©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Elements of Secretory Phospholipases A Involved in the Binding to M-type Receptors (*)

(Received for publication, November 1, 1994; and in revised form, January 9, 1995)

Gérard Lambeau (1) Philippe Ancian (1) Jean-Paul Nicolas (1) Sigrid H. W. Beiboer (2) Danielle Moinier (1) Hubertus Verheij (2) Michel Lazdunski (1)(§)

From the  (1)Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France and the (2)University of Utrecht, Department of Enzymology and Protein Engineering, Center for Biomembranes and Lipid Enzymology, Transitorium III, Padualaan 8, De Uithof, 3508 TB Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Specific membrane receptors for secretory phospholipases A(2) (sPLA(2)s) have been initially identified with novel snake venom sPLA(2)s called OS(1) and OS(2). One of these sPLA(2) receptors (muscle (M)-type, 180 kDa) has a very high affinity for OS(1) and OS(2) and a high affinity for pancreatic and inflammatory-type mammalian sPLA(2)s, which might be the natural endogenous ligands of PLA(2) receptors. Primary structures of OS(1) and OS(2) were determined and compared with sequences of other sPLA(2)s that bind less tightly or do not bind to the M-type receptor. In addition, the binding properties of pancreatic sPLA(2) mutants to the M-type receptor have been analyzed. Residues within or close to the Ca-binding loop of pancreatic sPLA(2) are crucially involved in the binding step, although the presence of Ca that is essential for the enzymatic activity is not required for binding to the receptor. These residues include Gly-30 and Asp-49, which are conserved in all sPLA(2)s. Leu-31 is also essential for binding of pancreatic sPLA(2) to its receptor. Many other mutations have been considered. Those occurring in the N-terminal alpha helices and the pancreatic loop do not change binding to the M-type receptor. Conversion of pancreatic prophospholipase to phospholipase is essential for the acquisition of binding properties to the M-type receptor.


INTRODUCTION

Mammalian secretory PLA(2)s (^1)(sPLA(2)s) have been divided in two different structural groups(1, 2) , including group I (the pancreatic type) and group II (the inflammatory type) (reviewed in (3, 4, 5, 6) ). The pancreatic-type sPLA(2) is particularly well characterized. Its three-dimensional structure is known(7, 8) , and a detailed characterization of its catalytic properties has been described(3, 9) . This enzyme, which has long been considered exclusively as a digestive enzyme(10) , has now been localized in several tissues of non-digestive origin such as lung, spleen, and plasma(11, 12, 13) . The pancreatic-type sPLA(2) now appears to have a variety of other cellular functions. It plays a role in cell proliferation(14) , and in smooth muscle contraction(15, 16) . The group II sPLA(2) is present in abundance in synovial fluids and plasma of patients with diverse inflammatory diseases and has been proposed to play a key role in the pathogenesis of inflammatory diseases (reviewed in (17, 18, 19, 20, 21, 22) ). Its expression and secretion are induced by proinflammatory agents like interleukin-1, interleukin-6, and tumor necrosis factor and are inhibited by glucocorticoids (reviewed in (17, 18, 19, 20, 21, 22) ). Other members of mammalian sPLA(2)s have been discovered very recently(23, 24) .

