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.
Klezovitch
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Department of Medicine, University of Chicago, IL 60637, USA. oklezovi@medicine.bsd.uchicago.edu
Scanu
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D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism*; D053241:Apoprotein(a); D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D018082:Kringles; D017270:Lipoprotein(a); D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism
D001053:Apolipoproteins; D017270:Lipoprotein(a); D011994:Recombinant Proteins; D005340:Fibrinogen; D053241:Apoprotein(a); D008239:Lysine