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Nephrology Division, Department of Internal Medicine, University of Michigan, Ann Arbor, MI
Published, JLR Papers in Press, August 16, 2005. DOI 10.1194/jlr.M500248-JLR200
1 M. Hiraoka and A. Abe contributed equally to this work. 
2 Present address of M. Hiraoka: Ophthalmology Department, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan.
3 To whom correspondence should be addressed. e-mail: jshayman{at}umich.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We propose that one disulfide bond between C65 and C89 and free cysteine residues at C330 and C371 and the triad, serine-198, aspartic acid-360, and histidine-392, are required for the full expression of mLPLA2 activity.
Supplementary key words surfactant • macrophage • lipase • phospholipid • phosphatidylcholine • phosphatidylethanolamine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LPLA2 has both transacylase and phospholipase A2 activities under acidic conditions (1, 5). Divalent cations are not required for LPLA2 activity, although calcium slightly enhances enzyme activity (1). The purified LPLA2 is a water-soluble glycoprotein consisting of a single peptide chain with a molecular mass of 45 kDa (1). The primary structure of LPLA2, deduced from DNA sequences encoding LPLA2, is highly conserved between mammals, including mouse, rat, human, and bovine. The amino acid sequence of LPLA2 is 49% identical to that of LCAT (Fig. 1). Based on their primary structures, both enzymes are members of the 
ß-hydrolase superfamily and have the amino acid residues forming a catalytic triad (2, 6). In general, the triad consists of three amino acid residues, serine, aspartic acid/glutamic acid, and histidine, a structure that is essential for the hydrolysis reaction. We previously demonstrated that the serine-198 (S198) residue within a putative lipase motif sequence and within the catalytic triad is required for phospholipase A2 activity (2).
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Independent of the structural similarities between LCAT and LPLA2, there exist significant differences in the acceptor specificity for the transacylase activity. For example, LPLA2 is unable to use cholesterol as an acceptor in the transacylase reaction (2). In addition, LPLA2 and LCAT exhibit different pH optima for their enzymatic activities.
With the exception of S198, the functional importance of these other amino acid residues for LPLA2 activity was unknown. In the present study, we evaluated the role of the catalytic triad and cysteine residues in LPLA2 by measuring the enzyme activities of site-directed mutated mouse LPLA2s expressed in COS-7 cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of LPLA2 expression plasmids
The entire open reading frame of mouse LPLA2 (mLPLA2) was obtained by PCR from mouse kidney cDNA as described previously (2). The construct was then subcloned into the Hind III and XhoI sites of pcDNA3-c-Myc to generate C-terminal tagged LPLA2 proteins.
Site-directed mutations of the amino acid residues in the putative catalytic triad and conserved cysteine residues of mLPLA2 were generated by the overlap extension method (10). The amino acid residues in the triad and the conserved cysteine residues in mLPLA2 were replaced with alanines. In brief, two PCRs were used to amplify the overlapping fragments, and another PCR was used to fuse the fragments. To obtain the overlapping fragments, amplification primers (Table 1) and the template DNA consisting of the plasmid containing the mLPLA2 gene between the HindIII and XhoI sites (TOPO-mLPLA2) were applied to the PCR. For the generation of single mutations, including A196G, S198A, D351G, D351A, D360A, H392A, C65A (C1A), C89A (C2A), C330A (C3A), and C371A (C4A), a primer pair consisting of one mutagenic primer (forward) and the mLPLA2-XhoI primer or the same mutagenic primer (reverse) and the mLPLA2-Hind III primer was used with TOPO-mLPLA2 in the first PCR. The single mutated mLPLA2 was obtained by the second PCR using two overlapping fragments and mLPLA2-XhoI and mLPLA2-Hind III primers. The double mutations of the cysteine residues in mLPLA2, C12A (C65A and C89A), were generated by PCR of three fragments, being obtained from the first PCR of mLPLA2-Hind III and the C1A (reverse) primer pair, those obtained from the C1A (forward) and C2A (reverse) primer pair and C2A (forward), and those from the mLPLA2-XhoI primer pair with TOPO-mLPLA2 and the mLPLA2-XhoI and mLPLA2-Hind III primers. Another double mutation, C34A (C330 and C371 replaced with alanines), was generated similarly by PCR of three fragments, being obtained from the first PCR of mLPLA2-Hind III and the C3A (reverse) primer pair, the C3A (forward) and C4A (reverse) primer pair, and the C4A (forward) and mLPLA2-XhoI primer pair with TOPO-mLPLA2, and the mLPLA2-XhoI and mLPLA2-HindIII primers. The quadruple mutations, C1234A (C65, C89, C330, and C372 replaced with alanines), were generated by PCR of five fragments, being obtained from the first PCR of mLPLA2-HindIII and the C1A (reverse) primer pair, the C1A (forward) and C2A (reverse) primer pair, the C2A (forward) and C3A (reverse) primer pair, the C3A (forward) and C4A (reverse) primer pair and the C4A (forward) and mLPLA2-XhoI primer pair with TOPO-mLPLA2, and the mLPLA2-XhoI and mLPLA2-HindIII primers. The PCR product of the second PCR was ligated into pcDNA3-c-Myc. All mutant construct sequences were confirmed by sequencing in both directions.
