GLYCOSYLATION OF RECOMBINANT ANCROD FROM AGKISTRODON RHODOSTOMA AFTEREXPRESSION IN MOUSE EPITHELIAL-CELLS

The thrombin-like serine protease ancrod from the Malayan pit viper Ag kistrodon rhodostoma was expressed in mouse epithelial cells (C127). O ligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N-4-(N-acetyl-beta-glucosaminyl)asparagine a midase F. Neutral oligosaccharide alditols obtained after reduction an d enzymic desialylation were separated by two-dimensional HPLC and cha racterized by methylation analysis, liquid secondary-ion mass spectrom etry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contr ast to natural ancrod, the recombinant glycoprotein carries exclusivel y diantennary, triantennary and tetraantennary N-glycans with Gal beta 4GlcNAc beta (type-2) antennae which were, in part, further substitut ed by host-cell-specific structural elements such as Gal alpha 3 resid ues or N-acetyllactosamine repeats. As a characteristic feature, a sub stantial proportion of the oligosaccharides bears a GalNAc beta 4GlcNA c antenna. Studies at the level of individual N-glycosylation sites de monstrated that glycans with N,N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Henc e, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein ho rmone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.