The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N(158)) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N(158), N(956), N(1341), N(1350), and N(1496)), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by >50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d < 1.02 and d > 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.     

An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells

Of all ligands of the transforming growth factor beta superfamily, inhibins and activins are a physiologically relevant pair that are functional antagonists of each other. Activin stimulates whereas inhibin blocks follicle-stimulating hormone biosynthesis and secretion from pituitary gonadotrope cells, and together, inhibin and activin control the pituitary gonadal axis essential for normal reproductive function. Sharing a similar beta-subunit, the secretion of inhibin heterodimers (alpha/beta) or activin homodimers (beta/beta) as mature bioactive ligands depends, in part, on the proteolytic processing of precursor proteins. A short loop regulatory pathway controlling precursor processing and dimer secretion was discovered. Activin stimulates endogenous inhibin alpha- and betaB-subunit mRNA, protein, and proteolytic processing. Simultaneously, activin stimulated the proconvertase furin through a Smad2/3-dependent process. The data provide a mechanism where the regulation of furin and inhibin subunits cooperates in an important positive short feedback loop. This regulatory loop augments the secretion of bioactive mature activin B, as well as inhibin B dimers, necessary for local follicle-stimulating hormone beta regulation.     

Inhibitors of the biosynthesis and processing of N-linked oligosaccharides

A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Structural characterization of the N-linked oligosaccharides from tomato fruit.

The primary structures of the N-linked oligosaccharides from tomato fruit (Lycopersicon esculentum) have been elucidated. For the isolation of the protein fraction, two procedures were employed alternatively: a low temperature acetone powder method and ammonium sulfate precipitation of the tomato extract. After peptic digestion, the glycopeptides were purified by cation-exchange chromatography; the oligosaccharides were released by N-glycosidase A and fluorescently labelled with 2-aminopyridine. Structural characterization was accomplished by means of two-dimensional HPLC in combination with exoglycosidase digestions and MALDI-TOF mass spectrometry. Two varieties as well as two stages of ripening were investigated. In all the samples, the same sixteen N-glycosidic structures were detected; the two most abundant glycans showed identical properties to those of the major N-linked oligosaccharides of horseradish peroxidase and pineapple stem bromelain, respectively and accounted for about 65-78% of the total glycan amount; oligomannosidic glycans occurred only in small quantities (3-9%). The majority of the N-glycans were beta 1,2-xylosylated and carried an alpha 1,3-fucose residue linked to the terminal N-acetylglucosamine. This structural element contributes to cross-reactions among non-related glycoproteins and has been shown to be an IgE-reactive determinant (Tretter, Altmann, Kubelka, M?rz, & Becker, 1993). The presented study gives a possible structural explanation for reported immunological cross-reactivities between tomato and grass pollen extracts due to carbohydrate IgE epitopes (Petersen, Vieths, Aulepp, Schlaak, & Becker, 1996), thereby demonstrating the importance of the structural characterization of plant N-glycans for a more reliable interpretation of immunological data.     

Processing of N-linked oligosaccharides in soybean cultured cells

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.     

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.     

Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides were GalGlcNAcMan(6)GlcNAc-Fuc(0-1)GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man(5-7)GlcNAc(2) were the predominant species (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.     

N-linked oligosaccharides affect the enzymatic activity of CD39: diverse interactions between seven N-linked glycosylation sites

Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, has seven potential N-glycosylation sites at asparagine residues 73, 226, 291, 333, 375, 429, and 458. To determine their roles in the structure and function of CD39, we mutated these sites individually or in combination by replacing asparagine with serine or glutamine and analyzed the surface expression and the enzymatic activity of the mutants. The results indicate that rat CD39 can be glycosylated at all seven sites when expressed in COS7 cells. Glycosylation sites 73 at the N terminus, 333 in the middle, and 429 and 458 at the C terminus were principally required for cell surface appearance of enzymatically active CD39. Whereas deletion of these sites individually had modest effects on surface ATPase activity, some double deletions of these sites had major effects on both surface activity and expression. The importance of these N-glycosylation sites is recognizable in other members of the ectonucleoside triphosphate diphosphohydrolase family.     