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Immunoblotting
The soluble fraction was precipitated by the method of Bensadoun and Weinstein (11). The resulting pellet was dissolved with 30 µl of loading buffer plus 1.5 µl of 2 M Tris for SDS-polyacrylamide gel electrophoresis. Proteins were separated using a 10% or 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using transfer buffer (20 mM Tris and 150 mM glycine in 20% methanol) at a constant voltage (100 V for 3 h at 4°C). The membrane was incubated with an anti-mouse LPLA2 peptide (100RTSRATQFPD), rabbit serum, or monoclonal anti-c-Myc mouse ascites fluid. The antigen-antibody complex on the membrane was visualized with an anti-rabbit IgG horseradish peroxidase-conjugated goat antibody or an anti-mouse IgG horseradish peroxidase-conjugated goat antibody using diaminobenzidine and hydrogen peroxide.
Transacylase activity of LPLA2
NAS was used as an acyl group acceptor. The reaction mixture consisted of 40 mM sodium citrate (pH 4.5), 10 µg/ml BSA, 40 µM NAS incorporated into phospholipid liposomes [phosphatidylcholine (PC)/phosphatidylethanolamine/dicetyl phosphate/NAS (5:2:1:2 in molar ratio)], and the soluble fraction (2 µg) in a total volume of 500 µl. The reaction was initiated by adding the soluble fraction, maintained for 10, 20, and 30 min at 37°C, and terminated by the addition of 3 ml of chloroform-methanol (2:1) plus 0.3 ml of 0.9% (w/v) NaCl. The mixture was centrifuged for 5 min at room temperature. The resulting lower layer was transferred into another glass tube and dried down under a stream of nitrogen gas. The dried lipid was dissolved in 40 µl of chloroform-methanol (2:1), half of which was applied on a high-performance thin-layer chromatography plate and developed in a solvent system consisting of chloroform-acetic acid (9:1). The plate was dried down and soaked in 8% (w/v) CuSO4·5H2O, 6.8% (v/v) H3PO4, and 32% (v/v) methanol. The uniformly wet plate was briefly dried down with a hair dryer and charred for 15 min in a 150°C oven. The plate was scanned, and the content of the product (1-O-acyl-NAS) was estimated by NIH Image 1.63.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß-hydrolase superfamily of enzymes consists of three amino acid residues, serine, aspartic acid/glutamic acid, and histidine. The triad is an essential structure to allow the enzyme to catalyze a hydrolytic reaction by the catalytic nucleophile. Previously, S198 in mLPLA2, part of the putative lipase motif AXSXG (where X represents any amino residue), was shown to be essential for enzyme activity (2). The active site serine residue was proposed to form an acyl-LPLA2 intermediate during the hydrolysis reaction. In the present study, the amino acid residues of the putative triad in mLPLA2, S198, D360, and H392, were replaced with alanine. mLPLA2 and single mutated mLPLA2s were equally expressed in the soluble fraction of COS-7 cells (Fig. 2). The transacylase activity of LPLA2 was measured to ascertain the effects of amino acid substitutions. Site-directed mutations of the triad eliminated the transacylase activity (Fig. 2). On the other hand, the replacement of aspartic acid at position 351 with alanine or glycine in mLPLA2 did not affect the enzyme activity (data not shown) in spite of its proximate location to D360. These results indicate that each amino acid residue in the triad is crucial for LPLA2 activity. Interestingly, mLPLA2 activity was reduced to 10% of its original activity when A196 was substituted with glycine found in another lipase motif, GXSXG, which is also preserved in LCAT (data not shown).
