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Substrate preferences and redundancy of HACDs
1
The 3-hydroxyacyl-CoA dehydratases HACD1 and HACD2 exhibit functional redundancy and are active
in a wide range of fatty acid elongation pathways
Megumi Sawai‡1, Yu kik o Uchida‡1, Yus uke Ohno‡, Masatoshi Miyamoto‡, Chieko Nishioka§,
Shigeyoshi Itohara§, Taka y uki Sassa‡, and Akio Kihara‡2
From the ‡Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan and the
§RIKEN Brain Science Institute, 2-1 Hirosawa, Wako 351-0198, Japan
Running title: Substrate preferences and redundancy of HACDs
1Both authors contributed equally to this work.
2To whom correspondence should be addressed: Akio Kihara, Faculty of Pharmaceutical Sciences,
Hokkaido University, Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan, Telepho ne:
+81-11-706-3754; Fax: +81-11-706-4900; E-mail: kihara@pharm.hokudai.ac.jp
Keywords: endoplasmic reticulum, fatty acid, fatty acid elongation, fatty acid metabolism,
3-hydroxyacyl-CoA dehydratase, lipid, muscle, myopathy, very long-chain fatty acid
ABSTRACT
Differences among fatty acids (FAs) in chain
length and number of double bonds create lipid
diversity. FA elongation proceeds via a four-step
reaction cycle, in which the 3-hydroxyacyl-CoA
dehydratases HACD1–4 catalyze the third step.
However, the contribution of each HACD to
3-hydroxyacyl-CoA dehydratase activity in
certain tissues or in different FA elongation
pathways remains unclear. HACD1 is specifically
expressed in muscles and is a
myopathy-causative gene. Here, we generated
Hacd1 KO mice and observed that these mice
had reduced body and skeletal muscle weights. In
skeletal muscle, HACD1 mRNA expression was
by far the highest among the HACDs. However,
we observed only a ~40% reduction in HACD
activity and no changes in membrane lipid
composition in Hacd1 KO skeletal muscle,
suggesting that some HACD activities are
redundant. Moreover, when expressed in yeast,
both HACD1 and HACD2 participated in
saturated and monounsaturated FA elongation
pathways. Disruption of HACD2 in the haploid
human cell line HAP1 significantly reduced FA
elongation activities toward both saturated and
unsaturated FAs , and HACD1 HACD2 double
disruption resulted in a further reduction.
Overexpressed HACD3 exhibited weak activity
in saturated and monounsaturated FA elongation
pathways, and no activity was detected for
HACD4. We therefore conclude that HACD1 and
HACD2 exhibit redundant activities in a wide
range of FA elongation pathways, including those
for saturated to polyunsaturated FAs , with
HACD2 being the major 3-hydroxyacyl-CoA
dehydratase. Our findings are important for
furthering the understanding of the molecular
mechanisms in FA elongation and diversity.
http://www.jbc.org/cgi/doi/10.1074/jbc.M117.803171The latest version is at
JBC Papers in Press. Published on August 7, 2017 as Manuscript M117.803171
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Substrate preferences and redundancy of HACDs
2
Fatty acids (FAs) are major components of
lipids. Differences in chain length and the
number and position of double bonds generate
diversity in FAs and lipids. Most cellular FAs are
long-chain FAs (LCFAs; C11–C20), among
which C16 and C18 LCFAs are especially
abundant. FAs of length ≥C21 are called very
long-chain FAs (VLCFAs). Although the cellular
quantities of VLCFAs are much lower than those
of LCFAs, VLCFAs possess characteristic
functions that cannot be performed by LCFAs.
Mutations in yeast and mice resulting in defective
VLCFA synthesis are lethal (1-5). Most of the
VLCFAs are used as components of
sphingolipids but are almost absent in
glycerolipids. (6,7). The functions of VLCFAs
include stabilization of membrane curvature,
membrane microdomain formation, and
enhancement of hydrophobicity of lipids at the
molecular level, which are important for
maintenance of organelle structure and function,
cellular signaling, vesicular trafficking, and
formation of multi-layered lipid structure at the
cellular level (8-13). Physiologically, VLCFAs
play important roles in skin barrier formation,
maintenance of liver integrity, spermatogenesis,
neural functions, myelin maintenance and
formation, and retinal functions (7,13-21).
FAs are elongated via the FA elongation
cycle in the endoplasmic reticulum, using
acyl-CoA as a substrate. In each cycle, the FA
chain length is increased by two carbon units.
The FA elongation cycle consists of four distinct
reactions (condensation, reduction, dehydration,
and reduction), and the enzymes involved are
conserved among eukaryotes (6,7,22) (Fig. 1). In
the first reaction (condensation), acyl-CoAs
receive two carbon units from malonyl-CoA,
generating 3-ketoacyl-CoAs. This step is the
rate-limiting step of the FA elongation cycle and
is catalyzed by FA elongases (ELOVL1–7 in
mammals and Elo1, Fen1/Elo2, and Sur4/Elo3 in
yeast) (6,7,11,22-24). The 3-ketoacyl-CoAs are
then reduced to (R)-3-hydroxy (3-OH) forms by
3-ketoacyl-CoA reductases (KAR/HSD17B12 in
mammals and Ifa38/Ybr159w in yeast) (3,25).
Subsequently, 3-OH acyl-CoAs are dehydrated
by 3-OH acyl-CoA dehydratases
(HACD1/PTPLA, HACD2/PTPLB,
HACD3/PTPLAD1, and HACD4/PTPLAD2 in
mammals and Phs1 in yeast) (4,26). Finally, the
resulting trans-2-enoyl-CoAs are reduced to
acyl-CoAs, which now have two more carbons
than the original acyl-CoAs. This step is
catalyzed by trans-2-enoyl-CoA reductases
(TECR/TER in mammals and Tsc13 in yeast)
(2,25).
The enzymes responsible for the first and
third steps of the FA elongation cycle have
multiple isozymes in mammals (ELOVL1–7 and
HACD1–4). The substrate specificities of the FA
elongases ELOVL1–7 have already been
determined; each exhibits characteristic substrate
specificity toward acyl-CoAs with different chain
lengths and numbers of double bonds (6,7,11,22).
In contrast, the substrate specificities of
HACD1–4 are unknown, mainly due to
limitations in the commercial availability of
3-OH acyl-CoA species. We previously
demonstrated that purified HACD1–4 all exhibit
activity in vitro toward the only commercially
available 3-OH palmitoyl-CoA substrate (26). In
that assay, HACD2 showed the greatest activity,
and that of HACD4 was the lowest (~16-fold
lower than that of HACD2 in terms of Vmax).
HACD2 mRNA is ubiquitously expressed in
tissues (27), and HACD3 mRNA is expressed in
many tissues, such as brain, kidney, liver, and
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Substrate preferences and redundancy of HACDs
3
placenta (26). In contrast, expression of HACD1
and HACD4 mRNA is restricted to muscle tissue
(skeletal muscle and heart) and leukocytes,
respectively (26,28). Mutations in HACD1 cause
myopathy in humans and dogs (29,30). Hacd1
KO mice also exhibit a myopathic phenotype,
with reduced body weight, muscle mass, muscle
force, and muscle fiber diameter (31). During
myogenesis, myoblasts are fused into
multinucleated myotubes, followed by
maturation into myofibers. The fusion process is
retarded in Hacd1 KO myoblasts (31).
In the present study, we aimed to identify
as-yet-undetermined substrate specificities of
HACDs. For this purpose, we performed
lipidomics analyses on newly generated Hacd1
KO mice. Furthermore, the 3-OH acyl-CoA
dehydratase activity of each HACD was
investigated by FA elongation assay, in which
commercially available acyl-CoAs/FAs were
used as substrates, instead of direct measurement
of 3-OH acyl-CoA dehydratase activity using
3-OH acyl-CoA substrates. Our results indicate
that HACD1 and HACD2 exhibit broad substrate
specificities. They are active toward saturated,
monounsaturated, and polyunsaturated 3-OH
acyl-CoAs of long- to very long-chain FA s, with
HACD2 exhibiting greater activity than HACD1.
In contrast, HACD3 showed only weak activity
in saturated and monounsaturated FA elongation
pathways, and no HACD4 activity was detected.
RESULTS
Moderate reduction in 3-OH acyl-CoA
dehydratase activity in Hacd1 KO mice—To
reveal the substrate specificity of HACD1 and the
pathogenesis of the myopathy caused by HACD1
mutations, Hacd1 KO mice were created.
