U.S. patent application number 11/539141 was filed with the patent office on 2007-11-15 for enhanced medical treatments resulting from chemical identification of calcium influx factor, identity with the factor activating phospholipolysis and precipitating sudden death during myocardial infarction, and determination of similar activating mechanisms in multiple cell types through disinhibiti.
Invention is credited to Richard W. Gross, Christopher Jenkins.
Application Number | 20070264246 11/539141 |
Document ID | / |
Family ID | 38685377 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264246 |
Kind Code |
A1 |
Gross; Richard W. ; et
al. |
November 15, 2007 |
ENHANCED MEDICAL TREATMENTS RESULTING FROM CHEMICAL IDENTIFICATION
OF CALCIUM INFLUX FACTOR, IDENTITY WITH THE FACTOR ACTIVATING
PHOSPHOLIPOLYSIS AND PRECIPITATING SUDDEN DEATH DURING MYOCARDIAL
INFARCTION, AND DETERMINATION OF SIMILAR ACTIVATING MECHANISMS IN
MULTIPLE CELL TYPES THROUGH DISINHIBITION OF CALCIUM-INDEPENDENT
PHOSPHOLIPASE A2beta
Abstract
A method for treating a mammal comprises administering at least
one of a gene, enzyme and pharmaceutical which modulates the
concentration of iPLA.sub.2.beta. through transcriptional and/or
translational regulation or effectively modulate the inhibition of
iPLA.sub.2.beta. through calmodulin or derivatives thereof.
Inventors: |
Gross; Richard W.;
(Chesterfield, MO) ; Jenkins; Christopher;
(Richmond Heights, MO) |
Correspondence
Address: |
PATRICK W. RASCHE (15060);ARMSTRONG TEASDALE, LLP
ONE METROPOLITAN SQUARE
SUITE 2600
SAINT LOUIS
MO
63102-2740
US
|
Family ID: |
38685377 |
Appl. No.: |
11/539141 |
Filed: |
October 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60723685 |
Oct 5, 2005 |
|
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|
Current U.S.
Class: |
424/94.1 ;
514/1.9; 514/16.4; 514/18.8; 514/19.1; 514/19.3; 514/20.7; 514/4.8;
514/44R; 514/7.4 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
31/7052 20130101; A61P 3/04 20180101; A61P 9/12 20180101 |
Class at
Publication: |
424/094.1 ;
514/012; 514/044 |
International
Class: |
A61K 38/43 20060101
A61K038/43; A61K 31/7052 20060101 A61K031/7052; A61P 3/04 20060101
A61P003/04; A61P 9/12 20060101 A61P009/12; A61P 9/00 20060101
A61P009/00; A61K 38/16 20060101 A61K038/16 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made under contracts NIH 5P01HL57278 and
NIH 5R01HL41250. The government has certain rights in the
invention.
Claims
1. A method for treating a mammal, said method comprising
administering a gene, enzyme or pharmaceutical which modulates the
concentration of iPLA.sub.2.beta. through transcriptional and/or
translational regulation or effectively modulates the inhibition of
iPLA.sub.2.beta. through calmodulin or derivatives thereof.
2. A method in accordance with claim 1 wherein the mammal is a
living human.
3. A method in accordance with claim 1 further comprising
decreasing the intracellular content of acyl-CoA and the production
of toxic species derived therefrom.
4. A method in accordance with claim 1 further comprising treating
hypertension in the metabolic syndrome by increasing the hydrolysis
acyl-CoA thereby promoting the inhibition of iPLA.sub.2.beta. and
the entry of calcium into the smooth muscle myocyte.
5. A method for in accordance with claim 1 further comprising
reducing lipid accumulation or inflammation in the vessel wall by
hydrolysis of acyl-CoA and/or capacitative calcium influx.
6. A method for in accordance with claim 1 further comprising
treating obesity by facilitating the removal of fatty acids from
fat cells by increased acyl-CoA hydrolysis.
7. A method in accordance with claim 1 further comprising
strengthening the interaction between iPLA.sub.2.beta. and
calmodulin, resulting in a decrease in iPLA.sub.2.beta.
phospholipase A.sub.2 activity.
8. A method in accordance with claim 1 further comprising
disrupting the interaction between iPLA.sub.2.beta. and calmodulin,
resulting in an increase in iPLA.sub.2.beta. phospholipase A.sub.2
activity.
9. A method in accordance with claim 1 further comprising
performing measurements of blood fatty acid and/or lysophospholipid
levels by shotgun lipidomics analysis and determining that the
treatment was successful when and if fatty acid/lysophospholipid
levels are normalized or ischemic damage is decreased.
10. A method in accordance with claim 1 further comprising
performing measurements of blood insulin and determining that the
treatment was successful when and if blood glucose tolerance is
normalized.
11. A method in accordance with claim 1 further comprising
performing measurements utilizing positron emission tomography
(PET) with radiolabeled neurotransmitter, and determining that the
treatment was successful when and if neurotransmitter release is
normalized.
12. A method of regulating cellular processes dependent upon
Ca.sup.2+ influx or entry related to the activation or inhibition
of iPLA.sub.2.beta., the method comprising at least one of the
steps of reversing the interaction of iPLA.sub.2.beta. with
calcium-activated calmodulin in a subject, and enhancing the
interaction of iPLA.sub.2.beta. with Calcium-activated
calmodulin.
13. A method in accordance with claim 12 wherein the subject is a
living human.
14. A method in accordance with claim 12 wherein conditions
presented are capable of being reversed.
15. A method of reducing activation of iPLA.sub.2.beta. during
ischemia comprises promoting re-formation of the
iPLA.sub.2.beta.:CaM complex in a subject, and decreasing
phospholipid hydrolysis and associated ischemic damage.
16. A method in accordance with claim 15 wherein the subject is a
living human.
17. A method in accordance with claim 15 wherein conditions
presented are capable of being reversed.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. Provisional
Patent Application Ser. No. 60/723,685 filed Oct. 5, 2005, which is
hereby incorporated by referenced in its entirety.
BACKGROUND OF THE INVENTION
[0003] This invention relates generally to biomarker screening and
more particularly to identifying new targets for pharmacological
inhibition.
[0004] This invention also relates generally to analytical (assays)
methods for identifying compounds useful for promoting health in
living mammalian systems. In particular this invention relates to
assays and analytical tools for monitoring health in living
mammals.
[0005] The function of complex living biological organisms relies
on the meticulous control of cellular activity, including close
regulation of cell growth, proliferation and function. The family
of enzymes known as phospholipases A.sub.2 has been implicated in
the control of cellular activity by catalyzing the esterolytic
cleavage of fatty acids from phospholipids, thereby regulating the
release of lipid second messengers, cellular growth factors, and
the properties of the cellular membrane (Samuelsson et al., Annu.
Rev. Biochem. 47:997-1029, 1978; Moolenaar, W. H., Curr. Opin.
Cell. Biol. 7:203-10, 1995). In particular, by controlling the
production of second messengers such as arachidonic acid and its
biologically active eicosanoid metabolites, phospholipases A.sub.2
are involved in modulating such processes as cellular growth
programs, inflammation, vascular tone and ion channel function.
(Needleman et al., Annu. Rev. Biochem. 55:69-102, 1986).
[0006] Phospholipases A.sub.2 are a broad family of enzymes with
varying kinetic and physical properties, and distinct functions.
Early research focused on distinguishing broad classes of the
enzymes within the larger family. Several classes were
distinguished using in vitro activity assays, and are categorized
based on the dependence of their enzymatic activity on the presence
of calcium ion. (See e.g., Demel et al, Biochim. Biopliys. Acta
406:97-107, 1975). Thus, one class, the secretory phospholipases
A.sub.2 are distinguished by an obligatory dependence on high
(millimolar) concentrations of calcium ions, as well as low
molecular weights (14-18 kDa) and relative heat stability. (Demel
et al., supra; Tischfield, J. A., J. Biol. Chem. 272:17247-50,
1997). The activity of a second class, the cytosolic phospholipases
A.sub.2 is facilitated by the presence of nanomolar concentrations
of calcium ions, but the presence of the calcium ion is not
obligatory. (Loeb et al., J. Biol. Chem. 261:10467-70, 1986; Kramer
et al., Biochim. Biophys. Acta 878:394-403; Glover et al., J. Biol.
Chem. 270:15359-67, 1986). A third class of enzymes is entirely
calcium-independent in in vitro studies, and is also distinguished
by a finely tuned inhibition by
(E)-6-(bromomethylene)-3-(1-napthalenyl)-2H-tetrahydropyran-2-one
(BEL). (Wolf et al., J. Biol. Chem. 260:7295-303; Hirashima et al.,
J. Neurochem. 59:708-14; Lehman et al., J. Biol. Chem.
268:20713-16).
[0007] Application of molecular biological techniques has provided
some insights into the structure and function of founding members
in each class of phospholipases A.sub.2 and has provided a further
basis for distinguishing among the classes. (See, e.g. Demel et al,
supra; Evenberg et al., J. Biol. Chem. 252: 1189-96, 1977;
Tischfeld, J. A., J. Biol. Chem. 272: 17247-50, 1997). For example,
members of the secretory phospholipases A.sub.2 use a calcium ion
to polarize the carbonyl for attack by a histidine-activated
H.sub.2O molecule, while the intracellular phospholipases use a
nucleophilic serine. The calcium-facilitated phospholipases A.sub.2
have a GXSGS consensus lipase motif, in contrast to the iPLA.sub.2
group which has a GXSTG consensus motif. The calcium-independent
phospholipases A.sub.2 are also distinguished by a consensus
sequence for nucleotide binding. (Andrews et al., Biochem. J.
252:199-206, 1988; Tang et al., J. Biol. Chem. 272:8567-75, 1998).
These findings have clearly boosted progress toward identifying the
polypeptides responsible for catalyzing the synthesis of the
eicosanoid metabolites and toward understanding the regulatory
mechanisms of phospholipases A.sub.2 that are involved in normal
and disease states.
[0008] The more recent developments of intense genome sequencing
efforts have produced partial sequence data on the phospholipases
and have led to related structural insights. For example, two new
calcium-facilitated phospholipases have recently been described
based on data from protein and nucleotide databases. (Underwood et
al., J. Biol. Chem. 273: 21926-32, 1998; Pickard et al., J. Biol.
Chem. 274: 8823-31, 1999). Further, during sequencing of the long
arm of chromosome 7 in the Human Genome Sequencing Project, a
predicted protein product of 40 kDa was identified. The polypeptide
contained two 10 amino acid segments homologous to the lipase and
nucleotide-binding consensus sequences described for the founding
members of the iPLA.sub.2 family. (Tang et al., supra).
[0009] Earlier work has been done with respect to phospholipases
and certain disease conditions in animals. For example, intensive
study of reperfusion injury in myocardial tissue has led to the
hypothesis that pathology is ultimately generated because of
membrane phospholipid breakdown attributable to activation of
myocardial phospholipase A.sub.2 activity. (See e.g. Van der Vusse
et al., Hydrolysis of phospholipids and cellular integrity, In: H.
M. Piper (ed.) Pathophysiology of Severe Ischemic Myocardial
injury, Kluwer Academic Publishers, Dordrecht, The Netherlands,
1990, 167-93). Furthermore, calcium-dependent and
calcium-independent phospholipase A.sub.2 activities have also been
found to be present in the human cerebral cortex. Some reports have
suggested a possible link between the activity of both
calcium-dependent calcium independent phospholipases A.sub.2 and
cortical degenerative diseases such as Alzheimer's disease. (For
reviews, see e.g. Farooqui et al., Neurochem. Int. 30: 5 17-22,
1997; Farooqui et al., Brain Res. Bull. 49: 139-53, 1999).
[0010] Certain inhibitors of phospholipases A.sub.2 have been
identified as possible therapeutic candidates for treating
PLA.sub.2-mediated diseases. For example, fatty acid
trifluoromethyl ketones, bromoenol lactone, methyl arachidonyl
fluorophosphonate, benzenesulfonamides and other specific
inhibitors of phospholipases A.sub.2 have been shown to decrease
PLA.sub.2 activity and all have been considered for treating
inflammatory diseases thought to be mediated by PLA.sub.2. (See
e.g. Farooqui et al, 1999, supra). Nevertheless, as noted above,
the phospholipases A.sub.2, as well as the iPLA.sub.2 subfamily
itself, are a heterogeneous group of enzymes, with differing
molecular weights, substrates, and responses to inhibitors. Because
of this, the development of agents for treating diseases mediated
by these compounds is ideally based upon determining and
characterizing the structure and functional characteristics of the
particular iPLA.sub.2 involved in the disease process. Thus, it is
important to identify and characterize the phospholipases A.sub.2
family members.
[0011] During the last decade, excessive consumption of fat in high
caloric Western diets in conjunction with a sedentary life style,
has resulted in an epidemic of obesity in industrialized nations
(1, 2). Obesity is associated with insulin resistance,
hypertension, dyslipidemia, type 2 diabetes and atherosclerosis,
which collectively constitute the metabolic syndrome (3, 4, 5).
Despite the enormous proportions of this public health problem, the
biochemical mechanisms underlying the metabolic syndrome and its
end-organ sequelae are poorly understood.
[0012] With respect to diabetes, glucose utilization is necessary
for the body to be able to use sugar which is stored in the blood
as glucose. Insulin initiates the process of taking glucose from
the blood and moving it into the cells. However, when glucose
builds up in the blood instead of going into cells (e.g., insulin
resistance), it can cause serious life threatening problems which
results in type 2 diabetes. These include heart disease
(cardiovascular disease), blindness (retinopathy), nerve damage
(neuropathy), and kidney damage (nephropathy).
[0013] Type 2 diabetes is the most common form of diabetes. In this
condition the body does not produce enough insulin to cause cells
to transport glucose or the cells are not sensitive enough to the
insulin present. The concentration of blood glucose becomes and
remains high in the blood resulting in unnecessary and undesired
damage to the body. Thus glucose is not utilized, proteins are
covalently modified, inappropriate oxidation occurs and a change to
fatty acid substrate occurs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A and 1B depict palmitoyl-CoA Thioesterase Activity
of iPLA.sub.2.beta.. 1A depicts Substrate inhibition of
iPLA.sub.2.beta. palm-CoA hydrolase activity. 1B depicts
iPLA.sub.2.beta. catalyzed hydrolysis of palmitoyl-CoA guest in
host POPC/DOPS vesicles.
[0015] FIG. 2 depicts substrate Selectivity of iPLA.sub.2.beta.
Long-chain Acyl-CoA Hydrolase Activity in the Presence of POPC/DOPS
Vesicles.
[0016] FIG. 3 depicts mutagenesis of Ser-465 to Alanine Eliminates
iPLA.sub.2.beta. Phospholipase A.sub.2 and Palmitoyl-CoA Hydrolase
Activities.
[0017] FIG. 4 depicts inhibition of iPLA.sub.2.beta. Palmitoyl-CoA
Thioesterase Activity by (R)- and (S)-BEL.
[0018] FIG. 5 depicts selective acylation of iPLA.sub.2.beta.
Wild-type, S465A Mutant, and BEL pretreated Wild-type Proteins with
Various Long-chain Acyl-CoAs.
[0019] FIG. 6 depicts stoichiometry of iPLA.sub.2.beta. Acylation
by Oleoyl-CoA.
[0020] FIG. 7 depicts effect of Various Chemical Treatments on
iPLA.sub.2.beta. Autoacylation with [1-.sup.14C]-Oleoyl-CoA.
[0021] FIG. 8 depicts partial trypsinolysis of
[1-.sup.14C]-Oleoyl-iPLA.sub.2.beta..
[0022] FIG. 9 depicts Ca.sup.2+-CaM Does Not Inhibit the
Palmitoyl-CoA Thioesterase Activity of iPLA.sub.2.beta..
[0023] FIG. 10 depicts Ca.sup.2+-CaM Inhibits Covalent Acylation of
iPLA.sub.2.beta. by Oleoyl-CoA.
[0024] FIGS. 11A-11I depict reversal of Ca.sup.2+/CaM-Mediated
Inhibition of iPLA.sub.2.beta. Activity by Oleoyl-CoA.
DETAILED DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A and 1B--Purified iPLA.sub.2.beta. (His).sub.6 was
incubated with the indicated concentrations of
[1-.sup.14C]-palmitoyl-CoA for 1-2 min at 37.degree. C. Reactions
were terminated by vortexing with butanol and [1-.sup.14C]-palmitic
acid extracted into the butanol layer was resolved by TLC and
quantified by liquid scintillation spectrometry as described in
Experimental Procedures. B, iPLA.sub.2.beta. catalyzed hydrolysis
of palmitoyl-CoA guest in host POPC/DOPS vesicles. The indicated
mol % of [.sup.14C]-palmitoyl-CoA were incorporated into POPC/DOPS
(90:10) vesicles (100 .mu.M final vesicle lipid concentration).
Each data point represents the average.+-.S.E. for at least 4
separate determinations.
[0026] FIG. 2--Purified iPLA.sub.2.beta. (His).sub.6 was incubated
with equal amounts of the indicated [1-.sup.14C]-acyl-CoA guest (5
mol %) in POPC/DOPS (90:10) host vesicles for 2 min at 37.degree.
C. Released [1-.sup.14C]-fatty acid was extracted into butanol by
vortexing, resolved by TLC, and quantified by liquid scintillation
spectrometry as described in Experimental Procedures. Each data
point represents the average.+-.SE. from 8 separate
determinations.
[0027] FIG. 3--Equivalent amounts of purified iPLA.sub.2.beta.
(His).sub.6 (WT) or mutant iPLA.sub.2.beta. (His).sub.6 (S465) were
incubated with either
1-palmitoyl-2-[1-.sup.14C]-oleoyl-sn-glycero-3-phosphocholine (5
.mu.M) or [1-.sup.14C]-palmitoyl-CoA (5 .mu.M) for 2 min at
37.degree. C. Radiolabeled fatty acids from the reaction were
extracted into butanol, resolved by TLC, and quantified by liquid
scintillation spectrometry as described in Experimental Procedures.
Results are representative of the average.+-.S.E. of 4 separate
determinations.
[0028] FIG. 4--Purified iPLA.sub.2.beta. (His).sub.6 was
preincubated with the indicated concentrations of either enantiomer
of BEL or ethanol vehicle for 3 min at 23.degree. C. The enzyme was
then added to 100 mM Tris-HCl, pH 7.2 containing 1 mM EGTA and
[1-.sup.14C]-palmitoyl-CoA guest (5 mol %) in POPC (100 .mu.M) host
vesicles for 2 min at 37.degree. C. Following extraction into
butanol, liberated [1-.sup.14C]-palmitic acid was separated from
[1-.sup.14C]-palmitoyl-CoA by TLC and quantified by liquid
scintillation spectrometry as described in Experimental
Procedures.
[0029] FIG. 5--Equivalent amounts of the indicated
[1-.sup.14C]-acyl-CoA guests (5 mol %) were incorporated into host
POPC (100 .mu.M) vesicles and incubated for 1 hr at 37.degree. C.
with equal amounts of either wild-type (WT)
iPLA.sub.2.beta.(His).sub.6, S465A iPLA.sub.2.beta.(His).sub.6, or
BEL-pretreated WT iPLA.sub.2.beta.(His).sub.6 enzyme as described
in Experimental Procedures. Samples were resolved by SDS-PAGE (10%
gel), fixed, and dried before visualization by autoradiography.
[0030] FIG. 6--Purified iPLA.sub.2.beta.(His).sub.6 (2 .mu.M) was
incubated with the indicated concentrations of
[1-.sup.14C]-oleoyl-CoA present as guest in host POPC (100 .mu.M)
vesicles for 1 hr at 37.degree. C. Samples were electrophoresed in
parallel with standard amounts (0.5-10 nCi) of [.sup.14C]-BSA of
known activity (not shown). The fixed and dried gel was exposed to
film and the resultant signals from the [.sup.14C]-BSA were
utilized to generate a standard curve utilizing ID software from a
Kodak Imagestation to determine the incorporation of
[1-.sup.14C]-oleate into iPLA.sub.2.beta..
[0031] FIG. 7--Purified iPLA.sub.2.beta.(His).sub.6 was incubated
with [1-.sup.14C]-oleoyl CoA (10 mol %) incorporated as guest in
host POPC vesicles. N-ethylmaleimide and iodoacetamide samples were
pretreated with 5 mM of either reagent for 5 min at 30.degree. C.
before addition of [1-.sup.14C]-oleoyl-CoA. Samples were incubated
with 1 N HCl, 1 N NaOH, or 2 N neutral hydroxylamine for 1 hr at
30.degree. C. as indicated. All samples, except lane 1, were
precipitated with CHCl.sub.3/MeOH and washed with 70% acetone
before SDS-PAGE and autoradiography as described in Experimental
Procedures.
[0032] FIG. 8--[1-.sup.14C]-oleoyl-iPLA.sub.2.beta.(His).sub.6 was
prepared by incubation of the unmodified enzyme (10 .mu.M) with 50
.mu.M [1-.sup.14C]-oleoyl-CoA for 1 hr at 37.degree. C. Trypsin
(1:25 w/w) was added and incubated with
[1-.sup.14C]-oleoyl-iPLA.sub.2.beta.(His).sub.6 for the indicated
times. Following termination of proteolysis by addition of loading
buffer, tryptic peptides were resolved by SDS-PAGE and radiolabeled
fragments were visualized by autoradiography as described in
Experimental Procedures.
[0033] FIG. 9--Calcium-independent iPLA.sub.2.beta. was
pre-incubated in the presence of Ca.sup.2+ (1 mM) or Ca.sup.2+-CaM
(3 .mu.g) on ice before addition to 95 .mu.M POPC containing either
5 mol % [1-.sup.14C]-POPC or [1-.sup.14C]-palmitoyl-CoA in 100 mM
Tris-HCl, pH 7.2. After incubation at 37.degree. C. for 3 min,
reactions were terminated by vortexing in the presence of butanol
and released radiolabeled fatty acids were resolved by TLC and
quantitated by liquid scintillation spectrometry as described in
Experimental Procedures.
[0034] FIG. 10--Purified iPLA.sub.2.beta.(His).sub.6 was incubated
with [1-.sup.14C]-oleoyl CoA (10 mol %) incorporated as guest in
host POPC vesicles in the presence of EGTA (5 mM), Ca.sup.2+ (1
mM), CaM (3 .mu.g), or Ca.sup.2+-CaM for 1 h at 37.degree. C.
Samples were resolved by SDS-PAGE and the gel was soaked in Amplify
fluorographic reagent, dried, and exposed to film as described in
Experimental Procedures.
[0035] FIGS. 11A-11D--Purified iPLA.sub.2.beta.+/-Ca.sup.2+/CaM was
incubated with POPC/BODIPY-PC (95:5 mol %) host vesicles for 2 min
at 37.degree. C. with the indicated concentrations of guest
oleoyl-CoA. Relative fluorescence was recorded utilizing 495 nm
excitation and 515 nm emission wavelengths as described in
Experimental Procedures. FIGS. 11E-11H--ESI-MS analysis of
phosphatidylcholine molecular species from the reactions described
in panels A and D. Spectra were acquired in the positive ion mode
with the indicated internal standards as described in Experimental
Procedures. FIG. 11I--Purified iPLA.sub.2.beta. in the presence or
absence of Ca.sup.2+/CaM was incubated with
1-palmitoyl-2-[1-.sup.14C]-oleoyl-sn-glycero-3-phosphocholine (100
.mu.M) host vesicles with or without guest oleoyl-CoA (10 .mu.M)
for 3 min at 37.degree. C. Radiolabeled fatty acid was extracted
into butanol, resolved by TLC, and quantified by scintillation
spectrometry as described in Experimental Procedures.
BRIEF DESCRIPTION OF THE INVENTION
[0036] A method for treating a mammal, said method comprising
administering a gene, enzyme or pharmaceutical which modulates the
concentration of iPLA.sub.2.beta. through transcriptional and/or
translational regulation or effectively modulates the inhibition of
iPLA.sub.2.beta. through calmodulin or derivatives thereof.
[0037] A method of regulating cellular processes dependent upon
Ca.sup.2+ influx or entry related to the activation or inhibition
of iPLA.sub.2.beta., the method comprising at least one of the
steps of reversing the interaction of iPLA.sub.2.beta. with
calcium-activated calmodulin, and enhancing the interaction of
iPLA.sub.2.beta. with Calcium-activated calmodulin.
[0038] A method of activation of iPLA.sub.2.beta. during ischemia
comprises promoting re-formation of the iPLA.sub.2.beta.:CaM
complex in a subject, and decreasing phospholipid hydrolysis and
associated ischemic damage.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Without being bound by theory, the discovery comprises an
isolated and characterized oleoyl-iPLA.sub.2.beta. having Sequence
NO. 1. In an aspect the oleoyl-iPLA.sub.2.beta. comprises that of a
living human. In an aspect the oleoyl-iPLA.sub.2.beta. comprises
that of a living mouse.
[0040] Unless otherwise defined, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present discovery.
[0041] As used herein, the term "peptide" includes any of a group
of compounds comprising two or more amino acids linked by chemical
bonding between their respective carboxyl and amino groups. The
term "peptide" includes peptides and proteins that are of
sufficient length and composition to effect a biological response,
e.g. antibody production or cytokine activity whether or not the
peptide is a hapten. The term "peptide" includes modified amino
acids, such modifications including, but not limited to,
phosphorylation, glycosylation, prenylation, lipidization and
methylation.
[0042] As used herein, the term "polypeptide" includes any of a
group of natural or synthetic polymers made up of amino acids
chemically linked together such as peptides linked together. The
term "polypeptide" includes peptide, translated nucleic acid and
fragments thereof.
[0043] As used herein, the term gene includes "polynucleotide"
which includes nucleotide sequences and partial sequences, DNA,
cDNA, RNA variant isoforms, splice variants, allelic variants and
fragments thereof.
[0044] As used herein, the terms "protein", "polypeptide" and
"peptide" are used interchangeably herein when referring to a
translated nucleic acid (e.g. a gene product). The term
"polypeptide" includes proteins. The term "protein" includes any
large molecule composed of one or more chains of amino acids in a
specific order; the order is determined by the base sequence of
nucleotides in the gene that codes for the protein. The term
"protein" includes a fragment and functional fragments of
proteins.
[0045] As used herein, the term "nucleic acid" refers to
oligonucleotides or polynucleotides such as deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA) as well as analogs of either RNA
or DNA, for example made from nucleotide analogs any of which are
in single or double stranded form.
[0046] As used herein, the term "therapeutic agent" is any molecule
or atom which is conjugated, fused or otherwise affixed to an
antibody moiety to produce a conjugate which is useful for
therapy.
[0047] As used herein, the term "biological sample" includes
vascular tissue or blood, urine or other body fluids.
[0048] As used herein, the term "antisense" means a strand of RNA
whose sequence of bases is complementary to messenger RNA.
[0049] As used herein the term "oligo" includes oligonucleotides
which are polymers of nucleosides joined, generally, through
phosphoester linkages.
[0050] As used herein a "therapeutic amount" is an amount of a
moiety which produces a desired or detectable therapeutic effect on
or in a mammal administered the moeity.
[0051] As used herein, the term "sample" means a viable
(analyzable) sample of biological tissue or fluid. A biological
sample includes an effective amount of a representative section of
tissues or fluids of living animals, viable cells or cell
culture.
[0052] In an aspect, the DNA or genetic construct further comprises
an expression control sequence operably linked to a sequence
encoding (and expressing) the expression product.
[0053] As used herein, the terms "DNA construct" or "genetic gene
construct", "gene" or "cDNA" are used interchangeably herein to
refer to a nucleic acid molecule which may be one or more of the
following: regulatory regions, e.g. promoter and enhancer sequences
(that are competent to initiate and otherwise regulate the
expression of a gene product(s)); any other mutually desired
compatible DNA elements for controlling the expression and/or
stability of the associated gene product(s) such as polyadenylation
sequences; other DNA sequences which function to promote
integration of operably linked DNA sequences into the genome of the
host cell and any associated DNA elements contained in any nucleic
acid system (e.g. plasmid expression vectors) used for the
propagation, selection, manipulation and/or transfer of recombinant
nucleic acid sequences, sequences encoding proteins that are part
of the biosensor or proteins that are functional G protein coupled
receptors.
[0054] As used herein, the terms "regulatory DNA sequences" or
"regulatory regions" or "DNA sequences which regulate the
expression of" are used interchangeably herein to refer to nucleic
acid molecules which function as promoters, enhancers, insulators,
silencers and/or other similarly defined sequences which control
the spatial and temporal expression of operably linked and/or
associated gene products.
[0055] As used herein, the term "transgenic" refers to an organism,
or progeny derived from such organism(s) by germ cell transmission
or cloning, that contains exogenous genetic constructs that have
been purposefully introduced into the organism. Moreover, this
refers to organisms which may or may not have the introduced
genetic construct stably integrated into their genome, that is,
constructs which are maintained stably and can be propagated
through germ cell transmission (i.e. sexual reproduction) or
constructs which are expressed transiently by the organism.
[0056] As used herein the term "expression library" includes a
library of chemical moieties generally whose functions are unknown.
"Expression library" also includes a database, collection or
assemblage of moieties or a system of containing capably identified
moieties, cataloged or uncataloged, present or not present in the
collection or assemblage and illustratively includes expression
products of cDNA such as proteins, and enzymes including those
wherein one or more of identity and function or known or are
unknown.
[0057] As used herein, the term "expression" includes the
biosynthesis of a product as an expression product from a gene such
as the transcription of a structural gene into mRNA and the
translation of mRNA into at least one peptide or at least one
polypeptide. The term "expression" includes gene products such as
proteins and functional fragments thereof.
[0058] As used herein, the term "mammal" includes living animals
including humans and non-human animals such as murine, porcine,
canine and feline.
[0059] As used herein, the term "isolated polypeptide" includes a
polypeptide essentially and substantially free from contaminating
cellular components.
[0060] As used herein, the term "isolated protein" includes a
protein that is essentially free from contamination cellular
components normally associated with the protein in nature.
[0061] As used herein, the term "patient" and subject" are
synonymous and are used interchangeably herein.
[0062] We have identified the modulation (inhibition) of calcium
independent phospholipase A.sub.2 activity by calcium-activated
calmodulin. We have demonstrated that activation of
iPLA.sub.2.beta. activity in intact cells results from the
disinhibition of iPLA.sub.2.beta. during calcium pool depletion
either by agonist stimulation (leading to IP.sub.3 mediated
internal store calcium release) or by direct depletion of internal
stores by inhibition of the Sarcoplasmic Endoplasmic Reticulum
Ca.sup.2+-ATPase (SERCA) by thapsigargin (46). Recently, this
hypothesis has been reformulated to include the activation of the
calcium channels by lysophospholipids through the activation of
iPLA.sub.2.beta. mediated by CIF-induced dissociation of the
iPLA.sub.2.beta.-calmodulin complex. Thus, capacitative calcium
entry is mediated by the activation of iPLA.sub.2.beta.
accomplished through the dissociation of the inhibitory
calmodulin-iPLA.sub.2 complex. The resultant lysolipids produced by
iPLA.sub.2, and perhaps other mediators as well (e.g.,
eicosanoids), serve to open plasma membrane calcium channels to
accomplish capacitative calcium influx. This cellular signaling
pathway is known to occur in a wide variety of cell specific
contexts. However, the chemical identity of CIF is unknown.
Similarly, the molecular types and classes of molecules that serve
to disinhibit the calmodulin-iPLA.sub.2 complex have not been
directly identified or demonstrated. In this work, we demonstrate
that the class of acyl-CoAs can serve to activate iPLA.sub.2.beta.
by release of calmodulin mediated inhibition. Since fatty acids are
produced by depletion of internal calcium stores by either agonist
stimulation or indirectly through SERCA inhibition and
intracellular fatty acids are rapidly converted to acyl-CoAs these
results: 1) identify acyl-CoAs as the factor that activates
iPLA.sub.2.beta. by relief of calmodulin mediated inhibition; 2)
identify the sequential production of CIF first through calcium
depletion fatty acid release and subsequent thioesterificaton; and
3) provide a molecular basis for pharmacotherapy to modulate the
signals generated through capacitative calcium influx pathways for
salutary interventions in a wide variety of disease states. Such
diseases include, but are not limited to, inflammation,
hypertension, diabetes and insulin release, congestive heart
failure, atherosclerosis, myocardial infarction, arrhythmias,
cancer, stroke and disorders of calcium mediated motility in
general (e.g., GI, urinary, conception, birth control) or calcium
influenced processes (e.g., erectile dysfunction, wrinkles and hair
loss).
[0063] During cellular stimulation, multiple cell types activate
calcium influx pathways in the plasma membrane facilitating the
influx of extracellular calcium through non-voltage dependent
calcium channels. This influx of extracellular calcium is induced
by agonist-receptor interaction, activation of phospholipase C and
the subsequent release of IP.sub.3. The released IP.sub.3 diffuses
to its receptor in internal calcium stores in the endoplasmic
reticulum and results in the release of calcium from the IP.sub.3
gated calcium channels. Next, a second messenger of unknown
chemical identity is released from these internal stores after
calcium pool depletion. This second messenger diffuses to the
plasma membrane to activate calcium channels in a process known as
capacitative calcium influx. This calcium influx factor (CIF) has
shown to be a critical component for cellular signaling in multiple
cell specific contexts. However, the chemical identity of CIF is
unknown. Moreover, the influx of extracellular calcium is critical
both for signaling functions as well as for the repletion of
internal calcium stores.
[0064] Inclusion of fatty acyl-CoA guest in phosphatidyl-choline
host vesicles was demonstrated to disrupt or reverse
Ca.sup.2+-activated calmodulin inhibition of iPLA.sub.2.beta.
phospholipase A.sub.2 activity resulting in activation of the
enzyme iPLA.sub.2.beta.. Shotgun lipidomics identification of
cardiolipin decrease in diabetic hearts. Calcium-independent
phospholipase A.sub.2.beta. and iPLA.sub.2.gamma. were demonstrated
to catalyze hydrolysis of cardiolipin as guest in
phosphatidylcholine vesicles.
[0065] This invention could be used in the development of
pharmaceuticals which would target the activation of
iPLA.sub.2.beta. and iPLA.sub.2.gamma. during ischemia, diabetes,
heart disease, atherosclerosis, and obesity. Biomarkers for
diabetes include cardiolipin. Inhibition of iPLA.sub.2.beta. and
iPLA.sub.2.gamma. would be predicted to decrease cardiolipin
degradation and improve mitochondrial function.
[0066] Calcium-independent phospholipase A.sub.2.beta.
(iPLA.sub.2.beta.) participates in numerous diverse cellular
processes such as arachidonic acid release, insulin secretion,
calcium signaling, and apoptosis. Herein, we demonstrate the highly
selective iPLA.sub.2.beta.-catalyzed hydrolysis of saturated long
chain fatty acyl-CoAs (rank order: palmitoyl-CoA
myristoyl-CoA.apprxeq.stearoyl-CoA>>oleoyl-CoA.apprxeq.arachidonyl--
CoA) present either as monomers in solution or guests in a host
bilayer membrane. Site-directed mutagenesis of the catalytic serine
(S465A) of iPLA.sub.2.beta. completely abolished acyl-CoA
thioesterase activity, demonstrating that the same active site
serine residue catalyzes both phospholipid and acyl-CoA hydrolysis.
Remarkably, incubation of iPLA.sub.2.beta. with oleoyl-CoA, but not
other long-chain acyl-CoAs, resulted in robust stoichiometric
covalent acylation of the enzyme (.about.1:1
oleoyl/iPLA.sub.2.beta. mol ratio). Moreover, mutagenesis of
Ser-465 or pretreatment of wild-type iPLA.sub.2.beta. with
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one
(BEL) unexpectedly increased acylation of the enzyme, demonstrating
the presence of a second reactive nucleophilic residue that
participates in the formation of the fatty acyl-iPLA.sub.2.beta.
adduct. Partial trypsinolysis of oleoylated iPLA.sub.2.beta. and
MALDI-MS analysis localized the acylation site to a hydrophobic 25
kDa fragment (residues.about.400-600) spanning the active site to
the calmodulin (CaM) binding domain. Intriguingly,
calmodulin-Ca.sup.2+ blocked acylation of iPLA.sub.2.beta. by
oleoyl-CoA. Remarkably, addition of low micromolar concentrations
(5 .mu.M) of oleoyl-CoA resulted in significant reversal of
calmodulin-mediated inhibition of iPLA.sub.2.beta. phospholipase
A.sub.2 activity. These results collectively identify the robust
and molecular species specific iPLA.sub.2.beta. catalyzed
hydrolysis of acyl-CoA, demonstrate the presence of a second active
site that mediates iPLA.sub.2.beta. autoacylation, and identify
long-chain acyl-CoAs and their cogeners as potential candidates for
calcium influx factor.
[0067] Phospholipases A.sub.2 (PLA.sub.2s) catalyze the hydrolysis
of ester-linked fatty acids from glycerophospholipids, thereby
regulating numerous cellular processes through the generation of
lysophospholipids, free fatty acids (e.g. arachidonic acid) and
their downstream metabolites. In eukaryotes, PLA.sub.2s are broadly
categorized into three families: secretory (sPLA.sub.2), cytosolic
(cPLA.sub.2), and calcium-independent phospholipases A.sub.2
(cPLA.sub.2) (1). Secretory PLA.sub.2s are low molecular weight
(.about.12-15 kDa) enzymes secreted into the extracellular space
which require high micromolar to millimolar concentrations of
Ca.sup.2+ for catalysis (2, 3). Six cytosolic phospholipases
A.sub.2 (.alpha., .beta., .gamma., .delta., .epsilon., .xi.) have
been characterized at present, five of which (.alpha., .beta.,
.delta., .epsilon., .xi.) contain C2 domains that require
sub-micromolar Ca.sup.2+ for membrane association (4-7).
Calcium-independent PLA.sub.2s are intracellular, do not require
calcium ion for membrane association or catalysis, and currently
are comprised of seven family members (.alpha., .beta., .gamma.,
.delta., .epsilon., .xi., .eta.) (8-11), all of which contain
conserved nucleotide-binding (GXGXR/KG) and lipase (GXSXG)
consensus sequence motifs.
[0068] Long-chain fatty acyl-CoAs occupy a central position in
mammalian neutral lipid and phospholipid metabolism. The
biosynthesis of long-chain acyl-CoAs represents the first committed
step in the flux of fatty acids into either lipid storage pools or
towards energy (ATP) production. Extracellular non-esterified fatty
acids arriving at the inner leaflet of the plasma membrane are
trapped within the cell by thioesteriflcation to CoASH catalyzed by
acyl-CoA synthetases (12-14). Alternatively, fatty aeyl-CoAs can be
synthesized de novo from acetyl-CoA and malonyl-CoA by fatty acid
synthase (15), to produce palmitoyl-CoA, which can then be acted
upon by a long chain fatty acyl-CoA elongase to yield stearoyl-CoA
(16). Importantly, the generation of mono-unsaturated (e.g. oleic)
and polyunsaturated (e.g. arachidonic) fatty acyl species requires
.DELTA.5, .DELTA.6, and .DELTA.9 desaturases and elongases which
utilize specific long chain fatty acyl-CoAs as substrates.
Following synthesis, long chain acyl-CoAs are predominantly
delivered to one of many acyl-CoA transferases (e.g. for the
synthesis of phospholipids and triglycerides (17, 18)) or used in
the mitochondria or peroxisomes for .beta.-oxidation (19-21).
[0069] In addition to their fundamental lipid metabolic roles,
fatty acyl-CoAs can also participate in numerous regulatory
cascades such as ion channel function (22-24), membrane fusion
(25-27), and signal transduction (28, 29). Modulation of signaling
pathways by fatty acyl-CoAs can occur through either proximal
association (e.g. activation of K.sub.ATP-channels through
interactions with the Kir6.2 pore-forming subunit (23)), covalent
modification (e.g. through acylation of regulatory proteins by
palmitoyl acyl-CoA transferases (30)), or by regulating
transcriptional factor activity by acting as ligands for hepatic
nuclear factor 4.alpha. (HNF-4.alpha.) (31, 32).
[0070] Furthermore, acyl-CoAs have been implicated in peroxisome
proliferator activated receptor (PPAR) inhibition (33, 34) and,
conversely, non-esterified fatty acids have been shown to activate
PPAR-mediated transcription (35-37). Thus, long chain acyl-CoA
thioesterases may regulate cellular energy expenditure and
signaling by determining acyl-CoA substrate availability, by
metabolic "futile" cycling of acyl equivalents through acyl-CoA
hydrolysis, by production of lipid second messengers, and by
influencing transcription of PPAR and HNF-4.alpha. regulated genes
through controlling the ratio of cellular free fatty acids to their
CoA derivatives.
[0071] Multiple fatty acyl-CoA thioesterases have been purified
from mammalian cytosol, peroxisomes, and mitochondria and have been
cloned and characterized with respect to substrate selectivity,
enzyme kinetics, and sensitivity to various inhibitors (38). In
general, fatty acyl-CoA thioesterases have been tentatively
classified as those which are structurally related to those induced
by peroxisome proliferators (Type-I or Type-II thioesterases) and
those which do not share significant sequence homology with these
isoforms (38). Several other mammalian enzymes, such as
lysophospholipases (39), secretory phospholipase A.sub.2(40), and
palmitoyl-protein thioesterases (41-43), have also been shown to
exhibit acyl-CoA hydrolase activity. Interestingly, HNF-4.alpha.
has been recently demonstrated to hydrolyze fatty acyl-CoAs,
followed by binding of the fatty acid product to HNF-4.alpha.,
thereby allowing crosstalk between the acyl-CoA and free fatty acid
binding domains (44). Despite advances in identifying the proteins
and enzymes mediating acyl-CoA binding, hydrolysis, and
transcriptional signaling, the metabolic significance and
integrated functional roles of these chemical entities in different
cellular, subcellular, and tissue-specific contexts remains
incompletely understood.
[0072] Calcium-independent phospholipase A.sub.2.beta. has been
implicated as an important participant in multiple and diverse
cellular processes such as agonist-induced arachidonic acid release
(45, 46), apoptosis (47, 48), lymphocyte proliferation (49), fat
cell differentiation (50), insulin secretion (51) and lysolipid
production mediating capacitive calcium influx (52, 53). In
addition, recent tests utilizing transgenic mice selectively
overexpressing iPLA.sub.2.beta. in myocardium have provided
evidence that cardiac ischemia activates iPLA.sub.2.beta.,
precipitating ion channel dysfunction and resultant malignant
ventricular tachyarrythnias which can be suppressed by pretreatment
with the mechanism-based iPLA.sub.2 inhibitor,
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran2-one
(BEL) (54). Previously, we have demonstrated that iPLA.sub.2.beta.
activity is regulated through calmodulin-mediated inhibition of
phospholipase A.sub.2 activity in the presence of physiologic
concentrations of calcium (.about.200 nM) (55). Subsequent
structural studies identified the calmodulin binding domain of
iPLA.sub.2.beta., containing "1-9-14" and IQ sequence motifs,
located within a .apprxeq.15 kDa region approximately 50 amino acid
residues from the C-tenninus (56). During cellular stimulation and
the depletion of intracellular Ca.sup.2+ stores, activation of
iPLA.sub.2.beta. has been proposed to occur through disassociation
of the iPLA.sub.2.beta.-CaM complex (46, 53), potentially through
the actions of a low molecular weight cellular component known as
calcium influx factor (CIF) (53, 57). Although CIF was first
described and partially characterized more than 10 years ago as a
diffusible messenger released upon intracellular Ca.sup.2+ store
depletion which stimulated Ca.sup.2+ influx through the plasma
membrane (58, 59), the precise molecular identity of CIF has
largely remained a mystery and for this reason, its existence has
been controversial.
[0073] In addition to its calmodulin binding domain,
iPLA.sub.2.beta. possesses a conserved nucleotide binding motif
(GXGXR/KG) which binds ATP, resulting in both enzyme stabilization
and activation (60, 61). Thus, modulation of iPLA.sub.2.beta.
activity by ATP provides a potential mechanism through which
phospholipid hydrolysis and second messenger generation can be
regulated by the energy state (e.g. ATP/ADP ratio) of the cell.
From this perspective, we considered the possibility that
iPLA.sub.2.beta. might also bind and hydrolyze long-chain
acyl-CoAs, given the structural similarity of the
3'-phosphoadenosine moiety of CoA to ATP. In this manuscript, we
demonstrate that iPLA.sub.2.beta. catalyzes the hydrolysis of
saturated long chain acyl-CoAs present as either monomers or as
guests in host membrane vesicles at physiologic concentrations (1-5
mol %). Moreover, robust and highly selective covalent acylation of
iPLA.sub.2.beta. by oleoyl-CoA occurred at a second site(s) within
the catalytic domain which is protected from oleoylation by
calcium-activated calmodulin. Finally, oleoyl-CoA was found to
attenuate calmodulin-mediated inhibition of iPLA.sub.2.beta.
phospholipase A.sub.2 activity.
[0074] The above describes the previously unrecognized fatty
acyl-CoA thioesterase catalytic activity and fatty acyl-CoA
dependent covalent acylation of iPLA.sub.2.beta., thereby revealing
an additional level of chemical complexity in the integration and
participation of this multifunctional signaling enzyme in the
regulation of numerous cellular pathways.
[0075] In an aspect, pharmaceutical compositions and preparations
are made in a manner well known in the pharmaceutical art. One
exemplary preparation utilizes a vehicle of physiological saline
solution comprising at least one of a chemical agent, siRNA, and
penetrant that is combined with a pharmaceutically acceptable
carrier. A suitable buffer, such as sterile water, may be present
in the composition.
[0076] In an aspect, the carrier can also contain other
pharmaceutically-acceptable excipients and additives for modifying
or maintaining pH, osmolarity, viscosity, clarity, color,
sterility, stability, rate of dissolution, or odor of the
formulation. Similarly, the carrier may contain still other
pharmaceutically acceptable excipients for modifying or maintaining
release or absorption or penetration.
[0077] It is also contemplated that some formulations are more
conveniently administered orally in an effective amount and dosage.
Such formulations are preferably encapsulated and formulated with
suitable carriers in solid dosage forms.
[0078] The construction of a suitable vector can be achieved by any
of the methods well-known in the art for the insertion of exogenous
DNA into a vector. see Sambrook et al., 1989, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, N.Y.; Rosenberg et
al., Science 242:1575-1578 (1988); Wolff et al., PNAS 86:9011-9014
(1989). For Systemic administration with cationic liposomes, and
administration in situ with viral vectors, see Caplen et al.,
Nature Med., 1:39-46 (1995); Zhu et al., Science, 261:209-211
(1993); Berkner et al., Biotechniques, 6:616-629 (1988); Trapnell
et al., Advanced Drug Delivery Rev., 12:185-199 (1993); Hodgson et
al., BioTechnology 13:222 (1995).
[0079] The following Example illustrates the best currently-known
method of practicing this invention which is described in detail in
order to facilitate a clear understanding of the discovery. It
should be understood, however, that the detailed expositions of the
application of the invention, while indicating exemplary
embodiments, are given by way of illustration only and are not to
be construed as limiting the discovery since various changes and
modifications within the spirit of the discovery will become
apparent to those skilled in the art from this detailed
description. In the following examples, which illustrate the
invention, and throughout the specification, parts and percent are
by weight unless otherwise indicated.
EXAMPLE
[0080] Materials--[1-.sup.14C]-palmitoyl-CoA and
[1-.sup.14C]-oleoyl-CoA, and
1-palmitoyl-2-[1-.sup.14C]-oleoyl-sn-glycero-3-phosphocholine were
obtained from Perkin-Elmer. [1-.sup.14C]-Arachidonoyl-CoA,
[1-.sup.14C]-myristoyl-CoA, [1-.sup.14C]-stearoyl-CoA, and
[methyl-14C] human albumin were obtained from American Radiolabeled
Chemicals. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) were
purchased from Avanti Polar Lipids.
2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3.alpha.,4.alpha.-d-
iaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero3phosphocholine
(BODIPY-PC) was purchased from Invitogen. High purity bovine
calmodulin was obtained from Calbiochem. Most other materials were
obtained from either Sigma or Fisher Scientific. BEL was purchased
from Cayman Chemical and separated into individual enantiomers as
described previously (62).
[0081] Expression and Affinity Purification of
iPLA2.beta.(His).sub.6 from Sf9 Cells--Following infection of
3.times.100 ml cultures of Sf9 cells (1.5.times.10.sup.6 cells/ml)
with baculovirus encoding iPLA.sub.2.beta.(His).sub.6 for 48 hr,
cells were harvested by centrifugation (900.times.g for 10 mm),
washed once with Grace's Insect Medium without serum, and
resuspended in 30 ml of 25 mM sodium phosphate, pH 7.8, 20%
glycerol, 2 mM .beta.-ME, 5 .mu.g/ml aprotinin, 5 .mu.g/ml
leupeptin. After lysing the cells by sonication (30.times.1 s
bursts), the homogenate was centrifuged at 100,000.times.g for 1 h
to obtain the cytosol to which NaCl was added to a final
concentration of 250 mM. The cytosol was then mixed by inversion
with 3 ml of HIS-Select-Co2.sup.+ affinity resin (Sigma) for 1 hr
and the cytosol-resin suspension was poured into a Pharmacia
1.times.10 cm column. Following washing of the settled resin with
30 mL of Buffer A (25 mM sodium phosphate, pH 7.8 containing 500 mM
NaCl, 20% glycerol, and 2 mM .beta.-ME), bound protein was eluted
from the column at a flow rate of 0.25 ml/min utilizing a 250 mM
imidazole gradient in Buffer A (50 ml total volume) generated using
a Pharmacia FPLC system. Column fractions were assayed for
iPLA.sub.2 activity as described below, pooled and dialyzed
overnight against Buffer B (25 mM imidazole, pH 7.8 containing 20%
glycerol, 1 mM DTT, and 1 mM EGTA). The dialyzed sample was applied
to a 2.5 ml column of ATP agarose equilibrated with Buffer B and
washed with Buffer B containing 1 mM AMP and 50 mM NaCl. Bound
iPLA.sub.2.beta. (His).sub.6 was eluted with Buffer B containing 2
mM ATP and 50 mM NaCl, dialyzed against Buffer B (EGTA
concentration was reduced to 0.1 mM) containing 50 mM NaCl to
remove ATP, flash frozen in liquid nitrogen, and stored at
-80.degree. C. Approximately 1 mg (65% yield) of
iPLA2.beta.(His).sub.6 with a specific activity of 500 nmol oleic
acid min.sup.-1.cndot.mg.sup.-1 utilizing 5 .mu.M [.sup.14C]-POPC
as substrate was typically recovered from 300 ml of Sf9 cell
culture by this procedure.
[0082] Phospholipase A.sub.2 and Acyl-CoA Hydrolase Enzymatic
Assays--Purified recombinant iPLA.sub.2.beta.(His).sub.6 (0.1-1
.mu.g) was incubated with radiolabeled phospholipid or acyl-CoA in
25 mM Tris-HCl, pH 7.2 containing 1 mM EGTA (200 .mu.l final
volume) for 1-2 mm at 37.degree. C. In tests using acyl-CoAs as
guests in host phospholipid bilayers, radiolabeled acyl-CoAs were
incorporated into POPC/DOPS (90:10 mol %) vesicles before addition
to the reaction mix. Long-chain acyl-CoAs have been previously
demonstrated to integrate into lipid bilayers within seconds (63).
Incorporation of acyl-CoA into the POPC/DOPS vesicles employed was
determined as described previously (64). Reactions were terminated
by extraction of the released radiolabeled fatty acids into 100
.mu.l butanol, separation of fatty acids from unreacted substrate
by thin layer chromatography, and quantitation by scintillation
spectroscopy as previous described (65). For tests examining the
effects of acyl-CoAs on calmodulin-mediated inhibition of
iPLA.sub.2.beta., phospholipase A.sub.2 activity was continuously
measured utilizing a SPECTRAmax GEMINI XS Dual-Scanning Microplate
Spectrofluorometer (Molecular Devices). BODIPY-PC substrate (1.17
.mu.M in DMSO, 5 .mu.M final concentration) was co-sonicated (10
mm. at 40% power, 50% duty cycle) with POPC (95 .mu.M final
concentration) in 25 mM HEPES, pH 7.2. Oleoyl-CoA and CaCl.sub.2
were added at the indicated concentration to the lipid vesicles
before addition to iPLA.sub.2.beta. with or without CaM
(preincubated on ice for 10 mm) present in individual wells of a
black 96-well microtiter plate. Fluorescence readings were acquired
at 20 sec intervals for 5 mm at 37.degree. C. utilizing
excitation/emission wavelengths of 495/515 nm, respectively.
[0083] Covalent Modification of IPLA.sub.2.beta. with [.sup.14
C]-long chain Acyl CoAs--Purified recombinant iPLA.sub.2.beta.
(His).sub.6 was incubated with POPC vesicles containing 10 mol %
[1-.sup.14C]-acyl-CoA for 1 h at 37.degree. C. In some tests,
iPLA.sub.2.beta. (His).sub.6 was pre-incubated with BEL (3 min at
23.degree. C.), N-ethylmaleimide (5 min at 30.degree. C.) or
iodoacetamide (5 min at 30.degree. C.) prior to addition of
radiolabeled acyl-CoA. Chloroform/methanol precipitation of some
samples was performed as described (66) utilizing 15 .mu.g of
bovine serum albumin as carrier. In tests to examine the nature of
the covalent linkage between oleic acid and iPLA.sub.2.beta., acid
(HCl), base (NaOH), and hydroxylamine were added to the indicated
concentrations and the samples were incubated at 30.degree. C. for
1 h. Bovine serum albumin (15 .mu.g) and SDS-PAGE loading buffer
were then added to each sample prior to dialysis against 50 mM
Tris-HCl, pH 6.8 containing 10% glycerol and 1% SDS for 4 h.
