The making of Wnt: new insights into Wnt maturation, sorting and secretion

In this review, we look at the processes that occur `behind the scenes' in Wnt signalling, within the Wnt-producing cells. The Wnt community has long been focused upon events that occur downstream of Wnt binding to its receptors, but the recent discovery that the maturation of the Wnt protein may have a profound effect on its signalling properties has excited great interest. In the last 2 years, several key regulators of Wnt production have been discovered, but our global understanding of this process remains relatively poor. Several models that reconcile former and recent observations of Wnt modification, sorting and secretion, and which highlight the potential of this emerging field, are presented here.Wnt glycoproteins are extracellular ligands that can be found in many species, ranging from the sea anemone Nematostella vectensis to human(Kusserow et al., 2005). Wnts act as morphogens and control the patterning of the developing embryo by triggering concentration-dependent responses in cells located at a distance from the signal-sending domain (Neumann and Cohen, 1997; Strigini and Cohen, 2000; Zecca et al.,1996). During animal development, Wnt ligands regulate several key processes, such as cell proliferation, cell migration and cell differentiation(Cadigan and Nusse, 1997; Moon et al., 2002; Wodarz and Nusse, 1998). Furthermore, deregulation of the Wnt signalling pathway has been implicated in many pathological disorders, including colon cancer(Bienz and Clevers, 2000; Logan and Nusse, 2004). The intracellular mechanisms that transduce the Wnt signal downstream of its receptors and modulate the expression of its target genes have been extensively studied, and will not be discussed here (for review, see Cadigan and Liu, 2006; Cadigan and Nusse, 1997; He et al., 2004; Moon et al., 2002; van Es et al., 2003) (also see the Wnt Homepage http://www.stanford.edu/~rnusse/wntwindow.html). However, little is known about the different steps that control the synthesis and secretion of a functional Wnt protein, and which ensure its delivery to the responding cells along the morphogenetic field. In this review, we focus on Wnt-producing cells and discuss the mechanisms that lead to the production of a fully active Wnt. All known aspects of Wnt maturation will be addressed:from its post-translational modification in the endoplasmic reticulum (ER) to its sorting within the expressing cells and, finally, to its secretion as a long-range-acting protein. Although the influence of Wnt properties on the formation of an extracellular concentration gradient will be mentioned, the multiple mechanisms that regulate the spreading of Wnt will not be presented here. For overviews of these processes, the reader is referred to other reviews (Cadigan, 2002; Eaton, 2006; Strigini and Cohen, 2000; Vincent and Dubois, 2002).Except for Drosophila Wingless (Wg), Wnt3/5 and Wnt4, which are all larger proteins, the different Wnts generally have similar structural characteristics (Miller, 2002)(Table 1). Thus, most Wnts are approximately 350 amino acids long and have a molecular weight of about 40 kDa. Wnts contain several charged residues and an average of 23 to 25 cysteines, some of which are highly conserved between different species(Fig. 1). Several of these cysteines are involved in inter- and intra-molecular disulfide bonds,participating in Wnt folding and multimerization(Tanaka et al., 2002). Surprisingly, despite the presence of charged residues in their primary sequences, mouse Wnt3A and Drosophila Wg were found to be hydrophobic molecules (Willert et al.,2003; Zhai et al.,2004). This hydrophobicity confers a strong affinity for cell membranes, which raises questions as to how these proteins spread in the extracellular space. Wnts do not contain any defined domains, but do have an N-terminal hydrophobic signal sequence that targets them to the ER. Finally, further analysis of the Wnt sequences reveals the presence of a number of potential N-glycosylation sites, the functional relevance of which will be discussed below (Fig. 1).Many properties of Wnts, such as their high hydrophobicity despite the presence of charged residues, cannot be understood from this rough analysis of their primary sequence. Further biochemical characterization of maturing Wnt in its producing cells has brought some answers to these conflicting observations, as discussed below.When they reach the ER, immature Wnts undergo important posttranslational modifications. First, the oligosaccharyl transferase complex (OST) rapidly and efficiently attaches N-linked oligosaccharide chains to the appropriate residues on the peptide backbone (Asn108 and Asn414 in Wg) (Tanaka et al.,2002). However, the biological function of Wnt N-glycosylation remains unclear. Indeed, mutation of part or all of the potential glycosylation sites of mouse Wnt1 does not affect its function