19 WNT proteins have been identified in human cells. The WNTs are members of a conserved metazoan family of secreted morphogens that activate several signaling pathways in the responding cell: the canonical (beta-catenin) WNT signaling cascade and several non-canonical pathways, including the planar cell polarity (PCP), the regulation of intracellular calcium signaling and activation of JNK kinases. WNT proteins exist in a gradient outside the secreting cell and are able to act over both short and long ranges to promote proliferation, changes in cell migration and polarity and tissue homeostasis, among others (reviewed in Saito-Diaz et al, 2012; Willert and Nusse, 2012).
The WNTs are ~40kDa proteins with 23 conserved cysteine residues in the N-terminal that may form intramolecular disulphide bonds. They also contain an N-terminal signal sequence and a number of N-linked glycosylation sites (Janda et al, 2012). In addition to being glycosylated, WNTs are also lipid-modified in the endoplasmic reticulum by a WNT-specific O-acyl-transferase, Porcupine (PORCN), contributing to their characteristic hydrophobicity. PORCN-dependent palmitoylation is required for the secretion of WNT as well as its signaling activity, as either depletion of PORCN or mutation of the conserved serine acylation site results in the intracellular accumulation of WNT ligand (Takada et al, 2006; Barrott et al, 2011; Biechele et al, 2011; reviewed in Willert and Nusse, 2012).
Secretion of WNT requires a number of other dedicated factors including the sorting receptor Wntless (WLS) (also knownas Evi, Sprinter, and GPR177), which binds WNT and escorts it to the cell surface (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006). A WNT-specific retromer containing SNX3 is subsequently required for the recycling of WLS back to the Golgi (reviewed in Herr et al, 2012; Johannes and Wunder, 2011). Once at the cell surface, WNT makes extensive contacts with components of the extracellular matrix such as heparan sulphate proteoglycans (HSPGs) and may be bound by any of a number of regulatory proteins, including WIFs and SFRPs. The diffusion of the WNT ligand may be aided by its packing either into WNT multimers, exosomes or onto lipoprotein particles to shield the hydrophobic lipid adducts from the aqueous extracellular environment (Gross et al, 2012; Luga et al, 2012, Korkut et al, 2009; reviewed in Willert and Nusse, 2012).
Komekado H, Yamamoto H, Chiba T, Kikuchi A.; ''Glycosylation and palmitoylation of Wnt-3a are coupled to produce an active form of Wnt-3a.''; PubMedEurope PMCScholia
Barrott JJ, Cash GM, Smith AP, Barrow JR, Murtaugh LC.; ''Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome.''; PubMedEurope PMCScholia
Belenkaya TY, Wu Y, Tang X, Zhou B, Cheng L, Sharma YV, Yan D, Selva EM, Lin X.; ''The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network.''; PubMedEurope PMCScholia
Bartscherer K, Pelte N, Ingelfinger D, Boutros M.; ''Secretion of Wnt ligands requires Evi, a conserved transmembrane protein.''; PubMedEurope PMCScholia
Port F, Hausmann G, Basler K.; ''A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.''; PubMedEurope PMCScholia
Port F, Kuster M, Herr P, Furger E, Bänziger C, Hausmann G, Basler K.; ''Wingless secretion promotes and requires retromer-dependent cycling of Wntless.''; PubMedEurope PMCScholia
Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR, Nusse R.; ''Wnt proteins are lipid-modified and can act as stem cell growth factors.''; PubMedEurope PMCScholia
Buechling T, Chaudhary V, Spirohn K, Weiss M, Boutros M.; ''p24 proteins are required for secretion of Wnt ligands.''; PubMedEurope PMCScholia
Smolich BD, McMahon JA, McMahon AP, Papkoff J.; ''Wnt family proteins are secreted and associated with the cell surface.''; PubMedEurope PMCScholia
Kadowaki T, Wilder E, Klingensmith J, Zachary K, Perrimon N.; ''The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing.''; PubMedEurope PMCScholia
Kurayoshi M, Yamamoto H, Izumi S, Kikuchi A.; ''Post-translational palmitoylation and glycosylation of Wnt-5a are necessary for its signalling.''; PubMedEurope PMCScholia