Samples were electrophoresed by SDS-PAGE, fixed (40% methanol
containing 10% acetic acid), stained with Coomassie Blue R-250,
incubated in Amplify fluorographic reagent, dried, and exposed to
Kodak Biomax MR film for 2-5 days at -80.degree. C.
[0084] Partial Trypsinolysis of Oleoylated
IPLA.sub.2.beta.--Purified iPLA.sub.2.beta. (His).sub.6 (10 .mu.M)
was incubated with 50 .mu.M [1-.sup.14C]-oleoyl-CoA or unlabeled
oleoyl-CoA in 25 mM imidazole, pH 7.8 containing 50 mM NaCl, 0.1 mM
EGTA, 1 mM DTT, and 20% glycerol for 1 h at 37.degree. C. Excess
[1-.sup.14C]-oleoyl-CoA was removed by using a Micro Bio-Spin
(Bio-Rad) column equilibrated with the above buffer. Recovered
iPLA.sub.2.beta. was partially digested with trypsin (1:25 w/w) for
1-30 mm at 37.degree. C. Tryptic peptides were separated by
SDS-PAGE, fixed in 40% methanol/10% glacial acetic acid, stained
with Coomassie Blue, and destained in the fixation solution. Gels
containing the radiolabeled peptide fragments were soaked in
Amplify fluorogenic reagent (Amersham), dried, and exposed to film.
In parallel samples utilizing unlabeled oleoyl-CoA, the band
corresponding to the .about.25 kDa radiolabeled was excised, cut
into .about.1.times.1 mm pieces, and destained further by washing
with 50% acetonitrile at 37.degree. C. The gel pieces were then
dried in a Speed-Vac, resuspended in 50 mM ammonium bicarbonate
(100 .mu.L) containing 0.5 .mu.g of sequencing grade modified
trypsin (Promega), and incubated 12 h at 37.degree. C. After
aliquoting the supernatant solution to a separate tube, residual
peptides in the gel pieces were extracted into 50% acetonitrile,
20% isopropanol, 0.1% TFA, combined with the supernatant solution,
and concentrated utilizing a Speed-Vac.
[0085] MALDI-TOF of iPLA.sub.2.beta. Tryptic
Fragments--Concentrated peptide samples were diluted with 0.5% TFA,
absorbed to a C18 Zip-Tip (Millipore), and desorbed with a solution
comprised of 50% acetonitrile, 20% isopropanol, 0.1% TFA, and
containing in addition 5 mg/ml .alpha.-cyano-4-hydroxycinnamic
acid. Samples were applied to 192-spot sample plates (ABI) and
allowed to air dry. Mass spectral (MS) analysis was performed
utilizing an Applied Biosystems 4700 Proteomics Analyzer
(Framingham, Mass.), which possesses a 200 Hz Nd:YAG laser
operating at 355 nm. Mass accuracy of the instrument was externally
calibrated to the 4700 Proteomics Analyzer Calibration Mixture of
peptides. For MALDI-MS analysis, spectra were obtained by the
accumulation of 2500 consecutive laser shots at a collision energy
of 1 kV with air serving as the collision gas. Calculations of
predicted peptide and peptide fragment masses were performed using
programs developed at the UCSF Mass Spectrometry Facility
(http://prospector.ucsf.edu).
[0086] SDS-PAGE was performed according to Laemmli (67). Silver
staining of SDS-PAGE gels was performed as described (68). Protein
concentration was determined by a version of the Bradford protein
assay (Bio-Rad) with bovine serum albumin as a standard.
Results
[0087] iPLA.sub.2.beta. Hydrolyzes Fatty Acyl-CoAs as Either
Monomers or as Guests in Membrane Bilayers.
[0088] Calcium-independent phospholipase .DELTA.2.beta. contains a
highly conserved nucleotide binding domain (GxGxR/KG) which tightly
binds ATP, resulting in both stabilization and activation of the
enzyme (60, 61). Due to the structural similarity between ATP and
the 3'-phosphoadenosine moiety present in CoA, we hypothesized that
iPLA.sub.2.beta. could bind to, and potentially hydrolyze, the
thioester linkage of long chain fatty acyl-CoAs. Accordingly, we
overexpressed iPLA.sub.2.beta. (His).sub.6 in Sf9 cells and
purified the enzyme to apparent homogeneity (as determined by
SDS-PAGE and silver staining) by sequential cobalt and ATP affinity
chromatographies as described in Experimental Procedures. Initial
assays with iPLA.sub.2.beta. utilizing supramicellar concentrations
of palmitoyl-CoA (100 .mu.M) typically used for acylCoA
thioesterases revealed very low rates of iPLA.sub.2.beta.-catalyzed
palmitoyl-CoA hydrolysis (FIG. 1A). Remarkably, robust rates of
palmitoyl-CoA thioesterase activity catalyzed by iPLA.sub.2.beta.
were demonstrated at low micromolar concentrations of palmitoyl-CoA
with a maximal rate of .apprxeq.250 nmol palmitic acid x
min.sup.-1.times.mg.sup.-1 protein at 2.5 .mu.M palmitoyl-CoA (FIG.
1A). Similar requirements for low acyl-CoA concentrations due to
substrate-mediated inhibition were observed in previous studies of
a purified mitochondrial thioesterase from rabbit myocardium (69)
and peroxisomal acyl-CoA thioesterase 2 (70). It should be
recognized that significant palmitoyl-CoA-mediated inhibition of
iPLA.sub.2.beta. occurs below the critical micelle concentration of
palmitoyl-CoA and may imply the presence of a second acylCoA
binding site on the enzyme (vide infra).
[0089] iPLA.sub.2.beta. Catalyzes Hydrolysis of Palm-CoA Present as
Guests in a Host Phospholipid Bilayer.
[0090] Since iPLA.sub.2.beta. would likely be expected to encounter
acyl-CoAs in a membrane bilayer environment in vivo, we examined
whether the enzyme could hydrolyze palm-CoA present as a guest (at
a low mol %) in phospholipid host vesicles. Purified
iPLA.sub.2.beta. effectively hydrolyzed palmitoyl-CoA at
physiologically relevant concentrations of acyl-CoA (i.e. 1-5 mol
%) when present as a guest in
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC)/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) host
bilayers (9:1 mol ratio) (FIG. 1B). This was surprising since POPC
is an excellent substrate for iPLA.sub.2.beta. and would be
expected to efficiently compete with the palmitoyl-CoA as
substrate. To determine whether iPLA.sub.2.beta. could hydrolyze
acyl-CoAs other than palmitoyl-CoA, incubations were performed with
a series of different long-chain acyl CoA molecular species in host
POPC/DOPS bilayers. A dramatic selectivity for myristoyl- and
palmitoyl-CoA hydrolysis was observed (up to 20-fold) in comparison
to stearoyl-, oleoyl-, and arachidonyl-CoAs (FIG. 2). Thus,
iPLA.sub.2.beta. displays substantial selectivity for saturated
acyl-CoA substrates (14-16 carbons in length) in comparison to
longer unsaturated acyl-CoA molecular species (C18:1 and C20:4) in
the presence of membrane bilayers.
[0091] Determination of the Active Site Nucleophile Mediating Palm
itoyl-CoA Hydrolysis.
[0092] Site-directed mutagenesis of the lipase consensus motif
serine (GTS.sup.465TG) of iPLA.sub.2.beta. to alanine has been
previously demonstrated to ablate phospholipase A.sub.2 activity
(10). To determine if Ser-465 was equally crucial for
iPLA.sub.2.beta. acyl-CoA thioesterase activity, we mutated S465 to
alanine and compared the phospholipase A.sub.2 and palmitoyl-CoA
hydrolase activities of the affinity purified mutant protein to its
wild-type counterpart. Importantly, the S465A
iPLA.sub.2.beta.(His).sub.6 bound to ATP agarose (as determined by
Western analysis) demonstrating that the mutant protein was
properly folded near the region of nucleotide binding site
(.sup.431GGGVKG.sup.436) which is .apprxeq.30 amino acid residues
away from the lipase consensus site. As expected, substitution of
an alanine residue at Ser-465 abolished calcium-independent
PLA.sub.2 activity utilizing POPC as substrate (FIG. 3).
Importantly, hydrolysis of palmitoyl-CoA incorporated into
POPC/DOPS vesicles also was virtually eliminated in the S465A
mutant (FIG. 3), indicating that the active site serine hydroxyl
likely serves as the primary nucleophile for both phospholipase
A.sub.2 and acyl-CoA thioesterase reactions catalyzed by
iPLA.sub.2.beta..
[0093] Chiral Mechanism-Based Inhibition of Acyl-CoA Hydrolysis by
(R)- and (S)-BEL.
[0094] In previous work, we demonstrated that racemic
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one
(BEL) is a potent and highly selective inhibitor of both
iPLA.sub.2.beta. (IC.sub.50.about.0.2 .mu.M) and iPLA.sub.2.gamma.
(IC.sub.50.about.3 .mu.M) phospholipase A.sub.2 activities (11, 71,
72). Through resolving the enantiomers of BEL by chiral HPLC, we
have further shown that (S)- and (R)-BEL are selective for
iPLA.sub.2.beta. and iPLA.sub.2.gamma., respectively (62). To
determine if the distinct enantiomers of BEL had similar effects on
iPLA.sub.2.beta. palmitoyl-CoA thioesterase activity, (R) and (S)
BEL were pre-incubated with purified iPLA.sub.2.beta.(His).sub.6
prior to addition of radiolabeled palmitoyl-CoA as guest in
POPC/DOPS host bilayers. As seen in FIG. 4, (S)-BEL inhibited
iPLA.sub.2.beta. palm-CoA hydrolase activity with an IC.sub.50 of
approximately 0.1 .mu.M, whereas (R)-BEL was .apprxeq.8-fold less
effective (IC.sub.50=0.8 .mu.M). Thus, the selectivity of the BEL
enantiomers for inhibiting iPLA.sub.2.beta. palmitoyl-CoA
thioesterase activity is virtually identical to that previously
observed for inhibition of phospholipase A.sub.2 activity (62).
Collectively these results suggest that both long-chain acyl-CoA
and phospholipid substrates utilize the same mechanism and
hydrolytic site (binding domain and catalytic residue(s)) in
iPLA.sub.2.beta. for hydrolysis.
[0095] Identification of Specific Autoacylation of iPLA.sub.2.beta.
by Oleoyl-CoA.
[0096] Since prior work has demonstrated that various proteins such
as rhodopsin (73), G-protein .alpha. subunits (74, 75), and PKC
(76) are autoacylated in the presence of palmitoyl-CoA, we sought
to determine if iPLA.sub.2.beta. could become similarly acylated in
the presence of various saturated and unsaturated long-chain
acyl-CoA substrates. Remarkably, although incubations with
[1-.sup.14C]-myristoyl-CoA, [1-.sup.14C]-palmitoyl-CoA, and
[1-.sup.14C]-stearoyl-CoA demonstrated either no observable or only
diminutive acylation of iPLA.sub.2.beta. following SDS-PAGE, those
containing [1-.sup.14C]-oleoyl-CoA resulted in the dramatic
radiolabeling of iPLA.sub.2.beta. which was 10-100 fold greater
than their saturated counterparts (FIG. 5). Furthermore,
incubations with [1-.sup.14C]-arachidonoyl-CoA resulted in
25-fold-less signal intensity than with [1-.sup.14C]-oleoyl-CoA,
but iPLA.sub.2.beta. was still arachidonoylated under these
conditions (FIG. 5). To our surprise, similar tests with the
iPLA.sub.2.beta. S465A mutant displayed a shift in the selectivity
of acylation, i.e. autoacylation was greatest with stearoyl-CoA and
labeling with palmitoyl-CoA became clearly detectable (FIG. 5). In
addition, labeling of the S465A mutant with oleoyl-CoA was notably
reduced relative to wild-type iPLA.sub.2.beta.. Similar incubations
of BEL-pretreated iPLA.sub.2.beta. with the different
[1-.sup.14C]-acyl-CoAs revealed marked increases in acylation with
palmitoyl-CoA and stearoyl-CoA that were not observed with the
wild-type protein (FIG. 5). Thus, inactivation of the catalytic
site through either site directed mutagenesis or pretreatment with
BEL does not abolish iPLA.sub.2 acylation supporting the existence
of a second active site which catalyzes autoacylation.
[0097] To determine the stoichiometry of iPLA.sub.2.beta. acylation
with oleoyl-CoA, we incubated increasing concentrations of
[1-.sup.14C]-oleoyl-CoA with iPLA.sub.2.beta. and compared the
intensity of radiolabeling to a standard curve generated from
adding incremental amounts of [methyl-.sup.14C]-human serum albumin
of known specific activity (FIG. 6). The results demonstrate that
approximately 1 mole of [1-.sup.14C]-oleic acid was incorporated
per mole of iPLA.sub.2.beta. in the presence of POPC vesicles
containing up to a 5-fold molar excess of [1-.sup.14C]-oleoyl-CoA
relative to iPLA.sub.2.beta.. One potential consequence of
iPLA.sub.2.beta. oleoylation is alteration of catalytic activity,
either toward phospholipid or acyl-CoA substrates. To address this
possibility, iPLA.sub.2.beta. was incubated with or without
oleoyl-CoA and then purified by Co.sup.2+ metal affinity
chromatography to remove residual oleoyl-CoA. Results from these
tests indicated that oleoylation did not significantly affect
either iPLA.sub.2.beta. mediated POPC or palmitoyl-CoA hydrolysis
(data not shown). Thus, iPLA.sub.2.beta. autoacylation with
oleoyl-CoA occurs at site which does not block accessibility of
substrate to, or inhibit release of products from, the active
site.
[0098] Although SDS-PAGE would be expected to remove the
overwhelming majority of non-covalently associated lipid, it was
remotely conceivable that the hydrophobicity of the
iPLA.sub.2.beta. putative substrate binding site could possibly
cause tight non-covalent association of radiolabeled oleoyl-CoA or
oleic acid to the denatured iPLA.sub.2.beta.. To exclude this
possibility, iPLA.sub.2.beta. was incubated with
[1-.sup.14C]-oleoyl-CoA, precipitated with chloroform/methanol and
the resultant pellet was extensively washed with 70% acetone (FIG.
7, lane 2) in which both oleic acid and oleoyl-CoA are readily
soluble. This treatment did not result in an appreciable decrease
in signal intensity, indicating that the [1-.sup.14C]-oleate is
covalently bound to iPLA.sub.2.beta.. Since esterification of fatty
acids to proteins has been demonstrated to occur through either
amide, oxyester, or thioester linkages which can be distinguished
through chemical treatment with strong acid (HCl), strong base
(NaOH), or neutral hydroxylamine, additional tests were performed
to determine the nature of the covalent linkage. In the case of
[1-.sup.14C]-oleoyl-iPLA.sub.2.beta., addition of either 1 NHC1 or
1-2 N neutral hydroxylamine did not result in a significant
decrease in radiolabeling, while addition of 1 NaOH completely
eliminated the majority of covalently bound [1-.sup.14C]-oleate
(FIG. 7). The insensitivity of [1-.sup.14C]-oleoyl-iPLA.sub.2.beta.
to hydroxylamine and HCl would indicate the absence of thioester
and oxyester linkages, respectively, while the disappearance of
radiolabeling in the presence of NaOH is consistent with an amide
linkage. Pre-treatment of iPLA.sub.2.beta. with N-ethylmaleimide
and iodoacetamide decreased radiolabeling with
[1-.sup.14C]-oleoyl-CoA (FIG. 7), indicating that free thiol
(cysteine) groups are important for either the formation of
oleoyl-iPLA.sub.2.beta. acylintermediate(s) or for its subsequent
transfer to the terminal nucleophilic acceptor residue(s) in
iPLA.sub.2.beta.. Remarkably, acylation of iPLA.sub.2.beta. was not
detectable utilizing
1-palmitoyl-2-[1-.sup.14C]-oleoyl-sn-glycero-3-phosphocholine (with
or without CoASH) (FIG. 7), suggesting that the acyl-enzyme
intermediates formed with oleoyl-CoA are fundamentally distinct
from those generated by POPC where the preferred nucleophile is
water (hydrolysis) while with oleoyl-CoA, endogenous nucleophilic
iPLA.sub.2.beta. amino acid residue(s) serve as the preferred
donors, resulting in production of a stable and isolatable acyl
enzyme.
[0099] Attempts at Mass Spectrometric Identification of the
Hydrophobic Acylation Site and Trypsin Footprinting Determination
of the Oleoylated Domain.
[0100] To identify the site(s) of oleoylation in iPLA.sub.2.beta.,
we utilized MALDI-MS to examine tryptic digests of the protein,
specifically searching for unique peptide peaks that were 264.245
mass units (i.e. C18:1-H.sub.2O greater than their respective
parent peak. Despite multiple attempts utilizing a wide range of
conditions (e.g. in-gel digests, solution digests, multiple
proteases, combinations of proteases, organic solvent and detergent
extraction/solubilization techniques, etc.), we were unable to
identify potential candidate peaks for MALDI-MS/MS analysis.
