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Journal of Lipid Research, Vol 36, 2329-2343, Copyright © 1995 by Lipid Research, Inc.
Two novel point mutations in the lecithin:cholesterol acyltransferase (LCAT) gene resulting in LCAT deficiency: LCAT (G873 deletion) and LCAT (Gly344-->Ser)
K Moriyama, J Sasaki, F Arakawa, N Takami, E Maeda, A Matsunaga, Y Takada, K Midorikawa, T Yanase and G Yoshino
Department of Internal Medicine and Biochemistry, School of Medicine, Fukuoka University, Japan.
We investigated the genetic defects in two patients with familial
lecithin:cholesterol acyltransferase (LCAT) deficiency. Their clinical
manifestations including corneal opacities, anemia, proteinuria, and
hypoalphalipoproteinemia were identical for familial LCAT deficiency. Their
LCAT activities and the cholesterol esterification rate (CER) were nearly
zero, and their LCAT masses were below 10% of normal control values.
Sequence analysis of the amplified DNA of case 1 revealed one base deletion
of G at base 873 (first position of Val264) in exon 6, leading to a
premature termination by frameshift. Sequence analysis of amplified DNA of
case 2 revealed a single G to A converting Gly (GGT) to Ser (AGT)
substitution at residue 344. When COS-1 cells were transfected with these
mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was
undetectable (< 0.01 microgram/ml). In contrast, LCAT activity in the
medium of COS-1 cells, transfected with wild-type LCAT, was 1.7 nmol/h per
ml and the LCAT mass was 0.09 micrograms/ml. The LCAT mass in the cell
lysates of the mutants was less than 12% of control for case 1 and 18% of
control for case 2. Northern blot analysis of the mRNA of COS-1 cells
transfected with the mutants showed the same amounts of LCAT mRNA as
compared with wild-type LCAT. Biosynthesis of mutant LCATs was analyzed by
pulse-chase and immunocytochemistry in transfected baby hamster kidney
cells. SDS- PAGE/fluorography demonstrated that wild-type LCAT was
synthesized as a high-mannose type of 56 kDa, which was very slowly
converted to a mature form of 67 kDa and was secreted into the media. In
contrast to the wild-type LCAT, the mutant precursors were not processed
into the mature form but slowly degraded along with chase times. On steady
and continuous labeling in the case of wild-type LCAT, the mature 67 kDa
form was observed in both the cell lysate and media, whereas no mature form
was detected in the cell lysates and media which were transfected mutant
LCATs. These data suggest that the mutant LCATs are actually synthesized in
an amount comparable to that of wild-type, but they are slowly degraded
without being processed into the mature form. The immunocytochemistry
revealed that mutant LCATs were mainly retained in the endoplasmic
reticulum. These data suggest that these two mutations may disrupt the
mutant LCATs' transport from the endoplasmic reticulum into Golgi
apparatus, resulting in LCAT deficiency.

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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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