Cysteine residues in LPLA2
LPLA2 is highly homologous with LCAT (Fig. 1). LCAT has six cysteine residues, four of which are involved in the formation of intramolecular disulfide bonds (7). The primary amino acid sequence of LPLA2 reveals that the cysteine residues involved in disulfide linkages in LCAT are conserved in LPLA2. The disulfide bonds in LCAT not only contribute stabilization of the protein but also play a crucial role in the interaction between LCAT and lipoprotein surfaces (8, 9). Unlike LCAT, LPLA2 is not able to catalyze the transacylation of an acyl group from the sn-2 position in PC to cholesterol (2), even though LPLA2 has both phospholipase A2 and transacylase activities, as does LCAT. The pH optima for LPLA2 and LCAT activities are acidic and neutral, respectively. Previously, it was shown that LPLA2 activity is not sensitive to either thiol compounds or thiol reagents (1). On this basis, we hypothesized that there might be a difference between LCAT and LPLA2 with respect to the formation of disulfide linkages. However, a disulfide linkage in an intact protein is not always accessible by thiol compounds or thiol reagents. To further evaluate the role of cysteine residues in LPLA2 activity, the four cysteine residues in mLPLA2 that are preserved in LCAT were substituted with alanines.
mLPLA2 and mutated mLPLA2s were equally expressed in the soluble fraction of COS-7 cells (Fig. 3). The transacylase activity of LPLA2 in each soluble fraction was measured to ascertain the effect of amino acid substitutions of each cysteine residue on LPLA2 activity. Quadruple mutations at C65, C89, C330, and C371, double mutations at C65 and C89, and a single mutation at C65 or C89 resulted in the elimination of LPLA2 activity (Fig. 3). On the other hand, double mutations at C330 and C371 and a single mutation at C330 or C371 resulted in a partial reduction of the activity (Fig. 3). The remaining LPLA2 activity in each mutated mLPLA2 was 29 ± 5.7% for the double mutations at C330 and C371, 4.8 ± 0.4% for the single mutation at C330, and 36 ± 5.7% for the single mutation at C371. Thus, the formation of a disulfide bond between C330 and C371 is not necessarily required for LPLA2 activity, suggesting that both of these cysteine residues in intact LPLA2 contain a free sulfhydryl group.
Although mouse, rat, and human LPLA2s have an additional cysteine residue located between the cysteine residues in the C-terminal region, bovine LPLA2 lacks the additional cysteine. Therefore, partially purified bovine LPLA2 was used to demonstrate the existence of the free cysteine residues in conserved cysteine residues of LPLA2 by application to an organomercury agarose column. LPLA2 activity was completely absorbed to the organomercury agarose column and eluted from the column with a buffer containing excess 2-mercaptoethanol (Fig. 4). The organomercury compound forms a covalent bond with a free sulfhydryl group that is cleaved in the presence of excess thiol compounds. These results indicate that at least two cysteine residues of the conserved cysteine residues in LPLA2 are free cysteine residues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß-hydrolase superfamily and that catalyzes the transfer of the acyl group at the sn-2 position of PC to an acceptor. As confirmed in the present study, the catalytic triad of LPLA2 is essential for enzymatic activity. The serine residue present in both the LCAT and the LCAT triads is contained within lipase motifs, 181GXSXG and 196AXSXG, respectively. Based on the secondary structure deduced from the primary structure, the serine of the active site is the nucleophile located in a keen turn structure in both cases (12). This strand-turn-helix structure is a conserved feature in the ß-hydrolase proteins (13) and may allow the nucleophile group the ease of access not only to the substrate but also to the acceptor molecule. The phenylalanine at residue 103 in LCAT is one of two amino acid residues involved in the formation of the oxyanion hole (14) and is conserved in mLPLA2 and other LPLA2s (Fig. 1). This homology suggests that the coordination of the enzyme-substrate intermediate at the catalytic site in the transition state is similar between LCAT and LPLA2. However, the difference in the lipase motif between LCAT and LPLA2 also suggests that the enzyme specificity and activity might be affected by such a small difference. Thus, when alanine-196 (A196) in mLPLA2 was substituted with glycine (G196), the mutated mLPLA2 showed both phospholipid-NAS 1-O-transferase and PLA2 activities, but the substitution resulted in a 10-fold reduction of the enzyme activity. Thus, the substitution of A196 with G196 of mLPLA2 changes the enzyme activity but not the enzyme specificity. A characteristic helical wheel alignment of homologous regions of mLCAT (residues 153–170) and mLPLA2 (residues 171–188) is predicted from their amino acid sequences (12) and is located in the proximity of the catalytic serine residue. Their helical wheels are amphipathic helices that may be involved in the binding of phospholipid at the active site (15). Mutagenesis studies have shown that the helical domain in LCAT plays a crucial role in the phospholipid substrate specificity as well as in phospholipid binding (16).
