Thrombin cleavage of apolipoprotein Bh of rabbit LDL: structural comparisons with human apolipoprotein B-100.
Rabbit plasma low density lipoprotein (LDL) contains one major apolipoprotein of apparent molecular weight of 320 kDa, designated apolipoprotein (apo) Bh, while another component termed apoB1 of apparent molecular weight of 220 kDa is found in chylomicrons. The fragments generated by thrombin digestion of the protein moieties of rabbit and human LDL were separated by polyacrylamide gradient gel electrophoresis and compared. As in the human species, the enzyme produced limited cleavage patterns of rabbit LDL apoB. Within the first 2 h, two fragments (Tr1 and Tr2, with apparent molecular weights 280,000 and 44,000, respectively) appeared. Longer incubations led to the production of two additional peptides, Tr3 and Tr4 (apparent molecular weights 180,000 and 96,000, respectively). Ten monoclonal antibodies, developed against rabbit LDL and designated P01 to P10, were found to react with rabbit apoB. Some also cross-reacted with human apoB. Epitope mapping, performed with these antibodies, showed that Tr3 and Tr4 were derived from the further degradation of Tr1. The rabbit is one of the most frequently used animals in atherosclerosis research. Its LDL receptor has been characterized and there exists a strain of homozygous LDL receptor-deficient rabbits referred to as WHHL rabbits. Despite this, little has been done to characterize the structure of rabbit apoB; only a short region has been sequenced and shown to be the carboxyl-terminal region, the rabbit apoB1. The molecular weight of human apoB (550,000) is much larger than rabbit apoBh. In both species, a primary and secondary thrombin cleavage occur, but the size of the fragments produced is very different between the two species. Identification of the thrombolytic fragments of the rabbit apoB have afforded the opportunity to compare the structures of both apoB species.
Leroy
A
A
SERLIA, Institut Pasteur et INSERM U 279, Lille, France.
Castro
G
G
Agnani
G
G
Saïle
R
R
Barkia
A
A
Fruchart
J C
JC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D006825:Hybridomas / Q000737:chemistry; D008077:Lipoproteins, LDL / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008297:Male; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D000939:Epitopes; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D013917:Thrombin