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Lipoprotein (a) as a risk factor for cardiovascular disease
231
Thursday June 29, 2000: Workshop Abstracts W:27
LIPOPROTEIN(a)
I ThW1:27 I Assembly of lipoprotein(a) S.P.A. McCormick. Department of Biochemistry, University of Otago, Dunedin, New Zealand The assembly of lipoprotein(a) [Lp(a)] is a two-step process. The first step is thought to be a noncovalent interaction between apolipoprotein (apo) B and apolipoprotein(a), while the second step comprises a disulphide link betweea apoBCys4326 and apo(a)Cys4057. Evidence for the initial noncovalent interaction comes from studies of two forms of apoB that lack apoBCys4326 (mouse apoB and human apoBCys4326) which can associate with apo(a) in the plasma. The protein interactions involved in the initial binding between apoB and apo(a) are not fully understood. The aim of our current research is to identify the apoB sequences that are important for its initial binding to apo(a). Recent work targeting the Cys4326 residue into the mouse apoB sequence in a human/mouse hybrid apoB has shown that variation in the carboxyl-terminal sequences of apoB affects the efficiency of Lp(a) formation. In addition, studies of Lp(a) assembly with carboxyl-terminally truncated human apoB proteins have indicted that apoB amino acids 4330-4397 are important for the initial interaction with apo(a). Within the 4330-4397 region, we have identified a stretch of amino acids (residues 4372-4392) that are highly conserved between human and mouse apoB. A synthetic apoB poptide spanning the 4372-4392 sequence has proved to be an effective inhibitor of Lp(a) assembly. Computer analysis of this region predicts an amphipathic alpha-belix containing two sets of paired lysine residues on opposite sides of the helix. Circular dichroism studies of the synthetic peptide have confirmed its alpha-helical nature. Our results indicate that apoB amino acids 4372-4392 play an important role in Lp(a) assembly. Additional studies are currently being performed to further characterise the structural features of this new putative apo(a) binding site.
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Lipoprotein (a) as a risk factor for cardiovascular disease
Lars Berglund. Columbia University, New York, NY, USA Lipoprotein(a), [Lp(a)], has been identified as a risk factor for cardiovascular disease (CAD) in numerous but not all prospective studies. Potentially, several different features of the Lp(a) particle, such as apo(a) protein properties and the apoB/lipid core could contribute to its atherogenicity, separately or in conjunction. Curiously, mean Lp(a) levels are twice as high in Blacks compared to Whites, but studies to date have failed to establish a significant association between elevated Lp(a) levels and CAD among Blacks. The lack of understanding of this racial difference has made it difficult to conclude with full confidence that Lp(a) is a risk factor for CAD. Further, presence of small apo(a) isoform size has been associated with CAD in Whites but not in Blacks. The majority of Whites with high Lp(a) levels also possess at least one small apo(a) isoform, but for Blacks, high mean Lp(a) levels are present over a wider range of apo(a) isoforms. The high correlation between elevated levels of Lp(a) and small apo(a) isoforms in Whites makes it difficult to ascertain whether one is a confounder for the other with regards to CAD. In Blacks, the more common combination of high Lp(a) levels with larger apo(a) sizes provides opportunities to test whether Lp(a) level or apo(a) size is more predicitive of CAD. We compared Lp(a) levels, apo(a) sizes and the level of Lp(a) particles carrying small apo(a) sizes in Blacks and Whites, and found that elevated levels of Lp(a) particles carrying a small apo(a) size were significantly associated with CAD in both groups. The results provide one basis for explaining the nature of the association between Lp(a) and CAD, as well as for the previously observed difference in association between Lp(a) and CAD among Blacks and Whites. The plasma level of Lp(a) carded by small-size apo(a) may be the atherogenic subpopulation of particles that determines the degree of risk for CAD contributed by Lp(a).