sPLA(2)s are also found in abundance in snake and bee venoms (25, 26) . These enzymes have conserved many important features with mammalian sPLA(2)s, including a common catalytic mechanism, the same calcium requirement, and very conserved primary and tertiary structures(2, 6, 27, 28) . In addition to their probable roles in the digestion of preys, snake venom sPLA(2)s have evolved into extremely potent toxins displaying neurotoxic, myotoxic, anticoagulant, and proinflammatory effects (reviewed in (25) and (26) ). The diversity of the pathophysiological effects of venom sPLA(2)s is probably linked to the presence of specific high affinity receptors for these enzymes(29, 30, 31, 32, 33) . A first type of receptors initially identified in brain is called N (for neuronal)-type PLA(2) receptors. It recognizes with high affinity a large number of toxic sPLA(2)s including OS(2), a new highly neurotoxic sPLA(2) purified from the Taipan snake venom, the bee venom sPLA(2), and the neurotoxic sPLA(2) CM-III from Naja mossambica mossambica(29) . Nontoxic venom sPLA(2)s as well as the porcine pancreatic sPLA(2) display very low affinities for these receptors(29) . A second type of receptors initially identified in rabbit skeletal muscle and thus referred to as M (for muscle)-type PLA(2) receptors recognizes with very high affinities OS(2) and OS(1), a new non toxic sPLA(2) also purified from the Taipan snake venom(30) . This M-type PLA(2) receptor does not bind the bee venom sPLA(2) or the CM-III sPLA(2) from N. mossambica mossambica(30) . This receptor binds the porcine pancreatic group I sPLA(2) as well as the human inflammatory group II sPLA(2), both with fairly high affinity(31) . It is now known that M-type as well as N-type receptors have different subunit constitutions and are not exclusively present in brain or muscle(32, 33) . It is believed that these receptors are normal binding targets for endogenous sPLA(2)s, which, by binding to them, could work as hormones or growth factors. Molecular cloning of rabbit (31) and bovine (34) M-type PLA(2) receptors has recently been achieved and has revealed that these receptors are structurally related to the macrophage mannose receptor, a protein involved in the endocytosis of mannose-bearing glycoproteins and microorganisms(35, 36) .

The purpose of the present paper is to identify the specific region in the structure of sPLA(2)s that is responsible for the interaction with M-type PLA(2) receptors, (i) using the structures of OS(1) and OS(2), the two venom sPLA(2)s that have initially served to discover PLA(2) receptors (29, 30) and (ii) using a series of mutants of the pancreatic sPLA(2), one of the endogenous sPLA(2)s that associates with M-type PLA(2) receptors.


EXPERIMENTAL PROCEDURES

Native sPLA(2)s

OS(1), OS(2), the CM-III sPLA(2) from N. mossambica mossambica, and the porcine pancreatic iso-PLA(2) were purified as described previously(29, 37) . Native porcine pancreatic sPLA(2) was purchased from Boehringer Mannheim.

Recombinant sPLA(2)s

Preparations of the different structural variants of porcine pancreatic recombinant sPLA(2)s used in this study have been described in detail elsewhere(38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48) . Protein concentrations were determined using the respective molar absorbance coefficients for each mutant.

Amino Acid Sequence Determinations of OS(1) and OS(2)

The amino acid sequencing of OS(1) and OS(2) first involved the generation of a small number of polypeptide fragments by digestion of reduced and Spyridylated sPLA(2)s with endoproteinase Lys-C, endoproteinase Glu-C, and cyanogen bromide. Briefly, each purified sPLA(2) (2 nmol of protein) was reduced with 2-mercaptoethanol and pyridylated with 4-vinylpyridine. S-Pyridylated sPLA(2)s were immediately desalted by high performance liquid chromatography onto a reverse phase support (Aquapore RP-300, 2.1 times 30 mm, Brownlee) and eluted as a single peak. For endoproteinase Lys-C digestion, 0.5 nmol of S-pyridylated sPLA(2)s were dissolved in 50 µl of 50 mM sodium phosphate buffer, pH 7.8, and were treated for 24 h at room temperature with 1 pmol of endoproteinase Lys-C (Promega Corp., Madison, WI). For endoproteinase Glu-C cleavage, 0.5 nmol of S-pyridylated sPLA(2)s were digested in 50 mM ammonium bicarbonate pH 7.8 at 37 °C for 24 h with 1 pmol of endoproteinase Glu-C (Promega). For cyanogen bromide peptides preparation, 1 nmol of sPLA(2) and 0.5 mg of CNBr dissolved in 70% formic acid (50 µl, final volume), were incubated in the dark at room temperature under N(2) for 24 h. The reaction mixture was evaporated by Speed-Vac centrifugation and redissolved in 0.1% trifluoroacetic acid. All peptide hydrolysates were then fractionated onto a C(18) reverse phase high performance liquid chromatography column (Aquapore RP300, 1 times 250 mm, Brownlee) by using an Applied Biosystems model 140A apparatus equipped with a spectrophotometer monitor 5000 UV detector (LDC Analytical). Initial chromatographic conditions were 0.1% trifluoroacetic acid in water with a flow rate of 100 µl/min at room temperature, and elution was performed by increasing the acetonitrile concentration to 70% in 0.1% trifluoroacetic acid using a linear gradient of 0.5%/min.