Coudreuse DY, Roël G, Betist MC, Destrée O, Korswagen HC.; ''Wnt gradient formation requires retromer function in Wnt-producing cells.''; PubMedEurope PMCScholia
MacDonald BT, Tamai K, He X.; ''Wnt/beta-catenin signaling: components, mechanisms, and diseases.''; PubMedEurope PMCScholia
Port F, Basler K.; ''Wnt trafficking: new insights into Wnt maturation, secretion and spreading.''; PubMedEurope PMCScholia
Bänziger C, Soldini D, Schütt C, Zipperlen P, Hausmann G, Basler K.; ''Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells.''; PubMedEurope PMCScholia
Korkut C, Ataman B, Ramachandran P, Ashley J, Barria R, Gherbesi N, Budnik V.; ''Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless.''; PubMedEurope PMCScholia
Simons M, Raposo G.; ''Exosomes--vesicular carriers for intercellular communication.''; PubMedEurope PMCScholia
Herr P, Hausmann G, Basler K.; ''WNT secretion and signalling in human disease.''; PubMedEurope PMCScholia
Coombs GS, Yu J, Canning CA, Veltri CA, Covey TM, Cheong JK, Utomo V, Banerjee N, Zhang ZH, Jadulco RC, Concepcion GP, Bugni TS, Harper MK, Mihalek I, Jones CM, Ireland CM, Virshup DM.; ''WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification.''; PubMedEurope PMCScholia
Franch-Marro X, Wendler F, Guidato S, Griffith J, Baena-Lopez A, Itasaki N, Maurice MM, Vincent JP.; ''Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex.''; PubMedEurope PMCScholia
Goodman RM, Thombre S, Firtina Z, Gray D, Betts D, Roebuck J, Spana EP, Selva EM.; ''Sprinter: a novel transmembrane protein required for Wg secretion and signaling.''; PubMedEurope PMCScholia
Yang PT, Lorenowicz MJ, Silhankova M, Coudreuse DY, Betist MC, Korswagen HC.; ''Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells.''; PubMedEurope PMCScholia
Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC.; ''Structural basis of Wnt recognition by Frizzled.''; PubMedEurope PMCScholia
Gasnereau I, Herr P, Chia PZ, Basler K, Gleeson PA.; ''Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling.''; PubMedEurope PMCScholia
Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, Buchanan M, Hosein AN, Basik M, Wrana JL.; ''Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.''; PubMedEurope PMCScholia
Biechele S, Cox BJ, Rossant J.; ''Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos.''; PubMedEurope PMCScholia
Proffitt KD, Virshup DM.; ''Precise regulation of porcupine activity is required for physiological Wnt signaling.''; PubMedEurope PMCScholia
Takada R, Satomi Y, Kurata T, Ueno N, Norioka S, Kondoh H, Takao T, Takada S.; ''Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion.''; PubMedEurope PMCScholia
Hofmann K.; ''A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling.''; PubMedEurope PMCScholia
Strating JR, Martens GJ.; ''The p24 family and selective transport processes at the ER-Golgi interface.''; PubMedEurope PMCScholia
Zhang P, Wu Y, Belenkaya TY, Lin X.; ''SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless.''; PubMedEurope PMCScholia
Xu Y, Hortsman H, Seet L, Wong SH, Hong W.; ''SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P.''; PubMedEurope PMCScholia
Dancourt J, Barlowe C.; ''Protein sorting receptors in the early secretory pathway.''; PubMedEurope PMCScholia
Palmer L, Vincent JP, Beckett K.; ''Wnts need a p(assport)24 to leave the ER.''; PubMedEurope PMCScholia
Gross JC, Chaudhary V, Bartscherer K, Boutros M.; ''Active Wnt proteins are secreted on exosomes.''; PubMedEurope PMCScholia
Ching W, Hang HC, Nusse R.; ''Lipid-independent secretion of a Drosophila Wnt protein.''; PubMedEurope PMCScholia
Galli LM, Burrus LW.; ''Differential palmit(e)oylation of Wnt1 on C93 and S224 residues has overlapping and distinct consequences.''; PubMedEurope PMCScholia
Herr P, Basler K.; ''Porcupine-mediated lipidation is required for Wnt recognition by Wls.''; PubMedEurope PMCScholia
Harterink M, Port F, Lorenowicz MJ, McGough IJ, Silhankova M, Betist MC, van Weering JRT, van Heesbeen RGHP, Middelkoop TC, Basler K, Cullen PJ, Korswagen HC.; ''A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion.''; PubMedEurope PMCScholia
van den Heuvel M, Harryman-Samos C, Klingensmith J, Perrimon N, Nusse R.; ''Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein.''; PubMedEurope PMCScholia
