on each chain become accessible for limited trimming to Man(5)GlcNAc(2) during glycan biosynthesis. This is the first study of the glycosylation of human serum IgD and IgE from nonmyeloma proteins.     

Identification of Lectin-Purified Neural Glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA Receptor Subunits: Conservation of Glycosylation at the Synapse

Abstract: The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for ∼50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.     

Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man3GlcNAc2 N-glycan core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc(2) structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5)GlcNAc(2) and GlcMan(5)GlcNAc(2) glycans, and to a lesser extent with Glc(2)Man(5)GlcNAc(2) glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man(3)GlcNAc(2) structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.