(Received for publication, November 29, 1995; and in revised form, January 2, 1996)
From the
A lysophospholipase was purified 506-fold from rat liver
supernatant. The preparation gave a single 24-kDa protein band on
SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed
lysophosphatidylcholine, lysophosphatidylethanolamine,
lysophosphatidylinositol, lysophosphatidylserine, and
1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8.
The purified enzyme was used for the preparation of antibody and
peptide sequencing. A cDNA clone was isolated by screening a rat liver
gt11 cDNA library with the antibody, followed by the selection of
further extended clones from a
gt10 library. The isolated cDNA was
2,362 base pairs in length and contained an open reading frame encoding
230 amino acids with a M
of 24,708. The peptide
sequences determined were found in the reading frame. When the cDNA was
expressed in Escherichia coli cells as the
-galactosidase
fusion, lysophosphatidylcholine-hydrolyzing activity was markedly
increased. The deduced amino acid sequence showed significant
similarity to Pseudomonas fluorescence esterase A and Spirulina platensis esterase. The three sequences contained
the GXSXG consensus at similar positions. The
transcript was found in various tissues with the following order of
abundance: spleen, heart, kidney, brain, lung, stomach, and testis
= liver. In contrast, the enzyme protein was abundant in the
following order: testis, liver, kidney, heart, stomach, lung, brain,
and spleen. Thus the mRNA abundance disagreed with the level of the
enzyme protein in liver, testis, and spleen. When HL-60 cells were
induced to differentiate into granulocytes with dimethyl sulfoxide, the
24-kDa lysophospholipase protein increased significantly, but the mRNA
abundance remained essentially unchanged. Thus a posttranscriptional
control mechanism is present for the regulation of 24-kDa
lysophospholipase.
Lysophospholipase hydrolyzes lysophosphatidylcholine (lyso-PC) ()to saturated fatty acid and sn-glycero-3-phosphocholine (GPC). This widely distributed
enzyme contains a number of isoforms and is known to be regulated. The
enzyme activity is modulated by lipid factors, such as acylcarnitine (1, 2) , arachidonic acid(3, 4) , and
phosphatidic acid(4) . An isoform of the enzyme was reported to
be inducible in HL-60 cells during granulocyte
differentiation(5, 6) . The physiological roles of
lysophospholipase have not been fully elucidated. A plausible function
is the control of the intracellular level of the physiologically active
lipid(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) ,
lyso-PC.
Lysophospholipase were purified from various sources, such as beef pancreas(18) , beef liver(19) , rabbit myocardium(1, 2) , human eosinophils(20) , macrophage cell lines, P388D1 (3) and WEHI 265.1 (21) , HL-60 cells(6) , rat liver(4) , and pig gastric mucosa(22) . Lysophospholipase can be classified into two large groups according to their molecular masses(23) . Large form enzymes (60 kDa) generally exhibit not only hydrolytic activity, but also transacylase activity, whereas small form enzymes (16.5-28 kDa) show only hydrolytic activity. Liver(19) , heart(1, 2) , and stomach (22, 24) contain both forms of enzyme. Furthermore, heart(1) , macrophages(3, 21) , HL-60 (6) , and stomach (22) contain two small form enzymes. Sunaga et al. (22) raised antibody against each of the two small form enzymes (22 and 23 kDa) purified from pig gastric mucosa and showed that the antibodies did not cross-react. Hence, the two small form enzymes of stomach are immunologically different. Furthermore, the major, 22-kDa enzyme (gastric enzyme I) hydrolyzed lyso-PC, lysophosphatidylethanolamine (lyso-PE), lysophosphatidylinositol (lyso-PI), lysophosphatidylserine (lyso-PS). In contrast, the minor, 23-kDa enzyme (gastric enzyme II) hydrolyzed only lyso-PC and lyso-PE. Thus the two enzymes show distinct substrate specificities.
