Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.     

Studies on the escape mutants of rabies virus which are resistant to neutralization by a highly conserved conformational epitope-specific monoclonal antibody #1-46-12

We investigated a virus-neutralizing conformational epitope of the rabies virus glycoprotein (G) that is recognized by an anti-G monoclonal antibody (mAb; #1-46-12) and shared by most of the laboratory strains of the virus. To investigate the epitope structure, we isolated escape mutants from the HEP-Flury virus (wild-type; wt) after repeated passages in culture in the presence of the mAb. Immunofluorescence studies indicated that the mutants could be classified into two groups; the Group I lacked the epitope, while Group II preserved the epitope. The latter was dominant under the passage conditions, since Group I disappeared during the continuous passages. G proteins showed different electrophoretic mobilities; G protein of Group I migrated at the same rate as wt G protein, while that of Group II migrated at a slower rate, which was shown to be due to acquisition of an additional oligosaccharide side chain. Nucleotide sequencing of the G gene strongly suggested that amino acid substitutions at Thr-36 by Pro and Ser-39 by Thr of the G protein are responsible for the escape mutations of Groups I and II, respectively. The latter is a unique mutation of the rabies virus that allows the G protein to be glycosylated additionally at Asn-37, a potential glycosylation site that is not glycosylated in the parent virus, in preserving the epitope-positive conformation. These results suggest that to keep the 1-46-12 epitope structure is of greater survival advantage for the virus to escape the neutralization than to destroy it, which could be achieved by acquiring an additional oligosaccharide chain at Asn-37.     

Glycosylation reduces the bioactivity of calcitonin

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.     

Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport.

When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gp160) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gp120 and gp41. The subunits gp120 and gp41 were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gp160 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gp120 envelope protein. Processing and transport of gp160 were blocked by the monovalent ionophore monensin, which at high concentrations (25 microM and above) was a potent inhibitor of the endoproteolytic cleavage of gp160; at lower concentrations (1 to 10 microM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 microM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gp120 that have a lower affinity for the CD4 receptor.     

Role of glycosylation in structure and stability of Erythrina corallodendron lectin (EcorL): a molecular dynamics study

The effect of glycosylation on structure and stability of glycoproteins has been a topic of considerable interest. In this work, we have investigated the solution conformation of the oligosaccharide and its effect on the structure and stability of the glycoprotein by carrying out a series of long Molecular dynamics (MD) simulations on glycosylated Erythrina corallodendron lectin (EcorL) and nonglycosylated recombinant Erythrina corallodendron lectin (rEcorL). Our results indicate that, despite the similarity in overall three dimensional structures, glycosylated EcorL has lesser nonpolar solvent accessible surface area compared to nonglycosylated EcorL. This might explain the experimental observation of higher thermodynamic stability for glycosylated EcorL compared to nonglycosylated EcorL. Analysis of the simulation results indicates that, dynamic view of interactions between protein residues and oligosaccharide is entirely different from the static picture seen in the crystal structure. The oligosaccharide moiety had dynamically stable interactions with Lys 55 and Tyr 53, both of which are separated in sequence from the site of glycosylation, Asn 17. It is possible that glycosylation helps in forming long-range contacts between amino acids, which are separated in sequence and thus provides a folding nucleus. Thus our simulations not only reveal the conformations sampled by the oligosaccharide, but also provide novel insights into possible molecular mechanisms by which glycosylation can help in folding of the glycoprotein by formation of folding nucleus involving specific contacts with the oligosaccharide moiety.     

Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system.

The baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector to produce hepatitis B virus surface antigen with and without the pre-S domain. The S gene product was expressed as both fusion and nonfusion polypeptides. No difference was observed in the posttranslational modification of the fusion and nonfusion polypeptides. The S proteins were not secreted into the medium but were inserted into the endoplasmic reticulum, glycosylated, and partially extruded into the lumen of the endoplasmic reticulum as 22-nm lipoprotein particles. The oligosaccharide chains on the insect cell-derived S protein were of the N-linked high-mannose form, in contrast to the complex-type oligosaccharides detected on plasma-derived hepatitis B virus surface antigen. The pre-S-S polypeptides were inserted into the endoplasmic reticulum, glycosylated, and modified by fatty acid acylation with myristic acid. A procedure was developed to purify the S protein from cellular membranes by using detergent extraction and immunoaffinity chromatography. The purified S protein was in the form of protein-detergent micelles and was highly antigenic and immunogenic.     