Asynchronous transport to the cell surface of intestinal brush border hydrolases is not due to differential trimming of N-linked oligosaccharides

Intestinal brush border enzyme glycoproteins are transported to the microvillar membrane at different rates in the differentiated intestinal cell line Caco-2. This asynchronism is due to at least two rate-limiting events, a pre- and an intra-Golgi step (Stieger B., Matter, K., Baur, B., Bucher, K., H?chli, M., and Hauri, H.P. (1988) J. Cell Biol. 106, 1853-1861). A possible cause for the asynchronous protein transport might be differential trimming of N-linked oligosaccharide side chains. The effects of two trimming inhibitors on the intracellular transport of sucrase-isomaltase, a slowly migrating hydrolase, and dipeptidylpeptidase IV, a rapidly migrating hydrolase, are described. 1-Deoxymannojirimycin, an inhibitor of Golgi alpha-mannosidase I, had no influence on the rate of appearance of these hydrolases in the brush border membrane as assessed by subcellular fractionation. In the presence of N-methyl-1-deoxynojirimycin, an inhibitor of glucosidase I, 30-40% of the newly synthesized molecules appeared at the cell surface, and half-time for appearance of this pool was identical to that found in control cells. The reduced maximal transport to the cell surface observed with N-methyl-1-deoxynojirimycin may suggest that proper glycosylation is necessary for an efficient transport from the Golgi apparatus to the microvillar membrane. Inhibition of glucosidase I does not prevent the acquisition of endoglycosidase H resistance. Furthermore, evidence is presented that the processing in the presence of N-methyl-1-deoxynojirimycin leads to glycosylated endoglycosidase H-resistant glycoproteins.     

Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.     

The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. Site-directed mutagenesis of the two asparagine-linked glycosylation sites of hCG alpha was used to study the function of the individual oligosaccharide chains in secretion and subunit assembly. Expression vectors for the alpha genes (wild-type and mutant) and the hCG beta gene were constructed and transfected into Chinese hamster ovary cells. Loss of the oligosaccharide at position 78 causes the mutant subunit to be degraded quickly and less than 20% is secreted. However, the presence of hCG beta stabilizes this mutant and allows approximately 45% of the subunit in the form of a dimer to exit the cell. Absence of carbohydrate at asparagine 52 does not perturb the stability or transport of the alpha subunit but does affect dimer secretion; under conditions where this mutant or hCG beta was in excess, less than 30% is secreted in the form of a dimer. Mutagenesis of both glycosylation sites affects monomer and dimer secretion but at levels intermediate between the single-site mutants. We conclude that there are site-specific functions of the hCG alpha asparagine-linked oligosaccharides with respect to the stability and assembly of hCG.     

Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction

The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.     

Four N-linked glycosylation sites in human toll-like receptor 2 cooperate to direct efficient biosynthesis and secretion

Most higher organisms have a system of innate immune defense that is mediated by a group of evolutionarily related, germ line-encoded receptors, so-called Toll-like receptors. In mammals Toll-like receptors signal in response to pathogen-associated microbial structures. For example, Toll-like receptor 2 appears to mediate responses to bacterial peptidoglycan and acylated lipoproteins and Toll-like receptor 4 to bacterial lipopolysaccharide. However, the structural principles that underlie recognition of these structures are poorly understood. Toll-like receptors have leucine-rich repeats in their extracellular domains and are thus believed to adopt solenoid structures, similar to that found in platelet glycoprotein Ib. Additionally, all Toll-like receptors contain N-linked glycosylation consensus sites, and Toll-like receptor 4 requires glycosylation for function. Toll-like receptor glycosylation is also likely to influence receptor surface representation, trafficking, and pattern recognition. Using circular dichroism spectroscopy, we show here that purified human Toll-like receptor 2 and 4 proteins have secondary structure contents similar to glycoprotein Ib. We have also analyzed where consensus glycosylation sites are located in the extracellular domains of other human Toll-like receptors. We found that there are significant differences in the location and degree of conservation between sites in different Toll-like receptors. Using site-directed mutagenesis, we have found that in Toll-like receptor 2 extracellular domain all four predicted glycosylation sites are substituted, although one site is inefficiently core-glycosylated and its removal drastically affects secretion. The remaining Toll-like receptor 2 glycosylation sites also contribute to efficient protein secretion, albeit to a lesser degree.