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Two distinct domains of the -subunit of glucosidase II interact with the catalytic -subunit
Abstract
Recent purification and cDNA cloning of the endoplasmic reticulum processing enzyme glucosidase II have revealed that it is
composed of two soluble proteins: a catalytic α‐subunit and a β-subunit of unknown function, both of which are highly conserved
in mammals. Since the β‐subunit, which contains a C-terminal His-Asp-Glu-Leu (HDEL) motif, may function to link the catalytic
subunit to the KDEL receptor as a retrieval mechanism, we sought to map the regions of the mouse β-subunit protein responsible
for mediating the association with the α-subunit. By screening a panel of recombinant β-subunit glutathione S‐transferase
fusion proteins for the ability to precipitate glucosidase II activity, we have identified two non-overlapping interaction
domains (ID1 and ID2) within the β-subunit. ID1 encompasses 118 amino acids at the N‐terminus of the mature polypeptide, spanning
the cysteine-rich element in this region. ID2, located near the C-terminus, is contained within amino acids 273–400, a region
occupied in part by a stretch of acidic residues. Variable usage of 7 alternatively spliced amino acids within ID2 was found
not to influence the association of the two subunits. We theorize that the catalytic subunit of glucosidase II binds synergistically
to ID1 and ID2, explaining the high associative stability of the enzyme complex.
... In fact, apart from the active site of the enzyme found in the β8α8 barrel domain, the domain primarily involved in binding GIIβ occupies the distal C-terminal side, interacting through the N-terminal one of GIIα-binding domain. In partic-ular, two non-overlapping interaction domains, ID1 and ID2, have been identified [364]. ID1 consists of 118 amino acids at the NH2-terminus of the coding region and includes the cysteine-rich element region of GIIα. ...
... ID1 consists of 118 amino acids at the NH2-terminus of the coding region and includes the cysteine-rich element region of GIIα. ID2 comprises amino acids 273-400 located at the COOH-terminus of GIIβ including a stretch of acidic residues [364]. ...
... Antibodies recognizing two different regions of the murine β-subunit were also generated [364]. ...
Glycans are one of the four fundamental macromolecular components of living matter, and they are highly regulated in the cell. Their functions are metabolic, structural and modulatory. In particular, ER resident N-glycans participate with the Glc3Man9GlcNAc2 highly conserved sequence, in protein folding process, where the physiological balance between glycosylation/deglycosylation on the innermost glucose residue takes place, according GANAB/UGGT concentration ratio. However, under abnormal conditions, the cell adapts to the glucose availability by adopting an aerobic or anaerobic regimen of glycolysis, or to external stimuli through internal or external recognition patterns, so it responds to pathogenic noxa with unfolded protein response (UPR). UPR can affect Multiple Sclerosis (MS) and several neurological and metabolic diseases via the BiP stress sensor, resulting in ATF6, PERK and IRE1 activation. Furthermore, the abnormal GANAB expression has been observed in MS, systemic lupus erythematous, male germinal epithelium and predisposed highly replicating cells of the kidney tubules and bile ducts. The latter is the case of Polycystic Liver Disease (PCLD) and Polycystic Kidney Disease (PCKD), where genetically induced GANAB loss affects polycystin-1 (PC1) and polycystin-2 (PC2), resulting in altered protein quality control and cyst formation phenomenon. Our topics resume the role of glycans in cell physiology, highlighting the N-glycans one, as a substrate of GANAB, which is an emerging key molecule in MS and other human pathologies.
... It forms the heterodimeric GII complex with glucosidase II α subunit (GIIα), which processes newly synthesized glycoproteins and is involved in ER protein quality control [24][25][26] . PRKCSH consists of multiple domains, including a signal sequence for translocation across the ER membrane, an N-terminal GIIα-binding (G2B) domain, a putative coiled-coil segment, a glutamic acid and proline-rich (E/ P) segment, and a C-terminal mannose 6-phosphate receptor homology (MRH) domain followed by an HDEL signal sequence for ER retention 27,28 . ER-translocation of PRKCSH is required for the expression and retention of GIIα in the ER lumen and maintaining optimal GII activity 29 . ...
... The inhibitory effect of the G2B domain on PRKCSH binding to IRE1α suggests that it regulates the specific binding of PRKCSH to IRE1α only when ER stress is present. Because the interaction between the GIIα subunit and PRKCSH is necessary for GII activity [24][25][26][27][28][29] , we examined the potential impact of ER stress on GII activity. Co-IP assay revealed that ER stress induced the dissociation of the GIIα-PRKCSH complex in both L02 and Huh-7 cells (Fig. 5g). ...
... Immunoprecipitation was performed by using anti-GIIα antibody and normal rabbit IgG. Two domains of PRKCSH contribute to its glucosidase activity: G2B, the N-terminal GIIα-binding domain, and MRH, the Cterminal N-glycan mannose recognition domain 27,28 . Binding of the G2B domain of PRKCSH to the GIIα subunit is required for maintaining the GIIα level in the ER and GII enzymatic activity 29 . ...
Unfolded protein response (UPR) is an adaptive mechanism that aims at restoring ER homeostasis under severe environmental stress. Malignant cells are resistant to environmental stress, which is largely due to an activated UPR. However, the molecular mechanisms by which different UPR branches are selectively controlled in tumor cells are not clearly understood. Here, we provide evidence that PRKCSH, previously known as glucosidase II beta subunit, functions as a regulator for selective activation of the IRE1α branch of UPR. PRKCSH boosts ER stress-mediated autophosphorylation and oligomerization of IRE1α through mutual interaction. PRKCSH contributes to the induction of tumor-promoting factors and to tumor resistance to ER stress. Increased levels of PRKCSH in various tumor tissues are positively correlated with the expression of XBP1-target genes. Taken together, our data provide a molecular rationale for selective activation of the IRE1α branch in tumors and adaptation of tumor cells to severe environmental stress.
... Subsequently, glucosidase II (GII) catalyzes two-step hydrolytic cleavage reactions of the Glc-α1,3-Glc and Glc-α1,3-Man glycosidic linkages, giving rise to Glc 1 Man 9 GlcNAc 2 and Man 9 GlcNAc 2 glycoforms, respectively (Arendt and Ostergaard 2000;Pelletier et al. 2000;Totani et al. 2008;Trombetta et al. 1996Trombetta et al. , 2001. In the ER quality-control system, the folding states of newly synthesized proteins are judged based on the presence or absence of a single terminal glucose residue on the D1 branch of N-glycan intermediates (Fig. 8.1b;Aebi et al. 2010;D'Alessio et al. 2010;Kamiya et al. 2012;Lederkremer 2009;Satoh et al. 2015;Takeda et al. 2009). ...
... GII consists of a catalytic α-subunit (GIIα) of approximately 110 kDa belonging to the GH family 31 and a 60 kDa multi-domain regulatory β-subunit (GIIβ; Arendt and Ostergaard 2000;Pelletier et al. 2000;Trombetta et al. 1996Trombetta et al. , 2001. Among the GH31 enzymes, only the GIIα operates with its binding partner GIIβ, which contains an N-terminal GIIα-binding (G2B) domain, a putative coiled-coil segment, and a C-terminal mannose-6-phosphate receptor homology (MRH) domain followed by an ER retention signal (Munro 2001;Quinn et al. 2009;Fig. ...
N-linked oligosaccharides attached to proteins act as tags for glycoprotein quality control, ensuring their appropriate folding and trafficking in cells. Interactions with a variety of intracellular lectins determine glycoprotein fates. Monoglucosylated glycoforms are the hallmarks of incompletely folded glycoproteins in the protein quality-control system, in which glucosidase II and UDP-glucose/glycoprotein glucosyltransferase are, respectively, responsible for glucose trimming and attachment. In this review, we summarize a recently emerging view of the structural basis of the functional mechanisms of these key enzymes as well as substrate N-linked oligosaccharides exhibiting flexible structures, as revealed by applying a series of biophysical techniques including small-angle X-ray scattering, X-ray crystallography, high-speed atomic force microscopy, electron microscopy, and computational simulation in conjunction with NMR spectroscopy.
... Unique to ER α-GluII α-subunit is its N terminus (residues 33-54), which reaches the brim of the catalytic pocket (red in Fig. 1 B-D) and supports a loop (residues 305-317 in Mmα-GluII) contributing to the +1 and +2 enzyme subsites ( Fig. 3 A and B). In addition to the α-subunit, our Mmα-GluII Tryps crystals contain two tandem low-density lipoprotein receptor class A (LDLRa) subdomains, the N-terminal and only portion of the β-subunit still associated with the α-subunit after trypsinolysis (light blue in Fig. 1 A-D and SI Appendix, Fig. S4A), in keeping with the predictions from previous biochemical studies (24)(25)(26)(27). Each β-subunit LDLRa module folds around an octahedrally coordinated calcium ion (SI Appendix, Figs. ...
