Characterization of the high mannose asparagine-linked oligosaccharides synthesized by microfilariae of <i>Dirofilaria immitis</i>

, Kang, Seung Won

doi:1994.32.2.101

Korean J Parasito > 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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