HACD/Phs1 family members contain the active
site residues Tyr and Glu within the fifth
transmembrane segment (32,33). The Hacd1 KO
gene was designed to replace exon 6, which
encodes the active site residues, with a
neomycin-resistant gene (Fig. 2A). Gene
disruption was confirmed by genomic PCR (Fig.
2B). The Hacd1 KO mice exhibited smaller body
size (Fig. 2C) and reduced body and skeletal
muscle (gastrocnemius) weight at one and six
months of age (Fig. 2D and E), compared to the
control mice, as has been reported previously
(31).
In WT skeletal muscle, expression levels of
HACD1 mRNA were much higher than those of
other HACDs (Fig. 3A). However, the reductions
in the 3-OH acyl-CoA dehydratase activity
toward 3-OH palmitoyl-CoA in skeletal muscles
of Hacd1 heterozygous KO (Hacd1+/–) and
Hacd1 homozygous KO (Hacd1–/–) mice relative
to WT mice were relatively small (~20%
reduction in Hacd1+/– mice and ~40% reduction
in Hacd1–/– mice; Fig. 3B). No compensatory
increases in HACD2, HACD3, or HACD4 mRNA
levels were observed in the Hacd1 KO skeletal
muscle (Fig. 3C).
Next, we examined the effect of Hacd1 gene
disruption on the product levels. Since the
acyl-CoA products elongated via the FA
elongation cycle are mainly used for membrane
lipid synthesis, we measured the levels of three
major lipid classes in skeletal muscle by liquid
chromatography (LC)–tandem mass
spectrometry (MS/MS). These were the
sphingolipid sphingomyelin (Fig. 3D) and the
glycerophospholipids phosphatidylcholine (Fig.
3E) and phosphatidylinositol (Fig. 3F). There
were no differences in the levels of these lipids
among WT (Hacd1+/+), Hacd1 heterozygous KO
(Hacd1+/–), and Hacd1 homozygous KO (Hacd1–
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Substrate preferences and redundancy of HACDs
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/–) mice, irrespective of chain lengths (Fig. 3D–
F).
To further examine the effect of Hacd1 gene
disruption on the FA elongation cycle, myoblasts
were prepared from WT and Hacd1 KO mice.
After differentiation into myotube cells, they
were incubated with deuterium (D)-labeled FAs
for 24 h, and their metabolism was traced by
LC-MS/MS analysis. Exogenously added FAs
are metabolized to other FAs via elongation
and/or desaturation within cells (Fig. 4A). When
cells were labeled with D31-palmitic acid
(C16:0-COOH) [palmitic acid with 31
deuteriums (D31)], D31-labeled saturated and
monounsaturated C16–C26 FAs were detected in
WT cells (Fig. 4B). Almost no differences were
observed in the compositions of D31-labeled FAs
between WT and Hacd1 KO cells. Furthermore,
tracer analyses using D9-oleic acid
(C18:1-COOH) indicated that D9-oleic acid
metabolism was indistinguishable between WT
and Hacd1 KO cells (Fig. 4C). Mammals cannot
produce n-6 and n-3 polyunsaturated FAs
endogenously, due to a lack of FA Δ12 desaturase
and FA Δ15 desaturase, and must therefore obtain
these FAs from foods. Food-derived
polyunsaturated FAs are subjected to repetitive
elongation and desaturation in cells, and are
converted to other polyunsaturated FAs (Fig. 4A).
D11-linoleic acid [C18:2(n-6)-COOH] (Fig. 4D)
and D5-α-linolenic acid [C18:3(n-3)-COOH]
labeling (Fig. 4E) experiments revealed that these
FAs were metabolized similarly in WT and
Hacd1 KO cells. Thus, disruption of Hacd1 gene
had no apparent effects on membrane lipid
composition or the FA elongation cycle.
Redundant substrate specificities of
HACD1 and HACD2 in saturated and
monounsaturated FA elongation
pathways—Relatively weak effects on the 3-OH
acyl-CoA dehydratase activity as a result of
Hacd1 gene KO (Fig. 3B) suggest redundancy
amongst the HACDs. We previously reported
that ectopic expression of HACD1 or HACD2 in a
PHS1-shutoff yeast strain complemented growth
defects, whereas neither HACD3 nor HACD4
exhibited such activity (26). In WT yeast S.
cerevisiae, the VLCFAs are almost exclusively
C26:0 (34), whereas the production of at least
some C24:0 VLCFAs is necessary for normal
growth (11). Consequently, the results of the
growth complementation analysis suggest that
both HACD1 and HACD2 possess the ability to
produce C24:0 or longer VLCFAs, and therefore
that there is some redundancy in their function.
We first examined the activity of HACD1
and HACD2 in the saturated FA elongation
pathway, using a yeast system. To minimize the
endogenous 3-OH acyl-CoA dehydratase
activity, we created phs1Δ htd2Δ cells expressing
human ceramide synthase CERS5. In these cells
both yeast HACD homolog PHS1 and the
mitochondrial 3-OH acyl carrier protein
dehydratase HTD2 were deleted. Htd2 is a
component of FA synthase type II (35). Since
VLCFA production is essential for yeast growth,
any genes involved in FA elongation, including
PHS1, cannot be deleted under normal conditions
(4). In yeast, most VLCFAs are used for
sphingolipid synthesis, and the lethality of the
VLCFA-deficient cells can be attributed to the
loss of sphingolipid production due to the
substrate specificity of yeast ceramide synthases.
Therefore, genes of the FA elongation machinery
can be deleted if yeast cells are engineered to
produce ceramides/sphingolipids, such as by
ectopic expression of the human ceramide
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Substrate preferences and redundancy of HACDs
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synthase CERS5, which uses LCFAs for
ceramide synthesis (36).
Phs1, HACD1, or HACD2 were expressed
as triple FLAG (3xFLAG)-tagged proteins in the
phs1Δ htd2Δ/CERS5 cells. Expression levels of
HACD2 were the highest, and those of Phs1 were
the lowest (Fig. 5A). Although the only
commercially available 3-OH acyl-CoA species
is 3-OH palmitoyl-CoA, several acyl-CoA
species can be obtained. Therefore, we performed
an in vitro FA elongation assay, in which
[14C]malonyl-CoA and acyl-CoA were used as
substrates for the FA elongation cycle, instead of
the 3-OH acyl-CoA dehydratase assay using a
3-OH acyl-CoA substrate. Membrane fractions
were prepared from the yeast cells and were
subjected to in vitro FA elongation assay using
stearoyl-CoA (C18:0-CoA) as an acyl-CoA
substrate. After the reactions, the products were
converted to FA methyl esters (FAME s),
separated by reverse-phase TLC, and detected
using autoradiography. In the control phs1Δ
htd2Δ/CERS5 cells bearing the vector,
C18:0-CoA was converted to 3-OH 20:0-CoA via
3-keto 20:0-CoA by endogenous FA elongases
(Fen1 and Sur4) and 3-ketoacyl-CoA reductase
Ifa38 (Fig. 5B). No further conversion was
observed, due to the lack of 3-OH acyl-CoA
dehydratase. When the PHS1 gene was added
back, the cells recovered the ability to produce
C20:0- to C26:0-CoAs. Note that 3-keto
acyl-CoA and trans-2-enoyl-CoA intermediates
were not observed in this assay, since the second
and fourth reactions in the FA elongation cycle
are rapid. Expression of HACD1 caused
production of C20:0- to C26:0-CoAs as well,
although their levels were lower than with Phs1
expression. Instead, more 3-OH acyl-CoA
intermediates were accumulated. Expression of
HACD2 resulted in production of C20:0- to
C26:0-CoAs with similar efficiency to Phs1. The
levels of 3-OH acyl-CoAs were higher than seen
with Phs1, but lower than with HACD1. These
results were assessed in more detail by
LC-MS/MS using the stable isotope 13C-labeled
malonyl-CoA. Again, C20:0- to C26:0-CoAs
were produced under expression of Phs1,
HACD1, or HACD2, and 3-OH acyl-CoA levels
were the highest in HACD1-expressing
membranes (Fig. 5C and D).
We also examined the activities of HACD1
and HACD2 in the monounsaturated FA
elongation pathway using [13C]malonyl-CoA and
oleoyl-CoA (C18:1-CoA) as substrates.
Expression of Phs1, HACD1, or HACD2 resulted
in production of C20:1- to C26:1-CoAs (Fig. 5E).
In most cases, the levels of the acyl-CoAs
produced by HACD1- and HACD2-expressing
membranes were slightly lower than those
produced by Phs1-expressing membranes. The
levels of 3-OH acyl-CoAs were higher in
HACD1- and HACD2-expressing membranes
than those in Phs1-expressing membranes, but
there were no significant differences between
those produced by HACD1- and
HACD2-expressing membranes (Fig. 5F).
