scholarly journals Characterization of the Glycosylation Occupancy and the Active Site in the Follow-on Protein Therapeutic: TNK-Tissue Plasminogen Activator

2010 ◽  
Vol 82 (14) ◽  
pp. 6154-6162 ◽  
Author(s):  
Haitao Jiang ◽  
Shiaw-Lin Wu ◽  
Barry L. Karger ◽  
William S. Hancock
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Protein Therapeutic ◽  
Tissue Plasminogen

scholarly journals The glycosylation of Bowes melanoma tissue plasminogen activator: lectin mapping, reaction with anti-L2/HNK-1 antibodies and the presence of sulphated/glucuronic acid containing glycans

1996 ◽  
Vol 316 (2) ◽  
pp. 427-437 ◽  
Author(s):  
A. J. JAQUES ◽  
G. OPDENAKKER ◽  
T. W. RADEMACHER ◽  
R. A. DWEK ◽  
S. E. ZAMZE
Keyword(s):  
Cell Line ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Glucuronic Acid ◽  
Gel Filtration ◽  
Lectin Affinity Chromatography ◽  
Tissue Plasminogen ◽  
A Cell ◽  
Neuroectodermal Origin

The glycosylation of tissue plasminogen activator (t-PA) obtained from the Bowes melanoma cell line was re-examined using methods of serial lectin affinity chromatography coupled with Bio-Gel P-4 gel filtration chromatography and exoglycosidase sequencing. This study clarified an earlier discrepancy in the literature and confirmed that the major complex N-linked glycans on Bowes t-PA that carry sialic acid as their sole charged group are bi-antennary, core fucosylated, with terminal N-acetylgalactosamine residues. We also report the characterization of a series of related and previously unidentified sialylated glycans. Further we show that Bowes t-PA expresses glucuronic acid/sulphate containing N-linked glycans and is recognized by anti-carbohydrate L2/HNK-1 monoclonal antibodies. The presence on Bowes t-PA of glycans associated primarily with the nervous system is consistent with its expression in a cell line of neuroectodermal origin.

scholarly journals Catabolism of human tissue plasminogen activator in mice

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 539-544 ◽  
Author(s):  
HE Fuchs ◽  
H Jr Berger ◽  
SV Pizzo
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Human Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Organ Distribution ◽  
Tissue Plasminogen ◽  
Iodine 125 ◽  
Distribution Studies

The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenylmethylsulfonyl-125I-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125I-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125I-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125I-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125I-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 125I-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.

scholarly journals Binding of tissue plasminogen activator to human endothelial cells. Importance of the B-chain as a ligand

1992 ◽  
Vol 287 (2) ◽  
pp. 407-413 ◽  
Author(s):  
X F Cheng ◽  
O Bäck ◽  
T K Nilsson ◽  
E Nylander Lundqvist ◽  
G Pohl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Binding Sites ◽  
Human Umbilical Cord ◽  
Cultured Endothelial Cells ◽  
Tissue Plasminogen ◽  
A Chain ◽  
Pai 1

The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.

Sign in / Sign up

Export Citation Format

Share Document