Doris A. Sparrow

Binding of a high reactive heparin to human apolipoprotein E: Identification of two heparin-binding domains

Ligand-blotting and dot-blotting procedures were used to investigate the binding of [125I]-heparin to apolipoprotein E, its thrombin fragments E22 (residues 1-191) and E12 (residues 192-299), and to nine apolipoprotein E synthetic fragments. E22 and E12 bound [125I] heparin indicating multiple heparin-binding domains. Synthetic peptides of apoE corresponding to residues 129-169, 139-169, and 144-169, but not 148-169, bound [125I] heparin suggesting that residues 144-147 (Leu-Arg-Lys-Arg) in E22 are important for binding. Peptide 202-243 and 211-243 but not 219-243 bound [125I] heparin suggesting that residues 211-218 (Gly-Glu-Arg-Leu-Arg-Ala-Arg-Met) comprise a portion of the E12 heparin-binding domain.

Interaction of Glycosaminoglycans with Lipoproteinsa

Apolipoproteins B-100 and E are protein constituents of human plasma chylomicrons, very low (VLDL), and low density lipoproteins (LDL). The interaction of lipoproteins with cell receptors is mediated by apoB and E. Lipoproteins also bind to the extracellular matrix, such as glycosaminoglycans (GAG), forming insoluble complexes in the presence of Ca2+. The purpose of this study was to identify the GAG-binding domains in apoB and E. By a combination of fragmentation of the intact proteins, peptide synthesis and quantitative GAG-binding, domains in apoB and apoE were identified and are shown below. These domains contain clusters of basic amino acids that we suggest are required for GAG-binding. table; see text.

Structure and orientation of apo B-100 peptides into a lipid bilayer.

Peptides corresponding to lipid binding domains of Apo B-100 were synthesized, purified, and incubated with dimyristoylphosphatidylcholine (DMPC) liposomes. The secondary structure of the apo B-100 peptide-lipid complexes was evaluated by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Those peptides belonging to the hydrophobic “core” domain of apo B-100 when associated with phospholipids were rich inΒ sheet structure; a predominantα helical conformation was shown to be associated with one peptide located in a surface region of apo B-100. IR dichroic spectra revealed, in the case of the “core” peptides, that theΒ sheet component is the only oriented structure with respect to the phospholipid acyl chains. This orientation of theΒ sheet was recently found in LDL particles after proteolytic digestion by trypsin (Goormaghtigh, E., Cabiaux, V., De Meutter, J., Rosseneu, M., and Ruysschaert, J. M., 1993,Biochemistry32, 6104–6110). Altogether, the data suggest thatΒ sheet, present in a high proportion in the native apo B-100, is probably another protein structure in addition to the amphipathic helix which strongly interacts with the lipid outer layer surrounding the LDL particle.

Plasma lipid transport in the preruminant calf, Bos spp: Primary structure of bovine apolipoprotein A-I

The preruminant calf (Bos spp.) is a model of considerable interest with regard to hepatic and intestinal lipoprotein metabolism (Bauchart et al., J. Lipid Res. (1989) 30, 1499-1514 and Laplaud et al., J. Lipid Res. (1990) 31, 1781-1792). As a preliminary step towards future experiments dealing with HDL metabolism in the calf, we have purified apoA-I from this animal and determined its complete amino acid sequence. Thus, approx. 10% of calf apoA-I was shown to contain a propeptide, with the sequence Arg-His-Phe-Trp-Gln-Gln. Enzymatic cleavage of apoA-I resulted in 10 proteolytic peptides. The complete apoA-I sequence was obtained after alignment of peptides on the basis of their homologies with those from rabbit apoA-I. Thus calf apoA-I consists of 241 amino acid residues, and exhibits high sequence homology with all mammalian apoA-Is studied to date. The bovine protein contained 10 hydrophobic amphipathic helical regions, occurring between residues 43-64, 65-86, 87-97, 98-119, 120-141, 142-163, 164-184, 185-206, 207-217 and 218-241. A computer-constructed phylogenetic tree showed that bovine apoA-I was more closely related to its dog counterpart, including the presence of a single methionine, than to the corresponding macaque and human proteins. Comparative predictions of the respective antigenic structures of human and bovine apoA-Is using the Hopp-Woods algorithm indicated similar positions for all 13 detectable antigenic sites, among which 7 were of identical, or closely related, amino acid composition. This finding was confirmed by demonstration of partial immunological identity between the two proteins upon immunodiffusion analysis, a result obtained using a monospecific rabbit antiserum against bovine apoA-I. Finally, comparison of sequence homology between bovine apoA-I and the lecithin:cholesterol acyl transferase (LCAT) activating region of human apoC-I suggests that several LCAT activating domains may be present in calf apoA-I.

Characterization and amino-terminal sequence of apolipoprotein AI from plasma high density lipoproteins in the preruminant calf, bos Spp

The major apolipoprotein of calf plasma high-density lipoproteins, apo-AI, has been isolated and characterized. Apolipoprotein AI (apo-AI) was separated from the protein moiety of high-density lipoproteins (d 1.090-1.180 g/ml) by preparative electrophoresis in SDS-polyacrylamide gels followed by electrophoretic elution. Purified calf apo-AI had an Mr of approx. 27,000-28,000 in SDS-polyacrylamide gels, resembling human apo-AI. The amino acid composition of calf apo-AI displayed an overall similarity to that of its human and other mammalian counterparts (baboon, dog, badger, rabbit, rat and mouse), but differed in having higher proportions of glutamic acid, alanine and isoleucine. Amino-terminal amino acid sequence analysis up to the 47th residue showed close homology between calf apo-AI and those of the mammals with which it was compared. However, residues 2, 7, 20 and 22 in calf AI (i.e. aspartic acid, serine, glutamic acid and isoleucine, respectively) were substituted by glutamic acid, proline or glutamine, aspartic acid, and valine or leucine respectively, in the other mammals.