Glycosylation of the thrombin-like serine protease ancrod fromAgkistrodon rhodostoma venom. Oligosaccharide substitution pattern at eachN-glycosylation site

The middle-sized (M) surface proteins of hepatitis B virus (HBV) and other orthohepadnaviruses contain a conserved N-glycan in their pre-S2 domain, which is essential for the secretion of viral particles. Recently, we also found O-glycans in the pre-S2 domain of M protein from woodchuck hepatitis virus (WHV) and HBV genotype D. Since the O-glycosylation motif is not conserved in all genotypes of HBV, the glycosylation patterns of HBV genotypes A and C were analysed. Pre-S2 (glyco)peptides were released from HBV-carrier-derived HBV subviral particles by tryptic digestion, purified by reversed-phase HPLC and identified by amino acid and amino-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion-exchange chromatography, methylation analysis and on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides in all genotypes examined. Pre-S2 O-glycans were characterized by on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS. The pre-S2 domain of M protein and, to a minor extent, of L (large) protein from HBV genotype C and D was partially O-glycosylated by Neu5Ac(a2-3)Gal(b1-3)GalNAca-or Gal(b1-3)GalNAca-units at Thr-37 within a conserved sequence context. Genotype A, containing no Thr at position 37 or 38, was not O-glycosylated. Analytical data further revealed that M protein is mostly amino-terminally acetylated in all examined genotypes and that the terminal methionine is partially oxidized. The findings may be relevant for the secretion and the immunogenicity of HBV.

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“…Oligosaccharides were separated from peptide residues by rHPLC as described previously (Pfeiffer et al, 1993).…”

Section: Methodsmentioning

confidence: 99%

Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus

Schmitt

Glebe

Tolle

et al. 2004

Journal of General Virology

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“…The digestion was repeated with 50 milliunits of enzyme. Oligosaccharides were separated from peptide residues by rHPLC as described previously (37).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Pre-S2 N- and O-Linked Glycans of the M Surface Protein from Human Hepatitis B Virus

Schmitt

Glebe²,

Alving³

et al. 1999

Journal of Biological Chemistry

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The surface antigen of hepatitis B virus comprises a nested set of small (S), middle (M), and large (L) proteins, all of which are partially glycosylated in their S domains. The pre-S2 domain, present only in M and L proteins, is further N-glycosylated at Asn-4 exclusively in the M protein. Since the pre-S2 N-glycan appears to play a crucial role in the secretion of viral particles, the M protein may be considered as a potential target for antiviral therapy. For characterization of the pre-S2 glycosylation, pre-S2 (glyco)peptides were released from native, patient-derived hepatitis B virus subviral particles by tryptic digestion, separated from remaining particles, purified by reversed-phase high performance liquid chromatography, and identified by amino acid and N-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion exchange chromatography, methylation analysis, and on target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides. In addition, the pre-S2 domain of M protein, but not that of L protein, was found to be partially O-glycosylated by a Gal(␤1-3)GalNAc␣-, Neu5Ac(␣2-3)Gal(␤1-3)GalNAc␣-, or GalNAc␣-residue. The respective O-glycosylation site was assigned to Thr-37 by digestion with carboxypeptidases in combination with MALDI-TOF-MS and by quadrupole time-of-flight electrospray mass spectrometry. Analytical data further revealed that about 90% of M protein is N-terminally acetylated. Hepatitis B virus (HBV),1 belonging to the virus family hepadnaviridae, is an important etiological agent of acute and chronic liver disease (1, 2). Chronic HBV infection may lead to liver cirrhosis and hepatocellular carcinoma, which result in about 1 million deaths per year worldwide. The virus replicates in the liver and is secreted in large amounts of up to 10 10 particles/ml into the blood (3). In addition to 42-nm DNA containing virions, infected hepatocytes produce subviral, noninfectious 22-nm spherical or filamentous particles in vast excess. The envelopes of virions and subviral particles contain varying amounts of three related HBV-encoded (glyco)protein species termed large (L), middle (M), and small (S) proteins, which are together referred to as HBV surface antigen (HBsAg). S protein is the major component of virions and both spherical and filamentous HBsAg particles, while filaments and virions contain more M and, in particular, more L proteins than spheres (4, 5). All envelope proteins are produced from a single open reading frame (see Fig. 1A) by the use of three different translation start sites, dividing this open reading frame into three domains: the amino-terminal pre-S1 domain, which occurs exclusively in the L protein; the pre-S2 domain, which is present in both M and L proteins and forms the amino-terminal end of the M protein; and the S domain, which is common to S, M, an...

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“…Separation of oligosaccharide alditols by anion-exchange HPLC on a Mikropak AX-10 (Varian) column, by gel filtration on a Bio-Gel P-4 (Bio-Rad) column, by high-pH anion-exchange HPLC (HPAEC) using Car-boPac PA-l or PA-100 columns (Dionex) and by HPLC using a column of LiChrosorb-NH2 (Merck) have been described in detail previously [11,13,141. Separation of oligosaccharides from peptides by reverse-phase HPLC using a column filled with ODs-Hypersil (3 pm; Shandon) has also been reported earlier [5]. Fractions were monitored for radioactivity by liquid scintillation counting.…”

Section: Isolation Of Tryptic Glycopeptides Recombinant Ancrodmentioning

confidence: 95%

“…("4 mg) was carboxymethylated and digested with trypsin (treated with N-tosyl-L-phenylalanylchloromethane ; Serva) as described previously [5]. For allocation of carbohydrate substituents, glycoproteins were separated by reverse-phase HPLC at pH 6.…”

Section: Isolation Of Tryptic Glycopeptides Recombinant Ancrodmentioning

confidence: 99%

“…In addition, a small proportion (about 2.5%) of the sugar chains was found to cany a single N,N'diacetyllactosediamine (GalNAcp4GlcNAcp) antenna exclusively linked to C2 of Man(a1-3) residues of the pentasaccharide core [4]. Studies at the level of individual N-glycosylation sites further demonstrated that each site displays its own, characteristic, oligosaccharide pattern with regard to branching and the presence of Galp4GlcNAcp (type-2), GalNAcp4GlcNAcp and incomplete antennae [5].…”

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confidence: 99%

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Glycosylation of Recombinant Ancrod from Agkistrodon rhodostoma after Expression in Mouse Epithelial Cells

Geyer

Jacobi

Linder

et al. 1996

European Journal of Biochemistry

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The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl -p-glucosaminy1)asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrixassisted laser desorptionhonization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Galp4GlcNAcp (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gala3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAcp4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N,N-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

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Glycosylation of the thrombin-like serine protease ancrod fromAgkistrodon rhodostoma venom. Oligosaccharide substitution pattern at eachN-glycosylation site

Cited by 19 publications

References 12 publications

Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus

Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus

Analysis of the Pre-S2 N- and O-Linked Glycans of the M Surface Protein from Human Hepatitis B Virus

Glycosylation of Recombinant Ancrod from Agkistrodon rhodostoma after Expression in Mouse Epithelial Cells

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