Automated Edman degradation of S-pyridylated sPLA(2)s and of purified peptides were performed with an Applied Biosystem sequencer (model 477A) equipped with an on-line phenylthiohydantoin-derivative analyzer (model 120A). C-terminal amino acid analysis was confirmed by carboxypeptidase Y cleavage according to the manufacturer's protocol (Boehringer Mannheim).

Molecular weights of OS(1) and OS(2) were first determined by SDS-polyacrylamide gel electrophoresis, indicating that OS(1) and OS(2) have apparent molecular weights of 14,900 and 15,500, respectively, under reducing conditions. Molecular weights of OS(1) and OS(2), as calculated from their sequences, are 14,108 and 13,331, respectively. Molecular weights of OS(1) and OS(2), as determined by mass spectrometry analysis using a laser desorption technique (Finnigan laser mat), are 14,088 and 13,325, respectively.

Binding Experiments

All binding experiments were performed at 20 °C in 1 ml of a buffer consisting of 140 mM NaCl, 20 mM Tris, pH 7.4, 0.1% bovine serum albumin, and 0.1 mM CaCl(2) except when specified in the legends to the figures. Incubations were started by addition of the rabbit skeletal muscle membranes and filtered after 90 min of incubation through GF/C glass fiber filters presoaked in polyethyleneimine as described previously(29) . Dilution of all sPLA(2)s were done in the above incubation buffer. I-OS(1) and rabbit skeletal muscle cell membranes were prepared as described(30) .


RESULTS

Properties of sPLA(2) Binding to the M-type Receptor and the Role of Ca

Fig. 1A shows binding experiments using I-OS(1). Results indicate that both OS(1) and OS(2) have very high affinities for the M-type PLA(2) receptor (K(0.5) values of 63 and 16 pM, respectively). The native wild-type porcine pancreatic sPLA(2) displays an apparent affinity of 14 nM. The CM-III sPLA(2) from N. mossambica mossambica venom has no measurable affinity.


Figure 1: Properties of sPLA(2)s binding to rabbit M-type PLA(2) receptors. Panel A, competition experiments involving I-OS(1) and unlabeled sPLA(2)s for binding to rabbit skeletal muscle myotube membranes. Membranes (2 µg of protein/ml) were incubated in the presence of I-OS(1) (25 pM) and various concentrations of unlabeled sPLA(2)s. All results are expressed as percentages of control made without unlabeled sPLA(2). 100% corresponds to a I-OS(1) specific binding of 0.9 pM. 0% corresponds to I-OS(1) binding measurement in the presence of 100 nM unlabeled OS(1), which was below 15% of the total binding. Panel B, Scatchard analysis of I-OS(1) binding to rabbit skeletal muscle myotube membranes in the absence or presence of various concentrations of unlabeled porcine pancreatic sPLA(2). Main panel, binding data obtained at various concentrations of I-OS(1) (5-350 pM) in the absence () or the presence of 10 (up triangle), 25 (), 50 (circle), and 100 (bullet) nM unlabeled porcine pancreatic sPLA(2) were plotted according to Scatchard. Inset, Cheng and Prusoff plot of the I-OS(1)K values obtained from the above Scatchard analysis as a function of porcine pancreatic sPLA(2) concentrations.



Scatchard plot analysis of saturation curves performed with increasing concentrations of I-OS(1) in the presence of various concentrations of porcine pancreatic sPLA(2) indicates that the porcine pancreatic sPLA(2) competitively inhibits the binding of I-OS(1) (Fig. 1B) since the maximal binding capacity is unchanged (B(max) = 1.56 ± 0.05 pmol/mg of protein), while the K(d) values for I-OS(1) vary from 22 pM (no addition of porcine pancreatic sPLA(2)) to 260 pM (addition of 100 nM unlabeled porcine pancreatic sPLA(2)). An apparent inhibitory constant (K(I)) of 8.7 nM for the pancreatic sPLA(2) was calculated from the inset of Fig. 1B.

