Intradermal DNA Immunization: Antisera Specific for the Membrane Lectin MR60/ERGIC-53

The trafficking of intracellular membrane proteins in Golgi apparatus, endoplasmic reticulum or intermediate compartment has not yet been fully elucidated. The human MR60/ERGIC-53 and the rat p58 proteins are one such protein; and to study them in cell-free and in situ systems, high quality monospecific antisera are required. Highly specific antisera have been obtained after immunization of mice with plasmids containing a gene encoding either the full length or a truncated protein. The best results were obtained after intradermal injections of a plasmid encoding a truncated protein comprising both the luminal carbohydrate recognition domain and the stem down to a cysteine residue close to the C-terminal end, but neither the transmembrane nor the cytosolic domains. Such antisera have a very high titer and are very efficient tools to visualize the MR60 protein in situ or to selectively precipitate the MR60 proteins from a whole cell lysate.     

Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.

Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.     

Oligomerization and interacellular localization of the glycoprotein receptor ERGIC-53 is independent of disulfide bonds

ERGIC-53 is a type I transmembrane lectin facilitating the efficient export of a subset of secretory glycoproteins from the endoplasmic reticulum. Previous results have shown that ERGIC-53 is present as reduction-sensitive homo-oligomers, i.e. as a balanced mixture of disulfide-linked hexamers and dimers, with the two cysteine residues located close to the transmembrane domain playing a crucial role in oligomerization. Here, we demonstrate, using sucrose gradient sedimentation, cross-linking analyses, and non-denaturing gel electrophoresis, that ERGIC-53 is present exclusively as a hexameric complex in cells. However, the hexamers exist in two forms, one as a disulfide-linked, Triton X-100, perfluoro-octanic acid, and SDS-resistant complex, and the other as a non-covalent, Triton X-100, perfluoro-octanoic acid-resistant, but SDS-sensitive, complex made up of three disulfide-linked dimers that are likely to interact through the coiled-coil domains present in the luminal part of the protein. In contrast to what was previously believed, neither of the membrane-proximal cysteine residues plays an essential role in the formation, or maintenance, of the latter form of hexamers. Subcellular fractionation revealed that the double-cysteine mutant was present in the endoplasmic reticulum-Golgi-intermediate compartment, indicating that the two cysteine residues are not essential for the intracellular distribution of ERGIC-53. Based on these results, we present a model for the formation of the two hexameric forms.     

Adhesive and lateral E-cadherin dimers are mediated by the same interface

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. To study cadherin-cadherin interactions that may underlie the adhesive process, a recombinant E-cadherin lacking free sulfhydryl groups and its mutants with novel cysteines were expressed in epithelial A-431 cells. These cysteine mutants, designed according to various structural models of cadherin dimers, were constructed to reveal cadherin dimerization by the bifunctional sulfhydryl-specific cross-linker BM[PE0]3. Cross-linking experiments with the mutants containing a cysteine at strand B of their EC1 domains did show cadherin dimerization. By their properties these dimers correspond to those which have been characterized by co-immunoprecipitation assay. Under standard culture conditions the adhesive dimer is a dominant form. Calcium depletion dissociates adhesive dimers and promotes the formation of lateral dimers. Our data show that both dimers are mediated by the amino-terminal cadherin domain. Furthermore, the interfaces involved in both adhesive and lateral dimerization appear to be the same. The coexistence of the structurally identical adhesive and lateral dimers suggests some flexibility of the extracellular cadherin region.     

Cysteines in the stalk of the nipah virus G glycoprotein are located in a distinct subdomain critical for fusion activation

