Deglycosylation Increases the Fibrinolytic Activity of a Deletion Mutant of Tissue-Type Plasminogen Activator

Summary

Δ₂₋₈₉ t-PA is a deletion mutant lacking the finger (F) and epidermal growth factor (EGF) domains; thus, the fibrin interaction of this molecule must be mediated solely by the kringle region. In the present study, the influence of the oligosaccharide side-chains on the activity of Δ₂₋₈₉ t-PA has been investigated. Δ₂₋₈₉ t-PA was secreted in two forms, designated I and II, which presumably differ by the lack of one asparagine-linked oligosaccharide in the kiiugle 2 domain of form TT, Forms I and II of Δ₂₋₈₉ t-PA weie puiified; form II displayed higher fibrinolytic activity than form I. When foini I was partially deglycosylated or treated to remove sialic acid, fibrinolytic activity was increased. Production of Δ₂₋₈₉ in the presence of timicamycin led to secielion of a glyean-free activator with higher activity. These findings suggest that certain oligusacchaiide side chains, particularly (hose containing sialic acid, can interfere with the interaction between the kringle region of t-PA and fibrin.

• References

• 1 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. On the regulation and control of fibrinolysis. J Biol Chem 1982; 257: 2912-9
• 2 van Zonnefeld A-J, Veerman H, Pannekoek H. Autonomous functions of structural domains on human tissue-type plasminogen activator. Proc Natl Acad Sci USA 1986; 83: 4670-4
• 3 Verheijen JH, Caspers MP M, Chang GT C, de Munk GA W, Pouwels PH, Enger-Valk BE. Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin. EMBO 1986; 5: 3525-30
• 4 Pohl G, Kallstrom M, Bergsdorf N, Wallen P, Jornvall H. Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences. Biochemistry 1984; 23: 3701-7
• 5 Einarsson M, Brandt J, Kaplan L. Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies. Biochim Biophys Acta 1985; 830: 1-10
• 6 Wittwer AJ, Howard SC, Carr LS, Harakas NK, Feder J, Parekh RB, Rudd RM, Dwek RA, Rademacher TW. Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibrolast derived tissue plasminogen activator. Biochemistry 1989; 28: 7662-9
• 7 Kalyan NK, Lee SG, Wilhelm J, Fu KP, Hum W-T, Rappaport R, Hartzell RW, Urbano C, Hung PP. Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH₂-terminal domains on its biochemical and biological properties. J Biol Chem 1988; 263: 3971-8
• 8 Collen D, Stassen J-M, Larsen G. Pharmacokinetics and thrombolytic properties of deletion mutants of human tissue-tvpe plasminogen activator in rabbits. Blood 1988; 71: 216-9
• 9 Larsen GR, Henson K, Blue Y. Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolyic characterization of genetic variants lacking the fibronectin fingerlike and/or the epidermal growth factor domains. J Biol Chem 1988 263. 1023-9
• 10 Hansen L, Blue Y, Barone K, Collen D, Larsen GR. Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator. J Biol Chem 1988; 263: 15713-9
• 11 Wilhelm J, Kalyan N, Lee SG, Hum WT, Hung PP. Influence of carbohydrate on the activity of t-PA mutant lacking the F and EGF domains. FASEB J 1988; 2: A344
• 12 Lee SG, Kalyan N, Wilhelm J, Hum W-T, Rappaport R, Cheng S-M, Dheer S, Urbano C, Hartzell R, Ronchetti-Blume M, Levner M, Hung PP. Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes. J Biol Chem 1988; 263: 2917-24
• 13 Dodd I, Fears R, Robinson JH. Isolation and preliminary characterization of active P-chain of recombinant tissue-type plasminogen activator. Thromb Haemostas 1986; 55: 94-7
• 14 Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959; 234: 1971-5
• 15 Plough J, Kjeldgaard NO. Urokinase an activator of plasminogen from human urine I. Isolation and properties. Biochim Biophys Acta 1957 24. 178-282
• 16 Pohl G, Kenne L, Nilsson B, Einarsson M. Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator. Eur J Biochem 1987; 170: 69-75
• 17 Vehar GA, Spellman MW, Keyt BA, Ferguson CK, Keck RG, Chloupek RC, Harris R, Bennett WF, Builder SE, Hancock WS. Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology. Cold Spring Harbor Symp Quant Biol 1986; 51: 551-62
• 18 Parekh RB, Dwek RA, Thomas JR, Opdenakker G, Rademacher TW, Wittwer AJ, Howard SC, Nelson R, Siegel NR, Jennings MG, Harakas NK, Feder J. Cell-type-specific and site-specific N-glvcosylation of type I and type II human tissue plasminogen activator. Biochemistry 1989; 28: 7644-62
• 19 Tarentino AL, Gomez CM, Plummer TH. Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 1985; 24: 4665-71
• 20 Vali Z, Patthy L. The fibrin-binding site of human plasminogen. Arginines 32 and 34 are essential for fibrin affinity of the kringle 1 domain. J Biol Chem 1984 259. 13690-4
• 21 Motta A, Laursen RA, Llinas M, Tulinsky A, Park CH. Complete assignment of the aromatic proton magnetic resonance spectrum of the kringle 1 domain from human plasminogen: structure of the ligand-binding site. Biochemistry 1987; 26: 3827-36
• 22 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heynecker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E coli. Nature 1983; 301: 214-21
• 23 Belin D, Vassalli J-D, Combepine C, Godeau F, Nagamine Y, Reich E, Kocher HP, Duvoisin RM. Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase-type plasminogen activator. Eur J Biochem 1985; 148: 1225-32