N-acyl phosphatidylethanolamine-specific phospholipase D
Encyclopedia
N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) is an enzyme
that catalyzes the release of N-acylethanolamine
(NAE) from N-acyl-phosphatidylethanolamine (NAPE). This is a major part of the process that converts ordinary lipid
s into chemical signals like anandamide
and oleoylethanolamine. In humans, the NAPE-PLD protein is encoded by the NAPEPLD gene
.
activity - a phospholipase
, acting on phospholipid
s found in the cell membrane
. It is not homology
but the chemical outcome of its activity that classes it as phospholipase D
. The enzymatic activity was discovered and characterized in a series of experiments culminating in the 2004 publication of a biochemical purification scheme from which peptide sequencing
could be accomplished. Researchers homogenized
(finely ground) hearts from 150 rats and subjected the resulting crude lysate
to sucrose sedimentation at 105,000 x g to separate out the cell membranes from the remainder of the cell. The integral membrane protein
s were then solubilized using octyl glucoside
and subjected to four column chromatography
steps (HiTrap SP HP cation-exchange column, HiTrap Q anion-exchange column, HiTrap Blue affinity column, Bio-Gel HTP hydroxyapatite column). Each of these separates the different types of membrane proteins into different sample containers when the proteins are eluted from the column over time, and by measuring the activity of samples in each container it was possible to track which ones received the active enzyme. Measurement of the enzyme activity was done by thin layer chromatography
of a radioactive substrate
sensitive to the NAPE-PLD enzymatic activity: Cleavage of the substrate affected where it appeared on the plate when the radiation was detected on a bioimaging analyzer.
The result of this extensive procedure was still not a pure protein, but it produced a limited number of bands by SDS-PAGE
, and one band of 46 kilodaltons was found to correlate in intensity with the enzymatic activity. This band was cut out from the gel and digested with trypsin
, and peptides from it were separated from one another by reverse phase high performance liquid chromatography. The resulting fragments were then microsequenced by an automated Edman degradation
. Three corresponded to vimentin
, an intermediate filament
protein of 56 kDa believed to be a contaminant, and the other two matched the cDNA clone subsequently identified as NAPE-PLD.
Once this clue had been obtained, the identification could be confirmed by a less onerous procedure: Overexpression of the putative NAPE-PLD cDNA in COS-7 cells
yielded a strong NAPE-PLD enzymatic activity, whose characteristics were shown to be similar to those of the original heart extract.
sequences in both mice and rats, which are 89% and 90% identical to that of humans. NAPE-PLD was found to have no homology to the known phospholipase D
genes, but can be classed by homology to fall into the zinc metallohydrolase
family of the beta-lactamase fold
. In particular, the highly conserved motif H
X(E/H
)XD(C
/R
/S
/H
)X50–70H
X15–30(C
/S
/D)X30–70H
was observed, which is, in general, associated with zinc
binding and hydrolysis
reaction in this class of proteins, leading the authors to propose that activity should be correlated with zinc content.
When recombinant
NAPE-PLD was tested in COS cells in vitro it had similar activity toward several radiolabeled substrates: N-palmitoylphosphatidylethanolamine, N-arachidonoylphosphatidylethanolamine, N-oleoylphosphatidylethanolamine, and N-stearoylphosphatidylethanolamine all reacted with a Km between 2-4 micromolar and a Vmax between 73 and 101 nanomole per milligram per minute as calculated by Lineweaver-Burk plot
. (These generate N-palmitoylethanolamine, anandamide
, N-oleoylethanolamine, and N-stearoylethanolamine, respectively) The enzyme also reacted N-palmitoyl-lyso-phosphatidylethanolamine and N-arachidonoyl-lyso-phosphatidylethanolamine with similar Km but at one-third to one-fourth the Vmax. These activities are consistent with the observation that many tissues produce a range of N-acylethanolamines
.
However, NAPE-PLD had no ability to produce detectable phosphatidic acid from phosphatidylcholine
or phosphatidylethanolamine
as is catalyzed by other phospholipase D
enzymes. It also lacks the transphosphatidylation activity of phospholipase D that allows the creation of phosphatidyl alcohols rather than phosphatidic acid in the presence of ethanol
or butanol
.
onto the amino group of phosphatidylethanolamine
. While NAPE-PLD contributes to the biosynthesis of several NAEs in the mammalian central nervous system
, this enzyme is not responsible for the formation of the endocannabinoid anandamide
, since NAPE-PLD knockout mice have wild-type levels of this lipid.
