(Received for publication, October 9, 1996, and in revised form, February 10, 1997)
From the Mass Spectrometry Resource, Divisions of Endocrinology, Diabetes, and Metabolism and of Laboratory Medicine, Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
Pancreatic islets express a
Ca2+-independent phospholipase A2
(CaI-PLA2) activity that is sensitive to inhibition by a
haloenol lactone suicide substrate that also attenuates glucose-induced hydrolysis of arachidonic acid from islet phospholipids and insulin secretion. A cDNA has been cloned from a rat islet cDNA library that encodes a protein with a deduced amino acid sequence of 751 residues that is homologous to a CaI-PLA2 enzyme recently
cloned from Chinese hamster ovary cells. Transient transfection of both COS-7 cells and Chinese hamster ovary cells with the cloned islet CaI-PLA2 cDNA resulted in an increase in cellular
CaI-PLA2 activity, and this activity was susceptible to
inhibition by haloenol lactone suicide substrate. The domain of the
islet CaI-PLA2 from amino acid residues 150-414 is
composed of eight stretches of a repeating sequence motif of
approximately 33-amino acid residues in length that is highly
homologous to domains of ankyrin that bind both tubulin and integral
membrane proteins, including several proteins that regulate ionic
fluxes across membranes. These findings complement previous
pharmacologic observations that suggest that CaI-PLA2 may
participate in regulating transmembrane ion flux in glucose-stimulated -cells.
Glucose-induced insulin secretion from pancreatic islet -cells
requires that glucose be transported into the
-cell and metabolized (1). Signals derived from glucose metabolism result in inactivation of
plasma membrane ATP-sensitive K+ channels
(KATP),1 membrane
depolarization, activation of voltage-operated Ca2+
channels, influx of Ca2+, and a rise in cytosolic
[Ca2+], which triggers insulin exocytosis (2).
Stimulation of islets with glucose also induces hydrolysis of
arachidonic acid from islet membrane phospholipids (3), and the
resultant accumulation of nonesterified arachidonic acid (4) may
facilitate Ca2+ entry into
-cells (5) and amplify
depolarization-induced insulin secretion (6).
Hydrolysis of arachidonic acid from membrane phospholipids in
glucose-stimulated islets appears to be mediated in part by a
phospholipase A2 (PLA2) enzyme that is
catalytically active in the absence of Ca2+ and that is
inactivated by a haloenol lactone suicide substrate (HELSS) (7-10).
Treatment of islets with HELSS results in attenuation of the
glucose-induced rise in -cell cytosolic [Ca2+] and in
inhibition of insulin secretion (8-10). The structure of islet
Ca2+-independent phospholipase A2
(CaI-PLA2) is not known, but a CaI-PLA2 enzyme
has recently been cloned from CHO cells (11) and its sequence
determined (GenBank accession number 115470). We report here the
cloning, expression, and sequence analysis of a homologous enzyme from
a rat pancreatic islet cDNA library (12).
Enhanced chemiluminescence detection reagents,
[32P]dCTP (3000 Ci/mmol), [35S]dATPS
(>1000 Ci/mmol), and
L--1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine
(50 mCi/mmol) were purchased from Amersham Corp. Male Harlan Sprague
Dawley rats (180-220 g) were obtained from Sasco (O'Fallon, MO);
collagenase was from Boehringer Mannheim; tissue culture media
(CMRL-1066 and MEM), penicillin, streptomycin, Hanks' balanced salt
solution, heat-inactivated fetal bovine serum, and
L-glutamine were from Life Technologies, Inc.; Pentex
bovine serum albumin (BSA, fatty acid-free, fraction V) was from Miles
Laboratories (Elkhart, IN); rodent Chow 5001 was from Ralston Purina
(St. Louis, MO); ampicillin and kanamycin were from Sigma; and
D-glucose was from the National Bureau of Standards. Media
included KRB (Krebs-Ringer bicarbonate buffer; 25 mM HEPES,
pH 7.4, 115 mM NaCl, 24 mM NaHCO3,
5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2); nKRB (KRB supplemented with 3 mM D-glucose); cCMRL-1066 (CMRL-1066 from Life
Technologies, Inc. supplemented with 10% heat-inactivated fetal bovine
serum, 1% L-glutamine and 1% (w/v) each of penicillin and
streptomycin); and Hank's balanced salt solution supplemented with
0.5% penicillin/streptomycin). The HELSS is
(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one and was purchased from Cayman Chemical (Ann Arbor, MI).
