The Differential Glycosylation of Human Pro-Urokinase from Various Recombinant Mammalian Cell Lines Does Not Affect Activity and Binding to PAI-1

Edoardo Sarubbi; M Luisa Nolli; Federico Robbiati; Adolfo Soffientini; Franco Parenti; Giovanni Cassani

doi:10.1055/s-0038-1651030

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1989; 62(03): 927-933
DOI: 10.1055/s-0038-1651030

Original Article

Schattauer GmbH Stuttgart

The Differential Glycosylation of Human Pro-Urokinase from Various Recombinant Mammalian Cell Lines Does Not Affect Activity and Binding to PAI-1

Authors

Edoardo Sarubbi

The Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy
M Luisa Nolli

The Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy
Federico Robbiati

The Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy
Adolfo Soffientini

The Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy
Franco Parenti

The Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy
Giovanni Cassani

The Merrell Dow Research Institute, Lepetit Research Center, Gerenzano (Varese), Italy

Abstract

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Summary

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function.

All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs.

No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the K_M and k_cat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1.

In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.

Keywords

Single-chain urokinase-type Plasminogen Activator - Prourokinase - Recombinant DNA - Glycosylation - Plasminogen Activator Inhibitor type-1

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Referenzen

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