... A conformational change has been reported by circular dichroism for rat liver α-GluII in the presence of the molecular crowding agent PEG 20,000; indeed, crowding did not affect the first cleavage, but it greatly enhanced the rate of the second cleavage (35). An arrangement in which the N-and C-terminal regions of the β-subunit were proximal in space has already been suggested on the grounds that the region β 273-400 has been reported to interact with the α-subunit (25). Notably, a subset of that region, residues β 357-390, seems to be protected from HDX exchange in our HDX experiment (SI Appendix, Fig. S6), although further experiments are necessary to determine if they contact the α-subunit or a different portion of the β-subunit. ...
Significance
Most pathogenic enveloped viruses crucially depend on the quality control (QC) machinery in the endoplasmic reticulum (ER) of the host cell. ERQC inhibitors therefore have the double potential benefit of targeting a wide variety of viruses (“broad-spectrum antivirals”) without the risk of losing efficacy due to escape mutations in the viral genome. Our recent work has proven that inhibition of the central enzyme of ERQC, α-glucosidase II (α-GluII), is sufficient for antiviral activity against dengue fever in vitro and in vivo. Here, we show how antiviral inhibitors bind to portions of α-GluII that are unique to this enzyme, and we open the way to the development of potent and selective antivirals against existing and emerging infectious disease.
... Unique to ER α-GluII α-subunit is its N terminus (residues 33-54), which reaches the brim of the catalytic pocket (red in Fig. 1 B-D) and supports a loop (residues 305-317 in Mmα-GluII) contributing to the +1 and +2 enzyme subsites ( Fig. 3 A and B). In addition to the α-subunit, our Mmα-GluII Tryps crystals contain two tandem low-density lipoprotein receptor class A (LDLRa) subdomains, the N-terminal and only portion of the β-subunit still associated with the α-subunit after trypsinolysis (light blue in Fig. 1 A-D and SI Appendix, Fig. S4A), in keeping with the predictions from previous biochemical studies (24)(25)(26)(27). Each β-subunit LDLRa module folds around an octahedrally coordinated calcium ion (SI Appendix, Figs. ...
... A conformational change has been reported by circular dichroism for rat liver α-GluII in the presence of the molecular crowding agent PEG 20,000; indeed, crowding did not affect the first cleavage, but it greatly enhanced the rate of the second cleavage (35). An arrangement in which the N-and C-terminal regions of the β-subunit were proximal in space has already been suggested on the grounds that the region β 273-400 has been reported to interact with the α-subunit (25). Notably, a subset of that region, residues β 357-390, seems to be protected from HDX exchange in our HDX experiment (SI Appendix, Fig. S6), although further experiments are necessary to determine if they contact the α-subunit or a different portion of the β-subunit. ...
... The mature protein contains two EF-Hand motifs (Ca 2+ binding loops), an acidic stretch of consecutive glutamic acids, several cysteine residues at the NH 2 and COOH termini (Trombetta et al. 1996), a mannose 6-phosphate receptor homology (MRH) domain (Munro 2001), and a COOHterminal HDEL sequence for ER retention/retrieval. Regions of the b-subunit that interact with the a-subunit have been mapped by co-immunoprecipitation (Arendt and Ostergaard 2000) and two nonoverlapping interaction domains, ID1 and ID2, were identified. ID1 consists of 118 amino acids at the NH 2 -terminus of the coding region and includes the cysteine-rich element region. ...
... ID1 consists of 118 amino acids at the NH 2 -terminus of the coding region and includes the cysteine-rich element region. Amino acids 273-400 of the coding region comprise ID2, which is located at the COOH-terminus and includes a stretch of acidic residues (Arendt and Ostergaard 2000). ...
The synthesis of N-linked glycans is initiated by the synthesis of a Glc3Man9GlcNAc2 lipid-linked precursor and the co-translational transfer to nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER) (Kornfeld and Kornfeld 1985). Immediately after transfer of the glycan to Asn side chains glucose (Glc), trimming is initiated by the cleavage of the terminal α1,2-Glc residue by α-glucosidase I (MOGS). Subsequent cleavage of the two internal α1,3-Glc residues is accomplished by the heterodimeric enzyme α-glucosidase II (GANAB/PRKCSH) (Burns and Touster 1982; Grinna and Robbins 1979, 1980) to produce the Man9GlcNAc2-Asn processing intermediate (Fig. 114.1).
... In addition to the C-terminal MRH domain, a domain near the N terminus that is conserved among GIIβ subunits of different species (G2B, Supplemental Figure S1) has been reported to be involved in the interaction of both GII subunits in mammalian cells and in the removal of the middle glucose in S. cerevisiae (Arendt and Ostergaard, 2000;Quinn et al., 2009). A series of mutations were introduced in the S. cerevisiae GIIβ G2B domain. ...
... G2B, a domain located close to the GIIβ N terminus, has been proposed to be involved in the GIIα-GIIβ interaction or, additionally, in G2M9 deglucosylation (Arendt and Ostergaard, 2000;Quinn et al., 2009). Our results support the former role because microsomes of cells lacking endogenous GIIβ but expressing GIIβ with a mutant G2B domain failed to correct the poor ability of GIIα to trim glucose residues from G1M9 in the absence of GIIβ. ...
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.
... MRH domains (Munro 2001) have been identified in a number of proteins including Yos9p and Erlectin (XTP3-B) and mutations in conserved residues in Yos9p have been shown to disrupt lectin activity (Bhamidipati et al. 2005;Szathmary et al. 2005;Quan et al. 2008). In addition to the Cterminal MRH domain, Gtb1p/GluIIβ also contains a highly conserved amino-terminal domain, hereafter described as the "G2B" domain (Glucosidase II Beta), which has been implicated in the interaction with the Gls2p/GluIIα subunit (Arendt and Ostergaard 2000;Pelletier et al. 2000). ...
... Two mutations in this domain, D125A and E132A, produced defects in Gtb1pdependent trimming. Interestingly, this domain has been implicated in the interaction with GluIIα (Arendt and Ostergaard 2000), and the reduced interaction observed for I125A in coimmunoprecipitation studies with Gls2p appears to support this idea. However, the E132A mutant protein still interacted with Gls2p ( Figure 4E) and yet has a very severe Glc1 trimming defect implying a further role for this domain. ...
Glucosidase II (GluII) is a glycan-trimming enzyme active on nascent glycoproteins in the endoplasmic reticulum (ER). It trims
the middle and innermost glucose residues (Glc2 and Glc1) from N-linked glycans. The monoglucosylated glycan produced by the first GluII trimming reaction is recognized by calnexin/calreticulin
and serves as the signal for entry into this folding pathway. GluII is a heterodimer of α and β subunits corresponding to
yeast Gls2p and Gtb1p, respectively. While Gls2p contains the glucosyl hydrolase active site, the Gtb1p subunit has previously
been shown to be essential for the Glc1 trimming event. Here we demonstrate that Gtb1p also determines the rate of Glc2 trimming.
In order to further dissect these activities we mutagenized a number of conserved residues across the protein. Our data demonstrate
that both the MRH and G2B domains of Gtb1p contribute to the Glc2 trimming event but that the MRH domain is essential for
Glc1 trimming.
... It has been demonstrated that 80K-H/bGII interacts with the aGII catalytic subunit. As such, 80K-H/bGII is proposed to function as an anchor of the aGII through its ER targeting signal (HDEL) found at the carboxyterminal end of 80K-H/bGII (D'Alessio et al. 1999;Arendt and Ostergaard 2000;Baldwin et al. 2000;Pelletier et al. 2000). Biochemical isolation of the aGII subunit alone showed it to be catalytically active, suggesting that the beta subunit is not required for GII activity (Hentges and Bause 1997). ...
... This observation corroborates the results of other groups who co-purified the aGII, as a 116-kDa protein, with the 80K-H/bGII (Trombetta et al. 1996;Arendt and Ostergaard 1997). The interaction between 80K-H/bGII and aGII is shown also in a GST pull-down experiment (Arendt and Ostergaard 2000). The bovine aGII was shown to migrate at 116 kDa (Hentges and Bause 1997), which is identical to our present observation. ...
We have characterized the localization of the protein termed VASAP-60 in different bovine tissues and cell lines, and have investigated if VASAP-60 interacts with other proteins. Monospecific polyclonal antibodies were raised against distinct fragments of VASAP-60: NH(2) (V(22) to Q(234)), central (A(246) to S(418)), and COOH (L(416) to L(533)). These three antibodies recognized an 88-kDa protein in immunoblotting analysis. The calculated Mr of VASAP-60 derived from its cDNA (60.1 kDa) was significantly lower than its Mr estimated by SDS-PAGE, and this was mainly attributed to the glutamic acid- and aspartic acid-rich composition of its central region (A(246) to S(418)). A 58-kDa proteolytically processed form of VASAP-60 was also identified. Immunocytochemistry demonstrated that VASAP-60 is found predominantly in the perinuclear region, colocalized with calnexin in the endoplasmic reticulum (ER), and partially colocalized with the endocytic marker DAMP. Immunohistochemical localization of VASAP-60 also demonstrated its presence within specialized vesicular structures not related to the ER. Immunoprecipitation using extracts prepared from S(35)Met/Cys metabolically labeled cells demonstrates that VASAP-60 interacts with 116-, 48.5-, and 26.5-kDa proteins. Therefore, VASAP-60 was found to be more widely distributed in the vacuolar system than anticipated, suggesting that VASAP-60 may function in intracellular transport events, rather than being an exclusive component of the quality control mechanism of newly synthesized proteins as thought previously.