Tog ether with the results obtained from the FA
elongation assay using C18:0-CoA, these results
indicate that both HACD1 and HACD2 exhibit
activities toward C20- to C26 saturated and
monounsaturated 3-OH acyl-CoAs. However, the
HACD1-mediated FA elongation cycle produces
more saturated 3-OH acyl-CoA intermediates
(but not monounsaturated 3-OH acyl-CoAs) than
the HACD2-mediated cycle.
Broad and redundant substrate
specificities of HACD1 and HACD2—Functions
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Substrate preferences and redundancy of HACDs
6
of HACD1 and HACD2 in the polyunsaturated
FA elongation cycle could not be determined by
the above yeast system, since yeast FA elongation
machinery cannot elongate polyunsaturated
acyl-CoAs. We therefore used a mammalian
system, in which the HACD gene(s) were
disrupted in the haploid human cell line HAP1 by
CRISPR/Cas9 genome editing. Quantitative
real-time RT-PCR analysis revealed that
expression levels of HACD3 mRNA were the
highest in HAP1 cells, followed by HACD1 and
HACD2 (Fig. 6A). No expression of HACD4 was
detected in HAP1 cells.
HACD1 KO, HACD2 KO, HACD3 KO, and
HACD1 HACD2 double KO (DKO) HAP1 cells
were created. These cells were incubated with
D31-C16:0-COOH for 6 h. Lipids were then
extracted and hydrolyzed to FAs by alkaline
treatment, and D31-labeled FA s were quantified
by LC-MS/MS analysis. Almost no differences
were observed in the compositions of
D31-labeled FAs between the control, HACD1
KO, and HACD3 KO cells (Fig. 6B). In contrast,
≥C18 saturated and monounsaturated FAs were
reduced in HACD2 KO cells, concomitant with
increases in C16 FAs. The HACD1 HACD2 DKO
caused a greater decrease in C18:0 to C22:0 FAs
and increase in C16 FAs than the HACD2 single
KO. Trace amounts of D31-labeled ≥C18
saturated and monounsaturated FAs were still
detected, even in the HACD1 HACD2 DKO cells,
suggesting that HACD3 or other unknown 3-OH
acyl-CoA dehydratases produced them under the
HACD1 and HACD2-null conditions.
When cells were labeled with
D9-C18:1-COOH, similar results were obtained
as with D31-C16:0-COOH labeling. There was a
decrease in ≥C20 monounsaturated FAs in
HACD2 KO cells and HACD1 HACD2 DKO
cells, whereas C18 monounsaturated FA was
increased in these cells (Fig. 6C).
D11-C18:2(n-6)-COOH (Fig. 6D) and
D5-C18:3(n-3)-COOH (Fig. 6E) labeling
experiments indicated that C20, C24, and C26
polyunsaturated FAs were decreased in HACD2
KO cells relative to control cells, and further
decreased in HACD1 HACD2 DKO cells in most
cases, whereas C18 polyunsaturated FAs were
increased. The exception was
C20:5(n-3)-COOH, which was slightly increased
in HACD2 KO cells relative to control cells (Fig.
6E). The levels of C20 polyunsaturated FAs are
determined by the balance between the increase
due to C18-to-C20 conversion and the decrease
due to C20-to-C22 conversion. HACD2
disruption appeared to have a stronger effect on
C20-to-C22 polyunsaturated FA conversion than
on C18-to-C20 polyunsaturated FA conversion,
which might explain the slight increase in
C20:5(n-3)-COOH levels in HACD2 KO cells.
Single gene disruption of HACD1 or HACD3 had
almost no effect on polyunsaturated FA
elongation (Fig. 6D and E). Thus, HACD2 is the
major 3-OH acyl-CoA dehydratase, not only for
saturated and monounsaturated FA elongation,
but also for polyunsaturated FA elongation, and
HACD1 has a redundant function, albeit weaker
than HACD2.
Weak activity of HACD3 in saturated and
monounsaturated FA elongation
pathways—HACD4 mRNA was not expressed in
HAP1 cells (Fig. 6A). Therefore, we could not
examine the activity of HACD4 via the above
assay. Furthermore, the activity of HACD3
remained unclear, since the high activity of
HACD2 might have masked the HACD3 activity.
To circumvent these problems, each of the
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Substrate preferences and redundancy of HACDs
7
3xFLAG-tagged HACDs was overproduced in
HACD1 HACD2 DKO cells. Expression of
3xFLAG-tagged HACD1–4 proteins was
confirmed by immunoblotting (Fig. 7A). Cells
were labeled with deuterium-labeled FAs
[D31-C16:0-COOH (Fig. 7B), D9-C18:1-COOH
(Fig. 7C), D11-C18:2(n-6)-COOH (Fig. 7D), and
D5-C18:3(n-3)-COOH (Fig. 7E)]. Replacing
HACD1 or HACD2 into HACD1 HACD2 DKO
cells reversed the decreased levels of FA
elongation products and increased substrate FA
levels (Fig. 7B–E). Expression of HACD3 had a
slight effect on the elongation of saturated and
monounsaturated FAs but had few effects on that
of polyunsaturated FAs . In the
D31-C16:0-COOH labeling experiment,
C18:0-COOH and C18:1-COOH were increased
relative to the control, but C16:0-COOH was
decreased (Fig. 7B). Similarly, in the
D9-C18:1-COOH labeling experiment,
C20:1-COOH and C22:1-COOH were increased
relative to the control, but C18:1-COOH was
decreased (Fig. 7C). No effect on FA elongation
was observed for HACD4 overexpression,
irrespective of FA species (Fig. 7B–E). These
results indicate that HACD3 exhibits weak
activity towards saturated and monounsaturated
3-OH acyl-CoAs, but that HACD4 exhibits no
such activity.
DISCUSSION
In the FA elongation cycle in mammals, there are
multiple isozymes able to catalyze the first step,
condensation (ELOVL1–7), and the third step,
dehydration (HACD1–4). Although knowledge
relating to the differences in the substrate
specificities and physiological functions of the
ELOVLs has been accumulated (6,7,11,22), such
information was limited for HACD1–4. While it
was known that HACD1–4 exhibits activities
toward 3-OH palmitoyl-CoA in vitro (26), their
activities toward other 3-OH acyl-CoA species,
as well as their actual activities within cells, had
not been determined.
ELOVL1–7 exhibit characteristic substrate
specificities: ELOVL1, saturated and
monounsaturated C20- to C24-CoAs; ELOVL2,
polyunsaturated C20- to C22-CoAs; ELOVL3,
C16- to C22-CoAs; ELOVL4, ≥C24-CoAs;
ELOVL5, polyunsaturated C18- and C20-CoAs;
ELOVL6, saturated and monounsaturated C12-
to C16-CoAs; and ELOVL7, C16- to C20-CoAs
(6,7,11,22,37). This study revealed that, in
contrast to the ELOVLs, the substrate
preferences of HACD1 and HACD2 are quite
broad; both were active in all steps of the FA
elongation pathways of saturated,
monounsaturated, and n-6 and n-3
polyunsaturated FAs (Figs. 5–7). In mouse
skeletal muscle, the expression levels of HACD1
mRNA were much higher (~75-fold) than those
of HACD2 mRNA (Fig. 3A). However, 3-OH
acyl-CoA dehydratase activity toward 3-OH
palmitoyl-CoA in Hacd1 KO skeletal muscle was
reduced only moderately (to ~60% of the
control), and the compositions of the membrane
lipids and metabolism of exogenously added FAs
were unchanged (Figs. 3 and 4). It is likely that
HACD2 was responsible for the remaining
activity in Hacd1 KO muscle. In HAP1 cells,
HACD2 was the predominant 3-OH acyl-CoA
dehydratase over HACD1 (Fig. 6B–E), although
the expression levels of HACD2 mRNA were
71% of those of HACD1 mRNA (Fig. 6A).
Tog ether with the ubiquitous tissue distribution
of HACD2 (27), these results suggest that
HACD2 is the major 3-OH acyl-CoA
dehydratase in the whole body.
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Substrate preferences and redundancy of HACDs
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The need for HACD1 is unclear, since
HACD2 has the same function as and higher
activity levels than HACD1. The only difference
that we observed between HACD1 and HACD2
in this study was in the levels of saturated 3-OH
acyl-CoA intermediates during the FA elongation
process: 3-OH acyl-CoAs were accumulated at
higher levels in HACD1-expressing membranes
than in HACD2-expressing membranes (Fig. 5B
and D). In the FA elongation assay, acyl-CoAs are
the main detectable products, although 3-OH
acyl-CoAs are also detected at low levels (38).