Paramyxoviruses initiate entry through the concerted action of the tetrameric attachment glycoprotein (HN, H, or G) and the trimeric fusion glycoprotein (F). The ectodomains of HN/H/G contain a stalk region important for oligomeric stability and for the F triggering resulting in membrane fusion. Paramyxovirus HN, H, and G form a dimer-of-dimers consisting of disulfide-linked dimers through their stalk domain cysteines. The G attachment protein stalk domain of the highly pathogenic Nipah virus (NiV) contains a distinct but uncharacterized cluster of three cysteine residues (C146, C158, C162). On the basis of a panoply of assays, we report that C158 and C162 of NiV-G likely mediate covalent subunit dimerization, while C146 mediates the stability of higher-order oligomers. For HN or H, mutation of stalk cysteines attenuates but does not abrogate the ability to trigger fusion. In contrast, the NiV-G stalk cysteine mutants were completely deficient in triggering fusion, even though they could still bind the ephrinB2 receptor and associate with F. Interestingly, all cysteine stalk mutants exhibited constitutive exposure of the Mab45 receptor binding-enhanced epitope, previously implicated in F triggering. The enhanced binding of Mab45 to the cysteine mutants relative to wild-type NiV-G, without the addition of the receptor, implicates the stalk cysteines in the stabilization of a pre-receptor-bound conformation and the regulation of F triggering. Sequence alignments revealed that the stalk cysteines were adjacent to a proline-rich microdomain unique to the Henipavirus genus. Our data propose that the cysteine cluster in the NiV-G stalk functions to maintain oligomeric stability but is more importantly involved in stabilizing a unique microdomain critical for triggering fusion.     

Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis- Golgi. To identify the targeting signals that mediate this recycling, N- glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER- ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC- cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.     

Molecular basis of sugar recognition by the human L-type lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.     

Mannose-dependent endoplasmic reticulum (ER)-Golgi intermediate compartment-53-mediated ER to Golgi trafficking of coagulation factors V and VIII.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is the site of segregation of secretory proteins for anterograde transport, via packaging into COPII-coated transport vesicles. ERGIC-53 is a homo-hexameric transmembrane lectin localized to the ERGIC that exhibits mannose-selective properties in vitro. Null mutations in ERGIC-53 were recently shown to be responsible for the autosomal recessive bleeding disorder, combined deficiency of coagulation factors V and VIII. We have studied the effect of defective ER to Golgi cycling by ERGIC-53 on the secretion of factors V and VIII. The secretion efficiency of factor V and factor VIII was studied in a tetracycline-inducible HeLa cell line overexpressing a wild-type ERGIC-53 or a cytosolic tail mutant of ERGIC-53 (KKAA) that is unable to exit the ER due to mutation of two COOH-terminal phenylalanine residues to alanines. The results show that efficient trafficking of factors V and VIII requires a functional ERGIC-53 cycling pathway and that this trafficking is dependent on post-translational modification of a specific cluster of asparagine (N)-linked oligosaccharides to a fully glucose-trimmed, mannose9 structure.     

Cargo selectivity of the ERGIC-53/MCFD2 transport receptor complex

Exit of soluble secretory proteins from the endoplasmic reticulum (ER) can occur by receptor-mediated export as exemplified by blood coagulation factors V and VIII. Their efficient secretion requires the membrane lectin ER Golgi intermediate compartment protein-53 (ERGIC-53) and its soluble luminal interaction partner multiple coagulation factor deficiency protein 2 (MCFD2), which form a cargo receptor complex in the early secretory pathway. ERGIC-53 also interacts with the two lysosomal glycoproteins cathepsin Z and cathepsin C. Here, we tested the subunit interdependence and cargo selectivity of ERGIC-53 and MCFD2 by short interference RNA-based knockdown. In the absence of ERGIC-53, MCFD2 was secreted, whereas knocking down MCFD2 had no effect on the localization of ERGIC-53. Cargo binding properties of the ERGIC-53/MCFD2 complex were analyzed in vivo using yellow fluorescent protein fragment complementation. We found that MCFD2 is dispensable for the binding of cathepsin Z and cathepsin C to ERGIC-53. The results indicate that ERGIC-53 can bind cargo glycoproteins in an MCFD2-independent fashion and suggest that MCFD2 is a recruitment factor for blood coagulation factors V and VIII.     

Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking

UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53.     

Profile-based data base scanning for animal L-type lectins and characterization of VIPL,a novel VIP36-like endoplasmic reticulum protein

Consensus profiles were established to screen data bases for novel animal L-type lectins. The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins. The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like. Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36. The cDNA of VIPL was cloned and expressed in cell culture. VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36. Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER. Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway. The results suggest that VIPL may function as a regulator of ERGIC-53.     

The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.     