tary. Early turnover studies in man revealed that individuals with high plasma Lp(a) levels exhibit a faster rate of synthesis whereas the FCR appeared to be uniform. There are currently two possibilities discussed on Lp(a) assembly: i) Apo(a) is secreted from liver and associates with LDL outside the liver cell mainly in circulating blood, and ii) Lp(a) is intraceUularly assembled. These latter findings were delineated from in vivo kinetics using stable isotopes. Concerning the catabolism of Lp(a) it appears that the liver is the major organ yet LDL-receptors may play an inferior role. There is still intensive work going on aimed at elucidating specific binding mechanisms of to the liver cell. Another organ, which might be important in the removal of Lp(a) from circulation, is the kidney. Not only that an arterio-venous concentration difference in Lp(a) in the order of >5% was found, Lp(a) might also bind to megalin, a member of the LDL-receptor family present in kidney cells. Another point underlining the role of kidney in Lp(a) catabolism is the presence of large apo(a) fragments in the urine. Although the total apo(a) immune reactivity found in urine accounts only for 1% catabolism/day, there is the possibility that small fragments not recognized by antibodies might be also secreted. One major question is which enzyme might be responsible for Lp(a) fragmentation and also what physiological significance might such a proteolytic cleavage have? There is little doubt that the proteases attacking Lp(a) belong to the family of metallo-proteinases of the elastase and collagenase type. Results of our ongoing studies will be presented aimed at elucidating the role of various enzymes in Lp(a) catabolism.
[ ThW4:27 ] Sequence changes in putative enhancer regions upstream of the apolipoprotein(a) gene L. Puckey, B. Knight. MRC Clinical Sciences Centre, London, UK
Objective: Plasma concentrations of the atherogenic lipoprotein(a) [Lp(a)] are almost entirely determined by sequences at or closely linked to the apolipoprotein(a) [apo(a)] gene locus. Two regions upstream of the apo(a) gene have been identified which enhance the activity of the apo(a) promoter in reporter-gene constructs in vitro, DHII, situated about 28kb, and DHIII, about 20kb upstream of the apo(a) gene. The aim of this study was to investigate whether sequence changes in these regions could influence Lp(a) concentrations. Methods: The enhancers were amplified and sequenced from subjects chosen to cover the whole range of Lp(a) concentrations. The sequence changes were introduced into reporter-gene constructs, the resulting change in apo(a) promoter activity measured and those that had an effect were tracked through families to discover any association between the different alleles and Lp(a) levels. Results: No base changes were found in the DHII region. In the DHIII region, 3 common base substitutions were found, an A to G change at position -1230, a C to A change at -1617 and a G to T substitution at -1712. The frequency of these sequence changes were 0.54 (A), 0,84 (C) and 0.89 (G) respectively in a Caucasian population. Changing the A to a G in reportergene constructs increased the activity of the downstream apo(a) promoter approximately 4-fold, while changing the C to a A and the G to a T decreased activity by 50% and 30% respectively. Family studies have shown that the G at -1230 is associated with significantly higher Lp(a) concentrations and the T at -1712 with significantly lower levels. Conclusions: These sequence changes could provide a significant contribution to the variation of plasma Lp(a) concentrations, but arc not solely responsible for determining the large range of concentrations seen in a Caucasian population. I ThW5:27 ] Immunohistoiogical Iocalisation of Lp(a) in the human
kidney ThW3:27 I LP(a) metabolism and in vivo proteolytic fragmentation
H. Dieplinger t, I. Leiter I , E. Trenkwalder I , W. Salvenmoser2, W. Hominger3, K. Lhotta4, P. Ktnig 4, F. Kronenberg] ./Institute of Medical Biology and
Graz, Austria
Human Genetics; 2Institute of Zoology; 3Department of Urology; #Department of Clinical Nephrology; University of lnnsbruck, Austria
The knowledge of specific features of the Lp(a) metabolism is still fragmen-