This indicates that the first (condensation) step
catalyzed by ELOVLs is rate limiting, and the
third (dehydration) step catalyzed by HACDs is
secondarily rate limiting in the FA elongation
cycle. Neither 3-ketoacyl-CoAs nor
trans-2-enoyl-CoAs are detected under normal
conditions (38), indicating that the second step,
catalyzed by KAR, and the fourth step, catalyzed
by TECR, are rapid. These fast reactions seem to
be achieved by the interplays between the
ELOVLs and KAR as well as those between the
HACDs and TECR, which enable direct transfer
of the FA elongation cycle intermediates from the
ELOVLs/HACDs to KAR/TECR without release
from the FA elongation machinery (38,39).
However, the detection of 3-OH acyl-CoA
intermediates in the FA elongation cycle (38)
(Fig. 5) implies that some 3-OH acyl-CoAs are
released from the FA elongation machinery and
used for other purposes. We hypothesize that
3-OH acyl-CoAs or their derivatives have certain
regulatory functions in skeletal muscle, such as
cell growth, differentiation, and fusion, and that
this may explain the high levels of HACD1 in
skeletal muscle.
Mutations in HACD1 cause myopathy in
humans and dogs (29,30). Hacd1 disruption also
causes a myopathic phenotype in mice (31),
although the molecular mechanism remains
largely unclear. We also observed decreased body
size, body weight, and skeletal muscle weight in
Hacd1 KO mice (Fig. 2C-E). Since HACD1 is
involved in FA elongation, it had been
hypothesized that membrane lipid compositions,
especially those of longer lipids, might be
affected by Hacd1 disruption in skeletal muscle.
However, we could not detect any differences in
the compositions of membrane lipids (Fig. 3D–
F). This suggests that certain changes in lipid
composition may occur during the developmental
stages, such as the mesoderm to muscle or
myoblast to myofiber differentiation stages.
Although the expression levels of HACD3
mRNA were the highest among the HACDs in
HAP1 cells, gene disruption of HACD3 had no
effect on FA elongation (Fig. 6). The activity of
HACD3 toward saturated and monounsaturated
3-OH acyl-CoAs was only detected when it was
overproduced in HACD1 HACD2 DKO cells
(Fig. 7). In this study, no HACD4 activity was
detected. To date, HACD4 activity toward 3-OH
C16:0-CoA has only been detected in vitro when
HACD4 was solubilized with the nonionic
detergent Triton X-100, although the Vmax value
was the lowest among the HACDs (26). In the
cell-based assay performed in this study, we
examined the activity of HACD4 toward 3-OH
acyl-CoAs with ≥C18, but not toward that with
C16, in the saturated FA elongation pathway.
Considering the low and zero activity of HACD3
and HACD4, respectively, in the cell-based
assay, we hypothesize that their natural substrates
are not 3-OH acyl-CoAs with ≥C18, but rather
specialized forms of 3-OH acyl-CoAs, such as
those containing a short or branched chain. Since
HACD4 is expressed specifically in leukocytes
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Substrate preferences and redundancy of HACDs
9
(26), it is possible that it functions in the
metabolism of pathogen-derived 3-OH short FAs.
For example, the lipid A portion of
lipopolysaccharides of the gram-negative
bacteria E. coli contains 3-OH C14:0-COOH
(40). Further studies are needed, however, to
elucidate the physiological functions and exact
substrates of HACD3 and HACD4.
EXPERIMENTAL PROCEDURES
Generation of Hacd1–/– Mice—The Hacd1
KO targeting vector, which contains the upstream
region (~7,000 bp) of exon 6, the Pgk-neo
(neomycin-resistant gene under the control of the
Pgk promoter) cassette flanked by two loxP
sequences, the downstream region (~2,000 bp) of
exon 6, and the Tk-DTA (diphtheria toxin A under
the control of thymidine kinase promoter)
cassette (Fig. 2A), was constructed using the
recombineering method (41) with the bacterial
artificial chromosome clone bMQ-431D1
(BACPAC Resources Center; Oakland, CA)
prepared from chromosomal DNAs of Mus
musculus AB2.2 (129S7/SvEvBrd-Hprtb-m2) in
ES cells (42). After transfection of the linearized
targeting vector into E14 ES cells, G418-resistant
clones were selected. Genomic DNAs were
prepared from each clone, and homologous
recombination was confirmed by genomic PCR
using primers p1 and p2 (Table 1 and Fig. 2A).
Recombination was also confirmed by Southern
blotting. Positive ES clones were injected into
C57BL/6J blastocysts, and the resulting chimera
mice were crossed with C57BL/6J mice to obtain
Hacd1+/– mice. After crossing Hacd1+/– mice with
C57BL/6J mice repeatedly (≥10 generations of
back-crossing), Hacd1–/– mice were generated by
intercrossing the Hacd1+/– mice. Genotyping was
performed by PCR using genomic DNAs and
primers p3 and p2 for detection of the Hacd1 WT
allele and p1 and p2 for detection of the Hacd1
KO allele (Table 1 and Fig. 2A). All mice were
kept at 23 ± 1 °C in a 12-h light/dark cycle with a
standard chow diet (PicoLab Rodent Diet 20;
LabDiet, St. Louis, MO) and water available ad
libitum. The animal experiments performed in
this study were approved by the institutional
animal care and use committees of Hokkaido
University and RIKEN Brain Science Institute.
Plasmids—The pAK1018 and pAK440
vectors are derivatives of the pRS426 yeast
expression vector (URA3 marker, 2 µ origin),
designed to produce an N-terminal tandemly
oriented His6, Myc epitope, and a triple FLAG
(HMF)-tagged protein under the
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH: TDH3) promoter and a C-terminal
3xFLAG-tagged protein, respectively. The pRF6
(PHS1-3xFLAG) plasmid was constructed by
cloning the PHS1 gene, together with its
promoter, into the pAK440 vector. The pYS10
(HMF-HACD1) and pTN28 (HMF-HACD2)
plasmids were constructed by cloning human
HACD1 and HACD2 into the pAK1018 plasmid,
respectively. The pCE-puro 3xFLAG-1 plasmid
is a mammalian expression vector, designed to
produce an N-terminally 3xFLAG tagged protein
under the human elongation factor 1α promoter
(43). The pCE-puro 3xFLAG-HACD1,
pCE-puro 3xFLAG-HACD2, pCE-puro
3xFLAG-HACD3, and pCE-puro
3xFLAG-HACD4 plasmids are derivatives of the
pCE-puro 3xFLAG-1 vector and were described
previously (26).
The all-in-one CRISPR/Cas9 vector
pYU417, which consists of a Cas9 D10A mutant
nuclease (Cas9 nickase), a guide RNA cloning
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Substrate preferences and redundancy of HACDs
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cassette, EGFP, and puromycin
N-acetyltransferase gene, was constructed from
the GeneArt CRISPR Nuclease Ve c tor with the
OFP plasmid (Thermo Fisher Scientific,
Waltha m, MA) by introducing D10A mutation
into the Cas9 nuclease, incorporating puromycin
N-acetyltransferase gene, and substituting the
ORF reporter with EGFP. Each forward (-F) and
reverse (-R) primer set for guide RNA expression
(for HACD1 KO, HACD1-CC1-F2,
HACD1-CC1-R2, HACD1-CC1-F3, and
HACD1-CC1-R3; for HACD2 KO,
HACD2-CC1-F1, HACD2-CC1-R1,
HACD2-CC1-F2, and HACD2-CC1-R2; for
HACD3 KO, HACD3-CC2-F1,
HACD3-CC2-R1, HACD3-CC2-F2, and
HACD3-CC2-R2; Tab le 1) was annealed and
inserted into the pYU417 vector, generating
pYU-HACD1-CC1-2, pYU-HACD1-CC1-3,
pYU-HACD2-CC1-1, pYU-HACD2-CC1-2,
pYU-HACD3-CC2-1, and pYU-HACD3-CC2-2
plasmids. The all-in-one CRISPR/Cas9 plasmid
(pX330A-puro-HACD1/HACD2) for HACD1
HACD2 DKO was constructed using the
pX330A-puro-1x4, pX330S-puro-2,
pX330S-puro-3, and pX330S-puro-4 vectors in
the Multiplex CRISPR/Cas9 Assembly System
Kit (Addgene, Cambridge, MA) as described
previously (44,45). The primers used for the
construction were HACD2-CC1-F1,
HACD2-CC1-R1, HACD2-CC1-F2,
HACD2-CC1-R2, HACD1-CC1-F2,
HACD1-CC1-R2, HACD1-CC1-F3, and
HACD1-CC1-R3 (Table 1).