Since Ca is an essential cofactor for the enzymatic activity of sPLA(2)s(3, 7, 9) , it was of interest to analyze its effect on sPLA(2) binding to M-type PLA(2) receptors. Fig. 2A shows that the binding of I-OS(1) is unchanged in the absence or in the presence of Ca. These results contrast with those previously obtained for N-type PLA(2) receptors for which binding activity was found to be highly dependent on the presence of Ca(29) . The binding of the pancreatic sPLA(2) to M-type PLA(2) receptors is also essentially Ca-independent (Fig. 2B) since the K(0.5) values for the inhibition of I-OS(1) binding are similar in the presence (2 mM CaCl(2)) or absence (2 mM EDTA) of free Ca.


Figure 2: Role of Ca in sPLA(2) binding to M-type PLA(2) receptors. Panel A, effects of free Ca ions on I-OS(1) binding to rabbit skeletal muscle myotube membranes. Free Ca concentrations were adjusted by buffering with 2 mM EDTA. Therefore, 0 Ca-free concentration corresponds to addition of 2 mM EDTA in the binding assay. Total binding (bullet) and nonspecific binding (circle) are shown. Binding conditions are as in Fig. 1. Panel B, competition experiments with I-OS(1) and the porcine pancreatic sPLA(2) in the presence of 2 mM CaCl(2) or EDTA. Binding conditions are as in Fig. 1.



Can Sequence Comparisons Tell Us about StructureFunction Relationships in Binding to M-type PLA(2) Receptors?

OS(1) and OS(2), the two venom sPLA(2)s that have, because of their very high affinity, initially served for the identification of M-type PLA(2) receptors, are composed of 127 and 119 amino acids, respectively (Fig. 3). Both enzymes contain the amino acid residues glycine 30, histidine 48, aspartic acid 49, tyrosine 52, tyrosine 73, and aspartic acid 99, which have been shown to be essential for sPLA(2) activity(9, 28) . The position of the half-cystine residues indicates that both OS(1) and OS(2), as expected from their elapid venom origin, belong to group I sPLA(2)s(1) . OS(1), which has a pancreatic-like loop, belongs to group IB, whereas OS(2), which does not have this loop, is a member of group IA(2) .


Figure 3: Sequence alignment of OS(1), OS(2), porcine pancreatic sPLA(2)(49) and CM-III sPLA(2) from N. mossambica mossambica(50) . The common numbering system defined by Renetseder et al. is used(51) . Amino acid residues found in all active group I sPLA(2)s sequenced so far are indicated in the consensus sequence(2, 27) .



Sequences of OS(1) and OS(2) are aligned with those of the porcine pancreatic sPLA(2) and of the CM-III sPLA(2) in Fig. 3. These four sPLA(2)s are all group I sPLA(2)s but have very different binding properties for M-type PLA(2) receptors (Fig. 1A). The highest similarity between these four sPLA(2)s is in the Ca-binding loop (residues 25-35) and in the second large alpha-helical segment (residues 40-57), which contains 2 out of the 4 amino acids implicated in the catalytic network(9, 28) . Another large region of homology is found in a region between residues 91 and 111, which contains aspartic acid 99, another perfectly conserved residue involved in the catalytic network(9, 28) . In addition, all these four sPLA(2)s have the amino acid residues (Fig. 3) found conserved in all group I sPLA(2)s sequenced so far(2, 27) . A detailed comparison of these four sPLA(2)s failed to reveal obvious amino acid residues that could be responsible for the binding to M-type PLA(2) receptors. The overall homology is almost the same between OS(1) and the porcine pancreatic sPLA(2) (50% identity), OS(2) (54% identity), or the CM-III sPLA(2) (51% identity). Therefore, the identification of amino acid substitutions that determine the binding of sPLA(2)s to M-type PLA(2) receptors requires different approaches.