Seaman MN.; ''The retromer complex - endosomal protein recycling and beyond.''; PubMedEurope PMCScholia
Liu J, Pan S, Hsieh MH, Ng N, Sun F, Wang T, Kasibhatla S, Schuller AG, Li AG, Cheng D, Li J, Tompkins C, Pferdekamper A, Steffy A, Cheng J, Kowal C, Phung V, Guo G, Wang Y, Graham MP, Flynn S, Brenner JC, Li C, Villarroel MC, Schultz PG, Wu X, McNamara P, Sellers WR, Petruzzelli L, Boral AL, Seidel HM, McLaughlin ME, Che J, Carey TE, Vanasse G, Harris JL.; ''Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974.''; PubMedEurope PMCScholia
Lippincott-Schwartz J, Roberts TH, Hirschberg K.; ''Secretory protein trafficking and organelle dynamics in living cells.''; PubMedEurope PMCScholia
N-linked glycosylation is the most important form of post-translational modification for proteins synthesized and folded in the Endoplasmic Reticulum (Stanley P et al, 2009). An early study in 1999 revealed that about 50% of the proteins in the Swiss-Prot database at the time were N-glycosylated (Apweiler R et al, 1999). It is now established that the majority of the proteins in the secretory pathway require glycosylation in order to achieve proper folding. The addition of an N-glycan to a protein can have several roles (Shental-Bechor D and Levy Y, 2009). First, glycans enhance the solubility and stability of the proteins in the ER, the golgi and on the outside of the cell membrane, where the composition of the medium is strongly hydrophilic and where proteins, that are mostly hydrophobic, have difficulty folding properly. Second, N-glycans are used as signal molecules during the folding and transport process of the protein: they have the role of labels to determine when a protein must interact with a chaperon, be transported to the golgi, or targeted for degradation in case of major folding defects. Third, and most importantly, N-glycans on completely folded proteins are involved in a wide range of processes: they help determine the specificity of membrane receptors in innate immunity or in cell-to-cell interactions, they can change the properties of hormones and secreted proteins, or of the proteins in the vesicular system inside the cell. All N-linked glycans are derived from a common 14-sugar oligosaccharide synthesized in the ER, which is attached co-translationally to a protein while this is being translated inside the reticulum. The process of the synthesis of this glycan, known as Synthesis of the N-glycan precursor or LLO, constitutes one of the most conserved pathways in eukaryotes, and has been also observed in some eubacteria. The attachment usually happens on an asparagine residue within the consensus sequence asparagine-X-threonine by an complex called oligosaccharyl transferase (OST). After being attached to an unfolded protein, the glycan is used as a label molecule in the folding process (also known as Calnexin/Calreticulin cycle) (Lederkremer GZ, 2009). The majority of the glycoproteins in the ER require at least one glycosylated residue in order to achieve proper folding, even if it has been shown that a smaller portion of the proteins in the ER can be folded without this modification. Once the glycoprotein has achieved proper folding, it is transported via the Cis-golgi through all the Golgi compartments, where the glycan is further modified according to the properties of the glycoprotein. This process involves relatively few enzymes but due to its combinatorial nature, can lead to several millions of different possible modifications. The exact topography of this network of reactions has not been established yet, representing one of the major challenges after the sequencing of the human genome (Hossler P et al, 2006). Since N-glycosylation is involved in an great number of different processes, from cell-cell interaction to folding control, mutations in one of the genes involved in glycan assembly and/or modification can lead to severe development problems (often affecting the central nervous system). All the diseases in genes involved in glycosylation are collectively known as Congenital Disorders of Glycosylation (CDG) (Sparks SE et al, 2003), and classified as CDG type I for the genes in the LLO synthesis pathway, and CDG type II for the others.