in a cell culture-based assay (Mason et al., 1992). In cell culture, the secretion of many Wnts by their producing cells is inefficient and most newly synthesized Wnt is retained in the ER (Burrus and McMahon,1995; McMahon and Moon,1989; St-Arnaud et al.,1989). Within this compartment, Wnt has been found to associate with the immunoglobulin heavy-chainbinding protein BiP, a member of the Hsp70 family of heat-shock proteins (Burrus and McMahon, 1995; Kitajewski et al., 1992). BiP acts as a chaperone for numerous proteins, and allows the further sorting and secretion of mature and functional proteins only (Gething and Sambrook,1990; Hurtley et al.,1989; Kassenbrock et al.,1988). Thus, BiP can sequester certain misfolded or unassembled proteins within the ER. Inhibition of Wnt N-glycosylation does not increase the relative amount of Wnt bound to BiP, suggesting that this does not severely affect Wnt folding and structure(Burrus and McMahon, 1995). Finally, similarly to other secreted proteins, Wnt N-glycosylation may be one of the determinant signals for its apical secretion, which may explain the absence of effect when glycosylation is inhibited in a non-polarized cell assay (Mason et al.,1992). However, this hypothesis has not been experimentally proven, and the exact role of N-glycosylation in the production and/or activity of Wnt remains to be elucidated.The recent purification of mouse Wnt3A from cell culture medium identified an additional post-translational modification(Willert et al., 2003). Specifically, a palmitate group is attached to a cysteine located in the N-terminal part of the protein (C77) (see Fig. 1). Wg was also found to be lipid-modified (Zhai et al.,2004), and the high conservation of the cysteine involved suggests that this palmitoylation is a common feature of all Wnts. The function of this second modification is also unclear. The hydrophobic lipid group may promote Wnt targeting to the ER membrane and subsequently allow efficient N-glycosylation by the membrane-associated OST complex. However, this cannot be the sole function of the palmitate moiety because, in contrast to loss of N-glycosylation of Wnt1, the absence of palmitoylation abrogates Wnt3A function in cell culture(Willert et al., 2003). Alternatively, Wnt palmitoylation may protect the modified cysteine from forming a disulfide bond, thereby preventing the aberrant folding of the protein and its retention in the ER by BiP. Palmitate groups are also known to act as intracellular sorting signals(Bijlmakers and Marsh, 2003). Thus, Wnt lipid-modification may allow the proper routing of the protein through different compartments of the Wnt-producing cells, where Wnts acquire their signalling potential. Finally, Wnt3A hydrophobicity has been found to depend largely upon the presence of the palmitate moiety(Willert et al., 2003). Therefore, this lipid-modification may play a role after secretion by targeting Wnt to the membranes of cells along the morphogenetic field. Such a mechanism may control the spatial distribution of Wnt and increase its local concentration at its receptors, allowing efficient signalling. This model is indeed supported by the capacity of a mutant form of Wnt3A that lacks the palmitoylated cysteine (C77A) to activate a Tcf reporter gene when the potential drop in local concentration is compensated for by overexpression(Willert et al., 2003). However, such a model implies that another mechanism exists that allows Wnt to spread away from its production site by partially balancing the hydrophobicity that is provided by the lipid group.There is some evidence that the protein Porcupine, which resides in the ER and is a member of the membrane-bound O-acyltransferase family(MBOAT), has an influence on both of these post-translational modifications. With respect to the N-glycosylation, in the absence of Porcupine, the attachment of the oligosaccharide chains is impaired. Conversely, Porcupine overexpression results in ectopic glycosylation of Wg(Tanaka et al., 2002). The precise role of Porcupine in this process is unknown. Interestingly, the Porcupine C-terminal region binds an N-terminal domain of Wg that contains conserved cysteines. Furthermore, Wg N-glycosylation competes with the formation of intracellular disulfide bonds, as suggested by the enhanced glycosylation observed after treatment by a reducing agent such as Dithiotreitol (DTT) (Tanaka et al.,2002). Porcupine may therefore protect these residues from forming disulfide bonds. This theory was initially supported by the absence of additional effects of Porcupine overexpression under fully reducing conditions. However, Porcupine does not affect the formation of disulfide bonds (Tanaka et al., 2002),although a transient effect delaying this process, which would promote Wg glycosylation, cannot be ruled out. Another model can be envisaged where Porcupine targets Wnt to the ER membrane, facilitating its modification by the OST complex. This may be directly achieved by the physical binding of Wnt to membrane-bound Porcupine, or indirectly by the Porcupine-dependent acylation of Wnt (see below). Consistent with this model, a membrane-tethered form of Wg is more efficiently glycosylated (Tanaka et al., 2002). In addition to targeting Wnt to the ER membrane,the binding of Porcupine may have an effect on Wnt conformation, allowing optimal access of the OST complex to the different N-glycosylation sites.Despite the lack of direct evidence, the acyltransferase Porcupine has also been suggested to catalyze the palmitoylation of Wnt. First, Porcupine is required for Wg hydrophobicity and membrane association(Zhai et al., 2004). Second,the Porcupine-binding domain of Wg spans the region where the modified cysteine is located (Fig. 1)(Tanaka et al., 2002). Furthermore, similarly to loss of palmitoylation, loss of Porcupine abrogates Wg signalling (Riggleman et al.,1990; van den Heuvel et al.,1993). However, in Drosophila embryos, Wg secretion is inhibited in the absence of Porcupine, whereas Wnt3A(C77A), lacking the palmitoylated cysteine, is secreted normally in mammalian cell culture(Kadowaki et al., 1996; van den Heuvel et al., 1993; Willert et al., 2003),indicating that catalyzing the palmitoylation of this conserved cysteine is not the only function that Porcupine may play in the Wnt-maturation process. Recent studies may, nevertheless, confirm Porcupine as the Wnt-acylating enzyme. In addition to being palmitoylated at this N-terminally located cysteine, Burrus and colleagues found that Wnt is lipid-modified at other positions in a Porcupine-dependent manner as well. Indeed, Wnt3A(C77A)was shown to still harbour one or more lipid modifications despite lacking the palmitoylated cysteine. Additionally, overexpression of Porcupine further increased the fraction of Wnt3A(C77A) found in the detergent phase in phase This suggests that residues other can be modified by Porcupine and can Interestingly, the group of has shown that mutation of another amino in all lipid modification and results in the retention of the protein in the ER The different lipid groups may therefore have The identified palmitoylation may affect Wnt function by allowing its sorting to compartments where Wnt or by its signalling potential at the of the responding cells. may be required for the proper sorting of Wnt of the ER and may therefore affect its All modifications to be on Porcupine, the retention of Wnt within its producing cells when Porcupine function is being the extracellular Wnts within the producing cells from the ER to the and from through different et al., is to that fully modified and functional Wnt can the ER and the thereby its expressing cells secretion However, some results that Wnt may an secretion First, in cells, a fraction of Wg has been found to in the in as as in that from and are to the et al., et al., 2002; van den Heuvel et al., This may from the direct of secreted Wg by the producing cells Alternatively, Wg may be to this within the expressing cells, Second,the of Wnt with also the hypothesis of a secretion Wnt and are morphogens that several properties at the structural are and functional 2003). recent that does not the et al., by in its producing cells, the found that away from a secreted form of Wnt may a similar Finally, Wg has also been found to be with lipid in cell culture (Zhai et al.,2004). are of lipid within the of the membrane and are known to be involved in multiple aspects of membrane is to that can act as that of proteins from other secreted proteins, them sorting and et al., is known about the signals involved in the potential intracellular sorting of Wnt. N-glycosylation, as as lipid modification, may play a role in this process. Indeed, palmitoylation of Wg is required for its with lipid et al., 2004). Furthermore, a Wnt at the membrane of the may Wnt and direct its to the lipid have been shown to the of sorting receptors and molecules the forming and The of such a has not been However, the protein which is required within Wnt-producing cells, may be a for this mechanism (see below). Although a for additional intracellular sorting events cannot be ruled is to the in its of its producing sorting of Wnt to different compartments may play a key role in its maturation process, and a precise of Wnt in the producing cells will be to its exact secretion Indeed, efficient and Wnt signalling may depend on more one secretion Furthermore, a about the for Wnt to the recent on the of Wg and with discussed may some this has been for Wg in and Cohen, 1997; Strigini and Cohen, 2000; Zecca et al., in other species, Wnts also act as morphogens and can reach responding cells that are located several cell away from the Wnt et al., 2006; and The migration of Wnt along the morphogenetic field, and this extracellular or has long been a of and Dubois, 2002). 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Furthermore, of Wnt3A acylation results in its retention in the ER, whereas Wg is the expressing cells in the absence of Finally, N-glycosylation of which may as a signal for its secretion, was found to be in the absence of et al., of Wnt may, regulate as modifications. Alternatively, may allow the optimal post-translational modification of Wnt by other the control of its Thus, may directly act as a chaperone and control the modification of residues that are for Wnt This be achieved by Wnt to the appropriate or by facilitating the access of the to the understanding of Wnt maturation allows only for and cannot be ruled that steps of Wnt production that are required for its of the biochemical properties of mature as as a of the by Wnt its production by the be to the of proteins such as additional of has been to this with the of the complex as a key in the production of a functional Wnt et al., The is a highly conserved the of which of the and et al., 1998). was initially in for its role in the intracellular sorting of the et al., et al., binds are in the and their to the of in is to the This is by the of the of with et al., In the absence of in the and is to the subsequently in the in the secretion of the secretion the mammalian which allows the of is to the by the complex et al.,2004). In mammalian cells, the complex was also shown to of the et al., recent both and suggests that the is also required within the Wnt-producing cells for proper Wnt signalling et Thus, loss of the protein abrogates signalling in (see and the of in mammalian cells and Wnt target gene et al., In function to be to Wnt signalling, as other signalling pathway is in of Interestingly, the was found to be required within the cells for the formation of an gradient of Consistent with these loss of the was shown to fully but to only affect signalling. Finally, studies in and biochemical in mammalian cells have shown that Wnt secretion is not inhibited in the absence of et al., models can be for the function of the in discussed Wnt post-translational such as and as modifications may also occur Wnt of these in compartments of the expressing cells, may be required for the formation of a Wnt and therefore for the signalling activity of Wnt. Thus, the may promote Wnt maturation by the different in their This may be achieved through the direct binding of the complex to the of membrane-bound or by the of this complex with a member of the Porcupine may be directly or indirectly by the complex. However, Porcupine is required for Wg secretion, whereas loss of function does not affect Wnt secretion, that the is not required for Porcupine the effect of loss of function is similar to the effect observed for Wg and when their with is et al., 2005). the absence of the Drosophila which is an of the in Wg signalling is more is Wg signalling. the binding of Wnt to within the after sorting of Wnt by a at can be envisaged for the complex. 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In addition to Wnt with may also depend on the N-terminal palmitate Thus, when the palmitoylated cysteine is a is similarly to form an gradient in on the sorting of Wnt to the secretion as as on the of a Wnt Furthermore, they that the of Wnt with which is for the spreading of Wnt along the morphogenetic field, is not the by which hydrophobic Wnt at the membrane of cells directly to its producing cells. Indeed, Wnt3A was found in in the medium of cells after the of by suggesting that does not to the membranes despite the potential absence of et Further studies are required to the exact role of the and the of the loss of function on the of these with and the complex may be involved in intracellular of Wnt within cells. is therefore to on models in which and the to allow the efficient maturation of Wnt and its with The molecular of a protein that binds suggests that may act as the discussed Wnt the of Wnt from the to the In this may directly or indirectly and the of the complex from the Alternatively, may regulate the of from the thereby the further of Wnt However, such models with the of Wnt secretion observed in the absence of whereas of Wnt to the cells when function is impaired. Further is therefore to reconcile these results and to the and the of Wnt with are different steps of the of the sorting and secretion of a Wnt protein has long been in of the of Wnt signal downstream of its However, has that the function of Wnt in and its capacity to spread and the developing embryo are on key processes that occur within Wnt-producing cells post-translational the sorting of Wnt to compartments and its with extracellular be at the of the of mechanisms that to the of an optimal Wnt protein. to these processes to be in to models and to some of the many questions and that we have in this The characterization of the properties that Wnt its as as an of Wnt are steps a understanding of its function in animal would to Burrus and for and for the This was by from the and the