Site of attachment of encephalomyocarditis virus on human erythrocytes.

Previous studies of the attachment of encephalomyocarditis (EMC) virus to human erythrocytes concluded that the glycophorins, a family of human erythrocyte sialoglycoproteins, act as EMC virus receptors. Evidence is presented that the major glycophorin species, glycophorin A, is the receptor for EMC virus attachment to human erythrocytes. Comparison of the structures of glycophorins A and B and sialoglycopeptides released by chymotrypsin and trypsin treatment of erythrocytes confirmed our previous suggestion (A. T. H. Burness and I. U. Pardoe, J. Gen. Virol. 64:1137-1148, 1983) that attachment of EMC virus to glycophorin A involves the region containing amino acids 35 to approximately 70 (numbered from the NH2 terminus), four of which (amino acids 37, 44, 47, and 50) are glycosylated. In addition, we provide evidence that the segment containing amino acids 35 to 39 with an oligosaccharide side chain on threonine-37 is particularly important for EMC virus attachment.     

All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase beta-subunit contain oligosaccharide

The beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.     

Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane

In this report, we have asked whether asparagine-linked oligosaccharides added to new sites in the polypeptide backbone of a model plasma membrane glycoprotein, the vesicular stomatitis virus G protein, can promote its intracellular transport. We modified the coding sequence of G protein lacking the two normal consensus sites for glycosylation by oligonucleotide-directed mutagenesis to create new consensus sites. The expression of the mutant proteins was then analyzed in transfected cells. Six of the eight new sites which were introduced were glycosylated, and an oligosaccharide at two of these new sites promoted transport of G protein which lacked the two normal sites. However, the efficiency of this process was reduced compared to the wild-type protein or to the proteins with only one oligosaccharide at either of the normal sites. In addition, an oligosaccharide at two of the other new sites caused inhibition of transport of the wild-type G protein. The data in this and the following report suggest that carbohydrate plays an indirect role in the intracellular transport of G protein.     

Intracellular transport and secretion of hepatitis B surface antigen in mammalian cells.

The oligosaccharide processing and secretion of hepatitis B surface antigen (HBsAg) was studied in Chinese hamster ovary cells stably transfected with the gene coding HBsAg. HBsAg was secreted from cells with a relatively long half time (ca. 5 h). This appeared to be a characteristic of HBsAg itself, since HBsAg-producing cells infected with vesicular stomatitis virus transported the viral envelope glycoprotein to the cell surface with normal kinetics (half time of ca. 30 min). The secreted HBsAg was comprised of both the unglycosylated (P20) and the glycosylated (G25) polypeptides, characteristic of HBsAg isolated from human serum or secreted from other cell lines (C. W. Crowley, C.-C. Liu, and A. D. Levinson, Mol. Cell. Biol. 3:44-55, 1983; M. F. Dubois, C. Pourcel, S. Rousset, C. Chang, and P. Tiollais, Proc. Natl. Acad. Sci. U.S.A. 77:4549-4553, 1980; C.-C. Liu, D. Yansura, and A. D. Levinson, DNA, 1:213-221, 1982; G. M. Macnab, J. J. Alexander, G. Lecatsas, E. M. Bey, and J. M. Urbanocvicz, Br. J. Cancer, 24:509-515, 1976; A. M. Moriarity, B. H. Hoyer, J. W.-K. Shih, J. L. Gerin, and D. H. Hamer, Proc. Natl. Acad. Sci. U.S.A. 78:2606-2610, 1981; D. L. Peterson, J. Biol. Chem., 256:6975-6983, 1981). The glycosylated polypeptide (GP25) contained complex oligosaccharide chains. Cell-associated HBsAg also was comprised of both an unglycosylated and a glycosylated polypeptide; however, the glycosylated form (GP23) contained only high-mannose oligosaccharide chains. No oligosaccharide processing of the high-mannose chains could be detected within the cells. Thus, most of the time before secretion of HBsAg from cells must have been spent in a pre-Golgi or early Golgi compartment. Glycosylation was inhibited completely by tunicamycin, although unglycosylated particles were still secreted from cells and were antigenic. The secretion and oligosaccharide processing of HBsAg were inhibited with high concentrations of monensin, but at lower concentrations of monensin HBsAg was still secreted, although only half of the oligosaccharide chains were processed to the complex form.     

