High Mannose Asparagine-linked Oligosaccharides Research Articles

Characterization of the high mannose asparagine-linked oligosaccharides synthesized by microfilariae of Dirofilaria immitis.

This report describes the structures of high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by the microfilariae of Dirofilaria immitis. Microfilariae of D. immitis were incubated in vitro in media containing 2-[3H] mannose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionation by chromatography on concanavalin A-Sepharose. Thirty eight percent of 2-[3H] mannose incorporated into the microfilariae of D. immitis glycopeptides was recovered in high mannose-type asparagine-linked oligosaccharides which were bound to the immobilized lectin. Upon treatment of 2-[3H] mannose labeled glycopeptides with endo-beta-N-acetylglucosaminidase H, the high mannosetype chains were released and their structures were determined by high performance liquid chromatography and exoglycosidase digestion. The major species of high mannosetype chains synthesized by microfilariae of D. immitis have the composition Man5GlcNAc2, Man6ClcNAc2, Man7GlcNAc2, and Man8GlcNAc2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by vertebrates.

Characterization of the high mannose asparagine-linked oligosaccharides synthesized by Schistosoma mansoni adult male worms

This report describes the structures of the high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult male worms. Adult male schistosomes were incubated in vitro in media containing either [2- 3H]mannose, [6- 3H]glucosamine or [6- 3H]galactose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with Pronase and fractionation by chromatography on concanavalin A-Sepharose. Eleven percent of [ 3H]mannose incorporated into the schistosome glycopeptides was recovered in high mannose-type Asn-linked oligosaccharides which bound to the immobilized lectin. Upon treatment of [ 3H]mannose-labeled glycopeptide with endo- β- N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography, methylation analysis, acetolysis and exoglycosidase digestion. The major species of high mannose-type chains synthesized by S. mansoni adult males have the composition Man 7GlcNAc 2, Man 8GlcNac 2 and Man 9GlcNA 2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by mammalian cells.

The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

Modification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 +/- 1.9 ml min-1 kg-1 to 3.5 +/- 0.6 ml min-1 kg-1 (mean +/- SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-beta-N-acetyl-glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 +/- 0.5 ml min-1 kg-1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gln) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

A study of the molecular pathology of sucrase-isomaltase deficiency. A defect in the intracellular processing of the enzyme.

The intestinal brush-border enzyme sucrase-isomaltase splits sucrose into its component monosaccharides, glucose and fructose. A deficiency of the enzyme leads to sucrose intolerance. We studied the synthesis and intracellular processing of sucrase-isomaltase, using human intestinal explants in organ culture. Pulse-chase experiments with [35S]methionine followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, and fluorography of labeled sucrase-isomaltase demonstrated that the molecule was initially recognized as a protein with a relative molecular weight (Mr) of 205,000. This was apparently converted to a species of 225,000 Mr within two hours. We studied the glycosylation of the protein using endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase digestion of oligosaccharide side chains of the two forms of sucrase-isomaltase. The results showed that the early-appearing 205-kd (kilodalton) molecule contained high-mannose asparagine-linked oligosaccharides, and that the later-appearing, 225-kd molecule contained highly processed (mature) carbohydrate chains. Studies in a patient with primary sucrase-isomaltase deficiency demonstrated normal translation and high-mannose glycosylation of the precursor but a failure in further processing of the oligosaccharides, with subsequent intracellular degradation of the glycoprotein and undetectable enzymatic activity of intestinal sucrase. Abnormal intracellular processing of the enzyme was the probable mechanism of enzyme deficiency in this patient.

Disruption of oligosaccharide processing in murine tumor cells inhibits their susceptibility to lysis by activated mouse macrophages.

The components of tumor cell surfaces that participate in the recognition and lysis of these cells by activated macrophages have not been identified. One plausible hypothesis is that these components are specific carbohydrate structures. As an initial test of this hypothesis, I have made use of the oligosaccharide processing inhibitors 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM). dNM is an inhibitor of the glucosidases involved in the initial steps of oligosaccharide processing. dMM inhibits mannosidase I. P815 cells incubated in the presence of 1-2 mM dNM for 24 hr synthesized mature glycoproteins that contained glucosylated high-mannose asparagine-linked oligosaccharides instead of complex forms. The glucosylated oligosaccharides were present in trypsin digests of the cell surface. The dNM treatment resulted in a diminution in the amount of surface galactose residues as evidenced by neuraminidase/galactose oxidase/NaB3H4 labeling of surface glycopeptides. It did not, however, inhibit protein synthesis or alter the surface polypeptide profile of the tumor cells. P815 and R1- cells incubated in the presence of 1-3 mM dNM for 24 hr were considerably less sensitive to lysis by interferon-gamma-activated macrophages than were cells incubated in control medium. At a dNM concentration of 3 mM, a 71% inhibition of P815 cell lysis was observed. Similarly, P815 and R1- cells incubated in the presence of 2 mM dMM were also less sensitive to macrophage-mediated lysis than were control cells. The inhibitors did not affect cell viability, growth, or gross morphology. These observations suggest that complex asparagine-linked oligosaccharides on tumor cell surfaces may participate in recognition and lysis by activated macrophages.

The effects of inhibitors of glucosidase I on the formation of Sindbis virus

We have examined the effects of deoxynojirimycin and castanospermine, compounds known to inhibit the removal of glucose from high mannose asparagine-linked oligosaccharides, on the formation of Sindbis virus. These drugs inhibited virion formation in baby hamster kidney (BHK) cells, 15B — the CHO cell line that lacks GlcNAc transferase activity, and chicken embryo fibroblasts, although our results with the latter cells were variable. We analyzed the [ 3H]mannose-labeled oligosaccharides from Sindbis virus infected 15B cells. Those from control cells were predominantly GlcNAc 2Man 5. Oligosaccharides from the treated cells were larger than the Man 5 species and as expected, were partially resistant to α-mannosidase. The growth of Sindbis virus was inhibited to a much greater extent at 37°C than at 30°C in BHK cells treated with either deoxynojirimycin or castanospermine. Both of these compounds also inhibited the proteolytic cleavage of the viral glycoprotein precursor, PE2, to the virion glycoprotein, E2, but did not prevent the migration of the glycoprotein to the cell surface. These results, taken together with our earlier studies with vesicular stomatitis virus (Schlesinger et al., 1984) provide strong evidence that the removal of glucose residues during the processing of asparaginelinked oligosaccharides is critical for some proteins to achieve a functional conformation.

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2- 3H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β- N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc 1Man 9GlcNAc, which was converted into Man 9GlcNAc, and then into Man 8GlcNAc, Man 7GlcNAc, Man 6GlcNAc, and Man 5GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man 9GlcNAc to Man 5GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man 9GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.