Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells

Abstract

The structure of the N-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo-beta-N-acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography. The results demonstrated that the glycoproteins decorating the virus shed from infected cells have N-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells. However, the E1 glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminal N-acetylglucosamine. The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at different N-glycosylation sites as in the case of E2, or at the same N-glycosylation site as in the case of E1. In the presence of 1-deoxymannojirimycin, no structural heterogeneities in the glycan chains were found. This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes.

Temperature-sensitive steps in the transport of Semliki Forest virus envelope proteins in mosquito C6/36 cells

“…As acylation of E~ takes place in the ER of C6/36 cells even at 8 °C [38] the two bands of E1 were tentatively identified as follows: Lower band: newly synthesized Ea containing a high mannose type glycan; upper band: acylated E1 with a high mannose type glycan. At higher temperatures, additional bands appear corresponding to the glycosylated and trimmed virion forms [29]. A similar interpretation has been made by Bonatti et al [1] for E1 (Sindbis virus, BHK cells) taking the complex gtycans of higher eukaryotes into account.…”

“…This was done by measuring the Endo-H-sensitivity of E 1 at different temperatures [28,29]. Infected C6/36 cells were incubated for 17 h and labeled for 5 min with [35S]methionine at 28 °C.…”

Retinal Glycoprotein Enrichment by Concanavalin A Enabled Identification of Novel Membrane Autoantigen Synaptotagmin-1 in Equine Recurrent Uveitis

“…Retinal glycoproteins were pre-enriched by taking advantage of lectin affinity, following a protocol based on a method previously described by Naim and Koblet [22]. Briefly, ConA-coupled sepharose beads were incubated with retinal tissue.…”

“…For retinal glycoprotein enrichment, we followed a protocol based on a previously described method [22]. We used Concanavalin-A (ConA) coupled sepharose beads (ConA Sepharose 4B, GE Healthcare, Freiburg, Germany), binding buffer (20 mM TrisHCl, 0.5 M NaCl, 1 mM Ca 2+ , 1 mM Mn + , pH 7) and elution buffer (20 mM TrisHCl, 500 mM D-mannose).…”

“…Our approach was based on pre-enriching glycoproteins from retinal tissue via affinity to Concanavalin A (ConA), which is a lectin widely used in glycoprotein enrichment and isolation techniques, especially known for its broad selectivity [21], [22]. This initial enrichment step was followed by a combination of proteomic methods, including Western blot and mass spectrometry and the subsequent successful validation of a candidate protein identified from the pre-enriched glycoprotein fraction.…”