The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

Summary

Elevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.

The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr¹²-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro¹²-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr¹² or Pro¹² in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (Desafib^TM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafib^TM-X was observed for the Pro¹²-subtype Lp(a).

• References

• 1 Dahlèn GH. Lp(a) lipoprotein in cardiovascular disease. Atherosclerosis 1994; 108: 111-126
• 2 Gaubatz JW, Heideman C, Gotto AM, Morrisett JD, Dahlèn GH. Human plasma lipoprotein(a): structural properties. J Biol Chem 1982; 258: 4582-4589
• 3 Koschinsky ML, Côté G, Gabel B, van der Hoek YY. Identification of the Cys residue in apolipoprotein(a) that mediates extracellular coupling with apolipoprotein-BlOO. J Biol Chem 1993; 268: 19819-19825
• 4 Brunner C, Kraft HG, Utermann G, Muller HJ. Cys⁴⁰⁵⁷ of apolipoprotein(a) is essential for lipoprotein(a) assembly. Proc Natl Acad Sci 1993; 90: 11643-11647
• 5 McLean JW, Tomlinson JE, Kuang WJ, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn R. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature 1987; 300: 132-137
• 6 Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature 1989; 339: 303-305
• 7 Miles LA, Fless GM, Levin EG, Scanu AM, Plow EF. A potential basis for the thrombotic risks associated with lipoprotein(a). Nature 1989; 339: 301-302
• 8 Harpel PC, Gordon BR, Parker TS. Plasmin catalyzes the binding of lipoprotein(a) to immobilized fibrinogen and fibrin. Proc Natl Acad Sci 1989; 86: 3847-3851
• 9 Loscalzo J, Weinfeld M, Fless FM, Scanu AM. Lipoprotein(a), fibrin binding and plasminogen activation. Arteriosclerosis 1990; 10: 240-245
• 10 Rouy D, Koschinsky ML, Fleury V, Chapman JM, Anglès-Cano E. Apolipoprotein(a) and plasminogen interactions with fibrin; a study with recombinant apolipoprotein(a) and isolated plasminogen fragments. Biochemistry 1992; 31: 6333-6339
• 11 Mulichak AM, Tulinsky A. Structure of the lysine fibrin binding subsite of human plasminogen kringle 4. Blood Coag Fibrinolysis 1990; 1: 673-679
• 12 Guevara J, Jan AY, Knapp R, Tulinsky A, Morisett JD. Comparison of ligand-binding sites of modeled apolipoprotein(a) kringle-like sequences in human lipoprotein(a). Arterioscler Thromb 1993; 13: 758-770
• 13 Lackner C, Cohen JC, Hobbs HH. Molecular definition of the extreme size polymorphism in apolipoprotein(a). Hum Mol Genet 1993; 2: 933-940
• 14 Van der Hoek YY, Wittekoek ME, Beisiegel U, Kastelein JJP, Koschinsky ML. The apolipoprotein(a) kringle IV repeats which differ from the major repeat kringle are present in variably-sized isoforms. Hum Mol Genet 1993; 2: 361-366
• 15 Van der Hoek YY, Kastelein JJP, Koschinsky ML. Analysis of structure function relationships in human apolipoprotein(a). Can J Physiol Pharmacol 1994; 72: 304-310
• 16 Kraft HG, Haibach C, Lingenhel A, Brunner C, Trommsdorf M, Kronenberg F, Müller HJ, Utermann G. Sequence polymorphism in kringle IV 37 in linkage disequilibrium with the apolipoprotein(a) size polymorphism. Hum Genet 1995; 95: 275-282
• 17 Scanu AM. Structural and functional polymorphism of lipoprotein(a): Biological and clinical implications. Clin Chem 1995; 41: 170-172
• 18 Utermann G, Menzel HJ, Kraft HG, Duba HC, Kemmler HG, Seitz C. Lp(a) glycoprotein phenotypes, inheritance and relation to the Lp(a)-lipoprotein concentration in plasma. J Clin Invest 1987; 80: 458-465
• 19 Amemiya H, Arinami T, Kikuchi S, Yamakawa-Kobayashi K, Li L, Fujiwara H, Hiroe M, Marumo F, Hamaguchi H. Apolipoprotein(a) size and pentanucleotide repeat polymorphism are associated with the degree of atherosclerosis in coronary heart disease. Atherosclerosis 1996; 123: 181-191
