Lys and fibrinogen binding of wild-type (Trp72) and mutant (Arg72) human apo(a) kringle IV-10 expressed in E coli and CHO cells.

In a previous study, we identified a lysine (Lys)-binding-defective form of human lipoprotein(a) and attributed this defect to the presence of a Trp72-->Arg mutation in apolipoprotein(a) [apo(a)] kringle IV-10. To document this relationship, we expressed both wild-type (wt) and mutant (mut) forms of kringle IV-10 in Escherichia coli (nonglycosylated form) and Chinese hamster ovary (CHO) cells (glycosylated form). The Arg72 mut was prepared by introducing the T-->A mutation in apo(a) kringle IV-10 amplified from human liver mRNA by the reverse-transcriptase polymerase chain reaction technique. All expressed kringles were tested for their ability to bind Lys and plasmin-modified fibrinogen (PM-fibrinogen). wt kringle IV-10 expressed in both E coli and CHO cells bound to Lys-Sepharose with comparable affinity. In contrast, the Arg72 mut expressed in both systems exhibited no Lys-binding capacity. Moreover, the wt kringle IV-10 expressed in both systems bound to PM-fibrinogen and exhibited two binding components, one Lys mediated (inhibitable by epsilon-amino-n-caproic acid) and one Lys insensitive, occurring in about the same proportions. Only the latter type of binding was present in the Arg72 mut expressed in E coli. We conclude that kringle IV-10 of human apo(a) has Lys- and PM-fibrinogen-binding capacities that are independent of glycosylation and require the presence of Trp72, one of the seven amino acids that constitute the Lys-binding site of kringle IV-10. Our results also show that the binding of kringle IV-10 to PM- fibrinogen is more complex than that to Lys, in that the former requires an additional binding site or sites outside the Lys-binding site.