© 1996 American Heart Association, Inc.
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Lys and Fibrinogen Binding of Wild-Type (Trp72) and Mutant (Arg72) Human Apo(a) Kringle IV-10 Expressed in E coli and CHO Cells
From the Departments of Medicine (O.K., A.M.S.) and of Biochemistry and Molecular Biology (A.M.S.) and the Committees on Genetics (A.M.S.) and Nutrition (A.M.S.), University of Chicago, Chicago, Ill.
Correspondence to Dr Olga Klezovitch, Department of Medicine, University of Chicago, 5841 S Maryland, MC5041, Chicago, IL 60637. E-mail oklezovi@medicine.bsd.uchicago.edu.
Abstract 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 TA 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 
-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.
Key Words: lysine and fibrinogen binding • Lp(a) • apo(a) kringle IV-10
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