Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II,an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc₃Man₉GlcNAc₂ transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.     

Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling

Kremen 1 and 2 (Krm1/2) are transmembrane receptors for Wnt antagonists of the Dickkopf (Dkk) family and function by inhibiting the Wnt co-receptors LRP5/6. Here we show that Krm2 functions independently from Dkks during neural crest (NC) induction in Xenopus. Krm2 is co-expressed with, and regulated by, canonical Wnts. Krm2 is differentially expressed in the NC, and morpholino-mediated Krm2 knockdown inhibits NC induction, which is mimicked by LRP6 depletion. Conversely, krm2 overexpression induces ectopic NC. Kremens bind to LRP6, promote its cell-surface localization and stimulate LRP6 signaling. Furthermore, Krm2 knockdown specifically reduces LRP6 protein levels in NC explants. The results indicate that in the absence of Dkks, Kremens activate Wnt/beta-catenin signaling through LRP6.     

Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning

A gradient of Wnt/beta-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopus embryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2 rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.     

Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the β subunit (GIIβ) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIβ MRH domain and that the N-terminal GIIβ G2B domain is involved in the GIIα-GIIβ interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIβ MRH domains with a higher specificity for glycans with high mannose content.     

Structural basis of sugar-recognizing ubiquitin ligase

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.     

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.     

The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology

Kremen (Krm) was originally discovered as a novel transmembrane protein containing the kringle domain. Both Krm1 (the first identified Krm) and its relative Krm2 were later identified to be the high-affinity receptors for Dickkopf (Dkk), the inhibitor of Wnt/beta-catenin signalling. The formation of a ternary complex composed of Krm, Dkk, and Lrp5/6 (the coreceptor of Wnt) inhibits Wnt/beta-catenin signalling. In Xenopus gastrula embryos, Wnt/beta-catenin signalling regulates anterior-posterior patterning, with low-signalling in anterior regions. Inhibition of Krm1/2 induces embryonic head defects. Together with anterior localization of Krms and Dkks, the inhibition of Wnt signalling by Dkk-Krm action seems to allow anterior embryonic development. During mammalian development, krm1 mRNA expression is low in the early stages, but gradually and continuously increases with developmental progression and differentiation. In contrast with the wide, strong expression of krm1 mRNA in mature tissues, expression of krm1 is diminished in a variety of human tumor cells. Since stem cells and undifferentiated cells rely on Wnt/beta-catenin signalling for maintenance in a low differentiation state, the physiological shutdown of Wnt/beta-catenin signalling by Dkk-Krm is likely to set cells on a divergent path toward differentiation. In tumour cells, a deficit of Krm may increase the susceptibility to tumourigenic transformation. Both positive and negative regulation of Wnt/beta-catenin signalling definitively contributes to diverse developmental and physiological processes, including cell-fate determination, tissue patterning and stem cell regulation. Krm is quite significant in these processes as the gatekeeper of the Wnt/beta-catenin signalling pathway.     

Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle

p97 (also called VCP), a member of the AAA ATPase family, is involved in several cellular processes, including membrane fusion and extraction of proteins from the endoplasmic reticulum for cytoplasmic degradation. We have studied the conformational changes that p97 undergoes during the ATPase cycle by cryo-EM and single-particle analysis. Three-dimensional maps show that the two AAA domains, D1 and D2, as well as the N-domains, experience conformational changes during ATP binding, ATP hydrolysis, P(i) release and ADP release. The N-domain is flexible in most nucleotide states except after ATP hydrolysis. The rings formed by D1 and D2 rotate with respect to each other, and the size of their axial openings fluctuates. Taken together, our results depict the movements that this and possibly other AAA ATPases can undergo during an ATPase cycle.     

Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD

The Htm1/EDEM protein has been proposed to act as a "degradation lectin" for endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. In this study, we provide genetic and biochemical evidence that Yos9 protein in Saccharomyces cerevisiae is essential for efficient degradation of mutant glycoproteins. Yos9 is a member of the OS-9 protein family, which is conserved among eukaryotes and shows similarities with mannose-6-phosphate receptors (MPRs). We found that amino acids conserved among OS-9 family members and MPRs were essential for Yos9 protein function. Immunoprecipitation showed that Yos9 specifically associated with misfolded carboxypeptidase Y (CPY*), an ERAD substrate, but only when it carried Man8GlcNAc2 or Man5GlcNAc2 N-glycans. Our experiments further suggested that Yos9 acts in the same pathway as Htm1/EDEM. Yos9 protein is important for glycoprotein degradation and may act via its MRH domain as a degradation lectin-like protein in the glycoprotein degradation pathway.     

Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling

Dickkopf1 (Dkk1) is a secreted antagonist of the Wnt/beta-catenin signaling pathway that acts by direct binding to and inhibiting the Wnt co-receptor LRP6. The related Dkk2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. We have recently identified the transmembrane proteins Kremen1 and -2 as additional Dkk receptors, which bind to both Dkk1 and Dkk2 with high affinity. Here we show that Kremen2 (Krm2) regulates Dkk2 activity during Wnt signaling. In human 293 fibroblasts transfected dkk2 activates LRP6 signaling. However, co-transfection of krm2 blocks the ability of Dkk2 to activate LRP6 and enhances inhibition of Wnt/Frizzled signaling. Krm2 also co-operates with Dkk4 to inhibit Wnt signaling, but not with Dkk3, which has no effect on Wnt signaling. Likewise, in Xenopus embryos, Dkk2 and Krm2 co-operate in Wnt inhibition leading to anteriorized embryos. Finally, we show that interaction with Krm2 is mediated by the second cysteine-rich domain of Dkks. These results suggest that Krm2 can function as a switch that turns Dkk2 from an activator into an inhibitor of Wnt/lRP6 signaling.     

Molecular chaperones stimulate the functional expression of the cocaine-sensitive serotonin transporter.

The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by the N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP.     

Folding of CFTR is predominantly cotranslational

The folding process for newly synthesized, multispanning membrane proteins in the endoplasmic reticulum (ER) is largely unknown. Here, we describe early folding events of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC-transporter family. In vitro translation of CFTR in the presence of semipermeabilized cells allowed us to investigate this protein during nascent chain elongation. We found that CFTR folds mostly during synthesis as determined by protease susceptibility. C-terminally truncated constructs showed that individual CFTR domains formed well-defined structures independent of C-terminal parts. We conclude that the multidomain protein CFTR folds mostly cotranslationally, domain by domain.     

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.     

Conformational changes of p97 during nucleotide hydrolysis determined by small-angle X-Ray scattering

Valosin-containing protein (VCP)/p97 is an AAA family ATPase that has been implicated in the removal of misfolded proteins from the endoplasmic reticulum and in membrane fusion. p97 forms a homohexamer whose protomers consist of an N-terminal (N) domain responsible for binding to effector proteins, followed by two AAA ATPase domains, D1 and D2. Small-angle X-ray scattering (SAXS) measurements of p97 in the presence of AMP-PNP (ATP state), ADP-AlF(x) (hydrolysis transition state), ADP, or no nucleotide reveal major changes in the positions of the N domains with respect to the hexameric ring during the ATP hydrolysis cycle. Nucleotide binding and hydrolysis experiments indicate that D2 inhibits nucleotide exchange by D1. The data suggest that the conversion of the chemical energy of ATP hydrolysis into mechanical work on substrates involves transmission of conformational changes generated by D2 through D1 to move N.     

Molecular determinants of Xolloid action in vivo

Xld (Xolloid) is a member of the Tolloid family of metalloproteases found in embryos of the frog Xenopus laevis. It cleaves Chordin, an inhibitory binding protein for BMP2/4, releasing fragments with reduced affinity for these important ventralizing signals. As a consequence, increasing Xld activity ventralizes Xenopus embryos. We have used this phenotype as an assay to determine the requirement for the C-terminal, nonprotease component of Xld for in vivo activity. This part of the protein is composed of five complement C1r/C1s-sea urchin epidermal growth factor-BMP1 (CUB) and two epidermal growth factor domains, which are thought to be involved in protein-protein interactions and may confer substrate specificity. Our results show that the protease coupled to CUB1 and CUB2 is the minimum domain structure required to ventralize Xenopus embryos and to block the dorsal axis-inducing activity of Chordin. Xld-CUB1-CUB2 cleaves Chordin, and a protease-inactive version co-precipitates Chordin. Our results indicate that the first and second CUB domains bind Chordin and present it to the protease domain. Protease-inactive Xld blocks the cleavage of Chordin by wild-type Xld and dorsalizes injected Xenopus embryos. We find that protease-inactive Xld-CUB1-CUB2 does not share this activity and that all of the C-terminal domains are required to generate the dorsalized phenotype.     

Structural investigation of glycan recognition by the ERAD quality control lectin Yos9

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between β-strands performing an inward movement and that this movement also affects the entire β-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.     

Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control

Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.     

Lynx1 Shifts α4β2 Nicotinic Receptor Subunit Stoichiometry by Affecting Assembly in the Endoplasmic Reticulum

Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and β2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)₃(β2)₂ pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein.