The structural domains of human apolipoprotein(a) [apo(a)] and its interaction with apolipoprotein B-100 (ape B-100) in the lipoprotein(a) [Lp(a)] particle were investigated by limited proteolysis with thermolysin and cathepsin D. We characterized the proteolytic products by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, followed by immunoblotting using different antibodies. For apo B-100 in Lp(a), the digestion patterns were found to be identical to those previously 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) resolved under reducing and nonreducing migrating conditions. Using an antibody 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 end. Various Lp(a)s isolated from several donors, and containing different isoforms, were used to study the structural domains of apo(a). Using the same procedure as for apo B-100, several common features were found for the different isoforms. (1) Apo(a) can be cleaved into two structural 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 digested apo(a) isoform, was not linked to apo B-100. Finally, when a recombinant 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) exists independently of the Lp(a) particle and is not due to interactions of apo(a) with apo B-100 or with lipids.