Secretory cargo destined to be secreted or to arrive at the plasma membrane (PM) leaves the ER via distinct exit sites. This cargo is destined for the Golgi apparatus.
Although the role of retromer in delivering WLS back to the Golgi is reasonably well established (reviewed in Johannes and Wunder, 2011; Willert and Nusse, 2012), the details of how the complex is disassembled at the TGN remain to be determined.
All WNT proteins except Drosophila WntD are lipid modified. Lipid modifications contribute to the hydrophobicity and poor solubility of all known WNT ligands with the exception of Drosophila WntD. Acylation is required for their secretion from the cell and their ability to bind to FRZ receptors (reviewed in MacDonald et al, 2009; Takada et al, 2006; Janda et al, 2012; Herr and Basler, 2012; Ching et al, 2008). Although an initial study suggested that conserved Cys77 in mouse Wnt3a was palmitoylated (Willert et al, 2003), further work showed that mutation of this residue had minimal effect on WNT secretion (Komekado et al, 2007). In contrast, addition of palmitoleic acid to mWnt3a Ser209 is essential for WNT secretion, and mutant S209A is largely retained in the ER (Takada et al, 2006; Galli and Burrus, 2011). This serine residue is conserved at this position in all known WNTs with the exception of Drosophila WntD (Ching et al, 2008; Herr and Basler, 2012). A recent crystal structure of Xenopus WNT8 in complex with a Frizzled cysteine-rich-domain shows a single lipid modification on the conserved serine residue, while the conserved cysteine participates in a disulphide bond (Janda et al, 2012). In addition to being required for secretion, the lipid at S209 also makes direct contact with a groove in the Frizzled receptor and is thus essential for binding (Janda et al, 2012).
Porcupine is a conserved multi-pass transmembrane ER protein that has an O-acyl-transferase domain (van den Heuvel et al, 1993; Kadowaki et al, 1996; Hofmann, 2000). First identified in Drosophila, Porcupine is a WNT-specific modulator that is required for Wingless processing and secretion (Kadowaki et al, 1996). In porcn-deficient cells, Wg and WNT3A have decreased palmitoylation at S209 and accumulate in the ER (Takada et al, 2006), and mutations in PORCN eliminate all WNT signalling and cause embryonic lethality in mice (Barrott et al, 2011; Biechele et al, 2011). Recent studies show that PORCN is required for activity of all human WNT ligands (Proffitt et al, 2012; Najdi et al, 2012).
WLS accompanies WNT through the secretory pathway to the cell surface, where the ligand is released into the extracellular space (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006).
All WNT ligands are predicted to be highly glycosylated. By similarity with WNT ligands in mouse, human WNTs are believed to undergo N-linked glycosylation at multiple asparagine residues and this glycosylation is critical for their secretion (Smolich et al, 1993; Willert et al, 2003, Komekado et al, 2006; Kurayoshi et al, 2007). The mechanism of N-linked glycosylation is not shown here. For a more detailed description, please refer to pathway "Asparagine N-linked glycosylation".
Vacuolar acidification is required but not sufficient for the release of WNT ligands from WLS at the cell surface. V-ATPase inhibitors cause the accumulation of WLS-WNT complexes both within the cell and at the plasma membrane (Coombs et al, 2010). Once in the extracellular space, the lipid-modified WNT ligand must be shielded to allow the morphogen to diffuse away from the plasma membrane. Possible mechanisms include interaction with HSPGs, exosomes, multimerization or incorporation into lipoprotein particles (reviewed in Eaton, 2006; Port and Basler, 2010).