Islets were isolated aseptically from male Harlan Sprague
Dawley rats, as described (13), by collagenase digestion of excised, minced pancreas, density gradient isolation, and manual selection under
microscopic visualization (10). Isolated islets were transferred to
Falcon Petri dishes containing 2.5 ml of cCMRL-1066, placed under an
atmosphere of 95% air, 5% CO2, and cultured overnight at
37 °C. Purified populations of islet -cells and non-
-cells were prepared by fluorescence-activated cell sorting of dispersed cells
obtained by treating islets with the enzyme dispase, as described
previously (5, 7, 10, 14, 15). COS-7 and CHO cells were cultured in
Dulbecco's modified Eagle's medium (MEM) containing 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate.
HIT-T15 cells were obtained from ATCC (Bethesda, MD) and were cultured
in Ham's F12K medium (Life Technologies, Inc.) containing 7 mM D-glucose, 10% dialyzed horse serum, and
2.5% fetal bovine serum.
Total RNA was isolated from cells and tissues after solubilization in guanidinium thiocyanate by extraction (phenol/chloroform/isoamyl alcohol) and precipitation (isopropyl alcohol) (16). First strand cDNA was transcribed with avian myeloblastosis virus reverse transcriptase (RT) obtained from Boehringer Mannheim. Polymerase chain reactions (PCR) were performed on a Perkin-Elmer DNA Thermal Cycler 480. Amplification steps (25-30 cycles) included denaturation (95 °C, 1 min), annealing (50-60 °C, 1 min), and extension (72 °C, 2 min) performed in Taq DNA polymerase buffer (Life Technologies, Inc.) containing 1.5 mM MgCl2; 1 µM of each primer; 200 µM each of dATP, dGTP, dCTP, and dTTP; and 25 units/ml Taq DNA polymerase. PCR products were analyzed by 1% agarose gel electrophoresis and visualized with ethidium bromide (16).
cDNA Cloning and SequencingA pair of PCR primers
(sense, 5-TATGCGTGGTGTGTACTTCC-3
and antisense,
5
-TGGAGCTCAGGGCGACAGCA-3
) were designed based on the cDNA
sequence of the CHO cell CaI-PLA2 (11) and used in RT-PCR
reactions with RNA from HIT-T15 insulinoma cells. These reactions
yielded a 820-bp product. A 32P-labeled form of this
product was prepared by randomly primed labeling and used to screen an
islet cDNA library prepared as described elsewhere (12) in the
Lambda ZAP Express system (Stratagene). Clones that hybridized with the
probe were isolated and further plaque-purified. The pBK-CMV plasmid
containing the CaI-PLA2 cDNA sequence as insert was
excised in vivo according to the manufacturer's protocol.
Insert sizes were determined after digestion with the restriction
enzymes EcoRI and XhoI. The cloned cDNA in
pBK-CMV was sequenced from the double strand by the method of Sanger
et al. (17) using a Sequenase version 2.0 DNA sequencing kit
(United States Biochemical Corp.) with T3 and T7 primers and specific synthetic oligonucleotide primers.