Overall, we were able to achieve 70% sequence coverage of
iPLA.sub.2.beta.. Addition of oleate would be expected to increase
the calculated water-octanol partition coefficient (log (P) value)
of the modified peptide by 2.23, representing a significant
increase in nonpolarity (for reference, log (P) for
phenylalanine=1.000). The relatively low MALDI-MS signal intensity
of hydrophobic peptides is well documented (77-79) and has been
recently discussed in The Journal (80). Accordingly, we performed
tests to identify the region of the protein which contained the
oleoyl moiety through partial in-solution trypsinolysis of the
[1-.sup.14C]-oleoyl-iPLA.sub.2.beta. and subsequent separation of
the radiolabeled peptide fragments by SDS-PAGE. Results from these
tests revealed that the majority of the radioactivity was contained
within a 25 kDa proteolytic fragment (FIG. 8). In-gel tryptic
digestion and subsequent MALDI-MS analysis of the resultant
peptides comprising this 25 kDa polypeptide determined that it
encompassed residues 408-578 which contains both the nucleotide
binding domain and the active site (Table I). Thus, acylation by
oleoyl-CoA occurs within the catalytic domain of iPLA.sub.2.beta.,
although it does not appear to inhibit or block substrate (i.e.
palmitoyl-CoA or POPC) access to the active site serine (Ser-465)
for catalysis.
[0101] Effects of Calcium Activated Calmodulin on iPLA.sub.2.beta.
Mediated Acyl-CoA Hydrolysis.
[0102] Calcium-bound calmodulin has been previously demonstrated to
bind to iPLA.sub.2.beta. and potently inhibit the phospholipase
A.sub.2 activity of the enzyme (55, 81). We were therefore
interested to determine if Ca.sup.+2-CaM would have a similar
effect on the acyl-CoA thioesterase activity of iPLA.sub.2.beta..
Although inclusion of Ca.sup.+2-CaM inhibited the PLA.sub.2
activity of recombinant iPLA.sub.2.beta. by .apprxeq.70-80%, the
palmitoyl-CoA thioesterase activity was relatively unaffected
(.about.10% inhibition) under similar conditions (FIG. 9). Thus,
while the phospholipase A.sub.2 activity of iPLA.sub.2.beta. is
responsive to changes in intracellular calcium (via calmodulin),
iPLA.sub.2.beta. would be expected to constitutively hydrolyze
acyl-CoA thioesters independent of calcium concentration or the
presence of calmodulin.
[0103] Calmodulin-Mediated Protection of iPLA.sub.2.beta. Against
Oleoylation by Oleoyl-CoA.
[0104] The proximity of the oleoylated iPLA.sub.2.beta. 25 kDa
tryptic fragment to the calmodulin binding domain next led us to
investigate whether Ca.sup.2+CaM could protect the enzyme against
covalent acylation by oleoyl-CoA. Although the addition of either
calcium ions or CaM in the presence of EGTA alone did not alter the
extent of oleoylation of iPLA.sub.2.beta. (FIG. 10), the
combination of Ca.sup.2+ and CaM significantly decreased
autoacylation of the enzyme. From these results, acyl-CoA mediated
acylation would be predicted to primarily occur after dissociation
of the iPLA.sub.2.beta./CaM complex.
[0105] Oleoyl-CoA Mediated Reversal of the Inhibition of
iPLA.sub.2.beta. by Calmodulin.
[0106] Depletion of intracellular calcium stores has been
previously demonstrated to initiate the activation of
iPLA.sub.2.beta. through a poorly understood process involving
reversal of calmodulin inhibition of the enzyme by an unknown
cellular metabolite referred to as calcium influx factor (CIF) (52,
53, 57-59, 82). To address the possibility that acyl-CoA could
mitigate the inhibition of iPLA.sub.2.beta. by CaM, we utilized a
real-time fluorescence assay employing the PLA.sub.2 substrate,
2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3.alpha.,4.alpha.-d-
iaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine
(BODIPY-PC), to measure the kinetic effects of oleoyl-CoA (guest in
POPC (95 mol %)/BODIPY-PC (5 mol %) host vesicles) on CaM
inhibition of iPLA.sub.2.beta. phospholipase A.sub.2 activity. In
the absence of calmodulin, iPLA.sub.2.beta. efficiently hydrolyzes
BODIPY-PC present at 5 mol % in a POPC background as demonstrated
by a robust time dependent increase in fluorescence intensity (FIG.
11A). The presence of calcium ion did not appreciably affect the
phospholipase A.sub.2 activity of iPLA.sub.2.beta. under these
conditions (data not shown). In contrast, inclusion of
Ca.sup.2+-bound CaM inhibited iPLA.sub.2.beta.-catalyzed hydrolysis
of BODIPY-PC by approximately 70-80% (FIG. 1 IA). Remarkably,
addition of 1 mol % oleoyl-CoA could activate CaM-inhibited
iPLA.sub.2.beta.(.apprxeq.40% of initial activity) (FIG. 11B) and
the presence of 2.5-5 mol % oleoyl-CoA completely eliminated CaM
mediated inhibition of iPLA.sub.2.beta. (FIG. 11C-D) under these
conditions.
[0107] To confirm that iPLA.sub.2.beta. was in fact hydrolyzing
BODIPY-PC and that the increase in fluorescence observed was not
due to either protein-fluorophore or acylCoA-fluorophore
interactions, the reaction substrates and products were extracted
into chloroform/methanol in the presence of internal standards and
subsequently quantified and analyzed by ESI-MS (83). As
anticipated, the production of
2-lyso-1-(O-(11-(4,4-difluoro-5,7-dimethyl-1-4-bora-3.alpha.,4.alpha.-dia-
za-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine,
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, and
1-lyso-2-oleoyl-sn-glycero-3-phosphoeholine was dependent upon the
presence of iPLA.sub.2.beta. and the amount of each product was
diminished (.about.80%) by the presence of Ca.sup.2+-bound CaM
(FIG. 11E-G). Importantly, addition of 5 mol % of oleoyl-CoA to the
POPC/BODIPY-PC vesicles in the presence of
Ca.sup.2+/CaM/iPLA.sub.2.beta. completely reversed the inhibition
of iPLA.sub.2.beta. by Ca.sup.2+/CaM as evidenced by the recovery
of similar amounts of 16:0-LysoPC, 18:1-LysoPC, and LysoBODIPY-PC
to that observed with iPLA.sub.2.beta. alone (FIG. 11H). Finally,
to establish that the effects of oleoyl-CoA on Ca.sup.2+/CaM
mediated inhibition of iPLA.sub.2.beta. was not dependent on the
presence of BODIPY-PC, similar tests were performed with
1-palmitoyl-2-[1-.sup.14C]-oleoyl-sn-glyeero-3-phosphocholine as
substrate. Under these conditions, CaM inhibited iPLA.sub.2.beta.
activity by approximately 70% and the presence of oleoyl-CoA alone
caused moderate inhibition (20%) of iPLA.sub.2.beta. activity (FIG.
11I) presumably due to interactions with iPLA.sub.2.beta. at or
near the substrate binding site. The presence of oleoyl-CoA
increased the activity of the CaM-inhibited iPLA.sub.2.beta. to
approximately 75% of the activity observed with oleoyl-CoA alone
(FIG. 11I). Thus, these results demonstrate the rescue of the
calmodulin-inhibited iPLA.sub.2.beta. activity by oleoyl-CoA by
three independent methods and identify fatty acyl-CoAs as potential
candidates for calcium influx factor.
Discussion
[0108] Since its initial identification (84), purification (65) and
cloning (9, 10), calcium-independent phospholipase A.sub.2.beta.
has been demonstrated to be an important enzymatic mediator of
multiple cellular regulatory processes which include
agonist-stimulated arachidonic acid release (45, 47, 85, 86),
lymphocyte proliferation (49, 87), store-operated Ca.sup.2+ entry
(46, 52, 53), insulin secretion (51, 88), ischemia-induced
myocardial phospholipid hydrolysis (89, 90), and malignant
ventricular tachyarrhythmias (54). Prior studies have identified
ATP (9) and calmodulin binding domains (56), ankyrin repeats (10,
91), multiple splice variants (92, 93), proteolytic products (94,
95), phosphorylation (9, 96), and interaction with calmodulin
kinase II .beta. (97) each of which collectively serve as potential
regulators of the pleiotropic signaling functions of
iPLA.sub.2.beta.. It has been demonstrated that iPLA.sub.2.beta.
efficiently catalyzes the hydrolysis of saturated fatty acyl-CoAs
at physiologically relevant concentrations, is selectively
autoacylated by oleoyl-CoA, is protected from autoacylation by
Ca.sup.2+-CaM, and is rescued from calmodulin-mediated inhibition
by oleoyl-CoA.
[0109] Optical antipodes of BEL and site-directed mutagenesis of
Ser-465 inhibit both iPLA.sub.2.beta. thioesterase and
phospholipase A.sub.2 activities to identical degrees, indicating
that the same active site and nucleophile (Ser-465) is utilized for
both reactions. In contrast, Ca.sup.2+-CaM significantly inhibited
only the phospholipase A.sub.2 activity of iPLA.sub.2.beta.,
whereas the acylCoA thioesterase activity of the enzyme was
unaffected under similar conditions. This suggests that the
"monoacyl" acyl-CoA substrate may have greater access to the
iPLA.sub.2.beta. active site (Ser-465) than the bulkier diacyl
phospholipid substrate in the presence of Ca.sup.2+-bound CaM.
Remarkably, native iPLA.sub.2.beta. is selectively and
stoichiometrically autoacylated by unsaturated acyl-CoAs
(particularly oleoyl-CoA), but not saturated acyl-CoAs, present in
a phospholipid bilayer. Conversely, autoacylation of
iPLA.sub.2.beta. by saturated acyl-CoAs is dramatically increased
by either mutagenesis of Ser-465 or pretreatment of the enzyme with
BEL. In contrast to its inability to inhibit acyl-CoA hydrolysis,
calmodulin in the presence of calcium blocked covalent acylation of
iPLA.sub.2.beta. by oleoyl-CoA. A combination of partial
trypsinolysis and MALDI-MS was utilized to localize the region of
acylation to amino acid residues .apprxeq.400-600 (adjacent to the
calmodulin binding domain) which includes the nucleotide and lipase
consensus sequence motifs. Clearly, the site of acylation is
distinct from the iPLA.sub.2.beta. active site serine (Ser-465) and
the proximal nucleophilic residue(s) which form(s) a covalent
adduct with BEL. In conjunction, these results demonstrate the
existence of a second nucleophilic site(s) in iPLA.sub.2.beta.
capable of cleaving the acyl-CoA thioester bond of both saturated
and unsaturated molecular species of acyl-CoA resulting in the
formation of a stable acyl-enzyme.
[0110] To our knowledge, we believe that iPLA.sub.2.beta. is the
only intracellular phospholipase A.sub.2 to exhibit substantial
amounts of long chain acyl-CoA thioesterase activity and represents
the first acyl-CoA thioesterase identified at the molecular level
shown to efficiently hydrolyze membrane-associated acyl-CoAs. In
contrast, in vitro assays with purified recombinant
cPLA.sub.2.gamma. (98) and iPLA.sub.2.gamma. (C. M. Jenkins and R.
W. Gross, unpublished observation).sup.2 in our hands did not
detect appreciable long-chain acyl-CoA hydrolase activities. A 54
kDa acyl-CoA hydrolase from rat intestinal microsomes was found to
cleave long chain acyl-CoAs in the presence of phosphatidylcholine
vesicles, although the sequence identity of this enzyme has not
been described since its original purification (99). Intriguingly,
iPLA.sub.2.beta. has been previously found to be localized to the
mitochondrial inner membrane (90) and the plasma membrane (96). One
possible role of iPLA.sub.2.beta. may be to prevent an excess of
palmitoyl-CoA accumulation at plasma membranes (e.g. the sarcolemma
which rapidly imports fatty acids and converts them to their
acyl-CoA derivatives) or at the mitochondrial inner membrane which,
through CPT-1, transfers palmitoyl-CoA to the matrix via a
palmitoyl-carnitine intermediate.
[0111] Furthermore, considerable work has underscored the
importance of both fatty acid and acyl-CoA molecular species as
modulators of ion channel function (100, 101). Arachidonic acid,
either added exogenously or generated in situ by phospholipase
A.sub.2, is believed to be an important regulator of ARC (102),
Kv1.1 (103), Kv1.5 (104), IK1 (105), and epithelial Na+ (106)
channels, among others. In addition, physiologically relevant
concentrations of acyl-CoAs in patch clamp as well as whole cell
studies have been shown to stimulate the activity of multiple
different types of K channels presumably through direct
interactions of the ion channel with the charged acyl-CoA moiety
(100, 101, 107). Accordingly, cells (e.g. cardiac myocytes) must
regulate ion channel function in the proximity of active fatty acid
import in which nascent acyl-CoAs are either hydrolyzed by
thioesterases (i.e. futile cycling) or transported out of the
sarcolemma through the process of diffusion or through specific
interactions with acyl-CoA binding proteins.
[0112] Considering the ability of iPLA.sub.2.beta. to translocate
to the plasma membrane in activated cells (96), modulate Kv1.1
electrophysiologic function (103), and precipitate ventricular
arrythmias following ischemia (54), we discovered that
iPLA.sub.2.beta. may regulate ion channel function both by the
release of arachidonic acid from phospholipids and modulation of
the effective mole concentrations of palmitoyl-CoA and fatty acids
(which increase dramatically during ischemia) surrounding
sarcolemmal ion channels.
[0113] Depletion of intracellular calcium stores in smooth muscle
cells has been previously demonstrated to activate iPLA.sub.2.beta.
through a mechanism hypothesized to involve the dissociation of CaM
from the enzyme (46, 62). Store operated calcium (cation) channels
(SOCs) in the plasma membrane are then activated in response to
agonist stimulated intracellular calcium pool depletion for the
purpose of replenishing the emptied calcium stores. Recent work by
Bolotina and colleagues has provided additional details into this
process by showing that iPLA.sub.2.beta. is required for activation
of SOCs through generation of lysophospholipids (53). Furthermore,
the inhibitory complex between CaM and iPLA.sub.2.beta. could be
disrupted by a partially purified preparation of calcium influx
factor (CIF) (53). Although attempts to elucidate the molecular
identity of CIF over the past 10 years have not been successful,
these studies have determined various chemical properties of
calcium influx factor.
[0114] In general, CIF is believed to be a non-protein, diffusible,
phosphorylated "sugar nucleotide" of approximately 600 Da which is
resistant to heat, alkaline pH, protease treatment, and is retained
on a C18 reverse phase matrix (108). Although the molecular weight
of oleoyl-CoA (1030 Da) and other fatty acyl-CoAs are slightly
larger than that approximated for CIF, many of the other chemical
characteristics of these compounds are similar. In this work, we
demonstrate that oleoyl-CoA is able to mimic the properties of CIF
by restoring the phospholipase A.sub.2 activity of
Ca.sup.2+/CaM-inhibited iPLA.sub.2.beta..
[0115] Although acyl-CoAs are considered to be ubiquitous cellular
constituents, their acute production due to fatty acid influx
and/or increases in acyl-CoA synthetase activity in specific
membrane microenvironments proximal to complexes of
Ca.sup.2+/CaM-inhibited iPLA.sub.2.beta. would likely be sufficient
to mediate activation of iPLA.sub.2.beta. through displacement of
calmodulin in a temporal and spatially-specific manner. We
specifically point out that many other CIF-like cellular
constituents capable of reversing Ca.sup.2+/CaM-iPLA.sub.2.beta.
inhibition may exist and that other membrane components and
conditions which occur in vivo (proteins, membrane surface charge
and curvature, membrane electrochemical potential, etc.) may
facilitate this process.
[0116] The specificity of iPLA.sub.2.beta.-mediated acyl-CoA
hydrolysis for saturated vs. unsaturated acyl-CoA molecular species
(FIG. 2) and inverse selectivity for autoacylation (FIG. 5),
provides intriguing evidence to suggest their potential functions
in cellular lipid homeostasis. Recently, small interfering RNA
knockdown of iPLA.sub.2.beta. and mechanism-based inhibition of
iPLA.sub.2.beta. by BEL was demonstrated to inhibit the hormone
induced differentiation of 3T3-L1 pre-adipocytes (50). Although the
phospholipase A.sub.2 activity of iPLA.sub.2.beta. likely
contributes to its importance in 3T3-L1 differentiation (through
production of non-esterified fatty acids and lysophosphatidic
acid), the acyl-CoA thioesterase activity of iPLA.sub.2.beta. may
also be important. For example, iPLA.sub.2.beta. could facilitate
futile cycling of excess saturated acyl-CoAs, thereby attenuating
their inhibition of acetyl-CoA carboxylase and thereby promoting
lipogenesis.
[0117] In addition, accelerated palm-CoA hydrolysis by
iPLA.sub.2.beta. could assist 3T3-L1 differentiation by relieving
potential palmitoyl-CoA mediated inhibition of PPAR.gamma. (34). De
novo synthesized monounsaturated fatty acyl-CoAs, produced through
the action of stearoyl-CoA desaturases from their saturated
precursors, is believed to directly contribute to the high content
of monounsaturated fatty acids (primarily 18:1) in adipocyte
triglycerides, phospholipids, and cholesterol esters (109). The
resistance of oleoyl-CoA to hydrolysis by iPLA.sub.2.beta. and the
formation of a stable oleoylated enzyme suggest that
iPLA.sub.2.beta. may be an unsaturated acyl-CoA acyltransferase for
as yet undiscovered protein or lipid acceptors.
[0118] Multiple acyl-CoA thioesterases have been cloned from
mammalian sources and are classified on the basis of their
subcellular localization (cytosolic, mitochondrial, or
peroxisomal), sequence similarity, and ability to be induced by
peroxisome proliferators. The majority of these thioesterases, as
well as all known intracellular phospholipases A.sub.2, contain the
canonical lipase (esterase) consensus sequence motif G-X-S-X-G.
