Characterization of the N-terminal and C-terminal domains of human apolipoprotein(a): relevance to fibrin binding.
The structural domains of human apolipoprotein(a) (apo(a)) in the lipoprotein(a) (Lp(a)) particle have been recently investigated by limited proteolysis [Huby, T., Doucet, C., Dieplinger, H., Chapman, J., & Thillet, J. (1994) Biochemistry 33, 3335-3341]. We have shown that apo(a) can be cleaved into two structural domains: one was of constant size (170 kDa) and corresponded to the C-terminal (Cter) domain of apo(a). This domain was linked by a disulfide bond to apo B100. By contrast, the N-terminal (Nter) domain, whose size varied according to the digested apo(a) isoform, was not linked to apo B100. We now describe the purification of these apo(a) domains and their interaction with fibrin surfaces in an in vitro binding assay. The Nter domain of apo(a) was purified as a soluble protein in a two-step procedure which involved sequential use of a heparin-Sepharose column and a lysine-Sepharose column. The Cter domain of apo(a), which remained in disulfide linkage with apo B100 of Lp(a), was isolated as a lipoprotein particle by a combination of chromatographic steps on heparin-Sepharose and Q-Sepharose columns. This particle, termed "mini-Lp(a)", appeared homogeneous in nondenaturing polyacrylamide gels and exhibited a particle size (285 A) which was intermediate between that of Lp(a) (300 A) and LDL (265 A). The cleavage site between the respective apo(a) domains was determined by N-terminal sequencing of the purified Cter domain. Such cleavage occurred between residues 3532 and 3533, which are located in the interkringle region between apo(a) kringles 4(4) and 4(5).(ABSTRACT TRUNCATED AT 250 WORDS)
Huby
T
T
Institut National de la Santé et de la Recherche Médicale, INSERM U321, Paris, France.
Schröder
W
W
Doucet
C
C
Chapman
J
J
Thillet
J
J
D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D053241:Apoprotein(a); D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D017270:Lipoprotein(a) / Q000097:blood / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013820:Thermolysin
D001053:Apolipoproteins; D017270:Lipoprotein(a); D010446:Peptide Fragments; D005337:Fibrin; D053241:Apoprotein(a); D013820:Thermolysin