Little is known about the molecular structures of lysophospholipases except for pancreatic and eosinophilic enzymes(25, 26) . Since the latter two enzymes have considerably different properties from other lysophospholipases, it is important to clone cDNAs for other lysophospholipases. In this paper, we report the purification and cDNA cloning of a small form lysophospholipase from rat liver. The enzyme had a molecular mass of 24 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and catalyzed the hydrolysis of a rather broad range of lysophospholipids, such as lyso-PC, lyso-PE, lyso-PI, and lyso-PS. The enzyme resembled pig gastric lysophospholipase I(22) , bovine liver lysophospholipase I(19) , and rabbit myocardial 23-kDa lysophospholipase(1) . The cloned cDNA encoded 230 amino acids with a calculated molecular mass of 24,708 Da. The deduced amino acid sequence showed significant sequence similarity to microbial esterases and contained the GXSXG consensus conserved in the active site of serine proteases, lipases, and esterases(27) . We also report the posttranscriptional control of the enzyme.
The second peak fractions
were collected, adjusted to 0.9 M NaCl, and loaded onto an
octyl-Sepharose column. The enzyme was eluted with a gradient of
decreasing NaCl concentration and increasing Triton X-100 and ethylene
glycol concentrations. The eluate was condensed by ammonium sulfate
precipitation and passed through Sephacryl S-300 in the presence of
Triton X-100. This step was very effective, resulting in more than
20-fold purification. The enzyme was further purified using
blue-Sepharose, G-butyl HPLC, and then Sephacryl S-200. The overall
purification of the enzyme was 506-fold with a yield of 9.6% (Table 1). The use of Triton X-100 was needed to avoid
aggregation of the enzyme. Glycerol and -mercaptoethanol
stabilized the enzyme. The enzyme was stored at -80 °C for at
least 6 months without an appreciable loss of activity. The final
enzyme preparation gave a single protein band on SDS-polyacrylamide gel
electrophoresis with a molecular mass of 24 kDa (Fig. 1A). The molecular mass of the native enzyme was
estimated to be 25 kDa on the Sephacryl S-200 column. Thus the enzyme
appeared to be a monomeric protein. The final specific activity was
0.87 µmol/min/mg. This was increased to 2 µmol/min/mg by the
addition of 0.4% (w/v) albumin to the assay. The N terminus of the
enzyme was found to be blocked.
Figure 1:
SDS-polyacrylamide gel electrophoresis,
tissue distribution, and antibody cross-reactivity. A,
SDS-PAGE of purified 24-kDa lysophospholipase. Purified enzyme (100 ng)
was electrophoresed and silver-stained as described under
``Experimental Procedures.'' The arrows indicate the
locations of molecular mass markers: glycerophosphate dehydrogenase, 36
kDa; carbonic anhydrase, 29 kDa; trypsin inhibitor, 20.1 kDa;
-lactoalbumin, 14.2 kDa. B, tissue distribution of 24-kDa
lysophospholipase. Purified enzyme (100 ng) and the 100,000
g supernatant (30 µg of protein) of different rat tissues
were separated by SDS-PAGE, followed by immunoblot analysis using
anti-24-kDa lysophospholipase serum as described under
``Experimental Procedures.'' Lane 1, purified
enzyme; lane 2, liver; lane 3, spleen; lane
4, lung; lane 5, heart; lane 6, kidney; lane
7, stomach; lane 8, testis; lane 9, brain. The arrows indicate the locations of prestained molecular mass
markers (Bio-Rad): carbonic anhydrase, 41.9 kDa; trypsin inhibitor, 32
kDa; lysozyme, 17.9 kDa. C, antibody cross-reactivity.
Purified gastric lysophospholipases I (lane 1), II (lane
2), and 24-kDa lysophospholipase (lane 3) were separated
by SDS-PAGE and then subjected to immunoblot analysis using anti-24-kDa
lysophospholipase serum as described under ``Experimental
Procedures.'' Molecular mass markers used are as in B.
We next examined the
substrate specificity of the enzyme using a substrate concentration
(0.4 mM) over 2 times higher than the K value determined for 1-palmitoyl-GPC. For limited availability of
labeled substrates, we used the previously developed calorimetric assay
method (4) with some modifications to improve sensitivity. As
shown in Table 2, the enzyme utilized various lysophospholipids,
including lyso-PC, lyso-PE (1-oleoyl-GPE), lyso-PS, and lyso-PI. The
acyl analog of platelet activating factor, 1-oleoyl-2-acetyl-GPC, was
also a good substrate. Lysophosphatidic acid and diacylphospholipids
were poor substrates. The enzyme showed little lipase and general
esterase activities.
