STRUCTURAL DOMAINS OF APOLIPOPROTEIN(A) AND ITS INTERACTION WITH APOLIPOPROTEIN B-100 IN THE LIPOPROTEIN(A) PARTICLE

The structural domains of human apolipoprotein(a) [apo(a)] and its int eraction with apolipoprotein B-100 (ape B-100) in the lipoprotein(a) [ Lp(a)] particle were investigated by limited proteolysis with thermoly sin and cathepsin D. We characterized the proteolytic products by sodi um dodecyl sulfate-polyacrylamide gradient gel electrophoresis, follow ed by immunoblotting using different antibodies. For apo B-100 in Lp(a ), the digestion patterns were found to be identical to those previous ly described [Chen et al. (1989) J. Biol. Chem. 264, 14369-14375; Chen et al. (1991) J. Biol. Chem. 266, 12581-12587] for apo B-100 in LDL. Thus, we compared the digestion patterns of apo B-100 in Lp(a) resolve d under reducing and nonreducing migrating conditions. Using an antibo dy specific for a synthetic peptide of apo B-100 (residues 4004-4021), we confirmed that apo B-100 was linked to apo(a) by its C-terminal en d. Various Lp(a)s isolated from several donors, and containing differe nt isoforms, were used to study the structural domains of apo(a). Usin g the same procedure as for apo B-100, several common features were fo und for the different isoforms. (1) Apo(a) can be cleaved into two str uctural domains: one was of constant size (170 kDa) and was linked to apo B-100. Using an antibody specifically directed against kringle V, we demonstrated that this fragment corresponded to the C-terminal part of apo(a). (2) The other domain, whose size varied according to the d igested apo(a) isoform, was not linked to apo B-100. Finally, when a r ecombinant apo(a) was used instead of Lp(a), it was also cleaved into two domains. This result could indicate that the structure of apo(a) e xists independently of the Lp(a) particle and is not due to interactio ns of apo(a) with apo B-100 or with lipids.