The N-acylethanolamine
s released by this enzyme become potential substrates for fatty acid amide hydrolase (FAAH), which hydrolyzes the free fatty acids from ethanolamine
. Defects in this enzyme can cause NAPE-PLD products such as anandamide to build up to levels 15-fold higher than normally observed.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that catalyzes the release of N-acylethanolamine
N-acylethanolamine
An N-acylethanolamine is a type of compound formed when one of several types of acyl group is linked to the nitrogen atom of ethanolamine. These amides conceptually can be formed from a fatty acid and ethanolamine with the release of a molecule of water, but the known biological synthesis relies...
(NAE) from N-acyl-phosphatidylethanolamine (NAPE). This is a major part of the process that converts ordinary lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
s into chemical signals like anandamide
Anandamide
Anandamide, also known as N-arachidonoylethanolamide or AEA, is an endogenous cannabinoid neurotransmitter. The name is taken from the Sanskrit word ananda, which means "bliss, delight", and amide. It is synthesized from N-arachidonoyl phosphatidylethanolamine by multiple pathways...
and oleoylethanolamine. In humans, the NAPE-PLD protein is encoded by the NAPEPLD gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
.
Identification and purification
NAPE-PLD is an enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
activity - a phospholipase
Phospholipase
A phospholipase is an enzyme that hydrolyzes phospholipids into fatty acids and other lipophilic substances. There are four major classes, termed A, B, C and D, distinguished by the type of reaction which they catalyze:*Phospholipase A...
, acting on phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...
s found in the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
. It is not homology
Homology (chemistry)
In chemistry, homology refers to the appearance of homologues. A homologue is a compound belonging to a series of compounds differing from each other by a repeating unit, such as a methylene group, a peptide residue, etcetera....
but the chemical outcome of its activity that classes it as phospholipase D
Phospholipase D
Phospholipase D is an enzyme which is located in the plasma membrane and catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid , releasing the soluble choline headgroup into the cytosol...
. The enzymatic activity was discovered and characterized in a series of experiments culminating in the 2004 publication of a biochemical purification scheme from which peptide sequencing
Edman degradation
Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues....
could be accomplished. Researchers homogenized
Homogenization (chemistry)
Homogenization or homogenisation is any of several processes used to make a chemical mixture the same throughout.-Definition:Homogenization is intensive blending of mutually related substances or groups of mutually related substances to form a constant of different insoluble phases to obtain a...
(finely ground) hearts from 150 rats and subjected the resulting crude lysate
Crude lysate
A crude lysate is the solution produced when cells are destroyed by disrupting their cell membranes, often with detergent or other chaotropic agent, in a process known as cytolysis. This releases the contents within the cell...
to sucrose sedimentation at 105,000 x g to separate out the cell membranes from the remainder of the cell. The integral membrane protein
Integral membrane protein
An integral membrane protein is a protein molecule that is permanently attached to the biological membrane. Proteins that cross the membrane are surrounded by "annular" lipids, which are defined as lipids that are in direct contact with a membrane protein...
s were then solubilized using octyl glucoside
Octyl glucoside
Octyl glucoside is a detergent frequently used to dissolve integral membrane proteins for studies in biochemistry...
and subjected to four column chromatography
Column chromatography
Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.The main advantage of column chromatography is the relatively low cost and disposability...
steps (HiTrap SP HP cation-exchange column, HiTrap Q anion-exchange column, HiTrap Blue affinity column, Bio-Gel HTP hydroxyapatite column). Each of these separates the different types of membrane proteins into different sample containers when the proteins are eluted from the column over time, and by measuring the activity of samples in each container it was possible to track which ones received the active enzyme. Measurement of the enzyme activity was done by thin layer chromatography
Thin layer chromatography
Thin layer chromatography is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose...
of a radioactive substrate
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
sensitive to the NAPE-PLD enzymatic activity: Cleavage of the substrate affected where it appeared on the plate when the radiation was detected on a bioimaging analyzer.