Exploitation of glycosylation in enveloped virus pathobiology

Glycosylation is a ubiquitous post-translational modification responsible for a multitude of crucial biological roles. As obligate parasites, viruses exploit host-cell machinery to glycosylate their own proteins during replication. Viral envelope proteins from a variety of human pathogens including HIV-1, influenza virus, Lassa virus, SARS, Zika virus, dengue virus, and Ebola virus have evolved to be extensively glycosylated. These host-cell derived glycans facilitate diverse structural and functional roles during the viral life-cycle, ranging from immune evasion by glycan shielding to enhancement of immune cell infection. In this review, we highlight the imperative and auxiliary roles glycans play, and how specific oligosaccharide structures facilitate these functions during viral pathogenesis. We discuss the growing efforts to exploit viral glycobiology in the development of anti-viral vaccines and therapies.     

Effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease

We have investigated the effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. The sensitivity of nonglycosylated RNase A to trypsin and chymotrypsin was compared with three glycosylated species of RNase B which differed with respect to the size of the carbohydrate chain. Two forms of glycosylated RNase B were isolated by concanavalin A-Sepharose affinity chromatography, and each was shown to contain a single carbohydrate chain composed of GlcNAc2Man1 (RNase B") or GlcNAc2Man5-8 (RNase B). A third form (RNase B'), with oligosaccharide composed of GlcNAc2Man4, was prepared by partial digestion of RNase B with alpha-mannosidase. Fully glycosylated RNase B was found to be 6-10 times more resistant to trypsin digestion than nonglycosylated RNase A. RNase B' and B", with intermediate chain sizes, were 3.0- and 1.3-fold more resistant to trypsin digestion than RNase A, respectively. With chymotrypsin, however, differences in rates of digestion were much less marked, with a maximum difference of 3-fold between RNase A and B. In addition, we found that the specificity of the primary trypsin (Arg 33-Asp 34 bond) or chymotrypsin (Tyr 25-Cys 26 bond) cleavage site was not affected by the presence or size of the oligosaccharide chain. These results are consistent with the view that the size of the oligosaccharide chain and its proximity to the primary or rate-limiting cleavage site are important for expression of the carbohydrate protection against proteolytic degradation, which thus appears to be mediated by steric hindrance.     

Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker insertion mutants

Glycoprotein B (gB) of Herpes simplex virus type 1 (HSV-1) plays an essential role in viral entry. A set of more than 100 HpaI (GTTAAC) linker insertion mutations and their derivatives were isolated in plasmids specifying the gB coding and flanking sequences. Mutations including addition, deletion and nonsense mutations at 34 independent sites were identified by DNA sequence analysis of 48 plasmids. A map was constructed for the ability of addition mutants to complement a gB-null virus. The expression of gB activity for some plasmids was temperature-dependent. Many complementation-negative plasmids inhibited the complementation activity of a plasmid specifying wild-type gB, suggesting an interaction between active and inactive molecules to form oligomers. The interaction was localized to 328 of the total of 904 amino acids comprising gB. Partial Endo H digestion of nonsense polypeptides revealed that five of the six potential N-linked oligosaccharide sites are glycosylated; the most C-terminal site appears not to be glycosylated. A number of mutations, including some on the cytoplasmic side, were identified that blocked processing, transport and secretion. Addition mutations that blocked processing of membrane polypeptides also blocked processing and secretion when combined into a nonsense mutant that by itself was processed and secreted. The previously predicted membrane spanning domain and the membrane orientation of the N-terminal portion of gB were confirmed.     

Effect of the threonine analog beta-hydroxynorvaline on the glycosylation and secretion of alpha 1-acid glycoprotein by rat hepatocytes

The threonine analog beta-hydroxynorvaline (Hnv) is an inhibitor of asparagine-linked glycosylation. In the presence of the analog hepatocytes synthesized immunoreactive alpha 1-acid glycoprotein with 0-6 oligosaccharide chains. Pulse-chase experiments were conducted to compare the rates of secretion of alpha 1-acid glycoprotein from untreated, tunicamycin-treated, and Hnv-treated cells. Partially glycosylated (1-5 oligosaccharide chains) and unglycosylated (tunicamycin-inhibited) molecules exited the cells more slowly than native alpha 1-acid glycoprotein. In addition, secretion of fully glycosylated (6 oligosaccharide chains) alpha 1-acid glycoprotein was retarded in Hnv-treated cells when compared to controls. The slowest rate of secretion was exhibited by the unglycosylated form from Hnv-treated cells. These results suggest that Hnv-induced changes either in the extent of glycosylation or in the peptide sequence of alpha 1-acid glycoprotein can interfere with its transport through the cell. The major intracellular forms of alpha 1-acid glycoprotein from control and Hnv-treated cells were endoglycosidase H-sensitive and contained Man9-8 GlcNAc2 oligosaccharide structures. The oligosaccharide chains on the secreted molecules from control and Hnv-treated cells were entirely of the endoglycosidase H-resistant, complex type.     

Glycosylation of the overlapping sequons in yeast external invertase: effect of amino acid variation on site selectivity in vivo and in vitro.

Yeast invertase contains 14 sequons, all of which are glycosylated to varying degrees except for sequon 5 which is marginally glycosylated, if at all. This sequon overlaps with sequon 4 in a sequence consisting of Asn92-Asn93-Thr94-Ser95(Reddy et al., 1988, J. Biol. Chem., 263, 6978-6985). To determine whether glycosylation at Asn93is sterically hindered by the oligosaccharide on Asn92, the latter amino acid was converted to a glutamine residue by site-directed mutagenesis of the SUC2 gene in a plasmid vector which was expressed in Saccharomyces cerevisiae. A glycopeptide encompassing sequons 3 through 6 was purified from a tryptic digest of the mutagenized invertase and sequenced by Edman degradation, which revealed that Asn93 of sequon 5 contained very little, if any, carbohydrate, despite the elimination of sequon 4. When Ser and Thr were inverted to yield Asn-Asn-Ser-Thr carbohydrate was associated primarily with the second sequon, in agreement with numerous studies indicating that Asn-X-Thr is preferred to Asn-X-Ser as an oligosaccharide acceptor. However, when the invertase overlapping sequons were converted to Asn-Asn-Ser-Ser, both sequons were clearly glycosylated, with the latter sequon predominating. These findings rule out steric hindrance as a factor involved in preventing the glycosylation of sequon 5 in invertase. Comparable results were obtained using an in vitro system with sequon-containing tri- and tetrapeptides acceptors, in addition to larger oligosaccharide acceptors.     

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc₂, N‐acetyl galactosamine₁, Gal₁). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.     

Subcellular localization of glycoproteins encoded by the viral oncogene v-fms

The McDonough strain of feline sarcoma virus encodes a polyprotein that is cotranslationally glycosylated and proteolytically cleaved to yield transforming glycoproteins specified by the viral oncogene v-fms. The major form of the glycoprotein (gp120fms) contains endoglycosidase H-sensitive, N-linked oligosaccharide chains lacking fucose and sialic acid, characteristic of glycoproteins in the endoplasmic reticulum. Kinetic and steady-state measurements showed that most gp120fms molecules were not converted to mature forms containing complex carbohydrate moieties. Fixed-cell immunofluorescence confirmed that the majority of v-fms-coded antigens were internally sequestered in transformed cells. Dual-antibody fluorescence performed with antibodies to intermediate filaments (IFs) showed that the IFs of transformed cells were rearranged, and their distribution coincided with that of v-fms-coded antigens. No specific disruption of actin cables was observed. The v-fms gene products cofractionated with IFs isolated from virus-transformed cells and reassociated with IFs self-assembled in vitro. A minor population of v-fms-coded molecules (gp140fms) acquired endoglycosidase H-resistant, N-linked oligosaccharide chains containing fucose and sialic acid residues, characteristic of molecules processed in the Golgi complex. Some gp140fms molecules were detected at the plasma membrane and were radiolabeled by lactoperoxidase-catalyzed iodination of live transformed cells. We suggest that v-fms-coded molecules are translated as integral transmembrane glycoproteins, most of which are inhibited in transport through the Golgi complex to the plasma membrane.     