WLS endocytosis is a clathrin-dependent process. In Drosophila cells, internalization of WLS has been shown to depend on clathrin, AP-2, dynamin, Rab5 and HRS (Belenkaya et al, 2006; Port et al, 2008), while in HeLa cells, WLS colocalizes with endogenous AP-2, and depletion of AP-2 increases WLS levels at the cell surface (Yang et al, 2008). A recent study identified a conserved YEGL endocytosis motif in the third intracellular loop of WLS that is required for its clathrin- and dynamin-dependent internalization (Gasnereau et al, 2011).
Wntless (WLS) (Evi/Sprinter/GPR177) is a conserved transmembrane protein that is required for the secretion of WNT ligands from the cell (Bänzinger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006). Notably, WLS is not required for the secretion of Hedgehog, another acylated signaling molecule, and wls-mutants phenocopy wg/wnt mutants (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006), supporting the notion that WLS is a dedicated WNT pathway member. WLS binds directly to WNT ligands in the Golgi in a WNT-acylation dependent manner, as the interaction is abrogated by mutation of either PORCN or the conserved Ser209 residue (Coombs et al, 2010; Herr and Basler, 2012). WLS is thought to contain a lipocalin-family fold (Coombs et al, 2010), a lipid-interacting domain, which may play a role in binding to the lipid adduct on WNT.
Retromer is a conserved multi-protein complex that is required for retrograde transport of transmembrane proteins. It was initially characterized in yeast as a pentameric complex required for the recycling of the transmembrane receptor VPS10 to the trans-Golgi, and was subsequently shown to be conserved in flies, worms and humans. In humans, retromer consists of a cargo-recognition subcomplex made up of VPS35, VPS26 and VPS29 and a membrane-targeting subcomplex containing a heterodimer of SNX proteins (SNX1 or 2 paired with SNX5 or 6). The SNX proteins contain a BAR domain that is believed to promote membrane curvature, and SNX-BAR proteins are thought to aid in the formation of endosomal membrane tubules into which cargo is loaded (reviewed in Pfeffer, 2001; Seaman, 2012).
Retromer is required for the recycling of WLS to the Golgi to allow further rounds of WNT-ligand delivery to the plasma membrane (Coudreuse et al 2006; Belenkaya et al 2008; Port et al, 2008). In the absence of essential retromer component VPS35 or VPS26, WLS is diverted to the MVB and degraded, and WNT ligand accumulates inside the cell; overexpression of WLS is sufficient to rescue the vps35 defect in WNT signaling (Belenkaya et al, 2006; Franch-Marro et al, 2008). WLS and retromer colocalize on endosomal structures and WLS and VPS35 co-precipitate in pull down studies (Belenkaya et al, 2006; Port et al, 2008; Franch-Marro et al, 2008).
Several recent studies have suggested that WLS recycling depends on a WNT-specific retromer in which the SNX-BAR proteins of the classic complex are replaced by SNX3 (Zhang et al, 2011; Harterink et al, 2011; reviewed in Johannes and Wunder, 2011). Unlike SNX1/2/5/6, SNX3 does not contain a BAR domain, and WLS is suggested to accumulate in endocytic vesicles rather than in the tubular structures of the 'classic' retromer (Harterink et al, 2011; Zhang et al, 2011). SNX3 is recruited from the cytosol to the early endosome through the interaction of its PX domain with PIP3 in the membrane. Mutation of critical residues in the PX domain abolish the interaction with PIP3 and ablate endsomal recruitment of SNX3 (Xu et al, 2001; Zhang at al, 2011; Harterink et al, 2011). SNX3 has been shown to co-immunoprecipitate with VPS35 and VPS26, and some studies have also shown a direct interaction between SNX3 and WLS (Zhang et al, 2011; Harterink et al, 2011).
