6 October 1997: Posters Apolipoproteins
predominance of small-dense LDL. The small-dense LDL particles exhibit both high susceptibility to oxidation and low affinity for the LDL receptor, suggesting that these particles may be of higher atherogenic potential. The present investigation examines whether the variation in biological function is due to differences in apo B-100 conformation that alter the interaction with the cellular LDL-receptor. The microenvironments (pK) of Lys residues in surface-exposed domains of apo B-100 in small-dense (d. 1.039-1.044 g/ml) and in intermediate (d. 1,029-l ,035 g/ml) human LDL subspecies have been compared by 13C NMR. The ratio of active (pK 8.9) to normal (pK 10.5) Lys residues in apo B-100 is 0.2 and 0.5 in small-dense and intermediate LDL, respectively. Therefore, differences in protein conformation, as reflected in the Lys microenvironment?., exist in the two LDL subspecies. Quantitative, electrophoretic measurements indicate that the maximal charge difference between the two subspecies is 19e at pH 8.6. The small-dense LDL subspecies is the most negatively charged particle and has a surface charge of -55 f 5e. These results indicate that variations in the conformation of apo B-100 and in the surface charge between the two LDL subspecies are major determinants of their catabolic fate; the higher net negative particle charge reduces binding of small-dense LDL to the LDL receptor and prolongs its plasma residence time. Taken together, these key features underlie the elevated atherogenicity of small-dense LDL.
El1 P 16
Enzyme-linked mice serum
assay for apolipoprotein
Z. Maid, F. Gbaguidi, J.C. Fruchart, P.M. Sullivan, N. Maeda, H. Mezdour, J.C. Gesqui&re, J. Najib. Dc’partement d’AthCros&rose, Institut Pasteur de Lille, Universitd de Lille II, INSERM (1325, I, rue Calmette 59019 Lille Cedex, France; Department of pathology and Laboratoire of Medicine, University of North Carolina, USA Apolipoprotein E plays a major role in the transport and redistribution of lipids among tissues and cells. The mouse model is the best mammalian system for the study of apolipoprotein genes in determining atherosclerosis susceptibility. A non-competitive sandwich ELISA for apo E in mice serum was developed. Antibodies were raised in rabbits against synthetic peptide corresponding to a C-terminal region of mouse apo E 179-241. The monospecificity of these antibodies to this peptide was checked by Western blot and ELISA and no cross-reaction with human apo E was found. For the precision of the assay, the within-day variance was determined as the average of the variance obtained for each day. The coefficient of variation was 8.4%. This method was used to determine the distribution of apo E in mice which expressed only the human apoE isofotm generated by a gene replacement strategy. The results showed significant differences in the distribution of apo E between human apoE homozygous (3/3) mice and their wild type littermates (+/+).
Aging decreases rat apolipoprotein AI expression I 1 P 17 T. Nakamur& A.H. Taylor, N.C.W. Wong. Departments of Medicine & Medical Biochemistry, Univ. of Calgary, Calgary, AB, T2N4Nl, Canada Atherosclerosis of coronary vessels is the number one cause of premature death in modem societies. This acquired disease rises with age and may be associated with changes in the levels of apolipoprotein AI (apo AI). Apo AI is the major protein component of the anti-atherogenic particles, high density lipoprotein (HDL). We previously showed that rat apo AI mRNA decreases with age. Therefore, we examined potential mechanisms underlying these changes. Western and northern blot analysis were used to determine the correlation between levels of apo AI protein and mRNA, respectively in new born, 15 day, 2, 4 and 6 month old rata. Results showed that levels of apo AI protein decreased progressively with age such that the highest levels were observed in the new born animals. Abundance of the protein in 6 month old rats was roughly l/2 compared to that in the new born animals. Similarly, abundance of hepatic apo AI mRNA paralleled the observed reduction in the protein such that in 6 month old rats, the mRNA was only 12.3 f 4.4% of that in the new boms. Since cis-acting site B (-172 to -147) exerts a dominant positive effect on apo AI promoter activity, we measured the activity that binds to this motif in bepatonuclear extract from rats of various age. Results showed site B binding activity to be low in the new born but increased with aging. In summary, our studies show that both ape AI protein and hepatic mRNA levels decrease with age. This level of expression correlates inversely with site B binding activity and raises the possibility that an inhibitory mechanism modulates apo AI expression with age.