... The proteins corresponded to altered metabolism, increase in invasive potential, angiogenesis, modified molecular chaperones, and a decrease in tumoral immunogenicity (Table 1). Glutathione S-transferase, [37][38][39] tumor rejection antigen (Gp96) 1, [40][41][42][43] tryptophan-tRNA ligase, [44][45][46] phosphoglycerate kinase 1, [47] alpha-actinin-4, [48][49][50] tenascin, [51][52][53] and glucosidase 2 [54,55] have previously been identified as potential cancer biomarkers. The protein myosin regulatory light chain 12A is a novel biomarker. ...
Purpose:
To validate differences in protein levels between good and poor prognosis AJCC stage III melanoma patients and compile a protein panel to stratify patient risk.
Experimental design:
Protein extracts from melanoma metastases within lymph nodes in patients with Stage III disease with good (n = 16, > 4 years survival) and poor survival (n = 14, < 2 years survival) were analysed by selected reaction monitoring (SRM). Diagonal Linear Discriminant Analysis was performed to generate a protein biomarker panel.
Results:
SRM analysis identified 10 proteins that were differentially abundant between good and poor prognosis Stage III melanoma patients. The 10 differential proteins were combined with 22 proteins identified in our previous work. A panel of 14 proteins was selected by Diagonal Linear Discriminant Analysis that was able to accurately classify patients into prognostic groups based on levels of these proteins.
Conclusions and clinical relevance:
The 10 differential proteins identified by SRM have biological significance in cancer progression. The final signature of 14 proteins identified by SRM could be used to identify AJCC Stage III melanoma patients likely to have poor outcomes who may benefit from adjuvant systemic therapy. This article is protected by copyright. All rights reserved.
... GIIβ is the non-catalytic subunit of the α-glucosidase II (GII) [40]. GII enzyme is an ER resident protein, and its subunits alpha and beta are covalently linked [40,41]. The catalytic activity of the enzyme is conferred by the alpha subunit. ...
RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.
... Absolutely conserved among GIIβs is a C-terminal domain homologous to the mannose 6-phosphate receptor (MPR) lectin domain (MRH) [61]. Another GIIβ conserved domain (G2B domain) is involved in GIIα-GIIβ interaction [36,62,63] (see Figure 3). GIIβ has been suggested to be responsible for GIIα folding, maturation or stability in mammals, heterodimer localization in the ER (GIIα lacks any known ER retention signal), and/or enhancing GIIα N-glycan processing rates [40,41,54,55,[63][64][65][66][67]. ...
N-glycosylation in the endoplasmic reticulum (ER) consists of the transfer of a preassembled glycan conserved among species (Glc3Man9GlcNAc2) from a lipid donor to a consensus sequence within a nascent protein that is entering the ER. The protein-linked glycans are then processed by glycosidases and glycosyltransferases in the ER producing specific structures that serve as signalling molecules for the fate of the folding glycoprotein: to stay in the ER during the folding process, to be retrotranslocated to the cytosol for proteasomal degradation if irreversibly misfolded, or to pursue transit through the secretory pathway as a mature glycoprotein. In the ER, each glycan signalling structure is recognized by a specific lectin. A domain similar to that of the mannose 6-phosphate receptors (MPRs) has been identified in several proteins of the secretory pathway. These include the beta subunit of glucosidase II (GII), a key enzyme in the early processing of the transferred glycan that removes middle and innermost glucoses and is involved in quality control of glycoprotein folding in the ER (QC), the lectins OS-9 and XTP3-B, proteins involved in the delivery of ER misfolded proteins to degradation (ERAD), the gamma subunit of the Golgi GlcNAc-1-phosphotransferase, an enzyme involved in generating the mannose 6-phosphate (M6P) signal for sorting acidic hydrolases to lysosomes, and finally the MPRs that deliver those hydrolytic enzymes to the lysosome. Each of the MRH-containing proteins recognizes a different signalling N-glycan structure. Three-dimensional structures of some of the MRH domains have been solved, providing the basis to understand recognition mechanisms.
... In contrast, glucosidase II is heterodimer complex consisting of two polypeptides, alpha (GIIα) and beta (GIIβ) subunits (D'Alessio et al. 1999;Pelletier et al. 2000;Trombetta et al. 1996). GIIα, a soluble protein is retained in the ER via its interaction with ER membrane integrated GIIβ subunit (Arendt and Ostergaard 1997;Arendt and Ostergaard 2000;Treml et al. 2000;Trombetta et al. 1996). Immunoelectron microscopy data support the presence of GIIα in endoplasmic reticulum (Brada et al. 1990;Lucocq et al. 1986). ...
α-Glucosidase II (GII), a resident of endoplasmic reticulum (ER) and an important enzyme in the folding of nascent glycoproteins, is heterodimeric, consisting of α (GIIα) and β (GIIβ) subunits. The catalytic GIIα subunit, with the help of mannose 6-phosphate receptor homology domain of GIIβ, sequentially hydrolyzes two α1-3-linked glucose residues in the second step of N-linked oligosaccharide-mediated protein folding. The soluble GIIα subunit is retained in the ER through its interaction with the HDEL-containing GIIβ subunit. N-glycosylation and correct protein folding are crucial for protein stability and trafficking and cell surface expression of several proteins in the brain. Alterations in N-glycosylation lead to abnormalities in neuronal migration and mental retardation, various neurodegenerative diseases, and invasion of malignant gliomas. Inhibitors of GII are used to inhibit cell proliferation and migration in a variety of different pathologies, such as viral infection, cancer, and diabetes. Despite the widespread use of GIIα inhibitory drugs and the role of GIIα in brain function, little is known about its expression in brain and other tissues. Here, we report generation of a highly specific chicken antibody to the GIIα subunit and its characterization by Western blotting and immunoprecipitation using cerebral cortical extracts. By using this antibody, we showed that the GIIα protein is highly expressed in testis, kidney, and lung, with the lowest amount in heart. GIIα polypeptide levels in whole brain were comparable to those in spleen. However, a higher expression of GIIα protein was detected in the cerebral cortex, reflecting its continuous requirement in correct folding of cell surface proteins. © 2014 Wiley Periodicals, Inc.
... We also identified Um-12045 as a putative homolog of the S. cerevisiae glucosidase II b-subunit (Gtb1). This ORF conserves a region (Val-65 to Cys-84) present in the human glucosidase II b-subunit, involved in glucosidase II substrate binding, as well as a C-type lectin domain (Asn-86 to Cys-132) homolog to other glucosidases (Arendt and Ostergaard, 2000) (see Supplemental Figure 1 online). Finally, we found that Um-02227 shares significant identity to ER mannosidase I (Mns1) proteins from several organisms. ...
Secreted fungal effectors mediate plant-fungus pathogenic interactions. These proteins are typically N-glycosylated, a common posttranslational modification affecting their location and function. N-glycosylation consists of the addition, and subsequent maturation, of an oligosaccharide core in the endoplasmic reticulum (ER) and Golgi apparatus. In this article, we show that two enzymes catalyzing specific stages of this pathway in maize smut (Ustilago maydis), glucosidase I (Gls1) and glucosidase II β-subunit (Gas2), are essential for its pathogenic interaction with maize (Zea mays). Gls1 is required for the initial stages of infection following appressorium penetration, and Gas2 is required for efficient fungal spreading inside infected tissues. While U. maydis Δgls1 cells induce strong plant defense responses, Δgas2 hyphae are able to repress them, showing that slight differences in the N-glycoprotein processing can determine the extent of plant-fungus interactions. Interestingly, the calnexin protein, a central element of the ER quality control system for N-glycoproteins in eukaryotic cells, is essential for avoiding plant defense responses in cells with defective N-glycoproteins processing. Thus, N-glycoprotein maturation and this conserved checkpoint appear to play an important role in the establishment of an initial biotrophic state with the plant, which allows subsequent colonization.
... 42 The β-subunit seems to interact with the α-subunit through two distinct regions. 43 In vivo, the β-subunit is essential for the formation of an active enzyme. 37,38,41 An important question concerning the function of glucosidase II is whether the protein is capable of cleaving the innermost glucose while the substrate glycoprotein is bound to CNX and CRT, or whether it acts on the unbound form of the glycoprotein and thereby prevents renewed association with the lectin. ...
The endoplasmic reticulum (ER) contains a particularly wide range of molecular chaperones and other proteins that assist the
folding and quality control of newly synthesized protein. Some, like BiP/GRP78 and GRP94, belong to classical chaperone families.