Figure 2:
cDNA and the deduced amino acid sequence. Upper panel, cDNA clones. The numbers below the
restriction sites indicate the nucleotide positions. The boxes denote the coding region and the thin lines the
untranslated regions. A represents the
poly(A)
tail. The restriction sites used for
nucleotide sequencing are shown. Lower panel, nucleotide
sequence and the deduced amino acid sequence. Nucleotide positions are
indicated in the right margin. Amino acids are denoted by the
one-letter code. Partial amino acid sequences determined for lysyl
endopeptidase-digested enzyme are underlined. The
GXSXG consensus is dotted.
Figure 3: Sequence comparison. The deduced amino acid sequence of the enzyme (LYS) is compared with those of P. fluorescens esterase A (PSE) and S. platensis esterase (SPE). The numbers indicate the amino acid positions. Thick letters indicate the identical amino acids. The GXSXG consensus is dotted.
Figure 4: Expression of lysophospholipase cDNA in E. coli cells. A, enzyme activity. E. coli XL1-Blue cells were transformed with SG1-1-Ex (lane 1) or pBluescript (lane 2) and cultured for 6 h in the presence of 10 mM IPTG. Twenty µg of cell extracts were assayed for lyso-PC-hydrolyzing activity as described under ``Experimental Procedures.'' B, immunoblot analysis. 100 ng of the purified enzyme (lane 1) and 8 µg of extracts from E. coli transformed with SG1-1Ex (lane 2) or pBluescript (lane 3) were subjected to SDS-PAGE, followed by immunoblot analysis using anti-lysophospholipase serum as described under ``Experimental Procedures.'' The arrows indicate the locations of molecular mass markers: carbonic anhydrase, 41.9 kDa; trypsin inhibitor, 32 kDa; lysozyme, 17.9 kDa.
Figure 5:
Tissue
distribution of 24-kDa lysophospholipase mRNA. Poly(A) RNA was prepared from 10 µg of total RNA of various tissues
from a male Wistar rat and subjected to Northern blot analysis using
the cDNA probe as described under ``Experimental
Procedures.'' Lane 1, liver; lane 2, spleen; lane 3, lung; lane 4, heart; lane 5, kidney; lane 6, stomach; lane 7, testis; lane 8,
brain. The blot was also probed with
-actin cDNA as the control (lower panel). Arrows indicate the locations of 28
and 18 S rRNA.
We cultured HL-60 cells
in the presence of MeSO to induce differentiation. After
culturing for 6 days, morphological changes were noted. The cells were
harvested and lysophospholipase activity was determined. Activity was
mainly localized in the 10,000
g pellet. In agreement
with the previous investigations(5, 6) ,
lysophospholipase activity increased more than 3-fold by the
Me
SO treatment (Fig. 6A). Consistently, the
antibody against the 24-kDa lysophospholipase clearly recognized a 24
kDa-protein in Me
SO-treated HL-60 cells (Fig. 6B). Although its intensity was greatly increased
by Me
SO treatment, the mRNA level remained essentially
unchanged (Fig. 6C). These results clearly show that
24-kDa lysophospholipase is the same enzyme as the ``Peak 2''
lysophospholipase that was reported to be induced in differentiated
HL-60 cells(6) . Since the increase in the enzyme amount is not
associated with the change in mRNA abundance, the induction is due to a
posttranscriptional mechanism. Probably, stimulation of translation or
stabilization of the enzyme protein is involved.
Figure 6:
Induction of 24-kDa lysophospholipase in
HL-60 cells by MeSO. A, enzyme activity. The
10,000
g pellet (71 µg of protein) of HL-60 cells
cultured for 6 days without (lane 1) or with Me
SO (lane 2) were assayed for lysophospholipase activity as
described under ``Experimental Procedures.'' B,
immunoblot analysis. The 10,000
g pellet (7.1 µg
of protein) of HL-60 cells cultured without (lane 1) or with
Me
SO (lane 2) and 100 ng of purified 24-kDa
lysophospholipase (lane 3) were subjected to immunoblot
analysis using anti-lysophospholipase antiserum. The arrows indicate the locations of molecular mass markers: carbonic
anhydrase, 41.9 kDa; trypsin inhibitor, 32 kDa; lysozyme, 17.9 kDa. C, Northern blot analysis. Total RNA (10 µg) prepared from
HL-60 cells cultured without (lane 1) or with Me
SO (lane 2) were subjected to Northern blot analysis using the
cDNA probe as described under ``Experimental Procedures'' (upper panel). Arrows indicate the locations of 28
and 18 S rRNA. The blot was also probed with
-actin cDNA as the
control (lower panel).
In the present study, we purified a small form lysophospholipase from rat liver and cloned its cDNA. It is interesting to compare this lysophospholipase with previously reported small form lysophospholipases. At least two types of small form lysophospholipase occur in mammalian tissues. Sunaga et al. (22) purified two small form lysophospholipases from pig gastric mucosa and examined their substrate specificities. The major, 22-kDa enzyme (gastric enzyme I) was able to hydrolyze lyso-PC, lyso-PE, lyso-PI, lyso-PS, and the analog of platelet-activating factor, 1-acyl-2-acetyl-GPC. In contrast, the minor, 23-kDa enzyme (gastric enzyme II) hydrolyzed only lyso-PC and lyso-PE. The hepatic 24-kDa enzyme shows similar substrate specificity to gastric enzyme I. Its antibody reacted with gastric enzyme I, but not II. Consistently, the hepatic enzyme was recognized by anti-gastric enzyme I, but not by anti-gastric enzyme II. Thus this enzyme is concluded to be the same enzyme as gastric enzyme I. The present enzyme has a similar molecular weight to bovine liver lysophospholipase I reported by de Jong et al.(19) . The bovine enzyme I was shown to be active at pH 6-8 and efficiently hydrolyze 1-acyl-2-acetyl-GPC(44) . Thus the present enzyme resembles bovine liver lysophospholipase I. Furthermore, immunoblot analysis indicated that the present enzyme is abundant in the heart tissue. Probably, the enzyme is also closely related to the 23-kDa lysophospholipase reported by Gross and Sobel(1) .
Only a few papers have dealt with successful cloning of lysophospholipase cDNA except for cDNAs encoding pancreatic lysophospholipase (25) and eosinophilic lysophospholipase(26) . However, a sequence comparison revealed no significant sequence similarity between the present enzyme and these two lysophospholipases. The pancreatic enzyme cloned by Han et al. (25) is a secretory digestive enzyme and was later shown to be the same enzyme as cholesterol esterase by cDNA cloning(45) . The present results support the previous proposal that pancreatic lysophospholipase should be called carboxyl ester lipase(46, 47) . Eosinophilic lysophospholipase (Charcot-Leyden crystal protein) was predicted to have a molecular mass of 16.5 kDa from its cDNA(26) . This size is exceptionally small among lysophospholipases. The final specific activity was reported to be very low, 23.4 nmol/h/mg(20) . Thus pancreatic and eosinophilic enzymes are thought to be evolutionarily apart from other lysophospholipases. This idea would be strengthened by future cloning of other intracellular lysophospholipases, such as 60-kDa lysophospholipase-transacylase (4) and 23-kDa gastric lysophospholipase II(22) .
It was shown that arachidonic
acid release and lysophospholipase activity increased in differentiated
HL-60 cells(5, 48, 49) . One plausible
function of the induction of lysophospholipase would be the removal of
lyso-PC that is cytotoxic when accumulated(50) . However, this
may not be the sole function of lysophospholipase. Simultaneous
induction of phospholipase A and lysophospholipase would
cause an increase in not only unsaturated fatty acid, but also
saturated fatty acid. It is interesting to speculate that not only
unsaturated fatty acid(51) , but also saturated fatty acid
might be needed for differentiated HL-60 cells. Garsetti et
al. (6) showed that the increase of lysophospholipase
activity in HL-60 was due to the expression of a single
lysophospholipase isoform. The present study has extended their
observations and clearly identified the induced enzyme as 24-kDa
lysophospholipase. Furthermore, the enzyme induction was shown to
involve posttranscriptional mechanism, probably stimulation of
translation or stabilization of the enzyme. These results would be an
important clue to the full understanding of the control and
physiological roles of this enzyme.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D63885[GenBank].