36

Fresenius J Anal Chem (1992) 343 : 36 - © Springer-Verlag 1992

Molecular genetics of lipid metabolism disorders

Herbert Schuster

Medizinische Poliklinik der Universit/it, Pettenkoferstrasse 8a, W-8000 Miinchen 2, Federal Republic of Germany

Primary dyslipoproteinemias can be due to monogenetic dis- orders of lipoprotein metabolism. These diseases are rare compared to polygenetic disturbances where several genes and environmental factors act together leading to hyperlipidemia. Apolipoprotein E and lipoprotein (a) are two of the major genetic determinants of cholesterol concentration in plasma, nutrition is the most influent exogenic factor. The increasing knowledge of the genetic background of hyperlipoproteinemias and the modern tools of molecular biology have lead to a classification of lipoprotein disorders based on pathophysiology of enzymes, lipoprotein receptors, transport proteins and apolipoproteins. Genetically determined effects of enzymes, re- ceptors and apolipoproteins lead to defects of synthesis, catabol- ism or turnover of a single lipid and its lipoprotein. A classical example of receptor disease is familial hypercholesterolemia characterized by hypercholesterolemia due to a defective LDL- receptor; but hypercholesterolemia can be also generated by a defective ligand to the LDL-receptor, namely apolipoprotein B- 100, producing familial defective apolipoprotein B-100. A lack

of lipoprotein lipase leads to hyperchylomicronemia, a lack of cholesterol ester transfer protein produces hyperalplialipopro- teinemia, whereas familial combined hyperlipidemia seems to be the result of increased turnover of apolipoprotein B. Some of these diseases can be traced to the defective gene, some can only be clarified by examination of the family and linkage analysis with common restriction fragment length polymor- phisms (RFLPs). In the German population familial hypercho- lesterolemia is a heterogeneous disease which is caused by at least 10 different mutations. So far 3 single point mutations and a 4 kilobase insertion are known to cause a defective LDL- receptor in 10% of primary hypercholesterolemia. In 4% the 3 500 arginine to glutamine mutation in the apolipoprotein B- 100 as the underlying defect of familial defective apo B-100 can be identified using the polymerase chain reaction (PCR). Recombinant DNA technology is also used for genotyping common apoliproprotein E variants in diagnosis of familial dysbetalipoproteinemia.

References

Breslow J (1989) J Clin Invest 84 :373 -380 Schonfeld G (i 990) Atherosclerosis 81:81 - 9 3 Schuster H, Rauh G, Kormann B, Hepp T, Humphries S, Keller

C, Z611ner N (1990) Arteriosclerosis 10:577-581 Schuster H, Rauh G, Gerl C, Keller C, Z611ner N (1991) J Med

Genet 28 : 865 - 870

Fresenius J Anal Chem (1992) 343:36-37 - © Springer-Verlag 1992

Advances in the molecular biology of the vascular lipases and apolipoprotein B

Lawrence Chan

Baylor College of Medicine, Department of Cell Biology, 1 Baylor Plaza, Houston, TX 77030, USA

Elevated plasma levels of apolipoprotein (apo) B-containing lipoproteins are associated with accelerated arteriosclerosis and premature coronary artery disease. There are two major apoB species in plasma, apoB-100 and apoB-48. ApoB-100 is the major protein component of very low density lipoprotein (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and lipoprotein (a), and is synthesized in the liver. It is a physiological ligand for the LDL receptor. Genetic defects in. the LDL receptor cause familial hyper- cholesterolemia. Mutations in apoB-100 can result potentially in a similar phenotype. One apoB-100 mutation that has been characterized consists in a Gln for Arg substitution at residue 3500. It is the underlying defect of familial defective apoB-100, which has about the same gene frequency as familial hypercholesterolemia. ApoB-48 is an obligate component of chylomicrons and is synthesized in the small intestine.