Others, like protein disulfide isomerase, ERp57, and ERp72, belong to the family of thiol-disulfide oxidoreductases especially
well represented in the ER. The ER lectin chaperones calnexin (CNX) and calreticulin (CRT) have unique features that distinguish
them from other known molecular chaperones. They interact with proteins that carry N-linked glycans, and cooperate with a
number of accessory enzymes during the folding process. Here we review work on calnexin/calreticulin-assisted glycoprotein
folding in the ER, with an emphasis on recent molecular and structural studies.
... Cdc25C proteins with an N-terminal maltose-binding protein tag [MBP-Cdc25C (WT and S287A)], human 14-3-3ζ protein with an N-terminal glutathione-S-transferase protein tag (GST-14-3-3ζ), and constitutively-active rat MEK(QP) with an N-terminal glutathione-S-transferase protein tag [MEK(QP)] were expressed in Escherichia coli and purified as previously described. 4,14,15 Preparation of Xenopus egg extracts Cycling egg extracts. Egg extracts that undergo repeated cell cycles were prepared exactly as described previously. ...
In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway is known to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation of p42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrest of the cell cycle, depending on the timing of p42MAPK activation. Here, we examined the molecular mechanism by which activation of the p42MAPK pathway during interphase leads to cell cycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form of Cdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling egg extracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame the G2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk a kinase that is the target of p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and was able to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenous Cdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90(Rsk) bound 14-3-3zeta, whereas S287A could not. These data suggest that the link between the p42MAPK signaling pathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287, causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rsk phosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays induced by differentiation signals that occur later in embryonic development.
... In addition, GIIα in microsomes, as measured by pNPG hydrolysis, increased to wild type cell levels when MRH-mutated GIIβ was expressed in GIIβ null cells [54]. It should be mentioned that the surface mediating the interaction between both subunits maps to GIIβ N-terminus [76]. Surprisingly, a point mutation in a conserved N-terminal domain of S. cerevisiae GIIβ resulted in a reduced G2M9 trimming, suggesting that this region may be also important for glucose trimming [70]. ...
The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.
... Supernatants were collected and protein concentration was analyzed using the BCA assay kit (Pierce, Rockford, IL). The GII activities of cell lysates were measured as previously described (Arendt and Ostergaard 2000). Briefly, 10 μg of cell lysate was added to 125 μL of pNP-αGlc (1 mg/mL in PBS; Wako) and incubated at 37 • C for 15 h. ...
Glucosidase II (GII) is a glycan-processing enzyme that trims two alpha1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first alpha1,3-linked glucose from Glc(2)Man(9)GlcNAc(2) (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc(1)Man(9)GlcNAc(2) (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic alpha subunit (GIIalpha) and a tightly associated beta subunit (GIIbeta) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIbeta (GIIbeta-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIbeta-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIbeta-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIbeta-MRH binds most strongly to the glycans with the alpha1,2-linked mannobiose structure. GII with the mutant GIIbeta that lost the sugar-binding activity of GIIbeta-MRH hydrolyzes p-nitrophenyl-alpha-glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIbeta-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.
... Glucosidase II, an oligomer of two different subunits α and β, is necessary for the interaction of nascent glycoproteins bearing monoglucosylated carbohydrates with calnexin and calreticulin leading to proper protein folding [36]. The α subunit represents the catalytic domain [37]. Since Glucosidase II shares homology with many other glucosidases, its loss of function may be compensated by other glucosidases present in the cells. ...
A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (alpha-Syn) upon differentiation with retinoic acid was used to study the impact of alpha-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced alpha-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. alpha-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating alpha-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 alpha/beta in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that alpha-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.
... GII Enzymatic Assay-Determination of the GII enzymatic activity was performed as described previously (27). Briefly, samples were incubated with 5 mM p-nitrophenyl ␣-D-gluocopyranoside (Sigma) in phosphate-buffered saline, pH 7.2, for 16 -18 h at room temperature. ...
We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase
II (GII). GII consists of the catalytic α-chain and an associated β-chain. To gain insight into the basis of the association
between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the α-subunit is essential
for the interaction. Interestingly, only a higher molecular weight form of GIIα is capable of associating with CD45 in a competitive
situation where multiple GIIα isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing
the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The
interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and
can be inhibited with mannose. Taken together, these results suggest that GIIα acts as a lectin and binds to CD45 in an exon-dependent
manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune
function, possibly by regulating CD45 glycosylation.
... Critical conserved features ( Figure 3) include the 6 Cys residues forming three disulphide bonds (although the S. pombe protein lacks cysteines 1 and 2), the substrate recognition loop between the cysteines 5 and 6 and the Y and R residues implicated in ligand binding (Roberts et al., 1998). Interaction between mouse a-and b-subunits was mapped to the N-terminal 118 residues of the b-subunit, which are reasonably well conserved in all sequences, and to residues 273-400 (Arendt and Ostergaard, 2000) which are not. Figure 3 shows, however, that all sequences show a high percentage of acidic residues here. ...
rsw3 is a temperature-sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose. The rsw3 gene was identified by a map-based strategy, and shows high similarity to the catalytic alpha-subunits of glucosidase II from mouse, yeast and potato. These enzymes process N-linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding. Putative beta-subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER-retention signal) with beta-subunits in mammals and yeast. The genes encoding the putative alpha- and beta-subunits are single copy and, like the rsw3 phenotype, widely expressed. rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems. Most features of the rsw3 phenotype are shared with other cellulose-deficient mutants, but some--notably, production of multiple rosettes and a lack of secreted seed mucilage--are not and may reflect glucosidase II affecting processes other than cellulose synthesis. The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature. This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.
... Subsequently, the middle Glc is removed by Glc'ase II, giving rise to the monoglucosylated glycan GM9, which is capable of interacting with the ER chaperones CNX and CRT (vide infra). Glc'ase II is a soluble heterodimer that is composed of a catalytic a subunit belonging to glycoside hydrolase family 31 and a b subunit containing an EF hand, Man-6-phosphate receptor homology (MRH) domains, and a C-terminal His-Asp-Glu-Leu sequence (Arendt and Ostergaard 2000). This enzyme is also responsible for the cleavage of the Glca1-3Man linkage, giving rise to the M9 glycoform that can no longer interact with CNX or CRT. ...
Processing of the N-glycans is coupled with the fates of glycoproteins in cells. A series of processing intermediates of high-mannose-type glycans are generated by specific glycosidases and thereby express biological signals recognized by intracellular lectins operating as molecular chaperones, cargo receptors, and ubiquitin ligases. Consequently, these lectins govern the intracellular processes of folding, transport, and degradation of the carrier polypeptides. To understand the underlying mechanisms of glycoprotein-fate determination, structural information on modes of molecular recognition by these lectins and enzymes is undoubtedly important. This article overviews our current knowledge of the structural basis for quality control of glycoproteins in cells.
The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum. Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. This article is protected by copyright. All rights reserved.
The biosynthesis of enveloped viruses depends heavily on the host
cell endoplasmic reticulum (ER) glycoprotein quality control (QC)
machinery. This dependency exceeds the dependency of host
glycoproteins, offering a window for the targeting of ERQC for the
development of broad-spectrum antivirals. We determined smallangle
X-ray scattering (SAXS) and crystal structures of themain ERQC
enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex
with key ligands of its catalytic cycle and antiviral iminosugars,
including two that are in clinical trials for the treatment of dengue
fever. The SAXS data capture the enzyme’s quaternary structure and
suggest a conformational rearrangement is needed for the simultaneous
binding of a monoglucosylated glycan to both subunits. The
X-ray structures with key catalytic cycle intermediates highlight that
an insertion between the +1 and +2 subsites contributes to the
enzyme’s activity and substrate specificity, and reveal that the presence
of D-mannose at the +1 subsite renders the acid catalyst less
efficient during the cleavage of the monoglucosylated substrate. The
complexes with iminosugar antivirals suggest that inhibitors targeting
a conserved ring of aromatic residues between the α-GluII +1
and +2 subsites would have increased potency and selectivity, thus
providing a template for further rational drug design.
Protein quality control in the endoplasmic reticulum (ER) is an elaborate process conserved from yeast to mammals, ensuring that only newly synthesized proteins with correct conformations in the ER are sorted further into the secretory pathway. The ER discriminates between native and nonnative protein conformations, selectively transporting properly folded proteins to their final destinations through the secretory pathway, or alternatively, retrotranslocating misfolded proteins to the cytosol to be degraded by proteasomes. In the quality control process, high-mannose type N-glycans play important roles in protein-folding events. Proteins that fail to achieve proper folding or proper assembly are degraded in a process known as ER-associated degradation (ERAD). The ERAD pathway comprises multiple steps including substrate recognition and targeting to the retro-translocation machinery, retrotranslocation from the ER into the cytosol, and proteasomal degradation through ubiquitination. The quality-control system also surveys the ER lumen for terminally misfolded proteins. Polypeptides singled out by this system are ultimately degraded by the cytosolic ubiquitin-proteasome pathway. Key components of both the ER quality-control system and the ERAD machinery have been identified, but a connection between the two systems has remained elusive (Yoshida and Tanaka 2010). Recent studies have documented the important roles of sugar-recognition (lectin-type) molecules for trimmed high-mannose type N-glycans and glycosidases in the ERAD pathways in both ER and cytosol. Since the ER is distributed throughout the cytosol, studies suggest that the cytosolic face of the ER membrane serves as a “platform” for degradation of misfolded cytosolic proteins (Metzger et al. 2008). The fundamental system that monitors glycoprotein folding in the ER and the unique roles of the sugar-recognizing ubiquitin ligase and peptide:N-glycanase (PNGase) in the cytosolic ERAD pathway has been reviewed (Yoshida and Tanaka 2010).
Information on receptor ligand systems used by NK cells to specifically detect transformed cells has been accumulating rapidly. Killer cell lectin-like receptor subfamily K, member 1, also known as KLRK1, is the product of human gene. The KLRK1 has been designated as CD314 and contains a C-type lectin-like domain (CTLD). KLRK1 is also known as: KLR; NKG2D; NKG2-D; FLJ17759; FLJ75772; D12S2489E. Human NKG2D was originally identified in 1991 as an orphan receptor on NK cells (Houchins et al. 1991). Although genetically mapping near the C-type lectin receptors CD94 and NKG2A-E, the NKG2D activating NK cell receptor has little sequence homology with these receptors and is expressed as a homodimer that signals through DAP10 rather than CD94 (Chap. 30). NKG2D binds to two distinct families of ligands, the MHC class I chain-related peptides (MICA and MICB) and the UL-16 binding proteins (ULBP). These ligands are upregulated in cells that have undergone neoplastic transformation, and NK cytotoxicity on tumor cells correlates with tumor expression of MICA and ULBP. The NKG2D differs from other members of the NKG2 family in significant ways. They do not form heterodimers with CD94 on the cell surface. Instead, they are expressed as homodimers, and each homodimer associates noncovalently with a homodimer of the adaptor protein DAP-10. The cytoplasmic tail of DAP-10 carries a YxxM motif, which can recruit the regulatory subunit p85 of phosphatidylinositol-3 kinase and Grb2 (see also Chap. 30).
Congenital hepatic fibrosis (CHF), Caroli’s disease (CD), and polycystic liver disease (PLD) are the three major descriptive categories of fibrocystic liver disease. Caroli’s syndrome (CS) and CD probably represent different presentations of the same continuum. CS refers to CD in association with CHF. CHF/CS and PLD are often part of multisystem disorders associated with fibrocystic renal involvement. These are collectively referred to as “ciliopathies,” since the abnormal proteins involved function on the primary cilium or its basal body. The inheritance pattern of CHF/CS is autosomal recessive, with rare exceptions such as the CHF associated with X-linked oral-facial-digital syndrome type 1. The inheritance pattern of PLD is autosomal dominant; the majority of patients have autosomal dominant polycystic kidney disease (ADPKD) caused by mutations in the PKD1 or PKD2 genes. Autosomal dominant polycystic liver disease (ADPLD), in which PLD is not associated with renal cysts, refers to a genetically distinct entity caused by mutations in the PRKCSH or SEC63 genes. CHF/CS most commonly presents in association with autosomal recessive polycystic kidney disease (ARPKD) caused by mutations in the PKHD1 gene. Multisystem syndromes associated with CHF/CS include Meckel, Bardet–Biedl, and Joubert syndromes and related cerebello-hepatorenal syndromes, renal-hepatic-pancreatic-dysplasia, and ciliary skeletal dysplasias such as Jeune’s chondrodysplasia. Many syndromic ciliopathies display marked genotypic heterogeneity with multiple different genes causing the same disease. This chapter will review the molecular genetic bases of these disorders and provide an overview of novel targeted therapies.
Mutations in the gene encoding hepatocystin/80K-H (PRKCSH) cause autosomal-dominant polycystic liver disease (ADPLD). Hepatocystin functions in the processing of nascent glycoproteins as the noncatalytic beta subunit of glucosidase II (Glu II) and regulates calcium release from endoplasmic reticulum (ER) through the inositol 1,4,5-trisphosphate receptor (IP3R). Little is known, however, on how cells respond to a deficiency of hepatocystin. In this study, we demonstrate that knockdown of hepatocystin induces autophagy, the major intracellular degradation pathway essential for cellular health. Ectopic expression of wild-type hepatocystin, but not pathogenic mutants, rescues the siRNA-induced effect. Our data indicate that the induction of autophagy by hepatocystin deficiency is mediated through mammalian target of rapamycin (mTOR). Despite the resulting severe reduction in Glu II activity, the unfolded protein response (UPR) pathway is not disturbed. Furthermore, the inhibition of IP3R-mediated transient calcium flux is not required for the induction of autophagy. These results provide new insights into the function of hepatocysin and the regulation of autophagy.
Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta. The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between alpha and beta subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha(1)beta(1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the alpha(1)beta(1) complex was confirmed by its high susceptibility to proteases. The beta subunit could be proteolytically removed from the alpha(1)beta(1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the alpha subunit, which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha(1)beta(1) complex in the ER.
Autosomal dominant polycystic liver disease (ADPLD) is a distinct clinical and genetic entity that can occur independently from autosomal dominant polycystic kidney disease (ADPKD). We previously studied two large kindreds and reported localization of a gene for ADPLD to an approximately 8-Mb region, flanked by markers D19S586/D19S583 and D19S593/D19S579, on chromosome 19p13.2-13.1. Expansion of these kindreds and identification of an additional family allowed us to define flanking markers CA267 and CA048 in an approximately 3-Mb region containing >70 candidate genes. We used a combination of denaturing high-performance liquid chromatography (DHPLC) heteroduplex analysis and direct sequencing to screen a panel of 15 unrelated affected individuals for mutations in genes from this interval. We found sequence variations in a known gene, PRKCSH, that were not observed in control individuals, that segregated with the disease haplotype, and that were predicted to be chain-terminating mutations. In contrast to PKD1, PKD2, and PKHD1, PRKCSH encodes a previously described human protein termed "protein kinase C substrate 80K-H" or "noncatalytic beta-subunit of glucosidase II." This protein is highly conserved, is expressed in all tissues tested, and contains a leader sequence, an LDLa domain, two EF-hand domains, and a conserved C-terminal HDEL sequence. Its function may be dependent on calcium binding, and its putative actions include the regulation of N-glycosylation of proteins and signal transduction via fibroblast growth-factor receptor. In light of the focal nature of liver cysts in ADPLD, the apparent loss-of-function mutations in PRKCSH, and the two-hit mechanism operational in dominant polycystic kidney disease, ADPLD may also occur by a two-hit mechanism.
Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose α-subunit contains
the glycosyl hydrolase active site. The function of the β-subunit has yet to be defined, but mutations in the human gene have
been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization
of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the β-subunit of human glucosidase II. The Gtb1 protein was shown
to be a soluble glycoprotein (96–102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together
with the yeast α-subunit homologue Gls2p. Surprisingly, we found that Δgtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient
for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate
that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.
Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of
the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on
a Glcα13Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed
with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out
with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components.
Although optimal binding occurred with GlcManGlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to
the GlcManGlcNAc stage. The α16-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as GlcManGlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding
as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin
proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex
mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable
amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative
model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum
locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.
It has been proposed that in rat and murine tissues glucosidase II (GII) is formed by two subunits, GIIα and GIIβ, respectively,
responsible for the catalytic activity and the retention of the enzyme in the endoplasmic reticulum (ER). To test this proposal
we disrupted genes (gls2α
+ andgls2β
+) encoding GIIα and GIIβ homologs in Schizosaccharomyces pombe. Both mutant cells (gls2α and gls2β) were completely devoid of GII activity in cell-free assays. Nevertheless,N-oligosaccharides formed in intactgls2α cells were identified as Glc2Man9GlcNAc2 and Glc2Man8GlcNAc2, whereasgls2β cells formed, in addition, small amounts of Glc1Man9GlcNAc2. It is suggested that this last compound was formed by GIIα transiently present in the ER. Monoglucosylated oligosaccharides
facilitated glycoprotein folding in S. pombe as mutants, in which formation of monoglucosylated glycoproteins was completely (gls2α) or severely (gls2β and UDP-Glc:glycoprotein:glucosyltransferase null) diminished, showed ER accumulation of misfolded glycoproteins when grown
in the absence of exogenous stress as revealed by (a) induction of binding protein-encoding mRNA and (b) accumulation of glycoproteins bearing ER-specific oligosaccharides. Moreover, the same as in mammalian cell systems, formation
of monoglucosylated oligosaccharides decreased the folding rate and increased the folding efficiency of glycoproteins as pulse-chase
experiments revealed that carboxypeptidase Y arrived at a higher rate but in decreased amounts to the vacuoles of gls2α than to those of wild type cells.
Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.
Asparagine-linked oligosaccharides of glycoproteins undergo extensive modification or "processing" following their attachment to protein. A key step in post-glycosylation processing is the sequential removal of glucose residues from the protein-linked oligosaccharide. We have studied rat liver preparations which catalyze removal of glucose from Glc3Man9GlcNAc, Glc2Man9GlcNAc, and Glc1Man9GlcNAc. Detergent solubilization studies, inhibitor studies, and temperature-activity profiles indicate that at least two distinct glucosidases are present in the membranes. One of these glucosidases removes the distal glucose from Glc3Man9GlcNAc, and the other glucosidase sequentially removes glucose from Glc2Man9GlcNAc and Glc1Man9GlcNAc. The latter glucosidase has been solubilized from the microsomal memrbranes and purified 12-fold. The glucosidases, which are integral membrane proteins, are localized in the rough and smooth microsomes and appear to be located on the cisternal surface of the microsomal vesicles. These glucosidases are suggested to be of biological importance in catalyzing the initial events in the post-glycosylation processing of cellular glycoprotein.
Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.
Glycosylation of asparagine residues of glycoproteins occurs by the transfer of a glucose3mannose9N-acetylglucosamine2 (Glc3Man9GlcNAc2) oligosaccharide from a lipid carrier to the nascent protein. Normally, this transfer is quickly followed by the stepwise removal of the glucose residues which are arranged in the sequence: Glc1 leads to 2Glc1 leads to 3Glc1 leads to 3Man. We now report studies which demonstrate that a lectin-resistant mutant of the BW5147 mouse lymphoma cell line is deficient in the enzyme which removes the two inner glucose residues. This cell line (PHAR2.7) was selected for resistance to the cytotoxic effects of Phaseolus vulgaris leukoagglutinating lectin (Trowbridge, I. S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Glycopeptides prepared from cells equilibrium-labeled with either [2-3H]mannose or [6-3H]galactose were characterized using lectin affinity chromatography, treatment with specific endo- and exoglycosidases, sizing by paper chromatography, and methylation analysis. Approximately 50% of the radioactivity in [3H]mannose-labeled glycopeptides from the mutant cells is present as glucosylated high mannose-type oligosaccharides whereas parent cell glycopeptides labeled under similar conditions lack detectable amounts of these species. Using [3H]galactose labeling, the major glucosylated oligosaccharides were identified as Glc2Man9GlcNAc2 and Glc2Man8GlcNAc2. In vitro enzyme assays demonstrated that the mutant cells cannot remove either of the two inner 1 leads to 3-linked glucose residues. Removal of the outer 1 leads to 3-linked glucose is normal. We conclude from these data that the PHAR2.7 cell line is deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the oligosaccharides of newly glycosylated proteins.
The UDP-Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein-linked Man7-9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a unique property: it glucosylates misfolded, but not native, glycoproteins. It has been proposed that the glucosyltransferase participates, together with calnexin, in the control mechanism by which only properly folded glycoproteins can exit from the endoplasmic reticulum. In this paper it is demonstrated that the glucosyltransferase recognizes two elements in the acceptor substrates: the innermost N-acetylglucosamine unit of the oligosaccharide and protein domains exposed in denatured, but not in native, conformations. Both determinants have to be covalently linked. In many cases the first element is not accessible to macromolecular probes in native conformations. Concerning the protein domains, it is demonstrated here that the glucosyltransferase interacts with hydrophobic amino acids exposed in denatured conformations. More disordered conformations, i.e. those exposing more hydrophobic amino acids, were found to be those having higher glucose acceptor capacity. It is suggested that both accessibility of the innermost N-acetylglucosamine unit and binding to hydrophobic patches determine the exclusive glucosylation of misfolded conformations by the glucosyltransferase.
To determine the role of N-linked oligosaccharides in the folding of glycoproteins, we analyzed the processing of in vitro translated influenza hemagglutinin (HA) in dog pancreas microsomes. We found that binding to calnexin, a membrane-bound molecular chaperone, was specific to molecules that possessed monoglucosylated core glycans. In the microsomes, these were generated either by glucose removal from the original triglucosylated core oligosaccharide by glucosidases I and II or by reglucosylation of already unglucosylated high mannose glycans. Release of fully folded HA from calnexin required the removal of the remaining glucose by glucosidase II. The results provided an explanation for trimming and reglucosylation activities in the endoplasmic reticulum and established a direct correlation between glycosylation and folding.
Many soluble resident proteins of the endoplasmic reticulum share a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticulum (ER) by bulk flow and are recognized and sorted in the Golgi apparatus by binding to a specific KDEL-receptor, which returns them to the ER. Through biochemical, morphological and mutational analysis we have studied the mechanisms that determine the localization of calreticulin, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling established the ER localization of calreticulin in transfected and nontransfected COS cells. Although the ER cisternae in transfected cells were enormously dilated and heavily labelled by gold particles we found no significant label in any other compartment. In vivo pulse chase experiments with [35S]methionine followed by biochemical fractionation of calreticulin overexpressing COS cells (50- to 100-fold) revealed that only a minor part of labelled calreticulin leaves the ER. Retrieval from the Golgi was confirmed by a partial redistribution of the endogenous KDEL-receptor as shown by double immunofluorescence. These data suggest a KDEL-independent retention of calreticulin in the ER. Further supporting evidence has come from morphological in vivo studies using calreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasing the temperature-dependent transport block for the viral G-protein resulted in a small but significant appearance of calreticulin in a post-ER compartment. In contrast a calreticulin mutant, which lacked the Ca(2+)-binding domain but included the KDEL sequence, could escape from the ER to a much higher extent. Secretion of the nonmutated calreticulin was very low (1-2% of total calreticulin in 3 hours) compared to the mutated form (18% in 3 hours). Deletion of the KDEL sequence led to an increase in secretion to 29% over a 3 hour period, which is much less than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retention/retrieval mechanisms for calreticulin: one providing for direct retention in the ER with a very high capacity and having Ca(2+)-dependent properties; the other a KDEL-based retrieval system for escaped calreticulin present in the Golgi apparatus.
Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds
are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin
binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble
calnexin was created. Incubation of soluble calnexin with a mixture of Glc0–3Man9GlcNAc2 oligosaccharides resulted in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5–7GlcNAc2 oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue,
at least one of the terminal mannose residues was important for binding. To assess the involvement of oligosaccharide-protein
interactions in complexes of calnexin and newly synthesized glycoproteins, α1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested
with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without
disrupting the association with calnexin. Furthermore, the addition of 1 M α-methyl glucoside or α-methyl mannoside had no
effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide
binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.
We have identified a new human Ca(2+)-binding protein that is specifically localized in the endoplasmic reticulum (ER). The protein is termed ERC-55, i.e. ER calcium-binding protein of 55 kDa. ERC-55 is a single copy gene and is encoded by an approximately 1900-base mRNA, which shows a ubiquitous expression pattern. The ERC-55 protein comprises an amino-terminal signal sequence followed by six copies of the EF-hand Ca2+ binding motif. Ca2+ binding was demonstrated directly for recombinant ERC-55 using the 45Ca2+ overlay technique. The carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL) is required for retention of ERC-55 in the ER. Deletion of HDEL results in slow secretion into the medium. In pulse-chase experiments, approximately 50% of the HDEL deletion mutant is secreted, whereas no detectable secretion is observed with the wild-type protein. This represents the first example of an endogenous human protein that is retained in the ER by an HDEL rather than Lys-Asp-Glu-Leu (KDEL) carboxyl-terminal tetrapeptide. Comparative sequence analysis indicates that ERC-55, together with the recently identified protein reticulocalbin (Ozawa and Muramatsu, 1993), constitute a new subfamily of the EF-hand superfamily of Ca(2+)-binding proteins that are specifically located in the ER.
The erd2 protein is the receptor responsible for recycling proteins bearing the carboxyl-terminal sequence KDEL (single-letter amino acid code) to the endoplasmic reticulum, following their loss from that organelle by the process of forward transport. To study the interaction of erd2p with the sequence KDEL we have reconstituted binding of erd2p to its ligand in vitro. Binding in vitro exhibits the same sequence specificity as retention of lumenal proteins in vivo and is strikingly sensitive to pH. Our results raise the possibility that erd2p-mediated sorting of lumenal endoplasmic reticulum proteins is facilitated by the pH differences between compartments of the secretory pathway.
A novel Ca(2+)-binding protein, tentatively designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an M(r) of 44,000 as revealed by biochemical analysis and immunofluorescence staining. The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids. The protein has six repeats of a domain containing the high affinity Ca(2+)-binding motif, the EF-hand. Although oxygen-containing amino acids important for the positioning of Ca2+ are conserved in all six domains, the conserved glycine residues in the central portion of the EF-hand motif are absent in three of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells binds Ca2+. The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This is similar to the Lys-Asp-Glu-Leu sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells. A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into medium in transient expression assays.
Calnexin (CNX) and calreticulin (CRT) are molecular chaperones that bind preferentially to monoglucosylated trimming intermediates of glycoproteins in the endoplasmic reticulum. To determine their role in the maturation of newly synthesized glycoproteins, we analyzed the folding and trimerization of in vitro translated influenza hemagglutinin (HA) in canine pancreas microsomes under conditions in which HA's interactions with CNX and CRT could be manipulated. While CNX bound to all folding intermediates (IT1, IT2 and NT), CRT was found to associate preferentially with the earliest oxidative form (IT1). If HA's binding to CNX and CRT was inhibited using a glucosidase inhibitor, castanospermine (CST), the rate of disulfide formation and oligomerization was doubled but the overall efficiency of maturation of HA decreased due to aggregation and degradation. If, on the other hand, HA was arrested in CNX-CRT complexes, folding and trimerization were inhibited. This suggested that the action of CNX and CRT, like that of other chaperones, depended on an 'on-and-off' cycle. Taken together, these results indicated that CNX and CRT promote correct folding by inhibiting aggregation, preventing premature oxidation and oligomerization, and by suppressing degradation of incompletely folded glycopolypeptides.
Trimming of glucoses from N-linked core glycans on newly synthesized glycoproteins occurs sequentially through the action of glucosidases I and II in
the endoplasmic reticulum (ER). We isolated enzymatically active glucosidase II from rat liver and found that, in contrast
with previous reports, it contains two subunits (α and β). Sequence analysis of peptides derived from them allowed us to identify
their corresponding human cDNA sequences. The sequence of the α subunit predicted a soluble protein (104 kDa) devoid of known
signals for residence in the ER. It showed homology with several other glucosidases but not with glucosidase I. Among the
homologues, we identified a Saccharomyces cerevisiae gene, which we showed by gene disruption experiments to be the functional catalytic subunit of glucosidase II. The disrupted
yeast strains had no detectable growth defect. The sequence of the β subunit (58 kDa) showed no sequence homology with other
known proteins. It encoded a soluble protein rich in glutamic and aspartic acid with a putative ER retention signal (HDEL)
at the C terminus. This suggested that the β subunit is responsible for the ER localization of the enzyme.
Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.
Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5-8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5-5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.
CD45 is an abundant, highly glycosylated transmembrane protein-tyrosine phosphatase expressed on hematopoietic cells. Herein
we demonstrate that two proteins of 116 kDa and 80 kDa copurify with CD45 from mouse T cells. Microsequence analysis of the
116-kDa protein revealed high similarity to an incomplete human open reading frame that has been suggested to correspond to
the catalytic α-subunit of glucosidase II. We determined the nucleotide sequence of the mouse cDNA and observed that it encodes
a protein product nearly identical to its human homologue and shares an active site consensus sequence with Family 31 glucosidases.
Amino acid sequencing of the 80-kDa protein, followed by molecular cloning, revealed high homology to human and bovine cDNAs
postulated to encode the β-subunit of glucosidase II. Antisera developed to the mouse β-subunit allowed us to demonstrate
that the interaction between CD45 and glucosidase II can be reconstituted in vitro in an endoglycosidase H-sensitive manner. The strong interaction between glucosidase II and CD45 may provide a paradigm for
investigating novel aspects of the biology of these proteins.
Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N-linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP-glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP-glucose enhanced the amount of folded transferrin and reduced the disulfide-linked aggregates. Analysis of transferrin folding in briefly heat-treated microsomes revealed that UDP-glucose was also effective in elimination of heat-induced misfolding. Incubation of the microsomes with an alpha-glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.
It has been proposed that in rat and murine tissues glucosidase II (GII) is formed by two subunits, GIIalpha and GIIbeta, respectively, responsible for the catalytic activity and the retention of the enzyme in the endoplasmic reticulum (ER). To test this proposal we disrupted genes (gls2alpha(+) and gls2beta(+)) encoding GIIalpha and GIIbeta homologs in Schizosaccharomyces pombe. Both mutant cells (gls2alpha and gls2beta) were completely devoid of GII activity in cell-free assays. Nevertheless, N-oligosaccharides formed in intact gls2alpha cells were identified as Glc(2)Man(9)GlcNAc(2) and Glc(2)Man(8)GlcNAc(2), whereas gls2beta cells formed, in addition, small amounts of Glc(1)Man(9)GlcNAc(2). It is suggested that this last compound was formed by GIIalpha transiently present in the ER. Monoglucosylated oligosaccharides facilitated glycoprotein folding in S. pombe as mutants, in which formation of monoglucosylated glycoproteins was completely (gls2alpha) or severely (gls2beta and UDP-Glc:glycoprotein:glucosyltransferase null) diminished, showed ER accumulation of misfolded glycoproteins when grown in the absence of exogenous stress as revealed by (a) induction of binding protein-encoding mRNA and (b) accumulation of glycoproteins bearing ER-specific oligosaccharides. Moreover, the same as in mammalian cell systems, formation of monoglucosylated oligosaccharides decreased the folding rate and increased the folding efficiency of glycoproteins as pulse-chase experiments revealed that carboxypeptidase Y arrived at a higher rate but in decreased amounts to the vacuoles of gls2alpha than to those of wild type cells.
Calnexin and calreticulin are lectin‐like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N‐linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N‐linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N‐glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin‐like interaction, and not a protein‐protein interaction, played the central role in stabilizing RNase‐calnexin/calreticulin complexes.
Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum, Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created, Incubation of soluble calnexin with a mixture of Glc(0-3)Man(9)GlcNAc(2) oligosaccharides resulted in specific binding of the Glc(1)Man(9)GlcNAc(2) species, Furthermore, Glc(1)Man(5-7)GlcNAc(2) oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding, To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha(1)-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin, Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability, These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction, However, it is likely that the binding of Glc(1)Man(9)GlcNAc(2) oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.
Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.
The microsomal glucosidases which act on the oligosaccharide that contains 2 N-acetylglucosamine, 9 mannose and 1–3 glucose residues have been studied. Two fractions were separated by differential solubility in detergent and phosphate solutions or by gel filtration. One of the fractions removed glucose from the oligosaccharide containing three glucose residues, and another fraction acted on the compounds containing one and two glucose residues. Both fractions were free of mannosidase. Some properties of the enzymes are described.
It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase.
The endoplasmic reticulum (ER) contains a family of luminal proteins (reticuloplasmins) that are normally excluded from the secretory pathway. However, reticuloplasmins are efficiently secreted when murine fibroblasts are treated with calcium ionophores. The secreted and cellular forms of endoplasmin are clearly distinguishable on the basis of gel mobility and endoglycosidase H sensitivity. Reticuloplasmin secretion leads to the depletion of the proteins from the ER and their accumulation in the Golgi apparatus. The stress response to calcium ionophore induces reaccumulation of reticuloplasmins in the ER and suppresses their secretion. Secretion is also associated with changes in the structure and distribution of the ER. These observations show that perturbation of cellular calcium levels leads to the breakdown of the mechanism for ER retention of reticuloplasmins and suggest a role for calcium ions in their sorting from secretory proteins.
An acidic phosphoprotein of Mr 80,000, the 80K protein, is a substrate for protein kinase C in fibroblasts and epidermal carcinoma cells. We purified the 80K protein from human squamous carcinoma Ca9-22 cells and fractionated it into two distinct molecular species, designated the 80K-L and 80K-H proteins. The amino acid sequences of the NH2-terminal region and cyanogen bromide-cleaved fragments of the 80K-H protein were determined and a corresponding oligonucleotide sequence was synthesized. Using this as a probe, two cDNA clones, lambda 80H-1 and lambda 80H-2, were selected from a lambda gt10 cDNA library from human A431 cells. The nucleotide sequence has an open reading frame of 1581 nucleotides encoding a protein of 527 amino acids. The deduced amino acid sequence revealed an extremely Glu-rich region. RNA blot analysis with the lambda 80H-1 cDNA clone detected two polyadenylated transcripts of 2.3 and 3.5 kb in Ca9-22 cells. Spot blot hybridization using flow-sorted human chromosomes provided evidence that the gene (G19P1) encoding 80K-H protein maps to human chromosome 19.
Glucosidase II removes the inner two alpha-linked glucose residues from freshly transferred Asn-linked oligosaccharide chains in the endoplasmic reticulum. This enzyme, whose activity could be measured by the hydrolysis of an artificial substrate (p-nitrophenyl alpha-D-glucopyranoside), was purified 240-fold from a rat liver microsome fraction by DEAE-cellulose, concanavalin A-Sepharose 4B, and hydroxylapatite chromatography. The apparent molecular weight of the active polypeptide was 123 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Glucosidase II has at least one high-mannose oligosaccharide chain that can be cleaved by endoglycosidase H. Trypsin readily cleaved the 123-kilodalton (kDa) form of glucosidase II into a fully active 73-kDa core. The pattern of this cleavage suggests a domain structure for this enzyme. We demonstrate that trypsin first removes a glycosylated 25-kDa domain to yield an apparently unglycosylated 98-kDa product which is further cleaved to yield the active 73-kDa core.
The processing of the oligosaccharide precursor chain, (GlcNAc) 2 (Man) 9 (Gle) 3 , of N ‐glycosylated glyco‐proteins starts with the action of glucosidase I which excises the terminal (α1–2)‐linked glucose residue. Glucosidase II removes the two inner (α1–3)‐linked glucose residues.
We have purified glucosidase II to homogeneity from pig kidney microsomes. The enzyme is a glycoprotein and contains a single type of subunit of molecular mass ∼ 100 kDa. The native enzyme is probably a tetramer. It cleaves glucosidic α1–3 and α1–4, but not α1–1, α1–2 or α1–6 bonds and lacks 2‐mannosidase and glucosidase I activity. The pH optimum is between 6.0 and 7.5. Specific antibodies against the native enzyme and the denatured subunit were prepared. By activity measurements and immune blotting, a similar enzyme was found in rat liver. In the fractionated rat liver, the enzyme was localized in the lumen of the endoplasmic reticulum, probably loosely bound to the inner face of the membrane. Purified Golgi fractions contained only low levels of the enzyme.
A glucosidase preparation with activity toward certain glucose-containing oligosaccharides was partially purified from calf liver membranes by Triton X-100 solubilization and DEAE-cellulose and hydroxylapatite chromatography. The enzyme preparation hydrolyzed the glucose residues from (glucose)1,(mannose)9(N-acetylglucosamine)1, and (glucose)2(mannose) 9(N-acetylglucosamine)1 but was totally inactive toward (glucose)3(mannose)9(N-acetylglucosamine) 1. In contrast, crude membrane preparations of the calf liver were active toward all three substrates. The partially purified enzyme had a pH optimum of 6.7 and was very unstable in the absence of added 20% glycerol. The rate of glucose release from the one-and two-glucose-containing oligosaccharides was significantly decreased when four or five of the mannose residues were first removed from the substrate. The release of glucose from (glucose)1(mannose)9(N-acetylglucosamine)1 was inhibited by p-nitrophenyl-α-d-glucoside much more effectively than by p-nitrophenyl-β-d-glucoside, suggesting that this glucose residue may be linked α to the mannose residue. We conclude that during oligosaccharide processing at least two different glucosidases are involved in glucose removal.
Further work on microsomal glucosidases of rat liver has confirmed that at least two enzymes are involved in the removal of glucose from the glucose-containing oligosaccharide. One acts on the oligosaccharide containing three glucose residues and another on the oligosaccharide which has one or two glucoses.
The glucosidase which acts on (Glc)2(Man)9(GlcNAc)2 could be purified with a ConcanavalinA—Sepharose column followed by electrofocusing. This purified preparation was active on the oligosaccharide containing one or two glucoses. Heat inactivation and inhibition by disaccharides was parallel for both activities. Inhibition of the glucosidase active on (Glc)3(Man)9(GlcNAc)2 was obtained with kojibiose which has an α 1–2 linkage, while the glucosidase acting on (Glc)1–2(Man)9-(GlcNAc)2 was inhibited by nigerose (α 1–3 linkage), maltose (α 1–4 linkage) and glucose at a higher concentration. None of the β anomers inhibited. These results are consistent with an α configuration of the three glucoses of the dolichyl-dinhosphate-linked oligosaccharide. Kojibiose was found to inhibit glucosidase action not only on the free oligosaccharide but also on protein-bound one.
Increasing evidence shows that calnexin, a membrane-bound chaperone in the endoplasmic reticulum, is a lectin that binds to newly synthesized glycoproteins that have partially trimmed N-linked oligosaccharides. It specifically attaches to core glycans from which two glucoses have been removed by glucosidases I and II. Several recent reports suggest, however, that it can also bind to proteins devoid of N-linked glycans. To investigate the extent of glycan-independent binding, we have analyzed two mutant cell lines (Lec 23 and PhaR2.7) that are unable to process the core glycans because they lack glucosidase I or glucosidase II, respectively. In contrast to parental cell lines, calnexin binding of substrate proteins was found to be virtually nonexistent in these cells. Neither cellular nor viral proteins associated with the chaperone. It was concluded that glycans are crucial for calnexin association and that the vast majority of substrate proteins are therefore glycoproteins.
We isolated full-length cDNA clones from size-fractionated cDNA libraries of human immature myeloid cell line KG-1, and the coding sequences of 40 genes were newly predicted. A computer search of the GenBank/EMBL databases indicated that the sequences of 14 genes were unrelated to any reported genes, while the remaining 26 genes carried some sequences with similarities to known genes. Significant transmembrane domains were identified in 17 genes, and protein motifs that matched those in the PROSITE motif database were identified in 11 genes. Northern hybridization analysis with 18 different cells and tissues demonstrated that 10 genes were apparently expressed in a cell-specific or tissue-specific manner. Among the genes predicted, half were isolated from the medium-sized cDNA library and the other half from the small-sized cDNA library, and their average sizes were 4 kb and 1.4 kb, respectively. As judged by Northern hybridization profiles, small-sized cDNAs appeared to be expressed more ubiquitously and abundantly in various tissues, compared with that of medium-sized cDNAs.
CD45 is a protein-tyrosine phosphatase expressed on all cells of hematopoietic origin. In an attempt to further characterize CD45 function, we set out to identify molecule(s) that specifically associate with CD45. A 116-kDa protein was detected in immunoprecipitates from CD45+ cells but not CD45- cells. The association between CD45 and this 116-kDa protein can be reconstituted by mixing lysates from CD45- cell lines with purified CD45. p116 appears to associate with CD45 through the external, transmembrane, or membrane-proximal region of CD45 since p116 is associated with a mutant form of CD45 possessing a truncated cytoplasmic domain. The association of p116 with CD45 is not isoform-specific as p116 associates equally well with various CD45 isoforms. We have determined that p116 is a tyrosine-phosphorylated glycoprotein and that it is associated with CD45 in all hematopoietic cells examined. Because of its broad distribution, it is possible that identification of p116 will provide additional insight into the function of CD45 in lymphoid as well as non-lymphoid hematopoietic cells.
Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.
Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.
Calnexin is a membrane protein of the endoplasmic reticulum that associates transiently with newly synthesized N-linked glycoproteins in vivo. Using defined components, the binding of ribonuclease B (RNase B) Man7-Man9 glycoforms to the luminal domain of calnexin was observed in vitro only if RNase B was monoglucosylated. Binding was independent of the conformation of the glycoprotein. Calnexin protected monoglucosylated RNase B from the action of glucosidase II and PNGase F but not from that of Endo H, which completely released the protein from calnexin. These observations directly demonstrate that calnexin can act exclusively as a lectin.
Glucosidase II is an ER resident glycoprotein involved in the processing of N-linked glycans and probably a component of the ER quality control of glycoproteins. For cloning of glucosidase II cDNA, degenerate oligonucleotides based on amino acid sequences derived from proteolytic fragments of purified pig liver glucosidase II were used. An unamplified cDNA library from pig liver was screened with a 760 bp glucosidase II specific cDNA fragment obtained by RT-PCR. A 3.9 kb glucosidase II cDNA with an open reading frame of about 2.9 kb was obtained. The glucosidase II sequence did not contain known ER retention signals nor hydrophobic regions which could represent a transmembrane domain; however, it contained a single N-glycosylation site close to the amino terminus. All studied pig and rat tissues exhibited an mRNA of approximately 4.4 kb with varying tissue expression levels. The authenticity of the identified cDNA with that coding for glucosidase II was proven by overexpression in CHO cells. Mouse lymphoma PHAR 2.7 cells, deficient in glucosidase II activity, were shown to be devoid of transcripts.
Glucosidase II is a processing enzyme of the endoplasmic reticulum that functions to hydrolyze two glucose residues in immature
N-linked oligosaccharides attached to newly synthesized polypeptides. We previously reported the cDNA cloning of the α- and
β-subunits of mouse glucosidase II from T cells following copurification of these proteins with the highly glycosylated transmembrane
protein-tyrosine phosphatase CD45. Subsequent examination of additional cDNA clones, coupled with partial genomic DNA sequencing,
has revealed that both subunits are encoded by gene products that undergo alternative splicing in T lymphocytes. The catalytic
α-subunit possesses two variably expressed segments, box A1, consisting of 22 amino acids located proximal to the aminoterminus,
and box A2, composed of 9 amino acids situated between the amino-terminus and the putative catalytic site in the central region
of the molecule. Box B1, a variably expressed 7 amino acid segment in the β-subunit of glucosidase II, is located immediately
downstream of an acidic stretch near the carboxyl-terminus. Screening of reverse transcribed RNA by polymerase chain reaction
confirms the variable inclusion of each of these segments in transcripts obtained from a panel of T-lymphocyte cell lines.
Thus, distinct isoforms of glucosidase II exist that may perform specialized functions.
- Sonnichsen