Yeas t Strains and Media—The yeast
Saccharomyces cerevisiae strains BY4741
(MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and
6615 (BY4741, htd2Δ::KanMX4) were described
previously (46,47). KMY81 [phs1Δ::LEU2
htd2Δ::KanMX4/pAB119 (3xFLAG-CERS5,
HIS3 marker)] was constructed by deletion of the
PHS1 gene in the yeast strain 6615 bearing the
pAB119 plasmid (36), using a phs1Δ::LEU2
fragment by homologous recombination. Cells
were grown in synthetic complete medium
(0.67% yeast nitrogen base, 2% D-glucose, and
nutritional supplements), but without histidine
and uracil, at 30 °C.
In Vitro 3-OH Acyl-CoA Dehydratase
Assay—Tot al cell lysates were prepared from
mouse gastrocnemius by homogenizing the
tissues, using a homogenizer, in buffer A [50 mM
HEPES/NaOH (pH 7.5), 150 mM NaCl, 10 %
glycerol, 1 × protease inhibitor mixture
(Complete, EDTA-free; Roche Diagnostics,
Basel, Switzerland), 1 mM PMSF, and 1 mM
DTT], followed by sonication and removal of cell
debris by centrifugation (400 × g, 3 min, 4 °C).
They were then subjected to centrifugation at
100,000 × g for 30 min at 4 °C, and the resulting
pellet (total membrane fraction) was suspended
in buffer A. The in vitro 3-OH acyl-CoA assay
was performed as described previously (32) in
reaction buffer I (total volume of 50 µl; buffer A
containing 1 mM CaCl2, 2 mM MgCl2, and 0.1%
digitonin). The total membrane fractions (20 µg
protein) were incubated with 0.01 µCi [14C]3-OH
palmitoyl-CoA (55 mCi/mmol; American
Radiolabeled Chemicals, St. Louis, MO) for 10
min at 37 °C. After terminating the reactions by
adding 25 µl of 75% KOH (w/v) and 50 µl of
ethanol, the lipids were saponified for 1 h at
70 °C. Samples were then acidified by adding
100 µl of 5 M HCl and 50 µl of ethanol. Lipids
were extracted with 700 µl hexane, dried,
suspended in 20 µl of chloroform, separated by
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11
TLC on Silica Gel 60 high performance TLC
plates (Merck, Darmstadt, Germany) with
hexane/diethyl ether/acetic acid (30:70:1, v/v) as
the solvent system, and detected using a
BAS-2500 image analyzer (GE Healthcare Life
Sciences, Little Chalfont, UK).
Real-Time Quantitative RT-PCR—Tot al
RNAs were isolated using the NucleoSpin RNA
II Kit (Takara Bio, Shiga, Japan) and then
converted to cDNA using the PrimeScript II 1st
strand cDNA Synthesis Kit (Takara Bio), both
according to the manufacturer's instructions.
Real-time quantitative PCR was performed using
KOD SYBR qPCR Mix (Toyobo, Osaka, Japan)
and primers (mouse Hacd1, mHacd1-F and
mHacd1-R; mouse Hacd2, mHacd2-F and
mHacd2-R; mouse Hacd3, mHacd3-F and
mHacd3-R; mouse Hacd4, mHacd4-F and
mHacd4-R; mouse Gapdh, mGapdh-F and
mGapdh-R; human HACD1, hHACD1-F and
hHACD1-R; human HACD2, hHACD2-F and
hHACD2-R; human HACD3, hHACD3-F and
hHACD3-R; and human HACD4, hHACD4-F
and hHACD4-R; Table 1) on a CFX96 Touch
Real-Time PCR Detection System (Bio-Rad,
Hercules, CA), according to the manufacturer’s
manual.
LC–MS/MS Analysis—Mouse
gastrocnemius muscles (16 mg) were chopped
and suspended in 144 µl of CHCl3/MeOH/formic
acid (100:200:1, v/v). Lipids were extracted by
mixing samples vigorously in tubes containing
zirconia beads for 1 min at 4 °C using Micro
Smash MS-100 (TOMY Seiko, Tokyo, Japan).
After centrifugation (1,000 × g, 10 min, 4 °C), the
supernatant was mixed with 48 µl of chloroform
and 86.4 µl of water, successively, with vigorous
mixing. Phases were separated by centrifugation
(9,100 × g, 1 min, room temperature), and the
organic phase was recovered and dried. Lipids
were dissolved in 100 µl of methanol for
LC-MS/MS analysis.
LC-MS/MS analyses were performed as
described previously (20,45). Lipids were
resolved and detected by ultra-performance LC
on a reverse-phase column (ACQUITY UPLC
CSH C18 column, length 100 mm; Waters ,
Milford, MA) coupled with electrospray
ionization tandem triple quadrupole MS (Xevo
TQ-S, Wat ers). The conditions of
ultra-performance LC and MS were as follows:
temperature, 55 °C; flow rate, 0.4 ml/min; binary
gradient system with mobile phase A
[acetonitrile/water (3:2, v/v) containing 10 mM
ammonium formate] and mobile phase B
[acetonitrile/2-pronanol (9:1, v/v) containing 10
mM ammonium formate]; elution gradient steps,
0 min, 10% B; 0–6 min, gradient to 40% B; 6–15
min, gradient to 70% B; 15–18 min, gradient to
100% B; 18–23 min, 100% B; 23–23.1 min,
gradient to 10% B; 23.1–25 min, 10% B; with
electrospray ionization capillary voltage, 3.0 kV;
sampling cone, 30 V; and source offset, 50 V for
positive ion mode (for sphingomyelin and FAs)
or capillary voltage, 2.0 kV; sampling cone, 30 V;
and source offset, 30 V for negative ion mode (for
phosphatidylcholine and phosphatidylinositol).
Lipid detection was performed via multiple
reaction monitoring, by selecting specific m/z for
the quadrupole mass filters Q1 and Q3 (Tables
2-6). MassLynx software (Waters) was used for
data analyses and quantification.
Immunoblotting—Immunoblotting was
performed as described previously (48).
Anti-FLAG M2 antibody (1 µg/ml; Agilent
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Substrate preferences and redundancy of HACDs
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Tec hnolo gies, Santa Clara, CA), anti-Pma1
antibody (1:2,000 dilution; Thermo Fisher
Scientific), or anti-GAPDH antibody (1:2,000
dilution; Thermo Fisher Scientific) was used as a
primary antibody. Horseradish
peroxidase-conjugated anti-mouse IgG F(ab’)2
fragment (1:7,500 dilution; GE Healthcare Life
Sciences) was used as a secondary antibody. The
chemiluminescence signal was detected using
Wester n Lightning Plus-ECL (PerkinElmer Life
Sciences, Wal th am, MA) or Pierce ECL Western
Blotting Substrate (Thermo Fisher Scientific).
FA Elongation Assay—Total membrane
fractions were prepared from yeast as described
previously (49). FA elongation assay using
[14C]malonyl-CoA was performed as described
previously (11,12). Briefly, total membrane
fractions (20 µg protein) were incubated with 20
µM stearoyl-CoA (Sigma) complexed with 0.2
mg/ml FA-free bovine serum albumin (Sigma)
and 75 nCi [14C]malonyl-CoA (American
Radiolabeled Chemicals) in reaction buffer II [50
mM HEPES/NaOH (pH 6.8), 150 mM NaCl,
10% glycerol, 1× protease inhibitor mixture, 1
mM PMSF, 1 mM DTT, 2 mM MgCl2, and 1 mM
CaCl2] containing the FA synthase inhibitor
cerulenin (200 µg/ml; Sigma) and 1 mM
NADPH. After the reaction, lipids were
saponified, acidified, extracted, dried, and
converted to FAME s as described previously
(11). FAM Es were extracted, dried, suspended in
20 µl of chloroform, separated by reverse-phase
TLC (Silica Gel 60 RP-18 F254s TLC plates;
Merck) with chloroform/methanol/water
(15:30:2, v/v), and detected using a BAS-2500
image analyzer.
FA elongation assay using
[13C]malonyl-CoA was performed as follows.