Identification of the Functional Binding Domain of sPLA(2)s to M-type PLA(2) Receptors Using Porcine Pancreatic sPLA(2) Mutants

This second approach uses a large number of recombinant porcine pancreatic sPLA(2) mutants. The localization of the corresponding mutated amino acids in the pancreatic sPLA(2) structure is summarized in Fig. 4. The affinity of these different mutants have been measured by their capacity to inhibit I-OS(1) binding to M-type PLA(2) receptors (Table 1). The binding properties of the porcine pancreatic proenzyme and of the porcine iso-PLA(2) have also been studied (Table 1).


Figure 4: Stereoview of the alpha-carbon backbone of the porcine pancreatic sPLA(2). Positions of the N-terminal (Nt) and C-terminal (Ct) residues are shown. All the amino acid positions that have been analyzed are marked using the one-letter code. Position of mutations (W3F, S7R, M8L, M20L, H17D, Y69F or -K, E71N, and Delta62-66) and of amino acid substitutions occurring in iso-PLA(2) (alanine 12, histidine 17, methionine 20, and glutamic acid 71 changed to threonine, aspartic acid, leucine, and asparagine, respectively) that are without effect on binding activity are indicated by small balls. Conservative mutations obtained by replacing all lysines for arginines which do not affect binding activity are not indicated by small balls because it is quite possible that if these lysines were changed to neutral or negatively charged residues, an effect on binding might occur. The mutant Delta62-66 was obtained by the deletion of residues 62-66 (residues shown in pink) and the simultaneous introduction of mutations D59S, S60G, and N67Y(44) . Position of mutations affecting the affinity of the pancreatic enzyme (G30S, L31R, -S, -T or -W, and D49K) are indicated by largerballs. Localization of Ca is indicated by a purple ball.





The first large N-terminal alpha-helix and the short alpha-helix that follows in the structure are both located at the surface of the sPLA(2) molecule (Fig. 4). This region of the pancreatic sPLA(2) molecule contains residues involved in interfacial binding, substrate specificity and formation of the hydrophobic channel (28, 53) . Analysis of the binding properties of four different mutants in this region of the enzyme and of the iso-PLA(2) suggests that this region does not contain structural determinants essential for the binding activity to M-type PLA(2) receptors (Table 1). Indeed, mutation of tryptophan 3 to phenylalanine (W3F; (38) ) does not strongly modify the K(0.5) value as compared with the wild-type recombinant sPLA(2) (Table 1). The presence of different residues at position 3 in OS(1), OS(2), and the CM-III sPLA(2) confirms that residues at this position have probably no role in the binding step. Likewise, a mutation of serine 7 to an arginine (S7R; (39) ) does not significantly change the K(0.5) value as compared with the wild-type recombinant enzyme (Table 1). This is consistent with the observation that serine 7 is replaced by an asparagine in OS(1) and a phenylalanine in OS(2), which bind well to M-type PLA(2) receptors, but also by an asparagine in the CM-III sPLA(2), which does not bind to this receptor. Similarly, a pancreatic mutant in which methionines 8 and 20 have been replaced by leucines (M8L/M20L; (40) ) still displays an affinity (K(0.5) = 18 nM) similar to that of the recombinant wild-type enzyme (K(0.5) = 15 nM), indicating that replacement of methionines by leucines at these positions does not change the binding properties (Table 1). OS(1) and OS(2) have a leucine or a methionine respectively at these positions; as expected, both bind similarly to M-type PLA(2) receptors, but this residue is not essential since the CM-III sPLA(2) also has a methionine in position 8 but does not bind to the receptor. The last mutant that provides some information on the role of this part of the pancreatic sPLA(2) structure in binding activity corresponds to a partial conversion of the pancreatic sPLA(2) major isoform to the iso-PLA(2) minor isoform by substitution of histidine 17 to an aspartic acid (together with a substitution of glutamic acid 71 to an asparagine). These mutations fail to produce drastic effects on the binding properties (Table 1). Finally, the native iso-PLA(2), which is different from the major isoform pancreatic sPLA(2) at four positions(37) , shows a K(0.5) value of 22 nM very similar to that of wild-type porcine pancreatic sPLA(2) (Table 1). Prophospholipase (pro-PLA(2)), the enzymatically inactive porcine pancreatic zymogen, has no measurable affinity for M-type PLA(2) receptors (Table 1).