After
removal of the 5-untranslated region, the full-length rat islet
CaI-PLA2 cDNA was subcloned in-frame into the
EcoRI and XhoI sites of pET-28c (Novagen) using
standard techniques. The fidelity of the construct was verified by
restriction analysis and sequencing. The pET28-CaI-PLA2
construct then was transformed into the bacterial expression host
Escherichia coli strain BL21(DE3) (Novagen). This system
yields a fusion protein with polyhistidine and T7 epitope tag sequences
joined to the sequence encoded by the insert. Cells transformed with
pET28c containing no insert served as negative controls. Expression was
induced by incubation of the cells with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside for 2 h and
assessed by 10% SDS-PAGE analysis of total protein followed by
Coomassie Blue staining. The expressed protein was affinity-purified by
nickel-chelate chromatography as described in the manufacturer's
protocol (Novagen). The protein was eluted with imidazole and analyzed
by 10% SDS-PAGE.
For Western blotting analyses, proteins were transferred to a nylon membrane, which was subsequently blocked (1 h, room temperature) with Tris-buffered saline-Tween (TBS-T, which contains 20 mM Tris-HCl, 137 mM NaCl, pH 7.6, and 0.1% Tween 20) containing 1% BSA and 3% milk powder. Following three washes with TBS-T, the blot was then incubated (1 h, room temperature) with a monoclonal antibody (1:50 dilution in TBS-T with 1% BSA) raised in a mouse against a synthetic peptide representing a 10-amino acid residue sequence (221TPLHLACQMG230) located at the C terminus of islet CaI-PLA2. The nylon membrane was washed three times in TBS-T and incubated (1 h, room temperature) with a goat anti-mouse IgG conjugated to horseradish peroxidase (Boehringer Mannheim) at a 1:3000 dilution in TBS-T containing 1% BSA. Detection of the secondary antibody was performed by enhanced chemiluminescence.
Examination of the Abundance of CaI-PLA2 mRNA by Competitive RT-PCRCompetitive RT-PCR (18, 19) was used to determine the abundance of CaI-PLA2 mRNA in various preparations. In this approach, a competitor DNA species is prepared that contains the same primer template sequences as the target cDNA but that contains an intervening sequence that differs from the target in size or in restriction sites so that PCR products from the target and competitor can be distinguished (18, 19). Using the competitor as an internal control, amounts of target cDNA can be determined by allowing known amounts of the competitor to compete with the target for primer binding during amplification (18, 19).
To prepare the competitor DNA, two composite primers were synthesized
(sense, 5-TGTGGGCAAGGTCATCC-3
and antisense, 5
-CTTGGAGGCCATGTAG-3
).
These primers contain the CaI-PLA2 primer sequence
(underlined) attached to sequences that hybridize to rat
glyceraldehyde-3-phosphate dehydrogenase cDNA (20). This pair of
primers was then used in PCR reactions with rat
glyceraldehyde-3-phosphate dehydrogenase cDNA as template. In these
reactions, the CaI-PLA2 primer sequences are incorporated
into the PCR product during amplification, and the intervening sequence
derives from glyceraldehyde-3-phosphate dehydrogenase. The resultant
PCR product (380 bp in length) was analyzed by agarose gel
electrophoresis, isolated with a QIAEX gel extraction kit (QIAGEN), and
used as the competitor DNA species in subsequent PCR experiments. In
these experiments, the primer pair (sense
5
--3
and antisense
5
--3
) was used. These primers
hybridize to the CaI-PLA2 cDNA sequence and to the
competitor DNA sequence. The PCR product derived from the
CaI-PLA2 cDNA is 528 bp in length and that from the
competitor DNA is 380 bp in length. The products were then analyzed by
1% agarose gel electrophoresis and visualized with ethidium bromide. Product band intensity was then determined with an IS-1000 Digital Imaging System.
With a fixed amount of target and competitor, varying the PCR cycle
number from 19 to 31 was found to yield a constant relative intensity
of the target and competitor PCR product bands. In subsequent experiments, 28 PCR cycles were used. To construct a standard curve,
the competitor DNA solution was diluted in the range of 102 to 10
6, and aliquots of each dilution
were added to a reaction mixture containing a fixed amount of
CaI-PLA2 cDNA. After PCR amplification, products were
analyzed by agarose gel electrophoresis, and product band intensity was
determined as above. The ratio of product band intensities was found to
correspond to the amount of input DNA over the range of tested
concentrations. Similar results were obtained in experiments in which
the amount of competitor DNA was fixed and the amount of target DNA was
varied.