The four conserved cysteine residues in mLPLA2 are located at C65, C89, C330, and C371. The present study supports a model in which one disulfide bridge is formed between conserved cysteine residues C65 and C89 and two free cysteine residues are located at C330 and C371. In both LPLA2 and LCAT, the region between the cysteine residues forming the disulfide bridge in the proximity of the N-terminal region is predicted to be the most hydrophobic region that is spanned as a loop containing an amphipathic helix (8, 9). The loop formed in LCAT is essential for the interaction between LCAT and lipoprotein surfaces and is thought to be indispensable for the interfacial activation of LCAT (17). By analogy, the loop in LPLA2 may also play a crucial role in the interaction of LPLA2 with hydrophobic structures such as phospholipid membranes. The substitution of C65 and/or C89 with alanine resulted in the elimination of LPLA2 activity, suggesting that a proper interaction between mutated LPLA2 and lipid membrane is not accomplished by disruption of the structural loop of this region when the disulfide bridge is absent. As proposed in LCAT and shown in other lipases of the ß-hydrolase superfamily, the loop in LPLA2 may act as a lid to regulate the access of substrate to the active site (6, 18, 19). Therefore, a degree of hydrophobicity may be required for substrates to access the active site. In support of this interpretation is the observation that the degradation rate of the artificial substrate p-nitro-phenyl butylate by LPLA2 under acidic conditions is 7-fold higher than that of p-nitro-phenyl acetate (data not shown). LPLA2 mutated at S198 does not hydrolyze these artificial substrates. This finding indicates that the triad in LPLA2 is essential for the hydrolysis reaction of the artificial substrates as well as for naturally occurring phospholipids. Similar properties have already been reported for LCAT (20).
Unlike LCAT, mLPLA2 may have two conserved free cysteine groups, C330 and C371, in the proximity of the C-terminal region. C330 and C371 are adjacent to the triad amino acids, D360 and H392. The catalytic triad structure in LPLA2 may be similar to that observed in other lipases, in which hydrogen bonds formed between the amino acid residues in the triad play a key role in enzyme activation and reaction (21). Although a single or double substitution of each cysteine residue (C330, C371) with alanine reduces the original enzymatic activity, each mutated LPLA2 still retains some enzyme activity. Thus, the free sulfhydryl groups at C330 and C371 are likely to be crucial for providing a proper tertiary structure for the catalytic triad. Although the tertiary structure of the active site in LPLA2 is thought to be similar to that of LCAT, differences in disulfide bridge formation between LCAT and LPLA2 may contribute to different substrate specificity and enzyme activity. For example, LPLA2 catalyzes the transacylation of the acyl group at sn-2 of PC to an acceptor with a hydroxyl group but is not able to use cholesterol as an acceptor (2). Our preliminary studies have shown that, in addition to NAS, LPLA2 prefers to use neutral and relatively hydrophobic compounds with a primary alcohol group rather than secondary and tertiary alcohol groups as the acceptor (unpublished data).
The presumed importance of the free sulfhydryl groups at C330 and C371 for enzyme activity could be questioned based on the failure of thiol reagents to decrease activity. When LPLA2 was initially purified from bovine brain and its activity characterized, N-ethylmaleimide only decreased the transacylase activity by 5%. This observation was initially interpreted as suggesting that the free sulfhydryl groups in LPLA2 were unimportant for activity. In the context of the present studies, another interpretation should be considered. The free sulfhydryl groups of the intact protein may not be readily accessible to thiol reagents. A similar result was observed in studies of the glycolipid transfer protein (22).
Further studies comparing the structure and function of LCAT and LPLA2 are likely to be informative. Such studies should include the construction of chimeric enzymes to confirm the importance of specific domains of each protein in determining substrate specificity. Such studies may also be informative regarding the evolutionary biology of both LPLA2 and LCAT. The genes encoding both proteins are members of an extensive and evolutionarily ancient gene family. Homologs of these genes are present in many ancient species that lack lungs (as might be predicted for LPLA2) and lipoproteins (as would be predicted for LCAT). LCAT appears to be the more rapidly diverging gene, and it is restricted to vertebrates. LPLA2 is more closely aligned to invertebrate homologs in the gene family. Thus, further studies on the protein structures of LCAT and LPLA2, including the catalytic domains and disulfide bonds, and the roles of their protein domains in determining the substrate specificity and activity of these closely related enzymes will be important in understanding the biology of these enzymes and their family members.
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ACKNOWLEDGMENTS |
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Manuscript received June 14, 2005 and in revised form July 27, 2005.
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REFERENCES |
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