Structural characterization of a specific glycopeptidolipid containing a novel N-acyl-deoxy sugar from mycobacterium intracellulare serotype 7 and genetic analysis of its glycosylation pathway

The nontuberculous Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is distributed ubiquitously in the environment and is an important cause of respiratory and lymphatic disease in humans and animals. These species produce polar glycopeptidolipids (GPLs), and of particular interest is their serotype-specific antigenicity. Structurally, GPLs contain an N-acylated tetrapeptide-amino alcohol core that is glycosylated at the C terminal with 3,4-di-O-methyl rhamnose and at the d-allo-threonine with a 6-deoxy-talose. This serotype nonspecific GPL is found in all MAC species. The serotype-specific GPLs are further glycosylated with a variable haptenic oligosaccharide at 6-deoxy-talose. At present, 31 distinct serotype-specific GPLs have been identified on the basis of oligosaccharide composition, and the complete structures of 14 serotype-specific GPLs have been defined. It is considered that the modification of the GPL structure plays an important role in bacterial physiology, pathogenesis, and host immune responses. In this study, we defined the complete structure of a novel serotype 7 GPL that has a unique terminal amido sugar. The main molecular mass is 1,874, and attached to the tetrapeptide-amino alcohol core is the serotype 7-specific oligosaccharide unit of 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-2-O-methyl-beta-hexose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->2)-alpha-l-6-deoxy-talose. Moreover, we isolated and characterized the serotype 7-specific gene cluster involved in glycosylation of the oligosaccharide. Nine open reading frames (ORFs) were observed in the cluster. Based on the sequence homology, the ORFs are thought to participate in the biosynthesis of the serotype 7 GPL.     

Characterization of oligosaccharide chains on mouse mammary tumor virus envelope proteins and the implications for the mechanism of their glucocorticoid regulated processing

Glucocorticoid hormone is required for complete posttranslational processing of the glycosylated mouse mammary tumor virus envelope precursor, Pr74env in the murine T-lymphosarcoma cell line, W7MG1. Metabolic labeling studies with [35S]methionine, [3H]galactose, and [3H]mannose, combined with enzymatic digestion analyses with a variety of endoglycosidases, demonstrated that both proteolytic processing and N-linked oligosaccharide maturation depended, either directly or indirectly, on glucocorticoid action. Pr74 is found in both control and hormone-treated cells. In both cases Pr74 molecules carry high mannose and/or hybrid, but not complex, oligosaccharide chains with very little or no sialic acid. When cells are grown with glucocorticoid, Pr74 is converted to gp52 and gp33 with greatly increased efficiency, and these mature glycoproteins carry complex oligosaccharides containing sialic acid. No O-linked carbohydrate was detected on any of these species. According to this evidence, the glucocorticoid-regulated step in this pathway must occur at or before the final mannose trimming step in the Golgi that is required for formation of complex carbohydrate chains.     

The glycan structure of albumin Redhill, a glycosylated variant of human serum albumin.

Although human serum albumin is synthesized without carbohydrate, glycosylated variants of the protein can be found. We have determined the structure of the glycan bound to the double-mutant albumin Redhill (-1 Arg, 320 Ala-->Thr). The oligosaccharide was released from the protein using anhydrous hydrazine, and its structure was investigated using neuraminidase and a reagent array analysis method, which is based on the use of specific exoglycosidases. The glycan was shown to be a disialylated biantennary complex type oligosaccharide N-linked to 318 Asn. However, a minor part (11 mol%) of the glycan was without sialic acid. The structure is principally the same as that of glycans bound to two other types of glycosylated albumin variants. Glycosylation can affect, for example, the fatty acid binding properties of albumin. Taking the present information into account, it is apparent that different effects on binding are caused not by different glycan structures but by different locations of attachment, with the possible addition of local conformational changes in the protein molecule.