Tot al membrane fractions (20 µg protein) were
incubated with 100 µM [13C]malonyl-CoA
(Sigma) and 10 µM acyl-CoA (stearoyl-CoA or
oleoyl-CoA; Ava nti Polar Lipids, Alabaster, AL)
complexed with 0.2 mg/ml FA-free bovine serum
albumin in 50 µl of reaction buffer III [50 mM
HEPES/NaOH (pH 7.5), 150 mM NaCl, 10%
glycerol, 1× protease inhibitor mixture, 1 mM
PMSF, 1 mM DTT, 2 mM MgCl2, and 1 mM
CaCl2] containing 200 µg/ml cerulenin and 1 mM
NADPH for 30 min at 30 °C. The reactions were
terminated by adding 25 µl of 75% KOH (w/v)
and 50 µl of ethanol, then saponified for 1 h at
70 °C, and acidified by adding 100 µl of 5 M
formic acid with 50 µl of ethanol. Lipids were
extracted with 750 µl of hexane and dried. FAs
were derivatized to
N-(4-aminomethylphenyl)pyridinium (AMPP)
amides using the AMP+ Mass Spectrometry Kit
(Cayman Chemical, Ann Arbor, MI), according
to the manufacturer’s protocol, and resolved by
LC-MS/MS as described above.
Cell Culture—HAP1 is a near-haploid
human cell line derived from myelogenous
leukemia (50) and was purchased from the
American Type Culture Collection (Manassas,
VA ) . HAP1 cells were grown in Iscove's
Modified Dulbecco's Medium (12440-053;
Thermo Fisher Scientific) containing 10% FBS,
100 units/ml penicillin, and 100 µg/ml
streptomycin. Transfections were performed
using Lipofectamine Plus Reagent (Thermo
Fisher Scientific), according to the
manufacturer’s instruction. The transfected cells
were subjected to selection in 2 µg/ml puromycin
for two days.
Mouse primary cultured myoblasts were
prepared essentially as described previously (51).
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Substrate preferences and redundancy of HACDs
13
The gastrocnemius, tibialis anterior muscles, and
thigh muscles were collected from the hindlimbs
of the one-month-old mice, shredded in PBS, and
incubated with 5 ml of cell separation solution I
[DMEM (D6429; Sigma) containing 10% FBS
and 1.4 units/ml collagenase D (Roche
Diagnostics)] for 1 h at 37 °C. The muscle cells
were then disaggregated by passing them through
a syringe with an 18 G needle and centrifuged
(650 × g, 3 min, room temperature). The resulting
pellets were suspended in 5 ml of cell separating
solution II [DMEM containing 10% FBS, 1.4
units/ml collagenase D, and 1.5 units/ml dispase
(Thermo Fischer Scientific)] and incubated for 30
min at 37 °C, and then passed through a syringe
with an 18 G needle. The cell solution was
filtered using a 70-µm cell strainer (Corning,
New York , NY) and a 35-µm cell strainer
(Corning). The mouse primary cultured
myoblasts thus obtained were grown in F-10
medium (Thermo Fischer Scientific) containing
20% FBS, 100 units/ml penicillin, 100 µg/ml
streptomycin (Sigma), and 5 ng/ml basic
fibroblast growth factor (PeproTech, Rocky Hill,
NJ). Cells were cultured in dishes coated with
Matrigel (Corning) diluted 10-fold with DMEM.
When the cells began differentiating into
myotube cells, the medium was replaced with
Skeletal Muscle Cell Differentiation Medium
(Takara Bio), and the cells were cultured.
Lipid Labeling Assay—Cells were
incubated with 1 µM deuterium-labeled FA
[D31-palmitic acid (Cayman Chemical),
D9-oleic acid (Avanti Polar Lipids), D11-linoleic
acid (Cayman Chemical), or D5-α-linolenic acid
(Cayman Chemical), where D31 means that the
FA contained 31 deuteriums] for 24 h at 37 °C.
Cells were then washed with PBS, detached from
the dish by incubating them in 0.05%
trypsin/EDTA solution, and resuspended in 100
µl of water. Lipids were extracted by mixing with
successive additions of 375 µl of
chloroform/methanol/formic acid (100:200:1,
v/v), 125 µl of chloroform, and 125 µl of water.
Phases were then separated by centrifugation,
and the organic phase was recovered and treated
with 71 µl of 0.5 M NaOH to hydrolyze the ester
bonds (release FAs from lipids) for 1 h at 37 °C.
After neutralization with 35.5 µl of 1 M formic
acid, lipids were extracted via successive
additions of 175 µl of chloroform and 250 µl of
water with mixing. Phases were separated by
centrifugation, and the organic phase was
recovered, dried, derivatized with an AMP+ Mass
Spectrometry Kit, and resolved by LC-MS/MS as
described above.
Construction of HACD KO Cells—To
obtain HACD1 KO, HACD2 KO, HACD3 KO,
and HACD1 HACD2 DKO cells, HAP1 cells
were transfected with the pYU417 vector (for the
control), the pYU-HACD1-CC1-2 and
pYU-HACD1-CC1-3 plasmids (for HACD1
KO), the pYU-HACD2-CC1-1 and
pYU-HACD2-CC1-2 plasmids (for HACD2
KO), the pYU-HACD3-CC2-1 and
pYU-HACD3-CC2-2 plasmids (for HACD3
KO), or the pX330A-puro-HACD1/2 plasmid
(for HACD1 HACD2 DKO). Twe nty-four hours
after transfection, cells were treated with 2 µg/ml
puromycin for two days. They were then cultured
in Iscove's Modified Dulbecco's Medium without
puromycin for an additional six days. Several
colonies were selected, and clones with
mutations in the genes of interest were used for
further study. The mutations in the selected
KO/DKO cells were as follows: HACD1 KO
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Substrate preferences and redundancy of HACDs
14
cells, 84-bp deletion (26-bp deletion in exon 1
and 58-bp deletion in intron 1); HACD2 KO cells,
33-bp deletion plus 77-bp insertion in exon 1;
HACD3 KO cells, 26-bp deletion in exon 4; and
HACD1 HACD2 DKO cells, 33-bp deletion
(23-bp deletion in exon 1 and 10-bp deletion in
intron 1) in HACD1 gene and 145-bp deletion
(53-bp deletion in exon 1 and 92-bp deletion in
intron 1) in HACD2 gene.
Acknowledgements
We thank Kohei Monobe and Saki Kuwahara for technical assistance.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Author contributions
MS and YU designed and performed the experiments. MS, YU, YO, and MM analyzed the data. YO, CN,
SI, and TS prepared the Hacd1 KO mice. AK planned and organized the project and wrote the manuscript.
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Substrate preferences and redundancy of HACDs
15
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FOOTNOTES
This work was supported by funding from the Advanced Research and Development Programs for Medical
Innovation (AMED-CREST) (to AK) of the Japan Agency for Medical Research and Development
(AMED), by a Grant-in-Aid for Scientific Research (A) 26251010 (to AK) from the Japan Society for the
Promotion of Science (JSPS), and by a Grant-in-Aid for Young Scientists (A) 15H05589 (to YO) from
JSPS.
The abbreviations used are: FA, fatty acid; LCFA, long-chain fatty acid; VLCFA, very long-chain fatty
acid; 3-OH, 3-hydroxy; LC, liquid chromatography; MS/MS, tandem mass spectrometry; D, deuterium;
3xFLAG, triple FLAG; FAM E, fatty acid methyl ester; DKO, double KO; HMF, His6, Myc epitope, and
triple FLAG; AMPP, N-(4-aminomethylphenyl)pyridinium.
FIGURE LEGENDS
FIGURE 1. FA elongation cycle. The four reactions in the FA elongation cycle and the enzymes involved
(red, yeast proteins; blue, mammalian proteins). In one round of the FA elongation cycle, the carbon chain
length of acyl-CoA is increased by two, where malonyl-CoA acts as a carbon donor.
FIGURE 2. Reduced body and skeletal muscle weights in Hacd1 KO mice. A, Schematic representation
of the Hacd1 region in WT (+/+) and Hacd1 KO (–/–) mice as well as the Hacd1 gene-targeting vector
construct. The positions of the primers (p1, p2, and p3) used for genomic PCR are denoted with arrows.
DTA, diphtheria toxin A gene for negative selection; neo, neomycin-resistant gene for positive selection. B,
Genomic DNAs prepared from the tails of Hacd1+/+, Hacd1+/–, and Hacd1–/– mice were subjected to PCR
using primers p1, p2, and p3. The amplified fragments were separated by agarose gel electrophoresis,
followed by staining with ethidium bromide. C, Hacd1+/+ and Hacd1–/– mice at three months old. D and E,
Body weight (D) and gastrocnemius weight (E) of female Hacd1+/+ and Hacd1–/– mice at one and six
months old. Val u e s represent the means ± S.D. from 5–8 mice. Statistically significant differences are
indicated (**, P < 0.01; *, P < 0.05; Student’s t-test). mo, month.
