Glycosylation of recombinant ancrod from Agkistrodon rhodostoma after expression in mouse epithelial cells.
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-beta-glucosaminyl) 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, matrix-assisted laser desorption/ionization 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 Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-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.
Geyer
H
H
Biochemisches Institut am Klinikum, Justus-Liebig-Universität, Giessen, Germany.
Jacobi
I
I
Linder
D
D
Stirm
S
S
Bialojan
S
S
Strube
K H
KH
Geyer
R
R
D017836:Agkistrodon / Q000378:metabolism*; D000721:Ancrod / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D003001:Cloning, Molecular; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D011134:Polysaccharides; D011994:Recombinant Proteins; D000721:Ancrod