The full-length islet CaI-PLA2 cDNA was subcloned into pcDNA3.1 (Invitrogen) in the forward orientation. Petri dishes (100 mm diameter) containing either COS-7 cells or CHO cells at about 60-80% confluence were transfected with 20 µg of plasmid DNA by calcium phosphate precipitation. After overnight culture, fresh MEM was added to each plate, and the cells were then cultured for an additional 2 days. At 72 h after transfection, cells were harvested after washing with ice-cold phosphate-buffered saline. For CaI-PLA2 enzyme activity assays, the cells were collected by centrifugation (Beckman table-top centrifuge, 3000 rpm, 2 min, room temperature). These cells were then resuspended and washed three times with ice-cold phosphate-buffered saline and once with homogenization buffer (250 mM sucrose, 40 mM Tris-HCl, pH 7.5). The cells were pelleted again, resuspended in homogenization buffer, and disrupted by sonication. The homogenates were centrifuged (170,000 × g, 60 min, 4 °C) to obtain a cytosolic supernatant. The protein content of the cytosolic fraction was measured by Bio-Rad assay. CaI-PLA2 activity was measured in aliquots of cytosol (100 µl, approximately 25 µg of protein) added to assay buffer (200 mM Tris-HCl, pH 6.0, 10 mM EGTA, total assay volume 200 µl). As described in the appropriate figure legends, effects of preincubating (2 min, room temperature) cytosolic fractions with various concentrations of HELSS on CaI-PLA2 activity were also determined, as were effects of varying the assay solution pH and effects of including nucleotide phosphates such as ATP in the assay solution. Reactions were initiated by injection of an ethanolic solution (5 µl) of L-a-1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine substrate (specific activity 50 mCi/mmol, final concentration 5 µM). Assay mixtures were then incubated (3 min, 37 °C, with shaking), and reactions were terminated by addition of butanol (100 µl) and vortexing (8 s). After centrifugation (2000 × g, 2 min), reaction products in 25 µl of the upper (butanol) layer were analyzed by Silica Gel G thin layer chromatography in the solvent system petroleum ether/ethyl ether/acetic acid (80/20/1). The region of the TLC plate containing free fatty acid (Rf 0.58) was identified with iodine vapor and scraped into scintillation vials, and the 14C-labeled content was then determined by liquid scintillation spectrometry. The amount of [14C]linoleate released was then converted to a PLA2 specific activity value (pmol/mg protein × min) as described elsewhere (7).
Dot Matrix Analysis of the Islet CaI-PLA2 Amino Acid Sequence and Comparison to Other SequencesDot matrix plots (21) were constructed with the UWGCG programs COMPARE and DOTPLOT using a window of 30 and stringency of 15. Segments of 30 amino acids from the horizontal axis were compared with segments from the vertical axis, and a dot was placed in the appropriate position whenever the total score of aligned sequences exceeded a value of 15. These plots were used to examine internal repeating motifs in the islet CaI-PLA2 sequence and to compare the sequences of ankyrin and of related proteins to the CaI-PLA2 sequence.
An oligonucleotide probe was generated by RT-PCR using HIT-T15 insulinoma cell RNA as template and primers designed from the CHO cell CaI-PLA2 sequence. Both CHO cells and HIT-T15 cells are hamster-derived lines, and HIT-T15 cells express a CaI-PLA2 activity similar to that in islets (22). A 32P-labeled form of the resultant 820-bp RT-PCR product was generated by randomly primed labeling and used to screen an islet cDNA library. Screening about 900,000 plaque-forming units resulted in the identification of five positive clones that were isolated, plaque-purified, and excised in vivo. The size of the cDNA inserts in the plasmids from each of the five clones was then determined after release of the inserts by digestion with the restriction enzymes EcoRI and XhoI. Each clone contained an insert of about 3.3 kilobases.