Retromer is believed to escort WLS from the early endosome back to the Golgi for subsequent rounds of WNT secretion (reviewed in Johannes and Wunder, 2011; Willert and Nusse, 2012 ).
This black box event represents the non-WNT-specific ER-to-Golgi trafficking step of protein secretion (reviewed in Dancourt and Barlowe, 2010; Lippincott Schwatz et al, 2000; for more details, please refer to the pathway "ER to Golgi transport"). Two recent screens in Drosophila have identified members of the p24 family as WNT-specific regulators of ER-to-Golgi transport, although the details have not been elucidated (Port et al, 2011; Buechling et al, 2011; reviewed in Strating and Martens, 2009). Depletion of the Drosophila p24 protein Opossum causes accumulation of WNT ligand in the ER, suggesting a role for Opm in ER-to-Golgi transport of WNTs. WNT-dependent reporter activity was reduced in HEK293 cells that were depleted for the human p24 homologue TMED5, supporting a conserved role for these proteins in WNT signaling (Buechling et al, 2011; reviewed in Palmer et al, 2012).
The WNTs are ~40kDa proteins with 23 conserved cysteine residues in the N-terminal that may form intramolecular disulphide bonds. They also contain an N-terminal signal sequence and a number of N-linked glycosylation sites (Janda et al, 2012). In addition to being glycosylated, WNTs are also lipid-modified in the endoplasmic reticulum by a WNT-specific O-acyl-transferase, Porcupine (PORCN), contributing to their characteristic hydrophobicity. PORCN-dependent palmitoylation is required for the secretion of WNT as well as its signaling activity, as either depletion of PORCN or mutation of the conserved serine acylation site results in the intracellular accumulation of WNT ligand (Takada et al, 2006; Barrott et al, 2011; Biechele et al, 2011; reviewed in Willert and Nusse, 2012).
Secretion of WNT requires a number of other dedicated factors including the sorting receptor Wntless (WLS) (also knownas Evi, Sprinter, and GPR177), which binds WNT and escorts it to the cell surface (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006). A WNT-specific retromer containing SNX3 is subsequently required for the recycling of WLS back to the Golgi (reviewed in Herr et al, 2012; Johannes and Wunder, 2011). Once at the cell surface, WNT makes extensive contacts with components of the extracellular matrix such as heparan sulphate proteoglycans (HSPGs) and may be bound by any of a number of regulatory proteins, including WIFs and SFRPs. The diffusion of the WNT ligand may be aided by its packing either into WNT multimers, exosomes or onto lipoprotein particles to shield the hydrophobic lipid adducts from the aqueous extracellular environment (Gross et al, 2012; Luga et al, 2012, Korkut et al, 2009; reviewed in Willert and Nusse, 2012).
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The addition of an N-glycan to a protein can have several roles (Shental-Bechor D and Levy Y, 2009). First, glycans enhance the solubility and stability of the proteins in the ER, the golgi and on the outside of the cell membrane, where the composition of the medium is strongly hydrophilic and where proteins, that are mostly hydrophobic, have difficulty folding properly. Second, N-glycans are used as signal molecules during the folding and transport process of the protein: they have the role of labels to determine when a protein must interact with a chaperon, be transported to the golgi, or targeted for degradation in case of major folding defects. Third, and most importantly, N-glycans on completely folded proteins are involved in a wide range of processes: they help determine the specificity of membrane receptors in innate immunity or in cell-to-cell interactions, they can change the properties of hormones and secreted proteins, or of the proteins in the vesicular system inside the cell.
All N-linked glycans are derived from a common 14-sugar oligosaccharide synthesized in the ER, which is attached co-translationally to a protein while this is being translated inside the reticulum. The process of the synthesis of this glycan, known as Synthesis of the N-glycan precursor or LLO, constitutes one of the most conserved pathways in eukaryotes, and has been also observed in some eubacteria. The attachment usually happens on an asparagine residue within the consensus sequence asparagine-X-threonine by an complex called oligosaccharyl transferase (OST).
