Journal of Lipid Research, Vol 24, 877-885, Copyright © 1983 by Lipid Research, Inc.
Limited proteolysis selectively destroys epitopes on apolipoprotein B in low density lipoproteins
KS Hahm, MJ Tikkanen, R Dargar, TG Cole, JM Davie and G Schonfeld
Apolipoprotein B (apoB), the major apoprotein in human low density
lipoprotein (LDL), is a large protein and complex immunogen, against which
seven monoclonal IgG antibodies have been produced in our laboratory. These
antibodies were used to define at least five individual epitopes on
holo-LDL. The aim of the present experiments was to ascertain whether
limited proteolysis of LDL would selectively affect the expression of the
epitopes. LDL was digested with staphylococcal protease (SP), trypsin (T)
or pronase; approximately 20% of LDL protein was lost by treatment with SP
and T, and approximately 60% by treatment with pronase. Structurally stable
SP- and T-LDL cores and soluble peptides were separated by gel permeation
chromatography and zonal ultracentrifugation. The cores resembled holo-LDL
in composition and flotation rates but apoB was fragmented into peptides
less than 100,000 in molecular weight. On analysis by competitive
radioimmunoassays some epitopes of the LDL cores were destroyed partially,
some completely, and some were spared. The enzymes each produced individual
patterns of epitope modulation. SP- and T-LDL cores retained most of their
abilities to be bound and degraded by normal human cultured fibroblasts.
Some soluble peptides also manifested both antigenicity and cell
reactivity. The retention of activities by the cores while active fragments
are lost is compatible with, but does not constitute unequivocal evidence
for, the hypothesis that apoB may consist of repeating structures.
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