Sequencing the inserts from both 5- and 3
-ends revealed that each
contained an identical 5
-sequence and an identical 3
-sequence ending
in a poly(A) tail. A putative initiation codon (ATG) was observed 475 bp downstream of the 5
-end. Nucleotide sequencing of the entire
3288-bp insert from one clone revealed a single long open reading frame
(2256 bp), which was predicted to encode a protein with 751 amino acid
residues. The insert also contained 474 bp of 5
-untranslated region
and 558 bp of 3
-untranslated region including a poly(A) tail. A
presumptive polyadenylation signal (AATAAA) was present 14 bp upstream
of the poly(A) tail (Fig. 1).
Deduced Amino Acid Sequence of the Rat Islet CaI-PLA2
The encoded protein has a calculated molecular mass of 83,591 daltons and a predicted isoelectric point of 6.6. The amino acid sequence of islet CaI-PLA2 deduced from the nucleotide sequence of the cDNA does not exhibit significant homology to the sequences of known Ca2+-dependent PLA2 enzymes, including the 85-kDa cytosolic PLA2 or secretory PLA2 (23). The islet CaI-PLA2 exhibits 90% identity of nucleotide sequence and 95% identity of amino acid sequence to the CaI-PLA2 recently cloned from CHO cells (11). One absolutely conserved region is that between His421 and Glu551, which flanks and includes a GXSXG lipase consensus sequence (11, 24, 25). This sequence occurs in the islet CaI-PLA2 as 462TT466.
Examination of the islet CaI-PLA2 amino acid sequence using
dot matix analysis revealed (Fig. 2A) that
the domain from amino acid residues 150-414 is composed of eight
strings of a repetitive sequence motif of approximately 33 amino acid
residues in length. The alignment of the repeats in the islet
CaI-PLA2 sequence is illustrated in Fig. 2B. A
BLAST search of the National Biomedical Research Foundation protein
data base revealed significant homology of the repetitive sequence of
the islet CaI-PLA2 to a number of ankyrin-related proteins
(ARP), including the Caenorhabditis elegans ARP (26) and
both murine (27) and human (28) red cell ankyrin. Dot matrix
analysis revealed that the repetitive sequence
motif of the islet CaI-PLA2 is highly homologous to that of
an 89-kDa domain of murine red cell ankyrin (Fig.
3A) that contains binding sites for tubulin
and for several integral membrane proteins (28-30). Alignment of the
repeating domain of islet CaI-PLA2 with the repeating domain of ankyrin indicated that both repeating motifs have very similar consensus sequences (Fig. 3B). The CHO cell
CaI-PLA2 is also known to contain ankyrin-like repeats
(11).
Bacterial Expression of the Protein Encoded by the Cloned Islet CaI-PLA2 cDNA
The open reading frame of the
putative islet CaI-PLA2 cDNA was subcloned in-frame
into the bacterial expression vector pET-28c (Novagen) to generate a
fusion protein containing the islet CaI-PLA2 sequence and
both polyhistidine and T7 epitope tag sequences. Upon
isopropyl-1-thio--D-galactopyranoside induction of
bacterial cultures transfected with vector containing the islet
CaI-PLA2 insert, strong expression of the expected 87-kDa
fusion protein occurred, and this material was not observed in control
cultures (Fig. 4). The expressed protein was recognized
by a monoclonal antibody raised against a synthetic peptide contained
within the islet CaI-PLA2 sequence.
