Oligosaccharides at each glycosylation site make structure-dependent contributions to biological properties of human tissue plasminogen activator.
The effect of altering oligosaccharide structures at sites 184 and 448 of tissue plasminogen activator (tPA) has been examined. Alteration to high-mannose forms at sites 184 and 448 was accomplished by the growth of cells in the presence of deoxymannojirimycin (dMM). Modification to neutral, unsialylated forms at these sites was achieved by neuraminidase treatment of control preparations of tPA. Oligosaccharides at site 117 were not markedly affected by either treatment because structures at this site are high-mannose and not sialylated in untreated preparations. The effect on enzymatic activity and on a related property, lysine affinity, was determined. dMM treatment was found to increase both the lysine affinity and catalytic activity of tPA. Neuraminidase treatment increased enzyme activity, but was without effect on affinity for lysine. To evaluate the effects of alterations at site 184 and site 448, the catalytic activity and lysine affinity of type I and type II tPA were monitored individually. In the dMM-treated sample, type I tPA (with sugars at sites 117, 184 and 448) was found to have 2- to 3-fold increased catalytic activity and an affinity for lysine which was greater than that of type I from untreated preparations, but less than that of control type II tPA (containing sugar only at sites 117 and 448). In neuraminidase-treated type I, catalytic activity was also enhanced but lysine affinity remained unchanged. Type II from dMM- and neuraminidase-treated preparations had catalytic activity that was increased approximately 1.5-fold compared to untreated controls, whereas affinity for lysine was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
Howard
S C
SC
Monsanto Corporate Research, Monsanto Co., St. Louis, MO 63167.
Wittwer
A J
AJ
Welply
J K
JK
D017485:1-Deoxynojirimycin; D005337:Fibrin / Q000494:pharmacology; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D006023:Glycoproteins; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D005337:Fibrin; D009439:Neuraminidase; D010959:Tissue Plasminogen Activator; D008239:Lysine; D005944:Glucosamine