FIGURE 3. Moderate reduction in 3-OH acyl-CoA dehydratase activity and unchanged membrane
lipid composition in Hacd1 KO mice. A, Total RNAs prepared from the gastrocnemius of female WT
mice at one month old were subjected to SYBR green-based real-time quantitative RT-PCR using specific
primers for Hacd1, Hacd2, Hacd3, Hacd4, and Gapdh. Val u e s represent the means ± S.D. relative to Gapdh
expression levels from three independent reactions. Statistically significant differences are represented (**,
P < 0.01; Tukey’s test). B, Total membrane fractions (20 µg protein) prepared from the gastrocnemius of
female Hacd1+/+, Hacd1+/–, and Hacd1–/– mice at one month old were incubated with 0.01 µCi [14C]3-OH
palmitoyl-CoA for 10 min at 37 °C. Lipids were saponified, acidified, extracted, separated by normal-phase
TLC, and detected using a BAS-2500 image analyzer. Values represent the means ± S.D. of the ratio of the
product lipid radioactivity to total lipid radioactivity from three independent reactions. Statistically
significant differences are represented (**, P < 0.01; Tukey’s test). C, Total RNAs prepared from the
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Substrate preferences and redundancy of HACDs
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gastrocnemius of female Hacd1+/+, Hacd1+/–, and Hacd1–/– mice at one month old were subjected to SYBR
green-based real-time quantitative RT-PCR using specific primers for Hacd2, Hacd3, Hacd4, and Gapdh.
Values represent the means ± S.D. relative to Gapdh expression levels from seven (Hacd1+/+ and Hacd1–/–)
or five (Hacd1+/–) independent experiments. No statistically significant differences were detected between
samples using the Tukey–Kramer method. D-F, Lipids were extracted from the gastrocnemius of Hacd1+/+,
Hacd1+/–, and Hacd1–/– mice at P0, and sphingomyelin (D), phosphatidylcholine (E), and
phosphatidylinositol (F) species were analyzed by LC-MS/MS. Value s are the means ± S.D. of the quantity
of each lipid species containing FAs with the indicated chain length and desaturation number from three
independent experiments. No statistically significant differences were detected between the samples by
Tukey’s test.
FIGURE 4. No FA metabolic change in the Hacd1 KO muscle. A, FA metabolic pathways in mammals.
FA elongation and desaturation reactions of saturated FAs (SFAs), monounsaturated FAs (MUFAs), and n-6
and n-3 polyunsaturated FAs (PUFAs) are illustrated. Δ, desaturase. B–E, Mouse primary cultured
myoblasts prepared from WT (+/+) and Hacd1 KO (–/–) mice at one month old were differentiated into
myotube cells for three days and labeled with 1 µM D31-palmitic acid (B), D9-oleic acid (C), D11-linoleic
acid (D), or D5-α-linolenic acid (E) for 24 h at 37 °C. Lipids were extracted, saponified, derivatized to
AMPP amides, and analyzed by LC-MS/MS. Va l ues are the means ± S.D. of the quantities of
deuterium-labeled FAs with the indicated chain length and desaturation number from three independent
experiments. No statistically significant differences were detected between WT and KO samples by
Student’s t-test.
FIGURE 5. HACD1 and HACD2 exhibit broad substrate specificities toward saturated and
monounsaturated 3-OH acyl-CoAs. A–F, Total membrane fractions were prepared from the yeast
KMY81 [phs1Δ htd2Δ/pAB119 (3xFLAG-CERS5)] cells bearing the pAK739 (vector), pRF6
(PHS1-3xFLAG), pYS10 (HMF-HACD1), or pTN28 (HMF-HACD2) plasmid and subjected to
immunoblotting with anti-FLAG and anti-Pma1 (membrane protein loading control) antibodies (A) and FA
elongation assays using [14C]malonyl-CoA (B) or [13C]malonyl-CoA (C-F). B, Total membrane fractions
(20 µg protein) were incubated with 75 nCi [14C]malonyl-CoA and 20 µM stearoyl-CoA for 30 min at
37 °C. After the reaction, lipids were saponified, converted to FAM Es, separated by reverse-phase TLC,
and detected using a BAS-2500 image analyzer. The acyl-CoA and 3-OH acyl-CoA intermediates of the FA
elongation cycle, starting from stearoyl-CoA (C18:0), are illustrated in the right panel. C–F, Tota l
membrane fractions (20 µg protein) were incubated with 100 µM [13C]malonyl-CoA and 10 µM
stearoyl-CoA (C and D) or oleoyl-CoA (E and F) for 30 min at 37 °C. After the reaction, lipids were
saponified, derivatized to AMPP amides, and analyzed by LC-MS/MS. Va l ues are the means ± S.D. of the
quantities of acyl-CoA derivatives (C and E) or 3-OH acyl-CoA derivatives (D and F) with the indicated
chain length and desaturation number from three independent experiments. Statistically significant
differences are calculated by Tukey’s test (**, P < 0.01; *, P < 0.05). The asterisks without indication bars
represent significant differences from the vector control. vec, vector.
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Substrate preferences and redundancy of HACDs
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FIGURE 6. HACD2 is the major 3-OH acyl-CoA dehydratase in HAP1 cells. A, Total RNAs prepared
from HAP1 cells were subjected to SYBR green-based real-time quantitative RT-PCR using specific
primers for HACD1, HACD2, HACD3, and HACD4. Absolute values of the mRNA levels were calculated
by comparison with the levels of the PCR products amplified from the corresponding plasmid encoding
HACD1, HACD2, HACD3, or HACD4. Va l u e s represent the means ± S.D. from three independent
reactions. Statistically significant differences are represented (**, P < 0.01; Tukey’s test). ND, not detected.
B–E, WT (control), HACD1 KO, HACD2 KO, and HACD1 HACD2 DKO HAP1 cells were treated with 1
µM D31-palmitic acid (B), D9-oleic acid (C), D11-linoleic acid (D), or D5-α-linolenic acid (E) for 6 h (B
and E), 2 h (C), or 4 h (D) at 37 °C. Lipids were extracted, saponified, derivatized to AMPP amides, and
analyzed by LC-MS/MS. Valu e s are the means ± S.D. of the quantities of deuterium-labeled FAs with the
indicated chain length and desaturation number from three independent experiments. Statistically
significant differences are calculated by Tukey’s test (**, P < 0.01; *, P < 0.05). Of the significant
differences found, only those from the control (without indication bars) and those between HACD2 KO and
HACD1 HACD2 DKO (with indication bars) are represented. Con, control; H, HACD.
FIGURE 7. Redundancy and broad substrate preferences of HACD1 and HACD2. A–E, HACD1
HACD2 DKO HAP1 cells were transfected with the pCE-puro 3xFLAG-1 (vector), pCE-puro
3xFLAG-HACD1, pCE-puro 3xFLAG-HACD2, pCE-puro 3xFLAG-HACD3, or pCE-puro
3xFLAG-HACD4 plasmid. Twenty-four hours after transfection, 2 µg/ml puromycin was added to the
culture medium to kill the non-transfected cells, and the culture was incubated for a further 24 h. Cells were
then treated with 1 µM D31-palmitic acid (A and B), D9-oleic acid (C), D11-linoleic acid (D), or
D5-α-linolenic acid (E) for 24 h at 37 °C. A, Proteins (10 µg) were prepared, separated by SDS-PAGE , and
detected by immunoblotting with anti-FLAG and anti-GAPDH (loading control) antibodies. B–E, Lipids
were extracted, saponified, derivatized to AMPP amides, and analyzed by LC-MS/MS. Value s are the
means ± S.D. of the quantities of deuterium-labeled FA s with the indicated chain length and desaturation
number from three independent experiments. Statistically significant differences from the control are
represented (**, P < 0.01; *, P < 0.05; Dunnett's test).