Expression of CaI-PLA2 Activity in COS-7 and CHO Cells After Transient Transfection with the Cloned Islet CaI-PLA2 cDNA
For eukaryotic expression, the islet
CaI-PLA2 cDNA was inserted into the vector pcDNA3.1
(Invitrogen), and this construct (pcDNA3.1-CaI-PLA2)
was used to transfect both COS-7 and CHO cells. At 72 h after
transfection with this construct or with vector containing no insert,
CaI-PLA2 activity was measured in cytosolic fractions
prepared from these cells. No measurable CaI-PLA2 activity was observed in the cytosol of either untransfected COS-7 cells (not
shown) or COS-7 cells transfected with vector containing no insert
(Fig. 5A). In contrast, COS-7 cells
transfected with the pcDNA3.1-CaI-PLA2 construct
exhibited substantial amounts of cytosolic CaI-PLA2
activity (Fig. 5A). This activity was inhibited by
pretreatment of cytosol with a suicide substrate (HELSS) (Fig. 5A) that has previously been demonstrated to inhibit
CaI-PLA2 activity in islet cytosol (7). Similar results
were obtained with CHO cells. Both untransfected CHO cells (not shown)
and CHO cells transfected with vector containing no insert exhibited
measurable CaI-PLA2 activity (Fig. 5B). This was
expected because a CaI-PLA2 enzyme has been cloned from CHO
cells (11). Transient transfection of CHO cells with the
pcDNA3.1-CaI-PLA2 construct, however, resulted in a
5.5-fold increase in cytosolic CaI-PLA2 activity, and this activity was inhibited by treatment of cytosol with HELSS (Fig. 5B).
Characterization of CaI-PLA2 Activity in COS-7 Cells Transiently Transfected with the Islet CaI-PLA2 cDNA Construct
The concentration dependence of inhibition by HELSS of
CaI-PLA2 activity in COS-7 cells transfected with the islet
CaI-PLA2 cDNA construct was found to be very similar to
that of the CaI-PLA2 activity in native HIT insulinoma cell
cytosol (Fig. 6). Like the CaI-PLA2 activity
in islet and HIT cell cytosol (7, 22), the CaI-PLA2
activity in cytosol from transiently transfected COS-7 cells was
optimal near neutrality and was stimulated by including 1 mM ATP in the assay solution (Fig. 7). The
effects of ATP to stimulate cytosolic CaI-PLA2 activity in
transiently transfected COS-7 cells, however, differed in some respects
from those observed with islet (7) or HIT cell (22)
CaI-PLA2 activity. First, the ATP concentration dependence
for stimulation of CaI-PLA2 activity from transiently
transfected COS-7 cells was bell-shaped, and the stimulatory effect
decreased at ATP concentrations exceeding 1 mM (not shown).
CaI-PLA2 activity in cytosol from islets (7) or HIT cells
(22) is stimulated by ATP concentrations as high as 10 mM.
Second, there was less nucleotide phosphate selectivity with the
CaI-PLA2 activity from transfected COS-7 cells than with islet (7) or HIT cell (22) activity. Although ATP was more effective
than AMP or ADP, ADP did exert some activating influence on
CaI-PLA2 activity from transfected COS-7 cells (not shown). ADP does not stimulate HIT cell CaI-PLA2 activity and
suppresses the stimulatory effect of ATP (22). In addition, GTP and UTP were nearly as effective as ATP in stimulating CaI-PLA2
activity from transfected COS-7 cells (not shown), whereas GTP has
little effect on HIT cell CaI-PLA2 activity (22). Finally,
the non-hydrolyzable ATP analog AMP-PCP stimulates CaI-PLA2
activity from transfected COS-7 cells less effectively than ATP (not
shown), whereas the two compounds are equipotent stimulators of HIT
cell CaI-PLA2 activity (22).
Expression of CaI-PLA2 mRNA in Rat Islet Cells
To determine whether islet CaI-PLA2 mRNA is
specifically expressed in -cells within islets, a competitive RT-PCR
(18, 19) method was developed. A competitor DNA was prepared that
shares with CaI-PLA2 cDNA sequences recognized by the
primers but that yields a smaller product than that derived from the
CaI-PLA2 cDNA (Fig. 8A). The
ratio of signals from the target and competitor was found to correspond
to the input DNA over a wide range of concentrations (Fig.