Analysis of Mutations in the Ca-binding Loop and of Residues Involved in Ca Binding

Glycine 30 and aspartic acid 49 ( Fig. 3and Fig. 4) have been found to be essential for the enzymatic activity and are conserved residues found in all sPLA(2)s(2, 3, 7, 27, 28, 53) . These 2 residues control Ca binding to the protein moiety and are directly involved in the catalytic mechanism. Table 1shows that these residues are also important for binding activity. When aspartic acid 49 in the porcine pancreatic sPLA(2) was replaced by a lysine, the corresponding mutant (D49K) had a tremendously decreased enzymatic activity of less than 0.004% as compared to the wild-type enzyme, a value that is of the order of the non-enzymatic hydrolysis of substrate (43) . Its binding activity was also drastically decreased by a factor of 100 with an affinity of 1.4 µM (Table 1). When glycine 30 was replaced by a serine (G30S), catalytic activity dropped to as low as 2% of the value found for the native enzyme (41) and the binding activity measured from the K(0.5) value was decreased by a factor of 8 (Table 1).

The effect of various mutations at position 31 has also been analyzed (42) . Leucine 31 in the pancreatic sPLA(2) is located in the Ca-binding loop (Fig. 4) but is not involved in the coordination of Ca. Three-dimensional structures of several sPLA(2)s have shown that residues at position 31 are located at the surface of the enzyme, at the entrance of the hydrophobic channel and of the active site(7, 51, 54, 55, 56, 57) . Leucine 31 of the pancreatic sPLA(2) also forms a part of the interfacial binding surface and is then involved in the binding of sPLA(2) to aggregated phospholipids(28, 53) . Site-directed mutagenesis experiments on porcine pancreatic sPLA(2) have shown that replacements of leucine 31 by a tryptophan, an arginine, an alanine, a threonine, a serine, or a glycine modified the affinity of the enzyme for monomeric and aggregated phospholipids as well as catalytic activity toward these substrates(42) . Replacements of leucine 31 in the porcine pancreatic sPLA(2) by a serine (L31S) or a threonine (L31T) resulted in a 100-fold decrease of the K(0.5) value relative to the wild-type enzyme. Replacement by an arginine (L31R) resulted in a total loss of binding activity (Table 1). Interestingly, the CM-III sPLA(2) has an arginine in position 31 (Fig. 3) and does not bind to M-type PLA(2) receptors (Fig. 1A). Conversely, a mutation to a tryptophan (L31W) did not result in a decrease of binding activity (Table 1).

Analysis of Mutations in the Pancreatic Loop Domain

One intriguing difference between pancreatic-type sPLA(2)s and many other sPLA(2)s is the presence of the so-called ``pancreatic loop'' domain in the pancreatic enzyme (residues 62-66 in Fig. 3and Fig. 4). Three-dimensional structures of sPLA(2)s with or without this loop are very similar(7, 8, 51, 54, 55, 56, 57) , the existence of this loop only resulting in a local modification of the sPLA(2) structure. This view was confirmed by x-ray crystallography data obtained with the Delta62-66 mutant of the pancreatic sPLA(2)(44) . The porcine pancreatic mutant Delta62-66 with a deletion of the loop displays almost the same affinity for M-type PLA(2) receptors as that of the wild-type porcine sPLA(2) (Table 1). This is not really surprising since OS(1), which contains the pancreatic loop, and OS(2), which lacks this loop (Fig. 3), both bind with a high affinity to M-type PLA(2) receptors (Fig. 1A).

Analysis of Other Mutations in the Porcine Pancreatic sPLA(2)

Among amino acid residues playing a role in sPLA(2) activity, the residue at position 69 (Fig. 4) has been found to contribute to the left wall of the hydrophobic channel of the active site(7, 8, 28, 53, 54, 55, 56, 57) . It is also implicated in the recognition of the phosphate group of phospholipid substrates(45, 46) . The K(0.5) values obtained for the binding of two mutants at this position 69 (Y69F and Y69K) to M-type PLA(2) receptors indicate no essential contribution of this residue in the binding of sPLA(2)s to these receptors (Table 1). The absence of role of this residue in binding is also indicated by the fact that sPLA(2)s with very high affinity (OS(1)), high affinity (wild-type porcine sPLA(2)), or no affinity (CM-III sPLA(2)) for M-type PLA(2) receptors have all a tyrosine residue at this position, whereas OS(2) has a phenylalanine and also binds with very high affinity.

