Human apolipoprotein B-100 heparin-binding sites.
Seven distinct heparin-binding sites have been demonstrated on human apolipoprotein (apo) B-100 by using a combination of digestion with cyanogen bromide or Staphylococcus aureus V-8 protease and heparin-Sepharose affinity chromatography. Based on fragment analysis, the approximate boundaries of the seven binding sites are as follows: site A, residues 5-99; site B, residues 205-279; site C, residues 875-932; site D, residues 2016-2151; site E, residues 3134-3209; site F, 3356-3489; and site G, residues 3659-3719. In sites E and F, two short regions enriched in basic amino acids have been identified, and it is likely that they are responsible for a major portion of the heparin-binding properties of these sites. The relative binding affinity of each of the seven sites was estimated in two ways. First, the affinity was assessed in a ligand blot assay using a 125I-labeled high-reactive heparin subfraction. Second, apoB-100 fragments generated by cyanogen bromide or S. aureus V-8 protease were separated into low- and high-affinity fractions by gradient salt elution of a heparin-Sepharose column. The distribution of the seven binding sites in the two fractions was determined in an immunoblotting assay using antibodies specific to each site, i.e. antibodies raised against synthetic peptide sequences found within each of the seven sites. The results of these two approaches demonstrate that site E and, to a somewhat lesser extent, site F bind to heparin with the highest affinity. Based on the analogy with apoE, in which the high-affinity heparin-binding site coincides with the domain of the protein that interacts with apoB,E (low density lipoprotein) receptors, the results of this study indicate that site E and site F, either singly or in combination, might constitute the receptor binding domain of apoB-100.
Weisgraber
K H
KH
Rall
S C
SC
D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D001057:Apolipoproteins E / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D008077:Lipoproteins, LDL / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D012697:Serine Endopeptidases
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D006493:Heparin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide