| We have previously found that some lepidopteran insect cell lines can synthesize N-linked complex-type oligosaccharides on human plasminogen (HPg) when using the baculovirus/based expression system, when it was formerly believed that insect derived proteins could only assemble high-mannose type glycans. The basis of this oligosaccharide processing pathway is yet unclear but was demonstrated to depend on post-baculoviral infection. To evaluate the contributions from the protein structure to the nature of the glycans obtained and to extend this analysis to other proteins, we studied the carbohydrate structures on recombinant HPg variants with the N-glycosylation site positioned in other regions of the protein. We also identified glycans in other proteins of the fibrinolytic system, viz., recombinant tissue- and, urinary-type plasminogen activators, using the insect expression system in Spodoptera frugiperda. The oligosaccharides obtained after infection with recombinant baculoviruses were characterized first by extensive lectin blotting and then released by hydrazinolysis and fluorescently labeled. The saccharides were then resolved according to charge and hydrophobicity by HPLC, by gel filtration combined with exoglycosidase sequencing using labeled standards for comparison, and by mass spectrometry. The HPg variants contained truncated high mannose glycans with/without fucose, sialylated N-linked complex type and O-linked type oligosaccharides. Recombinant tPA-derived glycans comprised N-linked high mannose and truncated high-mannose structures, with/without core fucosylation; additionally, a small amount (;The N-linked glycans of human blood coagulation Factor IX were also characterized and found to be of the complex-type only, including bi-, tri- and tetra-antennary saccharides with various degrees of sialylation. Its known O-linked glycans were isolated and used as standards. Glycans from plasma HPg were also released and used as controls for evaluation of the techniques.;The importance of protein structural requirements as determinants of glycan processing is discussed. |
