Characterization of the N-Linked Oligosaccharides in Glycoproteins Synthesized by Microfilariae of Dirofilaria immitis

Abstract

In this report, we describe studies on the structures of the N-linked oligosaccharides contained in glycoproteins synthesized by microfilariae of the canine heartworm, Dirofilaria immitis. Microfilariae were incubated in media containing either 2-[3H]mannose, 6-[3H]glucosamine, or 6-[3H]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were radiolabeled by both the mannose and glucosamine, whereas glycoproteins were not radiolabeled by the galactose. Glycopeptides from these total glycoproteins were fractionated and purified by serial lectin affinity chromatography, and the structures of the oligosaccharides in the isolated glycopeptides were analyzed by a variety of techniques. The N-linked oligosaccharides were shown to contain mannose (Man), fucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). However, they lacked sialic acid and galactose, which are commonly found in mammalian glycoproteins. GalNAc was shown to be in an unusual terminal position and beta-linked in the sequence GalNAc beta GlcNAc beta Man-R, where R is the typical branch of complex-type N-linked oligosaccharides. Similar structures were recently found by us to be synthesized by the helminthic parasite Schistosoma mansoni. These results demonstrate that glycoproteins synthesized by microfilariae of D. immitis have unusual carbohydrate moieties and may lead to a better understanding of the specific roles of glycoprotein oligosaccharides in host-parasite interactions.