Conformational differences in human apolipoprotein B-100 among subspecies of low density lipoproteins (LDL). Association of altered proteolytic accessibility with decreased receptor binding of LDL subspecies from hypertriglyceridemic subjects.
We asked at what point in the metabolic cascade of very low density lipoproteins (VLDL) to low density lipoproteins (LDL) the accessibility of proteolytic cleavage sites in B-100 changes, and we evaluated the effect of hypertriglyceridemia on the proteolytic accessibility, secondary structure, and receptor-binding affinity of B-100 in LDL subspecies of varying density. Limited proteolysis with Staphylococcus aureus V8 protease and cathepsin D identified the density (about 1.033 g/ml) between two LDL subspecies, designated LDL-1 and -2, as the transition point during VLDL metabolism of both normolipidemic (N-) and hypertriglyceridemic (HTG-) subjects at which accessibility to protease attack changed in three peptide regions of B-100. Hypertriglyceridemia greatly altered proteolytic accessibility of B-100 in the denser LDL subspecies. Specifically, B-100 in HTG-LDL exposed more cleavage sites than in N-LDL, including two novel sites, approximately 120 and approximately 130 kDa from the NH2 terminus in the small and dense subspecies (designated LDL-4, -4.5 or -5, d = 1.048-1.062 g/ml). Analysis of circular dichroic spectra indicated no difference in helical content between B-100 in N- and HTG-LDL but showed a greater content of beta-structure in HTG-LDL. Binding affinity for the LDL receptor of human fibroblasts decreased markedly with increasing density among HTG-LDL subspecies (by approximately 50% for LDL-4.5 or -5). We conclude that the changes in proteolytic accessibility observed between LDL-1 and -2 and in LDL-4, -4.5, or -5 indicate significant differences in local conformation of B-100 at specific peptide regions. The association of exposure of more cleavage sites, especially novel sites in the NH2-terminal regions, with greatly decreased receptor-binding affinity in LDL-4.5 or -5 suggests that altered local conformation in B-100 apart from the putative receptor-binding domain might affect interaction with the receptor.
Cardiovascular Research Institute, University of California, San Francisco 94143-0130.
G C
GC
Chen
W
W
Liu
P
P
Duchateau
J
J
Allaart
R L
RL
Hamilton
C M
CM
Mendel
K
K
Lau
D A
DA
Hardman
P H
PH
Frost
M J
MJ
Malloy
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D005260:Female; D006801:Humans; D006868:Hydrolysis; D015228:Hypertriglyceridemia / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008297:Male; D017433:Protein Structure, Secondary; D011973:Receptors, LDL / Q000378:metabolism*; D014461:Ultracentrifugation
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D011973:Receptors, LDL