Lectin control of protein folding and sorting in the secretory pathway

Glycan moieties are essential for folding, sorting and targeting of glycoproteins through the secretory pathway to various cellular compartments. The molecular mechanisms that underlie these processes, however, are only now coming to light. Recent crystallographic and NMR studies of proteins located in the endoplasmic reticulum (ER), Golgi complex and ER-Golgi intermediate compartment have illuminated their roles in glycoprotein folding and secretion. Calnexin and calreticulin, both ER-resident proteins, have lectin domains that are crucial for their function as chaperones. The crystal structure of the carbohydrate-recognition domain of ER-Golgi intermediate compartment (ERGIC)-53 complements the biochemical and functional characterization of the protein, confirming that a lectin domain is essential for the role of this protein in sorting and transfer of glycoproteins from the ER to the Golgi complex. The lectin domains of calnexin and ERGIC-53 are structurally similar, although there is little primary sequence similarity. By contrast, sequence similarity between ERGIC-53 and vesicular integral membrane protein (VIP36), a Golgi-resident protein, leaves little doubt that a similar lectin domain is central to the transport and/or sorting functions of VIP36. The theme emerging from these studies is that carbohydrate recognition and modification are central to mediation of glycoprotein folding and secretion.     

Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.     

The lectin ERGIC-53 is a cargo transport receptor for glycoproteins

Soluble secretory proteins are transported from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) in vesicles coated with COP-II coat proteins. The sorting of secretory cargo into these vesicles is thought to involve transmembrane cargo-receptor proteins. Here we show that a cathepsin-Z-related glycoprotein binds to the recycling, mannose-specific membrane lectin ERGIC-53. Binding occurs in the ER, is carbohydrate- and calcium-ion-dependent and is affected by untrimmed glucose residues. Binding does not, however, require oligomerization of ERGIC-53, although oligomerization is required for exit of ERGIC-53 from the ER. Dissociation of ERGIC-53 occurs in the ERGIC and is delayed if ERGIC-53 is mislocalized to the ER. These results strongly indicate that ERGIC-53 may function as a receptor facilitating ER-to-ERGIC transport of soluble glycoprotein cargo.     

N-glycosylation of human nicastrin is required for interaction with the lectins from the secretory pathway calnexin and ERGIC-53

The gamma-secretase complex, composed of four non-covalently bound transmembrane proteins Presenilin, Nicastrin (NCT), APH-1 and PEN-2, is responsible for the intramembranous cleavage of amyloid precursor protein (APP), Notch and several other type I transmembrane proteins. gamma-Secretase cleavage of APP releases the Abeta peptides, which form the amyloid plaques characteristic of Alzheimer's disease brains, and cleavage of Notch releases an intracellular signalling peptide that is critical for numerous developmental processes. NCT, a type I membrane protein, is the only protein within the complex that is glycosylated. The importance of these glycosylation sites is not fully understood. Here, we have observed that NCT N-linked oligosaccharides mediated specific interactions with the secretory pathway lectins calnexin and ERGIC-53. In order to investigate the role played by N-glycosylation, mutation of each site was performed. All hNCT mutants interacted with calnexin and ERGIC-53, indicating that the association was not mediated by any single N-glycosylation site. Moreover, the interaction with ERGIC-53 still occurred in PS1/2 double knockout cells as detected in immunoprecipitation as well as confocal immunofluorescence microscopy studies, which indicated that NCT interacted with ERGIC-53 prior to its association with the active gamma-secretase complex.     

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC.     

Vesicular-integral membrane protein, VIP36, recognizes high-mannose type glycans containing alpha1-->2 mannosyl residues in MDCK cells.

The 36 kDa vesicular-integral membrane protein, VIP36, has been originally isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes containing apical marker proteins, and its luminal domain shows homology to leguminous plant lectins and ERGIC-53. As the first step to identify the functional role of VIP36, the carbohydrate binding specificity of VIP36 was investigated using a fusion protein of glutathione- S -transferase and luminal domain of VIP36 (Vip36). It was found that VIP36 recognizes high-mannose type glycans containing alpha1-->2 Man residues and alpha-amino substituted asparagine. The binding of Vip36 to high-mannose type glycans was independent of Ca(2+)and theoptimal condition was pH 6.0 at 37 degrees C. The concentration at which half inhibition of the binding by Man(7-9).GlcNAc(2). N Ac. Asn occurred was 1.0 x 10(-9)M. The association constant between Man(7-9).GlcNAc(2)in porcine thyroglobulin and immobilized Vip36 was 2.1 x 10(8)M(-1)as determined by means of a biosensor based on surface plasmon resonance. These results indicate that VIP36 functions as an intracellular lectin recognizing glycoproteins which possess high-mannose type glycans, (Manalpha1-->2)(2-4).Man(5). GlcNAc(2).