Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies

Karen R. Murray, Maya P. Nair, Amir F. Ayyobi, John S. Hill, P. Haydn Pritchard, Andras G. Lacko

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Abstract

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

Original languageEnglish
Pages (from-to)267-275
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume385
Issue number2
DOIs
StatePublished - 15 Jan 2001

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Phosphatidylcholine-Sterol O-Acyltransferase
Antibodies
Enzymes
Mutation
Immunoassay
Epitopes
Plasma (human)
Mammals
Esterification
Lipoproteins
Conformations
Catalyst activity
Animals

Keywords

  • Functional domains
  • High-density lipoprotein
  • LCAT
  • Substrate binding

Cite this

Murray, Karen R. ; Nair, Maya P. ; Ayyobi, Amir F. ; Hill, John S. ; Pritchard, P. Haydn ; Lacko, Andras G. / Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies. In: Archives of Biochemistry and Biophysics. 2001 ; Vol. 385, No. 2. pp. 267-275.
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abstract = "Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.",
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Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies. / Murray, Karen R.; Nair, Maya P.; Ayyobi, Amir F.; Hill, John S.; Pritchard, P. Haydn; Lacko, Andras G.

In: Archives of Biochemistry and Biophysics, Vol. 385, No. 2, 15.01.2001, p. 267-275.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies

AU - Murray, Karen R.

AU - Nair, Maya P.

AU - Ayyobi, Amir F.

AU - Hill, John S.

AU - Pritchard, P. Haydn

AU - Lacko, Andras G.

PY - 2001/1/15

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N2 - Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

AB - Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

KW - Functional domains

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