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TABL E 1
Nucleotide sequences of primers used in this study
Primer
Nucleotide sequence
p1
5′-CATGCTCCAGACTGCCTTGGGAAAAGC-3′
p2
5′-CCTTGCGGGTGTCAGGCGACTCCGC-3′
p3
5′-CAAGTATTTGGAATAGCAAGGGAAGC-3′
mHacd1-F
5′-ATGGCGTCCAGTGAGGAGGACGGC-3′
mHacd1-R
5′-TTAATCGTCCTTCTCCGCGATC-3′
mHacd2-F
5′-TGCTATAGGGATTGTGCCATC-3′
mHacd2-R
5′-ACGGATAATTTCCGTGATTGTCC-3′
mHacd3-F
5′-GACGTGCAGAACCCTGCTATC-3′
mHacd3-R
5′-CTTCTGGACTGTGATGTTCACC-3′
mHacd4-F
5′-CAGCTCACAGAGAGAGTGATC-3′
mHacd4-R
5′-GAGTGTTTGACTGAGCCATGTC-3′
mGapdh-F
5′-TGAAGGTCGGTGTCAACGGATTTGGC-3′
mGapdh-R
5′-CATGTAGGCCATGAGGTCCACCAC-3′
hHACD1-F
5′-TGGTGTGGCTCATTACTCACAG-3′
hHACD1-R
5′-TGGCAAGTGGTCAAGAAGGC-3′
hHACD2-F
5′-TGGGCAGTAACACATAGCGTC-3′
hHACD2-R
5′-ACCTGGCCCATTTGATGAGG-3′
hHACD3-F
5′-GCGACCACTGTTTTTGGCTC-3′
hHACD3-R
5′-AGAGCCTTCGCTTTCCAGTC-3′
hHACD4-F
5′-CAGTTCTGTGGCCACTCTTG-3′
hHACD4-R
5′-GATGCGACTTTGCCAATCCG-3′
HACD1-CC1-F2
5′-GGTCATGGCGATGTCGTAGAGTTTT-3′
HACD1-CC1-R2
5′-TCTACGACATCGCCATGACCCGGTG-3′
HACD1-CC1-F3
5′-CTCGGGCCCTCCCCGCGCCGGTTTT-3′
HACD1-CC1-R3
5′-CGGCGCGGGGAGGGCCCGAGCGGTG-3′
HACD2-CC1-F1
5′-ACGCCGTGGCCAGGGGCCCCGTTTT-3′
HACD2-CC1-R1
5′-GGGGCCCCTGGCCACGGCGTCGGTG-3′
HACD2-CC1-F2
5′-TACA ATG TGGT GATGACA GCG TTT T-3′
HACD2-CC1-R2
5′-GCTGTCATCACCACATTGTACGGTG-3′
HACD3-CC2-F1
5′-AAGTCAGGAGCCAAAAACAGGTTTT-3′
HACD3-CC2-R1
5′-CTGTTTTTGGCTCCTGACTTCGGTG-3′
HACD3-CC2-F2
5′-GGATGAATCTGATGCGGAAAGTTTT-3′
HACD3-CC2-R2
5′-TTTCCGCATCAGATTCATCCCGGTG-3′
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TABL E 2
Selected m/z values for sphingomyelin species in LC-MS/MS analysis
FA chain length
Precursor ion
(Q1)a
Product ion (Q3)
Collision
energy (eV)
C16:0
703.40
184.10
60
C18:0
731.40
184.10
60
C18:1
729.40
184.10
60
C20:0
759.40
184.10
60
C22:0
787.50
184.10
60
C24:0
815.60
184.10
60
C24:1
813.60
184.10
60
aPrecursor ion is [M+H]+.
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TABL E 3
Selected m/z values for phosphatidylcholine species in LC-MS/MS analysis
FA chain lengths
(sn-1/sn-2)
Precursor ion (Q1)a
Product ion (Q3)
Collision
energy (eV)
C14:0-C16:0
750.40
227.00
50
C14:0-C18:1
776.40
227.00
50
C16:1-C16:1
774.40
253.00
50
C16:0-C16:0
778.40
255.00
50
C16:0-C18:2
802.40
255.00
50
C16:1-C18:1
802.40
253.00
50
C16:0-C18:1
804.40
255.00
50
C16:0-C18:0
806.40
255.00
50
C16:0-C20:4
826.40
255.00
50
C18:1-C18:3
826.40
277.00
50
C18:2-C18:2
826.40
279.00
50
C18:1-C18:2
828.40
281.00
50
C18:1-C18:1
830.40
281.00
50
C18:0-C18:2
830.40
283.00
50
C18:0-C18:1
832.40
283.00
50
C16:0-C22:6
850.40
255.00
50
C18:0-C20:4
854.00
283.00
50
C18:0-C22:6
878.40
283.00
50
aPrecursor ion is [M+HCOO]–.
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TABL E 4
Selected m/z values for phosphatidylinositol species in LC-MS/MS analysis
FA chain lengths
(sn-1/sn-2)
Precursor ion (Q1)a
Product ion (Q3)
Collision
energy (eV)
C16:1-C16:1
805.30
253.00
60
C18:0-C16:1
835.40
283.00
60
C18:0-C16:0
837.40
283.00
60
C16:0-C20:4
857.30
255.00
60
C18:1-C18:1
861.40
281.00
60
C18:0-C18:2
861.00
283.00
60
C18:1-C20:4
883.40
281.00
60
C18:0-C20:4
885.40
283.00
60
C18:0-C20:3
887.40
283.00
60
C18:0-C22:6
909.40
283.00
60
C18:0-C22:5
911.40
283.00
60
C18:0-C22:4
913.40
283.00
60
aPrecursor ion is [M–H]–.
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TABL E 5
Selected m/z values for [13C]malonyl-CoA metabolites in LC-MS/MS analysis
FA chain length
Precursor ion
(Q1)a
Product ion (Q3)
Collision
energy (eV)
C13:0
381.10
238.90
55
[13C]3-OH C20:0
497.10
229.08
55
[13C]C20:0
481.10
240.90
55
[13C]3-OH C22:0
527.10
229.08
55
[13C]C22:0
511.10
241.90
55
[13C]3-OH C24:0
557.20
229.08
55
[13C]C24:0
541.20
241.90
55
[13C]3-OH C26:0
587.20
229.08
55
[13C]C26:0
571.20
241.90
55
[13C]3-OH C20:1
495.10
229.08
55
[13C]C20:1
479.10
240.90
55
[13C]3-OH C22:1
525.10
229.08
55
[13C]C22:1
509.10
241.90
55
[13C]3-OH C24:1
555.10
229.08
55
[13C]C24:1
539.10
241.90
55
[13C]3-OH C26:1
585.10
229.08
55
[13C]C26:1
569.10
241.90
55
aPrecursor ion is [M+H]+.
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TABL E 6
Selected m/z values for the metabolites of deuterium-labeled FAs in LC-MS/MS analysis
FA chain length
Precursor ion
(Q1)a
Product ion (Q3)
Collision energy
(eV)
D31-C16:0
454.60
242.05
55
D31-C16:1
450.60
242.05
55
D31-C18:0
482.60
239.05
55
D31-C18:1
478.60
239.05
55
D31-C20:0
510.60
239.05
55
D31-C22:0
538.60
239.05
55
D31-C24:0
566.60
239.05
55
D31-C26:0
594.60
239.05
55
D9-C18:1
458.50
239.03
48
D9-C20:1
486.50
239.03
48
D9-C22:1
514.50
239.03
48
D9-C24:1
542.50
239.03
48
D9-C26:1
580.50
239.03
48
D11-C18:2(n-6)
458.30
239.03
44
D11-C18:3(n-6)
456.30
239.03
44
D11-C20:3(n-6)
484.30
239.03
44
D11-C20:4(n-6)
482.30
239.03
44
D11-C22:3(n-6)
512.30
239.03
44
D11-C22:4(n-6)
510.30
239.03
44
D11-C24:4(n-6)
538.30
239.03
44
D11-C24:5(n-6)
536.30
239.03
44
D11-C26:5(n-6)
564.30
239.03
44
D5-C18:3(n-3)
450.00
239.04
43
D5-C18:4(n-3)
448.00
239.04
43
D5-C20:3(n-3)
478.00
239.04
43
D5-C20:4(n-3)
476.00
239.04
43
D5-C20:5(n-3)
474.00
239.04
43
D5-C22:5(n-3)
502.00
239.04
43
D5-C24:5(n-3)
530.00
239.04
43
D5-C24:6(n-3)
528.00
239.04
43
D5-C26:6(n-3)
556.00
239.04
43
aPrecursor ion is [M+H]+.
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Shigeyoshi Itohara, Takayuki Sassa and Akio Kihara
Megumi Sawai, Yukiko Uchida, Yusuke Ohno, Masatoshi Miyamoto, Chieko Nishioka,
redundancy and are active in a wide range of fatty acid elongation pathways
The 3-hydroxyacyl-CoA dehydratases HACD1 and HACD2 exhibit functional
published online August 7, 2017J. Biol. Chem.
10.1074/jbc.M117.803171Access the most updated version of this article at doi:
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