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Sequence homology between Wingless/Wnt-1 and a lipid-binding domain in secreted phospholipase A2

1999; Elsevier BV; Volume: 9; Issue: 10 Linguagem: Inglês

10.1016/s0960-9822(99)80225-5

1879-0445

Frieda Reichsman, Helen M. Moore, Susan Cumberledge,

Cancer-related gene regulation

We have found that the carboxyl terminus of Wingless/Wnt-1 shares significant sequence homology with a lipid-binding domain found in class I and class II secreted phospholipase A2 (sPLA2) proteins. The Drosophila Wingless protein and its vertebrate ortholog, Wnt-1, belong to the Wnt family of secreted growth factors [[1]Cox RT Peifer M Wingless signaling: the inconvenient complexities of life.Curr Biol. 1998; 8: 140-144Abstract Full Text Full Text PDF Google Scholar]. Wnt activity is modulated by a number of extracellular factors, including cell-surface proteoglycans [[2]Cumberledge S Reichsman F Glycosaminoglycans and Wnts.Trends Genet. 1997; 13: 221-225Abstract Full Text PDF Scopus (46) Google Scholar] and two types of secreted antagonists, the Frizzled-related proteins [[3]Rattner A Hsieh JC Smallwood PM Gilbert DJ Copeland NG Jenkins NA Nathans J A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors.Proc Natl Acad Sci USA. 1997; 94: 2859-2863Crossref PubMed Scopus (472) Google Scholar] and Dickkopf-1 (Dkk-1) [[4]Glinka A Wu W Delius H Monaghan AP Blumenstock C Niehrs C Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction.Nature. 1998; 39: 357-362Google Scholar]. Lipids have been implicated in Wnt signaling before, when Aravind and Koonin [[5]Aravind L Koonin EV A colipase fold in the carboxy-terminal domain of the Wnt antagonists – the Dickkopfs.Curr Biol. 1998; 8: 477-478Abstract Full Text Full Text PDF PubMed Google Scholar] hypothesized that Dkk-1 inhibition of Wnt signaling involves membrane localization. Dkk-1 has been linked to lipids because of the presence of a putative colipase fold in the Dkks [[5]Aravind L Koonin EV A colipase fold in the carboxy-terminal domain of the Wnt antagonists – the Dickkopfs.Curr Biol. 1998; 8: 477-478Abstract Full Text Full Text PDF PubMed Google Scholar]. The colipase proteins assist pancreatic lipases in lipid digestion. When pancreatic lipases are unable to bind directly to a lipid interface (for example, in the presence of bile salts), colipases function as co-factors, promoting close contact between the lipase and the lipid interface. X-ray crystallographic studies [[6]van Tilbeurgh H Sarda L Verger R Cambillau C Structure of the pancreatic lipase-procolipase complex.Nature. 1992; 359: 159-162Crossref PubMed Scopus (303) Google Scholar][[7]van Tilbeurgh H Gargouri Y Dezan C Egloff MP Nesa MP Ruganie N Sarda L Verger R Cambillau C Crystallization of pancreatic procolipase and of its complex with pancreatic lipase.Mol Biol. 1993; 229: 552-554Crossref Scopus (28) Google Scholar] of the colipase–lipase complex have identified several conserved hydrophobic residues on the colipase that contact the lipid interface. The putative colipase fold present in the Dkks includes some of these conserved hydrophobic residues [[5]Aravind L Koonin EV A colipase fold in the carboxy-terminal domain of the Wnt antagonists – the Dickkopfs.Curr Biol. 1998; 8: 477-478Abstract Full Text Full Text PDF PubMed Google Scholar]. If Dkks and colipases share functional as well as structural homology, then Dkks might be expected to promote lipid–protein interactions. The amino acid sequence similarity between the carboxyl terminus of Wingless/Wnt-1 and the lipid-binding region of sPLA2s further implicates membrane interactions in Wnt signaling. The sPLA2 enzymes hydrolyze the headgroup of specific phospholipids at the sn-2 position [[8]Gelb MH Jain MK Hanel AM Berg OG Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2.Annu Rev Biochem. 1995; 64: 653-688Crossref PubMed Scopus (221) Google Scholar]. Four classes of sPLA2s have been categorized and the class I and II sPLA2s are highly conserved [9Dennis EA Diversity of group types, regulation, and function of phospholipase A2.J Biol Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 10Carredano E Westerlund B Persson B Saarinen M Ramaswamy S Eaker D Eklund H The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2.Toxicon. 