8B). Single cell suspensions were then prepared from
isolated islets and analyzed by fluorescence-activated cell sorting (5,
7, 10, 14, 15) to yield two populations of cells. One population
contains over 90%
-cells, and the second contains 10-15%
-cells and about 70%
-cells (5, 7, 10, 14, 15). RNA was then
prepared from the two populations of cells and used as template in
RT-PCR reactions with CaI-PLA2-specific primers and the
competitor DNA. At equivalent amounts of input RNA, a substantially
higher target to competitor ratio was observed with RNA from the
-cell-enriched population than with that from the
-cell-enriched
population (Fig. 9). This suggests that the RNA species
amplified in RT-PCR reactions with the
-cell-enriched population did
not arise from contaminating non-
-cells and that
-cells contain
CaI-PLA2 mRNA.
Expression of CaI-PLA2 mRNA was also examined in other tissues by competitive RT-PCR. At equivalent amounts of input RNA, the highest target to competitor ratio (TCR) among tissues examined was observed in brain (TCR 1.3). Target signal was also observed in lung (TCR 0.6), spleen (TCR 0.4), kidney (TCR 0.4), liver (TCR 0.2), heart (TCR 0.1), and skeletal muscle (TCR 0.1).
The studies described here indicate that pancreatic islet
-cells express an 84-kDa CaI-PLA2 enzyme that is highly
homologous to an enzyme recently cloned from CHO cells (11). The
occurrence of ankyrin-like repeats in the islet CaI-PLA2
sequence raises interesting possibilities about the function of this
protein in
-cells. The domain of ankyrin that contains similar
repeats is responsible for the binding of ankyrin to a number of
integral membrane proteins that govern ionic fluxes across cellular
membranes (29), including the Na+/K+-ATPase of
renal basolateral membranes (31-33), a renal amiloride-sensitive Na+ channel (34), the red cell anion exchanger (35, 36), a cerebellar inositol-trisphosphate receptor (37), and a
voltage-dependent Na+ channel in myelinated
neurons (29).
Regulation of ionic fluxes at the -cell plasma membrane is critical
in the control of insulin secretion, and both inactivation of
KATP channels and activation of voltage-operated
Ca2+ channels are required for the induction of insulin
secretion by glucose (2). One product of the action of PLA2
on islet phospholipids is nonesterified arachidonic acid. Arachidonic
acid is the vastly predominant sn-2 fatty acid substituent
in islet phospholipids and comprises 36% of the total fatty acyl mass
in islet phospholipids (9, 10). Nonesterified arachidonic acid facilitates Ca2+ entry into cells through both
voltage-operated (38) and receptor-operated (39) Ca2+
channels and induces a rise in
-cell cytosolic [Ca2+]
that is dependent on Ca2+ entry from the extracellular
space (5). Both exogenous PLA2 and the products of its
action suppress KATP channel activity in excised and
cell-attached patches of plasma membranes from HIT insulinoma cells
(40).
Taken together, the observations summarized in the preceding two
paragraphs raise the possibility that the ankyrin-like repeat domains
of the islet CaI-PLA2 might serve to attach the enzyme to
-cell KATP channels or to voltage-operated
Ca2+ channels or to anchor the enzyme in the membrane in
close proximity to such channels. The
-subunits of voltage-operated
Ca2+ channels exhibit striking similarities in sequence to
voltage-operated Na+ channels (41, 42) that interact with
ankyrin through its repeating domain (29). Ankyrin is distributed in
close proximity to voltage-operated Ca2+ channels in the
triad junctional structures of skeletal myocytes, although direct
interaction of ankyrin with these channels has not been demonstrated
(43).