Lysine residues have been implicated in the interfacial recognition site of the enzyme, in the process of interfacial activation, and in the ``penetrating power'' of sPLA(2)s into the lipid bilayer (see (48) ). A porcine pancreatic mutant was constructed in which all 9 lysines were replaced by arginines (All K-R). This mutant displayed 68% residual activity on micellar zwitterionic substrates, indicating that lysines are not essential for catalytic activity(48) . This mutant of the pancreatic sPLA(2) has nearly the same binding properties to M-type PLA(2) receptors as the wild type pancreatic sPLA(2) (Table 1). Sequence comparison at positions corresponding to lysines in the porcine pancreatic enzyme with other sPLA(2)s shown in Fig. 3also indicates that these lysines are not essential elements for the interaction with M-type PLA(2) receptors.


DISCUSSION

This paper presents sequences of the two sPLA(2)s isolated from the venom of Oxyranus scutellatus scutellatus and called OS(1) and OS(2), which have initially served to demonstrate the existence of high affinity receptors (in the 0.01-0.1 nM range) for sPLA(2)s(29, 30, 31, 32, 33) . Comparison of these sequences with the pancreatic sPLA(2), one of the probable endogenous ligand of the M-type PLA(2) receptor, which binds with an affinity in the 1-10 nM range (14, 31, 34) and with the CM-III sPLA(2), which does not recognize the M-type PLA(2) receptor(30) , failed to provide indications on residues involved in receptor recognition. Therefore, since pancreatic sPLA(2) binds competitively with the high affinity ligand OS(1) to the M-type receptor, since this enzyme as previously indicated is the probable endogenous ligand and since its three-dimensional structure is particularly well known, it appeared that the best way to analyze the structure-function relationships of the interaction of sPLA(2)s with their receptors was to use pancreatic sPLA(2) mutants.

Possible candidates for an interaction with the receptor were N-terminal helices, which are located at the surface of the sPLA(2) molecule. Mutations at different positions (W3F, S7R, M8L, M20L, and H17D) in this region of the pancreatic sPLA(2) failed to produce marked changes of the affinity of the pancreatic sPLA(2) to M-type PLA(2) receptors.

Another interesting property of pancreatic sPLA(2) is the existence of a pancreatic loop at residues 62-66, which is found in some other sPLA(2)s such as OS(1). Deletion of this loop does not significantly alter (a decrease by a factor of 3) the affinity of the pancreatic sPLA(2) to M-type PLA(2) receptors. The idea that this loop is not essential for binding activity is supported by the fact that OS(2), which lacks this loop, binds to M-type PLA(2) receptors with an affinity much better than that of the pancreatic sPLA(2) ( Fig. 1and Table 1).

Lysine residues are of course exposed at the surface of the pancreatic sPLA(2)(48) . Their replacement by arginines, i.e. other positively charged residues, does not significantly alter the affinity (a decrease by a factor of 2), indicating that these amino acid side chains are not crucially involved in the interaction. However, it cannot be eliminated that other types of mutations not conserving the charges on the side chains could have had a more drastic effect. Nevertheless, sequence comparisons shown in Fig. 3indicate that none of the lysines of the pancreatic sPLA(2) but lysine 121 is conserved in the structures of OS(1) and OS(2), which both bind extremely well to M-type PLA(2) receptors. It might be the change of lysine 121 plays some role (which would remain partially conserved after a mutation to an arginine) in the interaction with the receptor.

The beta-wing (residues 74-85, Fig. 3) of the pancreatic sPLA(2) is probably not involved in the interaction with the receptor since this part of the sPLA(2) differs considerably in the three-dimensional structures of group I (7, 8, 54, 57) and group II sPLA(2)s (51, 56) and since both groups of sPLA(2)s bind to M-type PLA(2) receptors including the human group II sPLA(2)(31) .