The biogenesis of apoB-48 is unique in that the production. of apoB-48 mRNA is based on a newly discovered genetic mechanism. It is the product of a C ~ U conversion ofnucleotide 6666 of apoB-100 mRNA, a process known as RNA editing. The subcellular compartment in which apoB mRNA is edited is unknown. We recently studied the site of endogenous apoB mRNA editing and correlated the extent of editing with mRNA maturation in the rat liver. Although RNA-editing activity was

demonstrated in both nuclear and cytoplasmic extracts, we found that in the rat liver apoB mRNA editing is not a cotranscriptional event. It occurs posttranscriptionally, but the process is essentially complete in the spliced polyadenylated apoB mRNA before it leaves the nucleus. Little, if any, additional editing occurs in the cytoplasmic compartment.

In the area of the structure-function relationships of apoB- 100, progress has been slow because o f the difficulty of working with this large protein. A new area of investigation was initiated by the establishment of transgenic mice that express an apoB minigene. Through its interaction with the LDL receptor, apoB- 100 is a major determinant of LDL metabolism and plasma cholesterol. Its receptor-binding ability is conformation-de- pendent and requires its expression on the right lipoprotein particles. The structural signal that targets apoB-100 to LDL is unknown. We have microinjected a human apoB-100 minigene construct comprising < 25% of the apoB-100 sequence driven by the natural apoB promoter to produce transgenic mice. The transgene product was expressed at a high level and was present exclusively in the LDL of these animals. Analysis of the respon- sible sequence (residues 2878 - 3925 ofapoB-100) reveals unique structural features that may be important in its role as an LDL- targeting domain. Recently, the full-length apoB-100 was also expressed in transgenic mice. It is anticipated that expression of site-specific mutants in this system will generate important new ideas about the structure-function relationships of apoB-100.

One factor that controls the level of plasma apoB-containing lipoproteins is regulated hydrolysis by the vascular lipases. Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HL) are two enzymes that work on a range of lipoprotein substrates: chylomicrons, VLDL and IDL for LPL, and IDL and high- density lipoproteins (HDL) for HL. The structure of human LPL was recently deduced from its cDNA sequence. It contains

eight serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both LPL and HL across all species studied. The high homology between LPL, HL and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism, and that one of the eight conserved serines in human LPL must play a catalytic role as does Serine 152 in the case of pancreatic lipase. We expressed wildtype and site-specific serine mutants of human LPL in Cos cells in vitro, and localized the nucleophilic residue essential for catalysis to Set 132 in human LPL. The observed specific activities of the variants involving the other seven highly conserved serines in human LPL are consistent with the interpre- tation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.

The advances in our knowledge of apoB and the vascular lipases are all made possible through the application of molec- ular biology techniques. They provide the necessary background for understanding the pathophysiological basis of the various

37

dyslipoproteinemias. Those who are interested in the potential role of apoB and the vascular lipases in these disorders should consult some recent articles for detailed information on these lipid related proteins [1 - 5].

References

1. Chan L, Dresel HA (1990) Lab Invest 62: 522-537 2. Chan L, Boerwinkle E, Li W-H (1990) In: Chien S (ed)

Molecular biology of the cardiovascular system. Lea and Febiger, Philadelphia PA, pp 183- 219

3. Yang C-Y, Gu Z-W, Weng S-A, Kim TW, Chen S-H, Pownall H J, Sharp PM, Li W-H, Gotto Jr AM, Chan L (1989) Arteriosclerosis 9 : 9 6 - 1 0 8

4. Faustinella F; Smith LC, Semenkovich CF, Chan L (1991) J Biol Chem 266: 9481 - 9485

5. Hide WA, Chan L, Li W-H (1992) J Lipid Res 33:167-178

Fresenius J Anal Chem (1992) 343:37 - © Springer-Verlag 1992

Lipoprotein(a) - A genetic risk factor for C H D

G. Utermann

Institut ffir Medizinische Biologie und Genetik der Universitfit, Sch6pfstrasse, A-6020 Innsbruck, Austria

Manuscript not received