Objective: Lipoprotein(a) [Lp(a)] is a genetically determined risk factor for
G.M. Kostner, S. Frank. Institute of Medical Biochemistry, University of
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29 2000
Thursday June 29, 2000: Workshop Abstracts W:27
LIPOPROTEIN(a)
I ThW1:27 I Assembly of lipoprotein(a) S.P.A. McCormick. Department of Biochemistry, University of Otago, Dunedin, New Zealand The assembly of lipoprotein(a) [Lp(a)] is a two-step process. The first step is thought to be a noncovalent interaction between apolipoprotein (apo) B and apolipoprotein(a), while the second step comprises a disulphide link betweea apoBCys4326 and apo(a)Cys4057. Evidence for the initial noncovalent interaction comes from studies of two forms of apoB that lack apoBCys4326 (mouse apoB and human apoBCys4326) which can associate with apo(a) in the plasma. The protein interactions involved in the initial binding between apoB and apo(a) are not fully understood. The aim of our current research is to identify the apoB sequences that are important for its initial binding to apo(a). Recent work targeting the Cys4326 residue into the mouse apoB sequence in a human/mouse hybrid apoB has shown that variation in the carboxyl-terminal sequences of apoB affects the efficiency of Lp(a) formation. In addition, studies of Lp(a) assembly with carboxyl-terminally truncated human apoB proteins have indicted that apoB amino acids 4330-4397 are important for the initial interaction with apo(a). Within the 4330-4397 region, we have identified a stretch of amino acids (residues 4372-4392) that are highly conserved between human and mouse apoB. A synthetic apoB poptide spanning the 4372-4392 sequence has proved to be an effective inhibitor of Lp(a) assembly. Computer analysis of this region predicts an amphipathic alpha-belix containing two sets of paired lysine residues on opposite sides of the helix. Circular dichroism studies of the synthetic peptide have confirmed its alpha-helical nature. Our results indicate that apoB amino acids 4372-4392 play an important role in Lp(a) assembly. Additional studies are currently being performed to further characterise the structural features of this new putative apo(a) binding site.
•
Lipoprotein (a) as a risk factor for cardiovascular disease
Lars Berglund. Columbia University, New York, NY, USA Lipoprotein(a), [Lp(a)], has been identified as a risk factor for cardiovascular disease (CAD) in numerous but not all prospective studies. Potentially, several different features of the Lp(a) particle, such as apo(a) protein properties and the apoB/lipid core could contribute to its atherogenicity, separately or in conjunction. Curiously, mean Lp(a) levels are twice as high in Blacks compared to Whites, but studies to date have failed to establish a significant association between elevated Lp(a) levels and CAD among Blacks. The lack of understanding of this racial difference has made it difficult to conclude with full confidence that Lp(a) is a risk factor for CAD. Further, presence of small apo(a) isoform size has been associated with CAD in Whites but not in Blacks. The majority of Whites with high Lp(a) levels also possess at least one small apo(a) isoform, but for Blacks, high mean Lp(a) levels are present over a wider range of apo(a) isoforms. The high correlation between elevated levels of Lp(a) and small apo(a) isoforms in Whites makes it difficult to ascertain whether one is a confounder for the other with regards to CAD. In Blacks, the more common combination of high Lp(a) levels with larger apo(a) sizes provides opportunities to test whether Lp(a) level or apo(a) size is more predicitive of CAD. We compared Lp(a) levels, apo(a) sizes and the level of Lp(a) particles carrying small apo(a) sizes in Blacks and Whites, and found that elevated levels of Lp(a) particles carrying a small apo(a) size were significantly associated with CAD in both groups. The results provide one basis for explaining the nature of the association between Lp(a) and CAD, as well as for the previously observed difference in association between Lp(a) and CAD among Blacks and Whites. The plasma level of Lp(a) carded by small-size apo(a) may be the atherogenic subpopulation of particles that determines the degree of risk for CAD contributed by Lp(a).