• 20 Kraft HG, Lingenhel A, Köchi S, Hoppicher F, Kronenberg F, Abe A, Miihlberger V, Schönitzer D, Utermann G. Apolipoprotein(a) kringle IV repeat number predicts risk for coronary heart disease. Arterioscler Thromb Vase Biol 1996; 16: 713-719
• 21 Hoover-Plow JL, Miles LA, Fless GM, Scanu AM, Plow EF. Comparison of the lysine binding functions of lipoprotein(a) and plasminogen. Biochemistry 1993; 32: 13681-13687
• 22 Fless GM, Furbee J, Snyder ML, Meredith SC. Ligand-induced conformational change of lipoprotein(a). Biochemistry 1996; 35: 2289-2298
• 23 Scanu AM, Edelstein C. Kringle-dependent structural and functional polymorphism of apolipoprotein(a). Biochim Biophys Acta 1995; 1256: 01-12
• 24 Ernst A, Helmhold M, Brunner C, Pethö Schramm, Armstrong VW, Müller H-J. Identification of two functionally distinct lysine-binding sites in kringle 37 and in kringle 32-36 of human apolipoprotein(a). J Biol Chem 1995; 270: 6227-6634
• 25 Huby T, Schroder W, Doucet C, Chapman J, Thillet J. Characterization of the N-terminal and C-terminal domains of human apolipoprotein(a): relevance to fibrin binding. Biochemistry 1995; 34: 7385-7393
• 26 Trieu VN, McConathy WJ. A two step model for lipoprotein(a) formation. J Biol Chem 1995; 270: 15471-15474
• 27 Van der Hoek YY. Lipoprotein (a). Structure/function analysis and metabolism. Thesis Univ Amsterdam. 1995. ISBN90-9008479-7
• 28 Kamboh MI, Ferrell RE, Kottke BA. Expressed hypervariable polymorphism of apolipoprotein(a). Am J Hum Genet 1991; 49: 1063-1074
• 29 Leus FR, Leerink CB, Prins J, van Rijn HJM. Influence of the apo(a) phenotype on lipoprotein(a) quantification. Evaluation of a nephelometric, immunoradiometric and enzyme-linked immunosorbent assay for Lp(a). Clin Biochem 1994; 27: 449-455
• 30 Scanu AM, Pfaffinger D, Lee JC, Hinman J. A single point mutation (Trp⁷²->Arg) in human apolipoprotein(a) kringle 4-37 associated with a lysine binding defect in lipoprotein(a). Biochim Biophys Acta 1994; 1227: 41-45
• 31 Scanu AM. Identification of mutations in human apolipoprotein(a) kringle 4-37 from the study of the DNA of peripheral blood lymphocytes: Relevance to the role of lipoprotein(a) in atherothrombosis. Am J Card 1995; 75: 58b-61b
• 32 Sangrar W, Marcovina SM, Koschinsky ML. Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells. Protein Engineering 1994; 7: 723-731
• 33 Trommsdorff M, Köchl S, Lingenhel A, Kronenberg F, Delport R, Vermaak H, Lemming L, Klausen IC, Faergeman O, Utermann G, Kraft HG. A pentanucleotide repeat polymorphism in the 5’ control region of the apolip- oprotein(a) gene is associated with lipoprotein(a) plasma concentrations in Caucasians. J Clin Invest 1995; 96: 150-157
• 34 Mancini FP, Mooser V, Guerra R, Hobbs HH. Sequence microheterogeneity in apolipoprotein(a) gene repeats an the relationship to plasma lipoprotein(a) levels. Hum Mol Genet 1995; 4: 1535-1542
• 35 Cohen JC, Chiesa G, Hobbs HH. Sequence polymorphisms in the apolipoprotein(a) gene. Evidence for dissociation between apolipoprotein(a) size and plasma lipoprotein(a) levels. J Clin Invest 1993; 81: 1630-1636
• 36 Mooser V, Mancini F, Bopp S, Pethö-Schramm A, Guerra R, Boerwinkle E, Muller H-J, Hobbs H. Sequence polymorphism in the apo(a) gene associated with specific levels of Lp(a) in plasma. Hum Mol Genet 1995; 4: 173-181
• 37 Guevara J, Knapp RD, Honda S, Northup SR, Morrisett JD. A structural assessment of the apo(a) protein of human lipoprotein(a). Proteins 1992; 12: 188-199
• 38 LoGrasso PV, Comell-Kennon S, Boettcher BR. Cloning, expression, and characterization of human apolipoprotein(a) kringle IV37. J Biol Chem 1994; 269: 21820-21877
• 39 Ernst A, Brunner C, Pethö-Schramm A, Helmhold M, Kraft HG, Utermann G, Armstrong VW, Müller HJ. 1995. Assembly and lysine binding of recombinant lipoprotein(a). Atherosclerosis 1995; 10: 879-883