1998; 36: 75-92Crossref PubMed Scopus (62) Google Scholar]. Sequence database analysis using the BLASTP [11Altschul SF Gish W Miller W Myers EW Lipman DJ Basic local alignment search tool.J Mol Biol. 1990; 215: 403-410PubMed Scopus (0) Google Scholar, 12Altschul SF Madden TL Schäffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (56802) Google Scholar] and FASTA [[13]Pearson WR Lipman DJ Improved tools for biological sequence comparison.Proc Natl Acad Sci USA. 1988; 85: 2444-2448Crossref PubMed Scopus (9201) Google Scholar] programs detected a 50 amino acid region of the class I and II sPLA2s that is 30–40% identical to the highly conserved carboxy-terminal domain of Wingless/Wnt-1. Figure 1 shows the conservation between the two protein families, as assessed using the ‘strong homology’ class of the Gonnet-PAM250 mutation data matrix [[14]Gonnet GH Cohen MA Benner SA Exhaustive matching of the entire protein sequence database.Science. 1992; 256: 1443-1445Crossref PubMed Scopus (626) Google Scholar]. The snake (Agkistrodon piscivorus piscivorus) class II sPLA2 (App-K49) and Wingless are 37% identical and 44% conserved in this 50 amino acid region, while the bovine pancreatic class I sPLA2 and Wingless are 28% identical and 35% conserved. This level of homology extends to other Wnt-1 proteins (Figure 1) and is similar among all members of the Wnt family. Within the 50 amino acid region, important structural features have been conserved between the two families. All catalytically active PLA2s contain two ‘signature’ consensus sequences [[15]Bairoch A Bucher P Hofmann K The PROSITE database, its status in 1997.Nucleic Acids Res. 1997; 25: 217-221Crossref PubMed Scopus (743) Google Scholar] that include active-site residues. One of these signature sequences, CCXXHXXC (in the single-letter amino acid code, where X is any amino acid), is present within the 50 amino acid region (Figure 1, alignment positions 39–47). A similar sequence (CXCXXHXXC) can be found at the corresponding position in Wingless and Wnt-1. Class I and II sPLA2s also have a conserved pattern of 12 cysteines [[16]Renetseder R Brunie S Dijkstra BW Drenth J Sigler PB A comparison of the crystal structure of phospholipase A2 from Bovine pancreas and Crotalus atrox venom.J Biol Chem. 1985; 21: 11627-11634Google Scholar]. Seven of these are within the 50 amino acid region shown in Figure 1, and all seven are conserved in the Wnt family (Figure 1). Note that two of the five gaps in the alignment shown are introduced by the standard alignment of class I and II sPLA2s [[16]Renetseder R Brunie S Dijkstra BW Drenth J Sigler PB A comparison of the crystal structure of phospholipase A2 from Bovine pancreas and Crotalus atrox venom.J Biol Chem. 1985; 21: 11627-11634Google Scholar]. Although no information is available, as yet, concerning the secondary or tertiary structure of Wnt proteins, several sPLA2 proteins have been crystallized and their structures determined. In the sPLA2 proteins, this 50 amino acid region forms a helix–loop–helix and is part of a conserved cleft that binds the phospholipid substrate (Figure 2a). The location of the conserved amino acids may be seen more clearly in a space-filling model of App-K49 sPLA2 (Figure 2b). The observation of sequence conservation in this region leads us to speculate that in Wnt proteins, these residues might participate in lipid binding. The region of homology to sPLA2 lies within the carboxy-terminal domain of Wingless and Wnt-1, a portion of Wingless that is essential for signaling. Wingless mutants lacking the carboxy-terminal 56 residues (for example, Wg412t; see Figure 1) are unable to signal (unpublished observations; [[17]Bejsovec A Wieschaus E Signaling activities of the Drosophila wingless gene are separately mutable and appear to be transduced at the cell surface.Genetics. 1995; 139: 309-320Crossref PubMed Google Scholar]). Single amino-acid substitutions within the carboxy-terminal domain can significantly reduce Wingless function. For example, a cysteine-to-serine substitution at amino acid 445 (residue 41 in Figure 1) results in reduced signaling and embryonic lethality [[15]Bairoch A Bucher P Hofmann K The PROSITE database, its status in 1997.Nucleic Acids Res. 1997; 25: 217-221Crossref PubMed Scopus (743) Google Scholar]. This cysteine is conserved in both the Wnt and sPLA2 families (Figure 1). In sPLA2s, this cysteine participates in a disulfide linkage near the lipid-binding site and appears to stabilize the helix that contains the active-site histidine. Thus, this conserved residue appears to play an important role in both protein families. The bovine pancreatic sPLA2 has been co-crystallized with a phospholipid analog and the residues that bind lipid have been identified [[18]Sekar K Kumar A Liu X Tsai MD Gelb MH Sundaralingam M Structure of the complex of bovine pancreatic phospholipase A2 with a transition-state analogue.Acta Crystallogr D Biol Crystallogr. 