Amino acid sequence alignments of iPLA.sub.2.beta. with the known
mammalian acyl-CoA thioesterases did not reveal any significant
sequence homology outside of the G-X-S-X-G consensus motif. This is
not completely unexpected given the diversity among the different
classes of acyl-CoA thioesterases. Some of the established acyl-CoA
thioesterase family members (e.g. MTE-I and CTE-I) possess
conserved putative nucleotide binding sequences (G-X-G-X-X-G),
however it is currently unknown whether these putative nucleotide
binding motifs interact with the CoA nucleotide. Interestingly,
calcium-independent phospholipase A.sub.2.beta. displays an
acyl-CoA substrate selectivity (C14-C20) similar to the cytosolic
Type-I thioesterase (CTE-I) (38). In addition, iPLA.sub.2.beta.,
like CTE-I, is not inhibited by high concentrations of CoASH,
indicating that these enzymes are probably not involved in
"sensing" and regeneration of free CoASH through acyl-CoA
hydrolysis as has been ascribed to peroxisomal acyl-CoA
thioesterase-2 (70). Calcium-independent phospholipase
A.sub.2.beta. and other acyl-CoA thioesterases likely serve both
metabolic and signaling functions. While the importance of fatty
acid synthesis and .alpha./.beta.-oxidation of fatty acids
(acyl-CoA-dependent processes) are evident, the metabolic role of
the multiple cellular fatty acyl-CoA thioesterase activities in
normal and disease states remain to be elucidated.
[0119] Covalent fatty acid acylation of eukaryotic proteins has
been previously demonstrated to occur either through
N-myristoylation, N-palmitoylation, or S-palmitoylation (110-112).
Intriguingly, although numerous investigators have described
protein palmitoylation utilizing palmitoyl-CoA, iPLA.sub.2.beta.
represents the first example to our knowledge of an enzyme which is
selectively acylated by oleoyl-CoA and arachidonyl-CoA (but not by
palmitoyl-CoA). The 54 kDa acyl-CoA hydrolase from rat intestinal
microsomes has been shown to be covalently modified by oleoyl-CoA,
likely through a thioester linkage, although the susceptibility of
this enzyme to acylation by other fatty acyl CoAs was not examined
(99). It would appear that this acyl-CoA hydrolase is unrelated to
iPLA.sub.2.beta. since it did not exhibit detectable phospholipase
or lysophopholipase activity (99).
[0120] We demonstrated our discovery that purified recombinant
iPLA.sub.2.beta. possesses robust palmitoyl-CoA hydrolase activity
in addition to its previously well-characterized lysophospholipase
and phospholipase A.sub.2 activities. Thus, iPLA.sub.2.beta. could
potentially have multiple effects on the production of lipid
metabolites (arachidonie acid and lysolipids), or alternatively
through removal of saturated acyl-CoAs from cellular membranes, and
regulate their metabolic fate. Importantly, calcium-independent
phospholipase A.sub.2.beta. is present in multiple subcellular
compartments, most notably cytosol, mitochondria, and nuclear
membranes (84, 90, 96). The presence of iPLA.sub.2.beta. in
mitochondria suggests that it may serve a similar function to the
mitochondrial acyl-CoA thioesterase which has been proposed to be
involved in UCP regulation. Moreover, iPLA.sub.2.beta. is
autoacylated in a highly substrate-specific fashion (by oleoyl--but
not palmitoyl-CoA) which occurs at a second active site distinct
from the hydrolytic lipase site (GXSXG). Collectively, these
results identify a complex interplay of enzymatic, biophysical, and
covalent modifications that likely contribute to the multiple
diverse signaling roles of iPLA.sub.2.beta. activity in cellular
functions. Multiple discrete chemical mechanisms (covalent and
non-covalent) likely regulate iPLA.sub.2.beta. activity, determine
iPLA.sub.2.beta. subcellular localization, and contribute to the
previously unappreciated biologic complexity of this critical
signaling enzyme. TABLE-US-00001 TABLE 1 25 kDa iPLA.sub.2.beta.
Fragment Tryptic Peptides Identified by MALDI-MS Analysis m/z
(Obs.) m/z (Calc.) Mod. Sequence Residues 1076.6085 1076.5376
(R)NYDAPEVIR(E) 547-555 1093.6473 1093.5795 pyroGlu (R)QPAELHLFR(N)
538-546 1110.6732 1110.6060 (R)QPAELHLFR(N) 538-546 1149.7157
1149.6454 (R)KPAFILSSMR(D) 408-417 1458.8209 1458.7341
(R)NYDAPEVIREPR(F) 547-558 1708.9492 1708.8546
(R)GSRPYESGPLEEFLK(R) 497-511 2184.2805 2184.1599
(K)VMLTGTLSDRQPAELHLFR(N) 528-546 2379.3950 2379.2573
(R)FNQNINLKPPTQPADQLVWR(A) 559-578
[0121] TABLE-US-00002 TABLE 2 Cardiolipin and Phospholipid
Hydrolysis Catalyzed by iPLA.sub.2.beta. and iPLA.sub.2.gamma. and
Inhibition by BEL. Purified calcium-independent phospholipase
A.sub.2.beta. (iPLA.sub.2.beta.) or calcium-independent
phospholipase A.sub.2.gamma. (iPLA.sub.2.gamma.) were incubated
with PAPC (100 .mu.M) vesicles containing 10 mol % tetra-18:1
cardiolipin (CL) for 20 min at 37.degree. C. Following addition of
internal standards, lipids were extracted utilizing a modified
Bligh-Dyer method prior to infusion and lipid analysis as described
in Experimental Procedures. nmol/reaction Blank iPLA.sub.2b +
iPLA.sub.2g + Lipid Species (-Ctl) iPLA.sub.2b BEL iPLA.sub.2g BEL
Tetra-18:1 CL 2.04 0.29 1.95 1.85 2.18 Tri-18:1 0.06 0.03 0.10 0.19
0.14 LysoCL Di-18:1 0.06 0.06 0.15 0.21 0.12 LysoCL 18:1 FA 0.20
6.32 0.61 1.07 0.54 16:0 FA 0.35 13.72 0.96 4.99 3.07 20:4 FA 0.15
8.53 0.50 0.94 0.54
[0122] TABLE-US-00003 TABLE 3 Cardiolipin and Phospholipid
Hydrolysis Catalyzed by iPLA.sub.2.beta.. Vesicles (100 .mu.M total
lipid) containing PLPC (60 mol %), plasmenyl-PE (30 mol %), and
tetra-18:1 cardiolipin (10 mol %) were incubated with purified
iPLA.sub.2.beta. for the indicated times at 37.degree. C. Following
addition of internal standards, lipids were extracted utilizing a
modified Bligh-Dyer method prior to infusion and lipid analysis as
described in Experimental Procedures. nmol/reaction Lipid Species 0
min 1 min 5 min PLPC 30.22 29.45 29.22 16:0 LPC 0.00 1.37 1.86 18:2
LPC 0.00 0.65 0.95 16:0 FA 0.00 0.65 1.30 18:2 FA 0.00 1.67 2.51
Plasm-PE 14.29 13.80 12.28 LPE 0.00 0.68 0.91 20:4 FA 0.00 0.72
1.13 Tetra 18:1-CL 6.06 5.96 5.84 Tri-18:1 LysoCL 0.00 0.03 0.05
18:1 FA 0.00 0.60 0.90
[0123] Cardiolipin Hydrolysis
[0124] Purified calcium-independent phospholipase A.sub.2
(iPLA.sub.2.beta.) and calcium-independent phospholipase
A.sub.2.gamma. were incubated with vesicles containing 10%
cardiolipin (tetra 18:1) for 20 min at 37.degree. C. For some
samples, BEL was added at 10 .mu.M final concentration. Following
addition of internal standards, lipids were extracted into
chloroform/methanol (modified Bligh-Dyer method) as previously
described (10.1) in the presence of internal standards
(14:1-14:1-PC (15 nmol/r.times.n), tetra 14:0-CL (2
nmol/r.times.n), and 16:0 d4 FA (5 nmol/r.times.n). Lipids from
each sample were extracted twice against 50 mM LiCl, back-extracted
once against 10 mM LiCl, dried under N.sub.2, and stored at
-20.degree. C. Samples were dissolved in 100 .mu.l l of 1:1 (v/v)
chloroform/methanol and diluted 20-fold immediately prior to
infusion and lipid analysis. TABLE-US-00004 SEQUENCE Human
Oleoyl-iPLA.sub.2.beta. SEQUENCE NO. 1
[MQFFGRLVNTFSGVTNLFSNPFRVKEVAVADYTSSDRVREEGQLILFQN
TPNRTWDCVLVNPRDSQSGFRLFQLELEADALVNFHQYSSQLLPFYESSP
QVLHTEVLQHLTDLIRNHPSWSVAHLAVELGIRECFHHSRIISCANCAEN
EEGCTPLHLACRKGDGEILVELVQYCHTQMDVTDYKGETVFHYAVQGDNS
QVLQLLGRNAVAGLNQVNNQGLTPLHLACQLGKQEMVRVLLLCNARCNIM
GPNGYPIHSAMKFSQKGCAEMIISMDSSQIHSKDPRYGASPLHWAKNAEM
ARMLLKRGCNVNSTSSAGNTALHVAVMRNRFDCAIVLLTHGANADARGEH
GNTPLHLAMSKDNVEMIKALIVFGAEVDTPNDFGETPTFLASKIGRQLQD
LMHISRARKPAFILGSMRDEKRTHDHLLCLDGGGVKGLIIIQLLIAIEKA
SGVATKDLFDWVAGTSTGGILALAILHSKSMAYMRGMYFRMKDEVFRGSR
PYESGPLEEFLKREFGEHTKMTDVRKPKVMLTGTLSDRQPAELHLFRNYD
APETVREPRFNQNVNLRPPAQPSDQLVWRAARSSGAAPTYFRPNGRFLDG
GLLANNPTLDAMTEIHEYNQDLIRKGQANKVKKLSIVVSLGTGRSPQVPV
TCVDVFRPSNPWELAKTVFGAKELGKMVVDCCTDPDGRAVDRARAWCEMV
GIQYFRLNPQLGTDIMLDEVSDTVLVNALWETEVYIYEHREEFQKLIQLL
LSP]-CO(CH.sub.2).sub.7CHCH(CH.sub.2).sub.7CH.sub.3* * Oleoylation
at any iPLA.sub.2.beta. residue
[0125] Shotgun lipidomics: multidimensional MS analysis of cellular
lipidomes The technical article SHOTGUN LIPIDOMICS: ELECTROSPRAY
IONIZATION MASS SPECTROMETRIC ANALYSIS AND QUANTITATIN OF CELLULAR
LIPIDOMES DIRECTLY FROM CRUDE EXTRACTS OF BIOLOGICAL SAMPLES,
Zianlin Han and Richard W. Gross, Washington University School of
Medice, St. Louis Mo., Mass. Spectrometry Reviews, 2005, 24,
367-412 is incorporated herein in its entirety by reference. This
article illustrates shotgun lipidomics.
[0126] Shotgun lipidomics comprises intrasource separation,
multidimensional mass spectrometry and computer-assisted array
analysis and is an emerging powerful technique in lipidomics.
Through effective intrasource separation of predetermined groups of
lipid classes based on their intrinsic electrical propensities,
analyses of lipids from crude extracts of biologic samples can be
directly and routinely performed. Appropriate multidimensional
array analysis of lipid pseudomolecular ions and fragments can be
performed leading to the identification and quantitation of
targeted lipid molecular species. Since most biologic lipids are
linear combinations of aliphatic chains, backbones and head groups,
a rich repertoire of multiple lipid building blocks present in
discrete combinations represent experimental observables that can
be computer reconstructed in conjunction with their pseudomolecular
ions to directly determine the lipid molecular structures from a
lipid extract. Through this approach, dramatic increases in the
accessible dynamic range for ratiometric quantitation and
discrimination of isobaric molecular species can be achieved
without any prior column chromatography and operator-dependent
supervision. At its current gate of development, shotgun lipidomics
can analyze over 20 lipid classes, hundreds of lipid molecular
species and more than 95% of the mass content of a cellular
lipidome. Thus, understanding the biochemical mechanisms underlying
lipid-mediated disease states will be greatly facilitated by the
power of shotgun lipidomics.
[0127] Expert Rev Proteomics 2(2), 253-264 (2005)
[0128] Lipidomics, the metabolomics of lipids, is a rapidly
expanding field following the tremendous progress that has been
made in genomics and proteomics [1.1, 2.1]. As such, lipidomics is
an essential component of systems biology [3.1, 4.1]. Specifically,
lipidomics is the large-scale study of organic solvent-soluble
lipids by integrating many different modern techniques (e.g., mass
spectrometry [MS]). The first essential step in lipidomics is to
determine a total lipid profile (i.e., lipidome). The total lipid
profile reflects the functional status of the cellular metabolic
history and the lipid-related protein expression and functional
profile of the cell resulting from metabolic, environmental or
nutritional clues [5.1, 6.1]. The lipidome provides information on
the biophysical state of cellular membranes [5.1], differences in
lipid pools and turnover rates by dynamic lipidomics) [7.1-10.1],
alterations in cellular energy supply [6.1], and lipid second
messenger levels reflecting cellular metabolic responses and
transcriptional programs [11.1]. The field of lipidomics has been
greatly advanced by the development and application of MS,
particularly electrospray ionization (ESI)/MS [1.1, 12.1-15.1].
Investigations in lipidomics are currently focused on identifying
alterations in cellular and/or body fluid lipid levels indicative
of pathology (e.g., the onset and progression of disease),
environmental perturbations (e.g., diet, toxins or drugs), or
response to treatment. Therefore, lipidomics is directly related to
drug discovery and evaluation of drug efficacy in addition to its
fundamental role in identifying the biochemical mechanisms of lipid
metabolism and the discovery of novel biomarkers.
[0129] Cellular lipidomes are highly complex and variable,
depending upon the species, cell type, internal organelles,
micro-domains (e.g., rafts) and growth conditions. Furthermore,
each cell type possesses different mole percentages of specific
lipid classes, subclasses and molecular species (that are comprised
of the differential lengths, degree of unsaturation and branching
of aliphatic chains). Tens of thousands of possible lipid molecular
species are predictably present in a cellular lipidome at the level
of attomole to nanomole of lipids per milligram of protein. Studies
in lipidomics by many investigators have focused on either one
class/subclass of lipids or one of the physical/chemical properties
of lipids [16.1-24.1], which is now referred to as targeted
lipidomics. However, the emergence of Intrasource separation with
multidimensional MS has allowed global lipid profiling and
quantitation directly from crude extracts of biologic samples [1.1,
6.1, 9.1, 15.1, 25.1-27.1]. These methods have now been referred to
as shotgun lipidomics, which was developed to exploit the synergy
between the uses of intrasource separation and multidimensional
MS.
[0130] Shotgun Lipidomics: Intrasource Separation
[0131] An essential point of ESI is the charge separation and
selective ionization that separated charges undergo at a high
electrical potential (typically .about.4 kV) in the ion source
[32.1-35.1]. Specifically, an electrospray ion source selectively
generates gas-phase cations in the positive-ion mode and results in
anions in the negative-ion mode if both inherently charged moieties
are present in the infused solution. If the analytes in the infused
solution do not carry net inherent charge(s), these compounds can
interact with small cation(s) or anion(s) available in the matrix
to yield adduct ions in positive- or negative-ionmode (i.e., in
conjunction with the imposed field), respectively. The ionization
efficiencies of these electrically neutral compounds depend on the
inherent dipoles of the compounds. The authors recognized this
physical process in the electrospray ion source in their earliest
study and used it to resolve lipid classes in a crude lipid extract
into different categories based on the intrinsic electrical
properties of each lipid class (see [1.1, 15.1] for reviews)
[36.1]. With regard to the separation of lipid classes, this
technique is analogous to using ion-exchange chromatography for
separation of lipid classes (as the authors have previously
employed [37.1]). However, this approach is rapid, direct,
reproducible and avoids artifacts inherent in chromatography-based
systems [38.1]. This new methodology has now been referred to as
intrasource separation [15.1, 26.1].
[0132] Although there are tens of thousands of potential lipid
molecular species present in a cellular lipidome, these species can
generally be classified into three main categories based upon their
electrical properties [15.1]. The lipid classes in the first
category are those carrying at least one net negative charge under
weakly acidic conditions and are therefore referred to as anionic
lipids, which can be directly analyzed from diluted lipid extracts
by negative-ion ESI/MS. Lipid classes in this category include
cardiolipin, phosphatidylglycerol, phosphatidylinositol and its
polyphosphate derivatives, phosphatidylserine, phosphatidic acid,
sulfatide, acyl-CoA and anionic lysophospholipids. The lipid
classes in the second category are those that are electrically
neutral under weakly acidic conditions, but become negatively
charged under alkaline conditions. Therefore, they are referred to
as weakly anionic lipids and can he analyzed in negative-ion ESI/MS
after addition of a small amount of LiOH (or other suitable bases).
Ethanolamine glycerophospholipid (PE), lysoPE, nonesterified fatty
acids and their derivatives, bile acids and ceramide are some
examples in this category. The remaining lipid classes belong to
the third category, which includes choline glycerophospholipid
(PC), lysoPC, sphingomyelin, cerebroside, acylcarnitine,
diacylglycerol, triacylglycerol, cholesterol and its esters. All of
these lipid classes in the third category can be analyzed in
positive-ion ESI/MS after addition of a small amount of LiOH to the
Infused solution as lipids in the first and second categories are
now anionic under these conditions. It should be pointed out that
the authors generally assess the content of cholesterol and its
esters in lipid extracts by employing a simple fluorometric method
[39.1, 40.1]. Individual molecular species of cholesterol esters
can be profiled by precursor-ion analysis as previously described
[41.1]. Alternatively, a method to quantitate cholesterol and its
derivatives by ESI tandem MS (MS/MS) after a simple one-step
chemical derivatization of cholesterol to cholesterol-3-sulfate by
a sulfur trioxide-pyridine complex may be employed [42.1].
[0133] The general strategy underlying the analyses of these
categories of lipids based on this approach is illustrated in FIG.