The repeat sequences in ankyrin are also responsible for its binding to the cytoskeletal component tubulin (29). Although the precise role of the cytoskeletal apparatus in insulin secretion is not clearly established, a number of agents that interfere with microtubule assembly or disassembly inhibit insulin secretion (44-47), and morphological evidence suggests a direct association of microtubules with insulin secretory vesicles (48). Electron microscopic evidence demonstrates secretory granules associated with microtubules and directed toward the plasma membrane (48). An association of the islet PLA2 with microtubular structures mediated by its ankyrin-like repeat domain might facilitate interaction of the enzyme with secretory granule membranes and/or plasma membranes. It is of interest in this regard that treatment of parotid acinar secretory granules with PLA2 greatly increases their tendency to fuse with plasma membranes (49), which is the final event in exocytosis.
The sensitivity of the islet CaI-PLA2 to inhibition by the
HELSS suggests that this enzyme could be the target within -cells that is responsible for the ability of HELSS to inhibit the
glucose-induced rise in
-cell cytosolic [Ca2+] and
insulin secretion (7-10), although HELSS has also recently been
demonstrated to inhibit an isozyme of phosphatidate phosphohydrolase (50). HELSS does not inhibit any known
Ca2+-dependent PLA2 of groups I
through IV (51-54), but Ca2+-independent PLA2
activities from several sources are sensitive to inhibition by HELSS.
These include the 84-kDa CHO cell CaI-PLA2 (11) and a
PLA2 from the macrophage-like P388D1 cell line (51, 52, 55,
56), which has recently been demonstrated to represent the mouse
homolog of the CHO cell CaI-PLA2 (61). In addition, Ca2+-independent, HELSS-sensitive PLA2
activities thought to reside in 40-kDa proteins have been described in
myocardium (53), HIT cells (22), and renal proximal tubular cells
(57).
It is not yet known whether these 40-kDa proteins are related to the 84-kDa CaI-PLA2 enzymes, although generation of the former from the latter by proteolytic processing is a formal possibility. It is also possible that the 40-kDa proteins are separate gene products that share sequence homology with the 84-kDa CaI-PLA2 enzymes, perhaps including the serine lipase consensus sequence that is likely to confer sensitivity to HELSS. The 40-kDa proteins from myocardium (58) and HIT cells (22, 59) are thought to be associated with 85-kDa protein subunits that are homologous to phosphofructokinase and that confer sensitivity to activation by ATP and to modulation of this effect by ADP. Experiments with the CaI-PLA2 enzymes from CHO cells (62) and P388D1 cells (51) have failed to demonstrate association with phosphofructokinase-like proteins, although both enzymes behave as 300-350-kDa multimers on gel filtration chromatography, as do CaI-PLA2 activities from myocardium (58) and HIT cells (59). Whether the 84-kDa CaI-PLA2 enzymes may exist as hetero-oligomers consisting of distinct catalytic and regulatory proteins in some cells but not others is a question that will require further examination.
Although the 84-kDa islet CaI-PLA2 expressed in transiently transfected COS-7 cells is sensitive to activation by ATP under the assay conditions employed in this study, the nature of this effect differs in several respects from that of CaI-PLA2 activities in islet and HIT cell cytosol and is in general quite similar to effects observed with the P388D1 cell CaI-PLA2 (51). The possibility that the differences in nucleotide phosphate sensitivity between the islet CaI-PLA2 expressed in transfected COS-7 cells and that in native islet cytosol might reflect the influence of an interacting protein that is expressed in islets but that is not abundantly expressed in COS-7 cells is under study. It is of interest that the CHO cell CaI-PLA2, after expression in an Sf9 cell host, has recently been demonstrated to interact avidly and selectively with both ATP affinity matrices and with calmodulin affinity matrices (60), suggesting that the enzyme may bind ATP and interact with other cytosolic proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U51898[GenBank].
We are grateful to Alan Bohrer, Bingbing Li, Zhiqing Hu, and Dr. Mary Mueller for excellent technical assistance.