The result of this extensive procedure was still not a pure protein, but it produced a limited number of bands by SDS-PAGE
SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
, and one band of 46 kilodaltons was found to correlate in intensity with the enzymatic activity. This band was cut out from the gel and digested with trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...
, and peptides from it were separated from one another by reverse phase high performance liquid chromatography. The resulting fragments were then microsequenced by an automated Edman degradation
Edman degradation
Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues....
. Three corresponded to vimentin
Vimentin
Vimentin is a type III intermediate filament protein that is expressed in mesenchymal cells. IF proteins are found in all metazoan cells as well as bacteria. IF, along with tubulin-based microtubules and actin-based microfilaments, comprise the cytoskeleton...
, an intermediate filament
Intermediate filament
Intermediate filaments are a family of related proteins that share common structural and sequence features. Intermediate filaments have an average diameter of 10 nanometers, which is between that of 7 nm actin , and that of 25 nm microtubules, although they were initially designated...
protein of 56 kDa believed to be a contaminant, and the other two matched the cDNA clone subsequently identified as NAPE-PLD.
Once this clue had been obtained, the identification could be confirmed by a less onerous procedure: Overexpression of the putative NAPE-PLD cDNA in COS-7 cells
COS cells
COS is a cell line often used by biologists when studying the monkey virus SV40. Cells from this line are also often transfected to produce recombinant proteins for molecular biology, biochemistry, and cell biology experiments...
yielded a strong NAPE-PLD enzymatic activity, whose characteristics were shown to be similar to those of the original heart extract.
Characteristics
The NAPEPLD cDNA sequence predicts 396 amino acidAmino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
sequences in both mice and rats, which are 89% and 90% identical to that of humans. NAPE-PLD was found to have no homology to the known phospholipase D
Phospholipase D
Phospholipase D is an enzyme which is located in the plasma membrane and catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid , releasing the soluble choline headgroup into the cytosol...
genes, but can be classed by homology to fall into the zinc metallohydrolase
Metalloproteinase
Metalloproteinases constitute a family of enzymes from the group of proteases, classified by the nature of the most prominent functional group in their active site. These are proteolytic enzymes whose catalytic mechanism involves a metal. Most metalloproteases are zinc-dependent, but some use...
family of the beta-lactamase fold
Metallo-beta-lactamase protein fold
The metallo-beta-lactamase protein fold is a protein domain contained in class B beta-lactamases and a number of other proteins.These proteins include thiolesterases, members of the glyoxalase II family, that catalyse the hydrolysis of S-D-lactoyl-glutathione to form glutathione and D-lactic acid...
. In particular, the highly conserved motif H
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
X(E/H
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
)XD(C
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
/R
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
/S
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
/H
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
)X50–70H
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
X15–30(C
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
/S
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
/D)X30–70H
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
was observed, which is, in general, associated with zinc
Zinc
Zinc , or spelter , is a metallic chemical element; it has the symbol Zn and atomic number 30. It is the first element in group 12 of the periodic table. Zinc is, in some respects, chemically similar to magnesium, because its ion is of similar size and its only common oxidation state is +2...
binding and hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
reaction in this class of proteins, leading the authors to propose that activity should be correlated with zinc content.
When recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
NAPE-PLD was tested in COS cells in vitro it had similar activity toward several radiolabeled substrates: N-palmitoylphosphatidylethanolamine, N-arachidonoylphosphatidylethanolamine, N-oleoylphosphatidylethanolamine, and N-stearoylphosphatidylethanolamine all reacted with a Km between 2-4 micromolar and a Vmax between 73 and 101 nanomole per milligram per minute as calculated by Lineweaver-Burk plot
Lineweaver-Burk plot
In biochemistry, the Lineweaver–Burk plot is a graphical representation of the Lineweaver–Burk equation of enzyme kinetics, described by Hans Lineweaver and Dean Burk in 1934.-Derivation:...
. (These generate N-palmitoylethanolamine, anandamide
Anandamide
Anandamide, also known as N-arachidonoylethanolamide or AEA, is an endogenous cannabinoid neurotransmitter. The name is taken from the Sanskrit word ananda, which means "bliss, delight", and amide. It is synthesized from N-arachidonoyl phosphatidylethanolamine by multiple pathways...