1.1. Through these methods, a comprehensive series of mass spectra
with respect to each of the aforementioned conditions can be
obtained for each category of lipids (FIG. 2.1). Each ion peak in
each of these mass spectra represents at least one lipid molecular
species. This set of three multiplexed truss spectra effectively
replace high-performance liquid chromatography (HPLC) column
separation by exploiting intrasource separation. Of course, each
pseudomolecular ion peak in each mass spectrum may contain nominal
isobaric species resulting from either members of the same lipid
class or from other class(es) in the category. Although product ion
PSI/MS analyses can be performed to identify the molecular species
underneath each ion peak at this stage (as the authors routinely
conducted previously [15.1]), It is labor Intensive and the results
of product ion analysis may be affected by the presence of
neighboring peaks. More effective and accurate deconvolution of
isobaric species can be accomplished through multidimensional MS
with appropriate array analysis.
[0134] Shotgun Lipidomics: Multidimensional MS
[0135] The inventors recognize that most classes of lipids in a
cellular lipidome are multiple discrete covalent assemblies of a
lipid backbone (typically glycerol) with linear combinations of
various aliphatic chains (typically 14-22 carbons long containing
variable degrees of unsaturation) with (or without) a wide variety
of polar head groups (e.g., choline, ethanolamine, serine and
inositol) (FIGS. 3.1 & 4.1]. Therefore, if one could
effectively and unambiguously identify the presence of each
building block of polar head groups and aliphatic chains (and
combinations thereof) in each pseudomolecular ion, the complexities
in the lipidome could be deconvoluted and readily solved. The
techniques of neutral loss and precursor-ion scanning each exploits
at least one of the structural characters of these building blocks
to provide the tools to efficiently profile each ion peak after
army construction, and deconvolution to identify the building
blocks present and identify the moieties from which they were
derived. Following this concept, a new technique, referred to as
multidimensional MS, has recently been developed [1.1, 9.1, 15.1,
25.1-27.1].
[0136] A coordinated series of sequential 2D mass spectra are the
basic components of multidimensional MS. In the first dimension,
each 2D mass spectrum contains the primary (molecular or
pseudomolecular) ions in the x axis of mass-to-charge ratio (m/z)
while the second dimension, in most cases, is comprised of the
individual building blocks (i.e., polar head groups and/or
aliphatic chains) of lipids (which are characterized by either
neutral loss scanning or precursor-ion scanning or both) in an axis
of mass (in the rare of neutral loss scanning) or m/z (in the case
of precursor-ion scanning) (FIG. 5.1). One feature of a 2D mass
spectrum is that each imaginary mass spectrum along a vertical line
through each m/z of the primary ion (see the broken lines in FIG.
5.1) represents a pseudo product ion mass spectrum of a precursor
ion at the primary ion mass spectrum crossed with the broken line.
This series of arrayed spectra is entirely analogous to a 2D-NMR
spectroscopy where axes are comprised of distinct frequency
domains.
[0137] Each 2D-ESI mass spectrum predictably varies with different:
[0138] Infused solution conditions (e.g., lipid concentration,
acidic/alkaline condition and solvent polarity, which can be
readily achieved by installation of a mixer in the front of a spray
capillary tube and can be controlled by operational software)
[0139] Ionization conditions (e.g., source temperature and spray
voltage) [0140] Fragmentation conditions (e.g., collision gas
pressure, collision energy, collision gas and MS/MS scanning
modes/settings)
[0141] These points aid in the identification, quantitation and
study of lipids. Each of these variables facilitates the
construction of additional dimensions that can be built upon each
2D mass spectrum foundation, which collectively constitutes a new
level of information directly obtainable from lipid mass
spectrometric analysis (i.e., multidimensional MS). Specifically,
multidimensional MS is defined as the aggregate of mass
spectrometric analyses conducted under a variety of instrumental
variables that collectively comprise an n-dimensional spectrum.
Each of these variables forms one dimension of the multidimensional
mass spectrum from which a 2D mass spectrum can be constructed for
ease of use and display. For example, FIG. 6.1 shows a 2D-ESI mass
spectrum of neutral loss of 50.0 u (i.e., loss of chloromethane
from the chlorine adducts of phosphocholine-containing molecular
species) from a diluted hepatic lipid extract under conditions with
a variety of collision energies in the second dimension. This 2D
mass spectrum illustrates the differential fragmentation kinetics
of chlorine adducts of hepatic PC and sphingomyelin molecular
species.
[0142] 2D mass spectrometric analysis for the identification of
lipid building blocks is different from MS/MS analysis, although a
2D mass spectrum for building block analysis includes of a
collection of MS/MS spectra from neutral loss and/or precursorion
scanning of numerous precursor ions in its arrayed format. The 2D
mass spectrum for building block analysis exploits array analysis
techniques integrating both the primary ion mass spectrum and
associated neutral loss/precursorion spectra to determine molecular
composition and amount of a lipid constituent from a single
automated platform. As previously mentioned, one very important
feature of a 2D mass spectrum is the presence of pseudo product-ion
mass spectra for each pseudomolecular ion in the primary ion mass
spectrum. Therefore, many of the characteristics of product-ion
analysis can be extracted from the 2D mass spectrometric analysis.
Regiospecific identification of each individual molecular species
[43.1] and quantitative analysis of isobaric species are two
important features of product-ion analyses (among others) that can
be readily achieved in 2D-MS analysis [9.1, 26.1]. Another very
important feature of a 2D mass spectrum is the increase of dynamic
range relative to a selected internal standard. Therefore,
quantitation and refinement of low-abundance molecular species with
a selected internal standard for each lipid class can also be
readily achieved by 2D [26.1, 27.1], but not by MS/MS analyses
where a set of internal standards must be employed [44.1-47.1].
Most Importantly, identification and quantitation of each
individual molecular species by multidimensional MS can be
automated, and thus multidimensional MS analysis of lipids
represents a high-throughout platform for global studies of the
cellular lipidome.
[0143] Shotgun Lipidomics: Quantitation of Individual Molecular
Species
[0144] Accurate quantitation of each individual molecular species
can be achieved by multidimensional MS through a two-stage
ratiometric process [26.1, 27.1]. First, the abundant molecular
species in a class are quantitated by comparison with a preselected
internal standard for the lipid class in the 1D (primary ion) mass
spectrum. Next, these quantified values are used as endogenous
internal standards in combination with the original exogenous
internal standard for ratiometric comparisons. This is performed to
quantitate or reline the mass content of low-abundance individual
molecular species from at least one representative MS/MS scan for
the class of interest in the 2D-MS for building block analyses.
[0145] The key advantage in this two-stage process is the increase
of dynamic range. There are many different measures of dynamic
range that it affords. For example, the dynamic range of
concentration in which the quantitative technique is linear. This
is the most commonly accepted meaning of the concept for dynamic
mange in the literature. The authors have demonstrated this measure
of dynamic mange in the low lipid concentration range in many of
their studies [25.1, 36.1, 48.1, 49.1]. Another measure of dynamic
range is the relative ratio of internal standard versus individual
molecular species of interest. A 100-fold dynamic range (from 0.1
to 10 of the ratio) can generally be achieved. However, this
dynamic range can suffer by the presence of background noise (i.e.,
chemical noise) and baseline drift (i.e., instrumental stability)
in some cases. Therefore, under adverse experimental conditions,
low-abundance molecular species can only be approximated (or not
quantitated at all) and require 2D analyses. Through MS/MS in a
2D-MS format, a 400-fold (even up to 1000-fold as long as the
concentration measures of dynamic range are linear over 1000-fold
in comparison with controls) increase can be obtained. The authors
find that this dynamic range can be achieved in almost all cases
since background noise is dramatically reduced and different
intensity peaks of the sane class can be found in the primary ion
spectra to serve as ratiometric makers for the quantitation of
low-abundant molecular species. However, the authors specifically
point out that these conditions must be validated and that
additional internal standards ray need to be employed in rare
cases.
[0146] The main advantage of this approach for the quantitation of
individual lipid molecular species in each lipid class is its
simplicity in comparison with the quantitation of lipid species by
MS/MS (in which multiple internal standards for each lipid class
must be selected to eliminate the effects of acyl chain length,
degrees of saturation and double bond locations on the kinetics of
pseudomolecular ion fragmentation [44.1, 45.1, 50.1, 51.1]). The
authors have demonstrated that the response factors of individual
molecular species in most of the polar lipid classes rarely depend
on the physical properties of aliphatic chains in biologic samples,
but rather on the electrical properties of the polar head groups
(i.e., dipole moments) under conditions that utilize low
concentrations of lipids (<10 pmol/.mu.l) so that aggregates do
not form in 1:1 CHCl.sub.3/MeOH (36.1, 48.1, 52.1). In all cases,
corrections for any differences in .sup.13C isotopomer peak
intensities must be made for accurate results [15.1, 25.1]. In this
study, the authors further examined the response factors of 11 PC
molecular species that possess different aliphatic chains in
equimolar mixtures of 1 pmol/.mu.l or less (each) and found that
the response factors of these PC species were essentially identical
within experimental error after correction for different .sup.13C
isotopomer distribution (FIG. 7.1). These results demonstrate that
individual molecular species of a polar lipid class can be
quantitated using one internal standard for the class. Somerharju
and colleagues independently examined the effects of acyl chain
length, unsaturation and lipid concentration on the response
factors of instruments and found that the response factors were
similar within experimental errors in the low lipid concentration
region, supporting the authors' previous observations [53.1].
Furthermore, if their data were corrected to account for the
different .sup.13C isotopomer distributions, the response factors
of molecular species containing different acyl chain lengths would
then yield virtually identical results to those previously
described [25.1, 49.1]. Thus, the response factors of individual
molecular species in a polar lipid class rarely depend on the
physical properties of acyl chains in the low concentration range
as determined in independent laboratories.
[0147] A set of endogenous internal standards from a given class in
addition to the original external standard are generally well
distributed in biologic samples regarding different aliphatic chain
lengths and degrees of unsaturation. Therefore, these endogenous
standards represent superior standards to human-selected internal
standards for lipid quantitation by MS/MS where the overlap of
added internal standard ions with endogenous molecular ions must be
considered, thereby limiting the candidates that can be selected
for exogenous internal standards. One weakness present in 2D-MS
analysis of lipids to quantitate and/or refine low-abundance
molecular species is that the endogenous set of standards are
secondary to the original internal standard and thus the
experimental errors of the mass content of these low-abundance
molecular species are amplified. However, the total mass content of
these low-abundance molecular species typically only account for
less than 5 mol % of the entire mass of the class. Therefore, the
amplified experimental error for the mass content of these
low-abundance species will not substantially affect the accuracy of
quantitation for the entire class of lipids. The authors would also
like to point out that the peaks composed of multiple isobaric
molecular species should not be selected as an endogenous internal
standard to minimize the effects of differential fragmentation on
quantitation as previously discussed [15].
Utilities
[0148] The first application of shotgun lipidomics after
intrasource separation and multidimensional MS was the quantitation
and fingerprinting of triacylglycerol (TAG) molecular species
directly from a crude lipid extract of a biologic sample [25.1].
Since there is no polar head group present in TAG molecular
species, the second dimension of a 2D mass spectrum for TAG
analysis represents the building blocks of TAG aliphatic chains
that can readily be identified by neutral loss scanning of all
naturally occurring fatty acids from lithiated or sodiated TAG
molecular ions as previously described [25.1, 54.1-56.1]. One
important feature of this methodology for TAG analysis is the ease
in identifying individual isobaric TAG molecular species due to the
abundance of multiple TAG molecular species present at each m/z
value in lipid extracts of biologic samples. To date, this
methodology represents the most sensitive, accurate and efficient
technique for individual TAG molecular species analysis. This
method has been extensively used in biologic, pathologic and
pathophysiologic studies in the last 3 years (e.g., [9.1, 11.1,
26.1, 57.1-60.1). It should be emphasized that the location of
double bonds in the constituent acyl chains is not identified by
this method. However, if that is desirable, the regiospecificity of
acyl chains in TAG species maybe identified in multidimensional MS
by varying collision energy.
[0149] Recently, multidimensional ESI/MS has been used to identify
the critical role of peroxisomal processing of fatty acids in
adipocyte lipid storage and metabolism [9.1]. 2D-ESI/MS analyses
demonstrated the accumulation of old chain length unbranched fatty
acids in all major lipid classes in 3T3-L1 differentiating
adipocytes, indicating the rapid .alpha.-oxidation of unbranched
fatty acids. Further studies identifying the double bond location
in odd chain length unbranched fatty acids found the exclusive
presence of .DELTA.9 olefinic species, suggesting the presence of
two critical processes in fatty acid handling in adipocyte lipid
storage and metabolism. First, monounsaturated fatty acids (e.g.,
oleic and palmitoleic acids) are not subject to .alpha.-oxidation,
resulting in the absence of .DELTA.8 unsaturated odd chain length
fatty acids. Second, .alpha.-oxidation of saturated fatty acid
substrate obeys the obligatory sequential ordering of
.alpha.-oxidation prior to .DELTA.9 desaturation [9.1].
[0150] Very recently, 2D-MS analysis has been exploited to
investigate the energy mobilization in modest caloric restriction
in mice and the mobilization of lipids in this process. Remarkably,
only brief periods of fasting (4 and 12 h) result in multiple
specific changes in the murine myocardial lipidome [6.1].
Specifically, substantial and specific depletion of PC and PE
species containing polyunsaturated acyl chains occurred in murine
myocardial, accounting for a total decrease of 39 nmol/mg protein
in these pools after 12 h fasting and representing approximately
25% of total phospholipid mass and approximately 20 cal of Gibbs
free energy/g wet weight of tissue. Furthermore, other myocardial
phospholipid pods such as phosphatidylserine and
phosphatidylinositol were not altered after fasting. No decrease in
TAG mass was observed in myocardium during fasting; however, during
12 h of refeeding, myocardial TAG increased nearly threefold and
returned to baseline levels after 24 h of refeeding. In contrast to
the lipid alterations in myocardium, no changes in phospholipid
mass were present in skeletal muscle and a dramatic decrease in
skeletal muscle (or skeletal muscle associated) TAG mass was
prominent after 12 h of fasting. These results identify
phospholipids as a rapidly mobilizable energy source during modest
caloric deprivation in murine myocardium while TAGs are a major
source of energy reserves in skeletal muscle.
SUMMARY
[0151] Shotgun lipidomics, based on intrasource separation.
multidimensional MS and array analysis, has recently emerged as a
powerful technique in the direct analysis of global cellular
lipidomes. Intrasource separation can largely replace ion-exchange
chromatography steps, allowing resolution of lipid classes based on
the electrical properties of individual lipid classes.
Multidimensional MS analysis facilitates an efficient
identification of each subsequent individual molecular ion peak
including potential nominal isobaric molecular species as well as
the polar head groups, acyl moieties and the regiospecificity of
each molecular species. The two-step quantitation process in 2D-MS
for the analysis of building blocks provides an expanded dynamic
range relative to a selected internal standard for each lipid class
and represents an efficient and accurate method to quantify
individual lipid molecular species. At the current stage of shotgun
lipidomics, the analyses of over 20 lipid classes, hundreds of
lipid molecular species and greater than 95% of the mass content of
a cellular lipidome can be readily achieved. Its broad applications
in biologic, pathologic and pathophysiologic studies have
demonstrated the power and utility of shotgun lipidomics. It is
anticipated that identification of many biochemical mechanisms
underlying lipid metabolism critical to disease states will be
uncovered through the use of shotgun lipidomics.
[0152] One key step to successfully perform shotgun lipidomics is
the preparation of the sample. Commonly, crude lipid extracts are
prepared by the Folch method [61.1] or the modified method of Bligh
and Dyer [62.1]. Small residual aqueousphase contaminants in the
extracts is inevitable, and thus back extraction or multiple
extractions against an aqueous phase with a low salt concentration
should be used to remove aqueous-soluable contaminants that
adversely effect spectral quality. Correct pH and ionic strength
conditions must be employed during sample preparation since acidic
conditions in the aqueous phase can improve the extraction
efficiency for acidic lipids (e.g., PtdH and acyl CoA) while
destroying others (note that vinyl ether-containing compounds
[i.e., plasmalogens] are acid labile). in addition, acidic/alkaline
conditions must be strictly maintained to facilitate the
selectivity of intrasource separation. The authors have found that
lipid extraction against a low concentration LiCl solution (a
weakly acidic condition) represents a suitable condition for
extracts of most tissues, fluids and cells [15.1]. Since extraction
recoveries of different lipid classes can vary, it is recommended
to re-extract multiple times (at least twice) to afford a nearly
complete extraction of all relevant lipids. The authors have found
that the effects of differences in molecular species in a class on
the extraction recoveries of these species are quite small. In
addition, it is emphasized that the internal standard for each of
the lipid classes should be added prior to lipid extractions for
lipid analyses. Accordingly, the extraction recoveries of lipid
classes are accounted for by comparisons with internal standards
even if a complete extraction cannot be achieved.
[0153] Response factors of different molecular species in a class
depend on the physical properties (i.e., length and saturation) of
aliphatic chains to only a small degree after correction of
isotopomer content when experiments are performed in the
appropriate (low) concentration region [38.1, 53.1, 63.1]. The
first consideration is the lipid concentration of the infused
solution. Lipids, unlike other analytes, are unique in terms of
their high hydrophobicity. When concentrations of lipids increase,
they tend to aggregate to form micelles, even in some organic
solvents [64.1]. It is well known that the longer the chain length
and the higher the degrees of saturation of a lipid species, the
lower the critical micellar concentration of the compound.
Therefore, molecular species containing short acyl chains and/or
polyunsaturation might show higher apparent response factors than
those containing long and/or saturated acyl chains at a high lipid
concentration if the lipid concentration exceeds approximately 10
pmol/.mu.l [53.1, 63.1]. However, at low concentrations (<10
pmol/.mu.l) in 1:1 (v/v) of chloroform/methanol, lipid-lipid
interactions are rare and ionization efficiency of lipid mixtures
largely depends on the electrical properties of each lipid
molecular species, which is predominantly determined by the dipole
in the polar head groups. Therefore, identical response factors for
different molecular species in a class can be obtained and have
been repeatedly and independently verified by multiple groups
[36.1, 38.1, 53.1, 65.1]. However, when the concentration of lipids
in the infusion solution increases to the point where lipid-lipid
interactions become apparent, these response factors are no longer
identical. Thus, concentration of lipids by straight or reversed
phase chromatography must be performed with extreme caution since
it promotes lipid-lipid interactions [38.1]. The maximal
concentrations of lipids at which lipid-lipid interactions are
small evidently depend on the solvent components used in the
infusion solution. Therefore, a solvent system containing water,
acetonitrile or a high percentage of methanol is not favored for
global lipid analysis by shotgun lipidomics, although such a
solvent system may be used for the analysis of a specific class of
lipids by ESI/MS. The second crucial consideration is the different
.sup.13C isotopomer intensity distributions as described previously
15.1, 25.1, 49.1]. These effects could cause considerable
differences between the apparent response factors of different
molecular species and must be corrected in comparison with a
selected internal standard as shown in FIG. 7.1. Alternatively
these effects can be eliminated by determining the peak intensities
after a deisotope calculation.