1 I th International
1. P 18
Production ceil system
L.E. Pvle, D. Sviridov, Victoria, Australia
AI In a bacukwIrusJbwct Research
To further investigate the antiatherogenic properties of high density lipoprotein, we have been developing the baculovirus/insect cell system in order to study the structure and function of recombinant apolipoprotein AI (apoA1). In viva, apoA1, the major protein component of high density lipoprotein, is translated as a preproprotein and we have previously expressed correctly processed proapoA1 via the above expression system. The function of the six amino acid residue pro-peptide of apoA1 has not been elucidated, but is known to be cleaved from mature apoA1 after secretion into the plasma or lymph. In order to achieve expression of mature apoA1 in the baculovirus/insect cell expression system, the section of cDNA encoding the pro-peptide of apoA1 was deleted from the full-length cDNA using the polymerase chain reaction. The apoAI-Apro cDNA was subcloned into BacPAKgR plasmid for use in co-transfection with BacPAK6R baculovirus into SF21 cells. All cultures were grown in the presence of 1 &ml leupeptin and 1 &ml pepstatin A added daily, to prevent the complete degradation of secreted apoA1. ApoAI secreted into the insect cell culture medium was detected by western blot, and protein transferred to PVDF membrane gave the correct N-terminal sequence Asp-Glu-Pro-Pro-Gln, for mature apoA1. As determined by ion spray mass spectrometry the molecular weight of the recombinant protein was 28081 Da approximately 3 Da different from the theoretical prediction. Preliminary results from large scale cultures of infected insect cells, indicated yields of approximately 5 mg/l, with maximum production between 96120 hours after infection. The amount of apoA1 retained in the cells determined by the ELISA technique, indicates that 50% of the total apoA1 protein remains unsecreted at 96 hours after infection. In conclusion, deletion of the pro-peptide does not prevent correct processing of the signal peptide, however there may be some retardation in secretion of mature apoA1.
1 .P 19
of apo B-100
Michiro Tamura, Yasushi Kobayashi, Akira Tanaka, Fuji0 Numano. Third Department of Internal Medicine, Tokyo Medical and Dental Universi& I-5-45 fiushima, Bunkyo-ku, Tokyo 113, Japan
Apolipoprotein B-48 (B48) and apolipoprotein B-100 (BlOO) has been known as essential proteins carrying triglyceride and cholesterol. Both proteins are encoded in the same gene and the regulation of each protein are caused by the mRNA editing. B48 is known to be produced mainly in small intestine and B 100 in liver. But we have previously reported BlOO is also expressed in intestinal epithelial cells in human immunostaining using monoclonal antibodies against B 100 specifically, which has been proven not to react with B48. To confirm this BlOO expression in small intestine in other way, we isolated epithelium from human small intestinal tissue by crypt isolation method, and analyzed the mRNA by RT-PCR method. The data showed that not only mRNA encoding B48 but also B 100 are expressed in human intestinal epithelium. We speculate the expression of BlOO from intestine might suggest tbe existence of dietary LDL, which could be related with postprandial hyperlipidemia.
Synthesis and secretion chick kidney
P. Tarugi, University
G. Ballarini, of Modena,
1 P 20
B. Pinotti, Italy
of apolipoprotein S. Calandra.
and A-I by
Dept. of Biomedical
Since previous studies have shown that apolipoprotein B-100 (apo B) is synthesized in chick kidney, we investigated whether kidney apo B synthesis was associated with the secretion of apo B-containing lipoproteins. By comparing apo B production (incorporation of 35S-labeled amino acids by tissue slices) by liver and kidney of the same animals we found that: a) total apo B production in kidney was 67% that found in the liver; b) the amount of apo B secreted in the medium by kidney slices was 85% that secreted by the liver. 35S-labeled apo B was found in VLDL, LDL and to some extent in HDL isolated from the medium of kidney slices. Chick kidney was found to synthesize and secrete also apo A-I which is incorporated not only in HDL but also in VLDL and LDL. Immunohistochemistry showed that both apo B and apo A-I were detectable in the cells of proximal and distal tubules. Since in the chick the kidney receives venous blood from the distal portion of the intestine via the inferior mesenteric vein (renal-portal system) we assumed that the renal apo B and apo A-I production was related to the uptake of lipids