The most evident domain of pancreatic sPLA(2) involved in the binding with the M-type receptor is the Ca-binding loop. This part of the enzyme, as its name indicates, is involved in Ca binding, which is essential for catalytic activity (28, 53) . It contains glycine 30 and aspartic acid 49, which are present in all active sPLA(2)s sequenced so far, including the evolutionary distant bee venom sPLA(2)(2, 27, 55) . Mutations of glycine 30 or of aspartic acid 49 drastically decreased binding activity by factors of 8 (G30S) to 100 (D49K) (Table 1). Clearly, residues 30 and 49 are essential for binding activity to M-type PLA(2) receptors, but their presence is not sufficient to confer binding since other sPLA(2)s such as the CM-III sPLA(2) and the bee venom sPLA(2), which do not bind to the M-type PLA(2) receptors ( Fig. 1and (30) ), also have these residues in their sequence ( Fig. 3and (55) ). Very drastic effects on binding activity have also been observed by mutation of leucine 31 to residues such as serine, threonine, or arginine (a decrease of the affinity by more than 100 times). Conversely, mutation of leucine 31 to a tryptophan did not result into a dramatic decrease of the affinity but rather into a moderate increase of the affinity (Table 1), suggesting that the presence of aliphatic or aromatic residues at this position 31 can also confer high affinity to M-type PLA(2) receptors. Several different residues are found in position 31 in other sPLA(2)s sequences(2, 27) . This position is occupied by a lysine in OS(1) and OS(2). It seems that while glycine 30 and aspartic acid 49 are essential for binding, the identity of the side chain in position 31 will finally determine whether this binding is possible or not. The CM-III sPLA(2) has an arginine in position 31; it does not bind to the M-type PLA(2) receptors, consistent with the observation that the pancreatic sPLA(2) mutant with an arginine in this position is inactive in binding (Table 1).

Since the Ca-binding loop is obviously involved in the sPLA(2)-M-type receptor interaction, it was of course important to determine whether Ca is important for the association. Clearly, results presented in this paper (Fig. 2) show that Ca is not important for the interaction. The receptor association of the ligand seems to be even better in the absence of Ca, suggesting that the preferred form of sPLA(2) for binding is the Ca-free form (Fig. 2). Conversely, the other type of PLA(2) receptors, i.e. the N-type receptor, requires Ca for sPLA(2) binding(29) .

One of the questions that immediately comes to mind is to know whether sPLA(2) catalytic activity is required for binding. At first sight, the two activities seem to be related (Table 1) since mutations that cause the largest decreases in catalytic activity also have a large impact in terms of binding. This is because the Ca-binding loop is necessary for both functions. However, catalytic activity is clearly not required, since it is absent without Ca while binding activity is preserved and even increased (Fig. 2).

The pancreatic sPLA(2) originates from its prophospholipase precursor. It is particularly interesting to see that while the pancreatic sPLA(2) as well as its variant iso-PLA(2) (with changes in positions 12, 17, 20, and 71) binds fairly avidly to M-type PLA(2) receptors, the prophospholipase does not. Clearly the precursor needs to be converted to acquire a conformation (58, 59, 60) that confers both catalytic activity and binding activity to M-type PLA(2) receptors.


FOOTNOTES

*
This work was supported by CNRS, ARC, and Grant DRET 93/122 from the Ministère de la Défense Nationale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-93-95-77-00; Fax: 33-93-95-77-04.

(^1)
The abbreviations used are: PLA(2), phospholipase A(2); sPLA(2), secretory phospholipase A(2); N-type; neuronal-type; M-type, muscle-type.


ACKNOWLEDGEMENTS

We thank P. W. Franken, A. C. A. P. A. Bekkers, and R. B. Lugtigheid for making available some of the mutants. We are very grateful to Dr. J. C. Doury and to Dr. H. Tolou (Institut de Médecine Tropicale du Service de Santé des Armées, CERMT, Marseille, France) for mass spectrometry analysis of OS(1) and OS(2). We thank M.-M. Larroque, C. Roulinat, F. Aguila, and M. Bordes for expert technical assistance.


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