Introduction of lysine and clot binding properties in the kringle one domain of tissue-type plasminogen activator.
Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in stimulation of plasminogen activation by fibrin or derivatives as CNBr fragments of fibrinogen. Hypothesizing that sequence differences are responsible for differences in function, we compared the amino acid sequences of K1 and K2 with a consensus kringle sequence. Six consecutive amino acids unique to K2 of t-PA were found, i.e. from Asn248 to Trp253. To test whether these residues are involved in lysine binding, fibrin binding, and fibrin-dependent plasminogen activation, we constructed a set of t-PA mutant proteins containing only a kringle and the protease (P) domain: K2P, K1P, and k1P. In the latter molecule the original amino acid residues Ala160-Ser165 from K1 were substituted by Asn248-Trp253 from K2. As expected, K2P showed enhanced plasminogen activation in the presence of CNBr fragments of fibrinogen, bound to lysine-Sepharose and to a forming fibrin clot. K1P did not show any of these features. In contrast, k1P could be stimulated by CNBr fragments of fibrinogen and bound to lysine-Sepharose and a forming fibrin clot. These results indicate that at least a part of the functional differences between K1 and K2 of t-PA can be localized to a stretch of 6 amino acid residues from Asn248 to Trp253 present in K2.
Gaubius Laboratory IVVO-TNO, Leiden, The Netherlands.
A H
AH
Bakker
W
W
van der Greef
E F
EF
Rehberg
K R
KR
Marotti
J H
JH
Verheijen
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003488:Cyanogen Bromide; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D008239:Lysine; D003488:Cyanogen Bromide