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Korean J Parasitol > Volume 32(2):1994 > Article

Original Article
Korean J Parasitol. 1994 Jun;32(2):101-110. English.
Published online Jun 20, 1994.  http://dx.doi.org/10.3347/kjp.1994.32.2.101
Copyright © 1994 by The Korean Society for Parasitology
Characterization of the high mannose asparagine-linked oligosaccharides synthesized by microfilariae of Dirofilaria immitis
Seung Won Kang*
Department of Parasitology, Veterinary Research Instituti, RDA, Anyang 430-016, Korea.
Received April 01, 1994; Accepted April 16, 1994.

Abstract

This report describes the structures of high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by the microfilariae of Dirofioria immitis. Microfilariae of D. immitis were incubated in vitro in media contaning 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 whech were bound to the immobilized lectin. Upon treatment of 2-[3H] mannose labeled glycopeptides with endo-β-N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography and exoglycosidase digestion. The major species of high mannose-type chains synthesized by microfilariae of D. immitis have the composition Man5 GlcNAc2, Man6 ClcNAc2, Man8 GlcNA2, and Man8 GlcNAc2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by vertebrates.

Figures


Fig. 1
Chromatography on Con A-Sepharose of radiolabeled glycopeptides. Radiolabeled microfilariae of D. immitis were homogenized and treated with Pronase to generate glycopeptides. The glycopeptides were applied to columns of Con A-Sepharose and fractions (2 ml) were collected. Bound glycopeptides were eluted first with 10 mM α-methylglucoside (α-m-Glc) followed by 100mM α-methylmannoside (α-m-Man) as described in MATERIALS AND METHODS. The resulting glycopeptide fractions were pooled and designated I, II, III as indicated. ecovery of applied radioactivity was greater than 90% for column.


Fig. 2
Descending paper chromatogran of the 2-[3H] mannose-labeled III glycopeptides after strong acid hydrolysis. An aliquot of the 2-[3H] mannose-labeled III glycopeptides was hydrolyzed in 2 N HCL at 100℃ for 4 hours and the released sugars were analyzed by descending paper chromatography as described in Materials and Methods. The migration positions of standards are indicated 1, mannose; 2, fucose.


Fig. 3
Chromatography on Bio-Gel P6 of 2-[3H] mannose labeled III glycopeptides before and after treatment with Endo H. 2-[3H] mannose-labeled glycopeptides (from Fig. 1) were applied to a column of Bio-Gel P6. The glycopeptides were then pooled, desalted by column chromatography on Sephadex G25, treated with Endo H as described in Materials and Methods, and re-applied to the Bio-Gel P6 column. 1 and 2 shown were determined by elution position of bovine albumin and galactose respectively. (○) Untreated; (●) Endo H treated.


Fig. 4
Descending paper chromatogram of the 2-[3H] mannose-labeled III glycopeptides after α-mannosidase treatment. After treatment with α-mannosidase, the samples were analyzed directly by descending paper chromatography in solvent III (○) Untreated; (●) Treated.


Fig. 5
Separation by HPLC of the 2-[3H] mannose-labeled oligosaccharides released by Endo H treatment. The elution position of standards are indicated as 1, Man5GlcNAc1; 2, Man6GlcNAc1 ;3, Man7GlcNAc1; 4, Man8GlcNAc1.


Fig. 6
Possible structures proposed for the high mannose-type N-linked oligosaccharides containing 5. 6, 7 and 8 mannose residues. M, mannose; G, N-acetyglucosamine; Asn, asparagine.

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