, N-oleoylethanolamine, and N-stearoylethanolamine, respectively) The enzyme also reacted N-palmitoyl-lyso-phosphatidylethanolamine and N-arachidonoyl-lyso-phosphatidylethanolamine with similar Km but at one-third to one-fourth the Vmax. These activities are consistent with the observation that many tissues produce a range of N-acylethanolamines
N-acylethanolamine
An N-acylethanolamine is a type of compound formed when one of several types of acyl group is linked to the nitrogen atom of ethanolamine. These amides conceptually can be formed from a fatty acid and ethanolamine with the release of a molecule of water, but the known biological synthesis relies...
.
However, NAPE-PLD had no ability to produce detectable phosphatidic acid from phosphatidylcholine
Phosphatidylcholine
Phosphatidylcholines are a class of phospholipids that incorporate choline as a headgroup.They are a major component of biological membranes and can be easily obtained from a variety of readily available sources such as egg yolk or soy beans from which they are mechanically extracted or chemically...
or phosphatidylethanolamine
Phosphatidylethanolamine
Phosphatidylethanolamine is a lipid found in biological membranes. It is synthesized by the addition of CDP-ethanolamine to diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the amine of phosphatidyl ethanolamine to yield phosphatidyl choline.Cephalin is a phospholipid,...
as is catalyzed by other phospholipase D
Phospholipase D
Phospholipase D is an enzyme which is located in the plasma membrane and catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid , releasing the soluble choline headgroup into the cytosol...
enzymes. It also lacks the transphosphatidylation activity of phospholipase D that allows the creation of phosphatidyl alcohols rather than phosphatidic acid in the presence of ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
or butanol
Butanol
Butanol or butyl alcohol can refer to any of the four isomeric alcohols of formula C4H9OH:*n-Butanol, butan-1-ol, 1-butanol, n-butyl alcohol;*Isobutanol, 2-methylpropan-1-ol, isobutyl alcohol;...
.
Pathway
This enzyme acts as the second step of a biochemical pathway initiated by the creation of N-acylphosphatidylethanolamine, by means of the transfer of an acyl group from the sn-1 position of glycerophospholipidGlycerophospholipid
Glycerophospholipids or phosphoglycerides are glycerol-based phospholipids. They are the main component of biological membranes.-Structures:...
onto the amino group of phosphatidylethanolamine
Phosphatidylethanolamine
Phosphatidylethanolamine is a lipid found in biological membranes. It is synthesized by the addition of CDP-ethanolamine to diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the amine of phosphatidyl ethanolamine to yield phosphatidyl choline.Cephalin is a phospholipid,...
. While NAPE-PLD contributes to the biosynthesis of several NAEs in the mammalian central nervous system
Central nervous system
The central nervous system is the part of the nervous system that integrates the information that it receives from, and coordinates the activity of, all parts of the bodies of bilaterian animals—that is, all multicellular animals except sponges and radially symmetric animals such as jellyfish...
, this enzyme is not responsible for the formation of the endocannabinoid anandamide
Anandamide
Anandamide, also known as N-arachidonoylethanolamide or AEA, is an endogenous cannabinoid neurotransmitter. The name is taken from the Sanskrit word ananda, which means "bliss, delight", and amide. It is synthesized from N-arachidonoyl phosphatidylethanolamine by multiple pathways...
, since NAPE-PLD knockout mice have wild-type levels of this lipid.
The N-acylethanolamine
N-acylethanolamine
An N-acylethanolamine is a type of compound formed when one of several types of acyl group is linked to the nitrogen atom of ethanolamine. These amides conceptually can be formed from a fatty acid and ethanolamine with the release of a molecule of water, but the known biological synthesis relies...
s released by this enzyme become potential substrates for fatty acid amide hydrolase (FAAH), which hydrolyzes the free fatty acids from ethanolamine
Ethanolamine
Ethanolamine, also called 2-aminoethanol or monoethanolamine , is an organic chemical compound that is both a primary amine and a primary alcohol . Like other amines, monoethanolamine acts as a weak base...
. Defects in this enzyme can cause NAPE-PLD products such as anandamide to build up to levels 15-fold higher than normally observed.