[0154] Caution should be exercised in employing ESI/MS/MS for
quantitation of individual molecular species of each class of
lipids, since the fragmentation patterns of each lipid molecular
species depend on both the applied energy for collision-induced
dissociation and on the structure of individual molecular species
(FIG. 6.1 [25.1, 38.1, 43.1, 49.1, 66.1]. Changes in applied
collision energy alter the kinetics of individual fragmentation
pathways and result in changes in the distribution of the observed
fragment ions. Thus, it is important to closely control
fragmentation energies and to utilize both appropriate internal
standards for each lipid class and molecular species as well as
ratiometrically quantify each individual species so that identical
physical parameters are compared.
[0155] Shotgun lipidomics is a rapidly evolving technology. The
authors believe the techniques described herein will be extended to
identify low-abundance concentration lipid classes through the
integration of enrichment techniques (e.g., nano-HPLC) and the
development of new MS/MS methods for the identification of these
classes. Additionally, the development of instruments with greatly
improved sensitivity and resolution will extend penetration into
the low-abundance region of cellular lipidomes. To this end,
enrichment approaches in conjunction with ESI Fourier transform ion
cyclotron resonance MS holds much promise [13.1]. Second,
high-efficiency direct-infusion techniques such as microfluidic
approaches will be integrated into shotgun lipidomics to
accommodate the need for high through put. Third, bioinformatics in
lipidomics through database development and automation of data
processing will play an essential role in the development and
utility of shotgun lipidomics. Finally, it appears likely that
affordable robust platforms for shotgun lipidomics will be made
available to the biomedical research community for even routine
clinical applications such as diagnosis and monitoring of drug
therapy. The authors speculate that the large flux of quantitative
lipidomics data integrated with genomic and proteomic studies will
significantly enhance our understanding of the role of lipids in
biologic systems. Advances in this field may also lead to enhanced
diagnosis of lipid-related disease states at earlier time points to
enhance therapeutic efficacy and tailor drug therapy in the next 5
years.
[0156] This work was supported by NIH grant PO1HL57278 and
RO1HL41250 as well as the U.S. Neurosciences Education and Research
Foundation.
[0157] In an aspect, the methods recited in Claims 21-28 herein
after, are carried out by screening a library of compounds in an
expression vector system (e.g., mammalian expression vector) and
identifying compounds that alter the specific activity of the
enzyme as assessed by shotgun lipidomics (or other techniques such
as thin layer chromatography and scintillation spectrometry), or
alter either the amount of the protein, or the structure of the
protein as determined by 1D or 2D Western blot analysis or by mass
spectrometry. Then determining if a compound has an effect on the
ratio of the specific activity of the reaction of hydrolysis of
AcylCoA or phosphatidylcholine. Alterations in the mass of the
protein can be identified by Western blot analysis when a change in
change in the molecular weight or the isoelectric point of the
enzyme has occurred. Other methods known to those skilled in the
art can be used in conjunction with the above approaches to
identify altered binding characteristics, subcellular localization
or covalent modifications. Such techniques include, but are not
limited to mass spectrometric study of the protein either as an
intact entity or through a bottom up mass spectrometric approach.
The physiologic effects of such identified compounds can then be
determined through comparisons of the effects of said agents in
models of the above identified disease state to demonstrate the
presence of physiologic benefit of said agent.
[0158] In one embodiment, a method is provided of altering the
interaction of iPLA.sub.2.beta. with its endogenous calmodulin
inhibitor through the actions of acyl-CoA or acyl-CoA like
compounds that increase the inhibition of enzymic activity
resulting in a decrease of iPLA.sub.2.beta. activity and a decrease
in the production of lipid second messengers.
[0159] In one embodiment, a method is provided of altering the
interaction of iPLA.sub.2.beta. with its endogenous calmodulin
inhibitor through the actions of an acyl-CoA or acyl-CoA-like
compound that decreases the inhibition of enzymic activity
resulting in an increase of iPLA.sub.2.beta. activity and an
increase in the production of lipid second messengers.
[0160] In one embodiment, a method is provided that identifies an
exocyte in an iPLA.sub.2.beta.-calmodulin complex capable of
modifying the interaction of iPLA.sub.2.beta. with calmodulin
resulting in a change of enzymic activity.
[0161] In one embodiment, a means is provided to identify effective
treatment modalities of calcium mediated or related disease
processes that depend, either directly or indirectly, on the
influence of capacitative calcium influx. These include, but are
not limited to inflammation, heart attack, congestive heart failure
and blood pressure.
[0162] In one embodiment, a method is provided for treating
inflammation in living mammals. The method administering a compound
that alters the interaction of iPLA.sub.2.beta. and its inhibitor
and subsequently determining if that compound alters the
inflammatory response.
[0163] In one embodiment, a method is provided for treating
platelet thrombosis in living mammals. The method comprises
administering a compound that alters the interaction of
iPLA.sub.2.beta. and its inhibitor and subsequently determining if
that compound alters platelet function.
[0164] In one embodiment a method is provided for screening a
library of compounds to identify modulators of the
iPLA.sub.2.beta.-calmodulin interaction by reconstitution of the
enzyme modulator interaction and use of calcium entry fluorescence
to screen for modulators of this interaction.
[0165] In one embodiment, a method is provided to screen natural
product or combinatorial libraries by calcium sensitive dyes to
identify modulators of the iPLA.sub.2.beta.-calmodulin
interaction.
[0166] In one embodiment, a mechanism is provided to screen for
compounds that alter the deleterious effects of congestive heart
failure by promoting calcium uptake into the sarcoplasmic reticulum
through modulating iPLA.sub.2.beta. activity and regulatory
interactions.
[0167] In one embodiment, a mechanism is provided to modulate
hormone or neurotransmitter release by intentionally altering
capacitive calcium influx by modulation of iPLA.sub.2.beta.
activity in a living mammal.
[0168] In one embodiment, a mechanism is provided to modulate the
local concentrations of calcium by modulating iPLA.sub.2.beta.
enzymic activity and subsequent capacitative calcium entry.
[0169] In one embodiment, a means is provided to attenuate
arrhythmias or damage from a heart attack by modulating the calcium
influx through regulation of iPLA.sub.2.beta..
[0170] In one embodiment, a means is provided of identifying those
living animals having disorders related to alteration of
capacitative calcium influx by determining alterations in the
iPLA.sub.2.beta.-calmodulin interaction.
[0171] In one embodiment, a method is provided of determining an
inhibitor of iPLA.sub.2.beta. or iPLA.sub.2.gamma.. The method
comprises obtaining an cardiolipin analysis by shotgun lipidomic
technology on a living mammalian subject, determining the results
of that analysis and determining the iPLA.sub.2.beta. or
iPLA.sub.2.gamma. is inhibited when the cardiolipin content of the
serum/biopsy sample is increased.
[0172] In one embodiment, a method is provided of treating a living
mammal having diabetic cardiomyopathy which comprising
administering an effective amount of an inhibitor to that subject,
performing a shotgun lipidomics analysis on the subject and
determining that the treatment was successful when and if serum or
tissue biopsy cardiolipin levels are increased and/or
lysocardiolipin levels are decreased.
[0173] In one embodiment, a pharmaceutical composition is provided
that is effective to treat cardiomyopathy. The composition
comprises a gene expressing a protein which synthesizes
cardiolipin, increases the synthesis of cardiolipin, or is in a
metabolic pathway of cardiolipin synthesis.
[0174] In one embodiment, a method is provided for treating
diabetes in living mammals. The method comprises administering a
gene, enzyme or pharmaceutical which decreases the concentration
iPLA.sub.2.beta. and/or iPLA.sub.2.gamma. through transcriptional
and/or translational regulation or effectively increases the
inhibition of iPLA.sub.2.beta. through calmodulin or derivatives
thereof.
[0175] In one embodiment, a method is provided of treating a living
mammal having diabetic cardiomyopathy. The method comprising
administering an effective amount of an inhibitor to that subject,
performing a shotgun lipidomics analysis on the subject and
determining that the treatment was successful when and if serum or
tissue biopsy cardiolipin levels are increased and/or
lysocardiolipin levels are decreased.
[0176] In one embodiment, a pharmaceutical composition is provided
that is effective to treat cardiomyopathy. The composition
comprises a gene capably expressing a protein which synthesizes
cardiolipin, increases the synthesis of cardiolipin, or is in a
metabolic pathway of cardiolipin synthesis.
[0177] In one embodiment, a method is provided for treating
diabetes in living mammals. The method comprises administering a
gene, enzyme or pharmaceutical which decreases the concentration
iPLA.sub.2.beta. and/or iPLA.sub.2.gamma. through transcriptional
and/or translational regulation or effectively increases the
inhibition of iPLA.sub.2.beta. through calmodulin or derivatives
thereof.
[0178] The article Global analyses of cellular lipidomes directly
from crude extracts of biological samples by ESI mass spectrometry:
a bridge to lipidomes, Xianlin Han, Richard W. Gross, Washington
University in St. Louis, Lipid Research, Inc. is incorporated
herein in its entirety by reference. Volume 44, 2003, used with
permission.
[0179] This discovery is important to use an effective research
tool because specific interactions are involved in almost any
physiological process. If mankind is ever to cure diabetes and
other debilitating diseases killing humans, thus mankind must have
and use bedrock effective diagnostic and treatment tools.
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[0341] While the invention has been described in terms of various
specific embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the claims.
Sequence CWU 1
1
13 1 752 PRT Homo sapiens 1 Met Gln Phe Phe Gly Arg Leu Val Asn Thr
Phe Ser Gly Val Thr Asn 1 5 10 15 Leu Phe Ser Asn Pro Phe Arg Val
Lys Glu Val Ala Val Ala Asp Tyr 20 25 30 Thr Ser Ser Asp Arg Val
Arg Glu Glu Gly Gln Leu Ile Leu Phe Gln 35 40 45 Asn Thr Pro Asn
Arg Thr Trp Asp Cys Val Leu Val Asn Pro Arg Asp 50 55 60 Ser Gln
Ser Gly Phe Arg Leu Phe Gln Leu Glu Leu Glu Ala Asp Ala 65 70 75 80
Leu Val Asn Phe His Gln Tyr Ser Ser Gln Leu Leu Pro Phe Tyr Glu 85
90 95 Ser Ser Pro Gln Val Leu His Thr Glu Val Leu Gln His Leu Thr
Asp 100 105 110 Leu Ile Arg Asn His Pro Ser Trp Ser Val Ala His Leu
Ala Val Glu 115 120 125 Leu Gly Ile Arg Glu Cys Phe His His Ser Arg
Ile Ile Ser Cys Ala 130 135 140 Asn Cys Ala Glu Asn Glu Glu Gly Cys
Thr Pro Leu His Leu Ala Cys 145 150 155 160 Arg Lys Gly Asp Gly Glu
Ile Leu Val Glu Leu Val Gln Tyr Cys His 165 170 175 Thr Gln Met Asp
Val Thr Asp Tyr Lys Gly Glu Thr Val Phe His Tyr 180 185 190 Ala Val
Gln Gly Asp Asn Ser Gln Val Leu Gln Leu Leu Gly Arg Asn 195 200 205
Ala Val Ala Gly Leu Asn Gln Val Asn Asn Gln Gly Leu Thr Pro Leu 210
215 220 His Leu Ala Cys Gln Leu Gly Lys Gln Glu Met Val Arg Val Leu
Leu 225 230 235 240 Leu Cys Asn Ala Arg Cys Asn Ile Met Gly Pro Asn
Gly Tyr Pro Ile 245 250 255 His Ser Ala Met Lys Phe Ser Gln Lys Gly
Cys Ala Glu Met Ile Ile 260 265 270 Ser Met Asp Ser Ser Gln Ile His
Ser Lys Asp Pro Arg Tyr Gly Ala 275 280 285 Ser Pro Leu His Trp Ala
Lys Asn Ala Glu Met Ala Arg Met Leu Leu 290 295 300 Lys Arg Gly Cys
Asn Val Asn Ser Thr Ser Ser Ala Gly Asn Thr Ala 305 310 315 320 Leu
His Val Ala Val Met Arg Asn Arg Phe Asp Cys Ala Ile Val Leu 325 330
335 Leu Thr His Gly Ala Asn Ala Asp Ala Arg Gly Glu His Gly Asn Thr
340 345 350 Pro Leu His Leu Ala Met Ser Lys Asp Asn Val Glu Met Ile
Lys Ala 355 360 365 Leu Ile Val Phe Gly Ala Glu Val Asp Thr Pro Asn
Asp Phe Gly Glu 370 375 380 Thr Pro Thr Phe Leu Ala Ser Lys Ile Gly
Arg Gln Leu Gln Asp Leu 385 390 395 400 Met His Ile Ser Arg Ala Arg
Lys Pro Ala Phe Ile Leu Gly Ser Met 405 410 415 Arg Asp Glu Lys Arg
Thr His Asp His Leu Leu Cys Leu Asp Gly Gly 420 425 430 Gly Val Lys
Gly Leu Ile Ile Ile Gln Leu Leu Ile Ala Ile Glu Lys 435 440 445 Ala
Ser Gly Val Ala Thr Lys Asp Leu Phe Asp Trp Val Ala Gly Thr 450 455
460 Ser Thr Gly Gly Ile Leu Ala Leu Ala Ile Leu His Ser Lys Ser Met
465 470 475 480 Ala Tyr Met Arg Gly Met Tyr Phe Arg Met Lys Asp Glu
Val Phe Arg 485 490 495 Gly Ser Arg Pro Tyr Glu Ser Gly Pro Leu Glu
Glu Phe Leu Lys Arg 500 505 510 Glu Phe Gly Glu His Thr Lys Met Thr
Asp Val Arg Lys Pro Lys Val 515 520 525 Met Leu Thr Gly Thr Leu Ser
Asp Arg Gln Pro Ala Glu Leu His Leu 530 535 540 Phe Arg Asn Tyr Asp
Ala Pro Glu Thr Val Arg Glu Pro Arg Phe Asn 545 550 555 560 Gln Asn
Val Asn Leu Arg Pro Pro Ala Gln Pro Ser Asp Gln Leu Val 565 570 575
Trp Arg Ala Ala Arg Ser Ser Gly Ala Ala Pro Thr Tyr Phe Arg Pro 580
585 590 Asn Gly Arg Phe Leu Asp Gly Gly Leu Leu Ala Asn Asn Pro Thr
Leu 595 600 605 Asp Ala Met Thr Glu Ile His Glu Tyr Asn Gln Asp Leu
Ile Arg Lys 610 615 620 Gly Gln Ala Asn Lys Val Lys Lys Leu Ser Ile
Val Val Ser Leu Gly 625 630 635 640 Thr Gly Arg Ser Pro Gln Val Pro
Val Thr Cys Val Asp Val Phe Arg 645 650 655 Pro Ser Asn Pro Trp Glu
Leu Ala Lys Thr Val Phe Gly Ala Lys Glu 660 665 670 Leu Gly Lys Met
Val Val Asp Cys Cys Thr Asp Pro Asp Gly Arg Ala 675 680 685 Val Asp
Arg Ala Arg Ala Trp Cys Glu Met Val Gly Ile Gln Tyr Phe 690 695 700
Arg Leu Asn Pro Gln Leu Gly Thr Asp Ile Met Leu Asp Glu Val Ser 705
710 715 720 Asp Thr Val Leu Val Asn Ala Leu Trp Glu Thr Glu Val Tyr
Ile Tyr 725 730 735 Glu His Arg Glu Glu Phe Gln Lys Leu Ile Gln Leu
Leu Leu Ser Pro 740 745 750 2 5 PRT Artificial Sequence Description
of Artificial Sequence Synthetic peptide MOD_RES (2) variable amino
acid 2 Gly Xaa Ser Gly Ser 1 5 3 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide MOD_RES (2)
variable amino acid 3 Gly Xaa Ser Thr Gly 1 5 4 6 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 6x His tag 4
His His His His His His 1 5 5 5 PRT Artificial Sequence Description
of Artificial Sequence Synthetic peptide 5 Gly Thr Ser Thr Gly 1 5
6 6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 6 Gly Gly Gly Val Lys Gly 1 5 7 11 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 7 Arg
Asn Tyr Asp Ala Pro Glu Val Ile Arg Glu 1 5 10 8 11 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 8 Arg
Gln Pro Ala Glu Leu His Leu Phe Arg Asn 1 5 10 9 12 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 9 Arg
Lys Pro Ala Phe Ile Leu Ser Ser Met Arg Asp 1 5 10 10 14 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 10 Arg Asn Tyr Asp Ala Pro Glu Val Ile Arg Glu Pro Arg Phe
1 5 10 11 17 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 11 Arg Gly Ser Arg Pro Tyr Glu Ser Gly
Pro Leu Glu Glu Phe Leu Lys 1 5 10 15 Arg 12 21 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 12
Lys Val Met Leu Thr Gly Thr Leu Ser Asp Arg Gln Pro Ala Glu Leu 1 5
10 15 His Leu Phe Arg Asn 20 13 22 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 13 Arg Phe Asn
Gln Asn Ile Asn Leu Lys Pro Pro Thr Gln Pro Ala Asp 1 5 10 15 Gln
Leu Val Trp Arg Ala 20
* * * * *
References