1998; 54: 334-341Crossref PubMed Scopus (19) Google Scholar]. In this regard, it is interesting to note that Wingless453, which contains a valine-to-glutamic acid substitution at amino acid 453, is also deficient in signaling and is embryonic lethal (unpublished observations; [[17]Bejsovec A Wieschaus E Signaling activities of the Drosophila wingless gene are separately mutable and appear to be transduced at the cell surface.Genetics. 1995; 139: 309-320Crossref PubMed Google Scholar]). The corresponding amino acid in the bovine pancreatic sPLA2 (residue 49 in Figure 1) contacts the bound phospholipid [[18]Sekar K Kumar A Liu X Tsai MD Gelb MH Sundaralingam M Structure of the complex of bovine pancreatic phospholipase A2 with a transition-state analogue.Acta Crystallogr D Biol Crystallogr. 1998; 54: 334-341Crossref PubMed Scopus (19) Google Scholar]. The identity of this residue varies between sPLA2s, presumably to accommodate specific lipid substrates [[19]Han SK Yoon ET Scott DL Sigler PB Cho W Structural aspects of interfacial absorption.J Biol Chem. 1997; 272: 3573-3582Crossref PubMed Scopus (86) Google Scholar]. Thus, within regions of homology between sPLA2 and Wingless/Wnt, single amino acid changes at residue 47 or residue 55 disrupt both Wingless and PLA2 activity. These data suggest that Wingless and sPLA2 share functional homology. The Wingless/Wnt-1 proteins lack the second PLA2 signature sequence critical for phospholipase activity and are thus not likely to hydrolyze lipids. Lipid hydrolysis is not required for some membrane-interactive functions of sPLA2s, however. Non-catalytic sPLA2 homologs (such as App-K49) can bind lipids, interact with liposomes, affect lipid bilayer organization [20Rufini S Cesaroni P Desideri A Farias R Gubensek F Gutierrez JM Luly P Massoud R Morero R Pedersen JZ Calcium ion independent membrane leakage induced by phospholipase-like myotoxins.Biochemistry. 1992; 31: 12424-12430Crossref PubMed Scopus (104) Google Scholar, 21Diaz C Gutierrez JM Lomonte B Gene JA The effect of myotoxins isolated from Bothrops snake venoms on multilamellar liposomes: relationship to phospholipase A2, anticoagulant and myotoxic activities.Biochim Biophys Acta. 1991; 1070: 455-460Crossref PubMed Scopus (110) Google Scholar, 22Gutierrez JM Lomonte B Phospholipase A2 myotoxins from Bothrops snake venoms.Toxicon. 1995; 33: 1405-1424Crossref PubMed Scopus (413) Google Scholar], and alter membrane permeability [[22]Gutierrez JM Lomonte B Phospholipase A2 myotoxins from Bothrops snake venoms.Toxicon. 1995; 33: 1405-1424Crossref PubMed Scopus (413) Google Scholar]. If the structural similarities between Wingless/Wnt-1 and the class I and II sPLA2s are reflected in protein function, then Wnt signaling may require interaction(s) with the plasma membrane; several observations now support this idea. Wnt proteins are known to be closely associated with the cell surface, both in vivo and in vitro[23Reichsman F Smith L Cumberledge S Glycosaminoglycans can mediate extracellular localization of the Wingless protein and promote signal transduction.J Cell Biol. 1996; 135: 819-827Crossref PubMed Scopus (252) Google Scholar, 24Besjovec A Martinez-Arias A Roles of wingless in patterning the larval epidermis of Drosophila.Development. 1991; 113: 471-485PubMed Google Scholar], and the Dkk-1 antagonist might affect Wnt interaction with membranes [[5]Aravind L Koonin EV A colipase fold in the carboxy-terminal domain of the Wnt antagonists – the Dickkopfs.Curr Biol. 1998; 8: 477-478Abstract Full Text Full Text PDF PubMed Google Scholar]. Finally, the homology between Wnts and sPLA2s suggests a membrane localization and perhaps a lipid-binding function for Wnts. We thank Alfonso Martinez-Arias and Jeffrey S. Rubin for helpful comments. F Reichsman, HM Moore and S Cumberledge, Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01002, USA.