The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator.
Tissue-type plasminogen activator (t-PA) is composed of structurally autonomous domains. From the N-terminus of t-PA, a finger-like domain (F), an epidermal growth factor-like domain (G), two kringle domains (K1 and K2) and a serine protease domain (P) can be discerned. The K2 domain of t-PA is known to be involved in lysine binding, fibrin binding and fibrin-dependent plasminogen activation. To study the functional autonomy of the K2 domain in t-PA we constructed, with the aid of a cassette t-PA gene [Rehberg et al. (1989) Protein Engng, 2, 371-377], mutant t-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2P and GK2K1P) in which the K2 domain was placed in two different positions in t-PA. The DNAs of wild-type t-PA and the t-PA variants were expressed in Chinese hamster ovary cells and the recombinant proteins were purified by affinity chromatography. All molecules were expressed in their single-chain form and could be converted to their two-chain form. With these molecules, lysine binding, fibrin binding and fibrin-dependent plasminogen activation were studied. All variants showed affinity for lysyl-Sepharose and aminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versus FGK2K1P and GK1K2P versus GK2K1P) resulted in a 23-47% weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose. Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versus GK2K1P) resulted in a 20-70% improvement of the interactions lysyl-Sepharose and aminohexyl-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D018082:Kringles / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; C030675:lysine-sepharose; C024903:aminohexyl-sepharose; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D012685:Sepharose; D010959:Tissue Plasminogen Activator