tary. Early turnover studies in man revealed that individuals with high plasma Lp(a) levels exhibit a faster rate of synthesis whereas the FCR appeared to be uniform. There are currently two possibilities discussed on Lp(a) assembly: i) Apo(a) is secreted from liver and associates with LDL outside the liver cell mainly in circulating blood, and ii) Lp(a) is intraceUularly assembled. These latter findings were delineated from in vivo kinetics using stable isotopes. Concerning the catabolism of Lp(a) it appears that the liver is the major organ yet LDL-receptors may play an inferior role. There is still intensive work going on aimed at elucidating specific binding mechanisms of to the liver cell. Another organ, which might be important in the removal of Lp(a) from circulation, is the kidney. Not only that an arterio-venous concentration difference in Lp(a) in the order of >5% was found, Lp(a) might also bind to megalin, a member of the LDL-receptor family present in kidney cells. Another point underlining the role of kidney in Lp(a) catabolism is the presence of large apo(a) fragments in the urine. Although the total apo(a) immune reactivity found in urine accounts only for 1% catabolism/day, there is the possibility that small fragments not recognized by antibodies might be also secreted. One major question is which enzyme might be responsible for Lp(a) fragmentation and also what physiological significance might such a proteolytic cleavage have? There is little doubt that the proteases attacking Lp(a) belong to the family of metallo-proteinases of the elastase and collagenase type. Results of our ongoing studies will be presented aimed at elucidating the role of various enzymes in Lp(a) catabolism.
[ ThW4:27 ] Sequence changes in putative enhancer regions upstream of the apolipoprotein(a) gene L. Puckey, B. Knight. MRC Clinical Sciences Centre, London, UK
Objective: Plasma concentrations of the atherogenic lipoprotein(a) [Lp(a)] are almost entirely determined by sequences at or closely linked to the apolipoprotein(a) [apo(a)] gene locus. Two regions upstream of the apo(a) gene have been identified which enhance the activity of the apo(a) promoter in reporter-gene constructs in vitro, DHII, situated about 28kb, and DHIII, about 20kb upstream of the apo(a) gene. The aim of this study was to investigate whether sequence changes in these regions could influence Lp(a) concentrations. Methods: The enhancers were amplified and sequenced from subjects chosen to cover the whole range of Lp(a) concentrations. The sequence changes were introduced into reporter-gene constructs, the resulting change in apo(a) promoter activity measured and those that had an effect were tracked through families to discover any association between the different alleles and Lp(a) levels. Results: No base changes were found in the DHII region. In the DHIII region, 3 common base substitutions were found, an A to G change at position -1230, a C to A change at -1617 and a G to T substitution at -1712. The frequency of these sequence changes were 0.54 (A), 0,84 (C) and 0.89 (G) respectively in a Caucasian population. Changing the A to a G in reportergene constructs increased the activity of the downstream apo(a) promoter approximately 4-fold, while changing the C to a A and the G to a T decreased activity by 50% and 30% respectively. Family studies have shown that the G at -1230 is associated with significantly higher Lp(a) concentrations and the T at -1712 with significantly lower levels. Conclusions: These sequence changes could provide a significant contribution to the variation of plasma Lp(a) concentrations, but arc not solely responsible for determining the large range of concentrations seen in a Caucasian population. I ThW5:27 ] Immunohistoiogical Iocalisation of Lp(a) in the human
kidney ThW3:27 I LP(a) metabolism and in vivo proteolytic fragmentation
H. Dieplinger t, I. Leiter I , E. Trenkwalder I , W. Salvenmoser2, W. Hominger3, K. Lhotta4, P. Ktnig 4, F. Kronenberg] ./Institute of Medical Biology and
Graz, Austria
Human Genetics; 2Institute of Zoology; 3Department of Urology; #Department of Clinical Nephrology; University of lnnsbruck, Austria
The knowledge of specific features of the Lp(a) metabolism is still fragmen-
Objective: Lipoprotein(a) [Lp(a)] is a genetically determined risk factor for
G.M. Kostner, S. Frank. Institute of Medical Biochemistry, University of
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29 2000