After being attached to an unfolded protein, the glycan is used as a label molecule in the folding process (also known as Calnexin/Calreticulin cycle) (Lederkremer GZ, 2009). The majority of the glycoproteins in the ER require at least one glycosylated residue in order to achieve proper folding, even if it has been shown that a smaller portion of the proteins in the ER can be folded without this modification.
Once the glycoprotein has achieved proper folding, it is transported via the Cis-golgi through all the Golgi compartments, where the glycan is further modified according to the properties of the glycoprotein. This process involves relatively few enzymes but due to its combinatorial nature, can lead to several millions of different possible modifications. The exact topography of this network of reactions has not been established yet, representing one of the major challenges after the sequencing of the human genome (Hossler P et al, 2006).
Since N-glycosylation is involved in an great number of different processes, from cell-cell interaction to folding control, mutations in one of the genes involved in glycan assembly and/or modification can lead to severe development problems (often affecting the central nervous system). All the diseases in genes involved in glycosylation are collectively known as Congenital Disorders of Glycosylation (CDG) (Sparks SE et al, 2003), and classified as CDG type I for the genes in the LLO synthesis pathway, and CDG type II for the others.
VPS29
VPS26Annotated Interactions
Porcupine is a conserved multi-pass transmembrane ER protein that has an O-acyl-transferase domain (van den Heuvel et al, 1993; Kadowaki et al, 1996; Hofmann, 2000). First identified in Drosophila, Porcupine is a WNT-specific modulator that is required for Wingless processing and secretion (Kadowaki et al, 1996). In porcn-deficient cells, Wg and WNT3A have decreased palmitoylation at S209 and accumulate in the ER (Takada et al, 2006), and mutations in PORCN eliminate all WNT signalling and cause embryonic lethality in mice (Barrott et al, 2011; Biechele et al, 2011). Recent studies show that PORCN is required for activity of all human WNT ligands (Proffitt et al, 2012; Najdi et al, 2012).
Once in the extracellular space, the lipid-modified WNT ligand must be shielded to allow the morphogen to diffuse away from the plasma membrane. Possible mechanisms include interaction with HSPGs, exosomes, multimerization or incorporation into lipoprotein particles (reviewed in Eaton, 2006; Port and Basler, 2010).
Retromer is required for the recycling of WLS to the Golgi to allow further rounds of WNT-ligand delivery to the plasma membrane (Coudreuse et al 2006; Belenkaya et al 2008; Port et al, 2008). In the absence of essential retromer component VPS35 or VPS26, WLS is diverted to the MVB and degraded, and WNT ligand accumulates inside the cell; overexpression of WLS is sufficient to rescue the vps35 defect in WNT signaling (Belenkaya et al, 2006; Franch-Marro et al, 2008). WLS and retromer colocalize on endosomal structures and WLS and VPS35 co-precipitate in pull down studies (Belenkaya et al, 2006; Port et al, 2008; Franch-Marro et al, 2008).
Several recent studies have suggested that WLS recycling depends on a WNT-specific retromer in which the SNX-BAR proteins of the classic complex are replaced by SNX3 (Zhang et al, 2011; Harterink et al, 2011; reviewed in Johannes and Wunder, 2011). Unlike SNX1/2/5/6, SNX3 does not contain a BAR domain, and WLS is suggested to accumulate in endocytic vesicles rather than in the tubular structures of the 'classic' retromer (Harterink et al, 2011; Zhang et al, 2011). SNX3 is recruited from the cytosol to the early endosome through the interaction of its PX domain with PIP3 in the membrane. Mutation of critical residues in the PX domain abolish the interaction with PIP3 and ablate endsomal recruitment of SNX3 (Xu et al, 2001; Zhang at al, 2011; Harterink et al, 2011). SNX3 has been shown to co-immunoprecipitate with VPS35 and VPS26, and some studies have also shown a direct interaction between SNX3 and WLS (Zhang et al, 2011; Harterink et al, 2011).
VPS29
VPS26VPS29
VPS26