Glycobiology, 2000, Vol. 10, No. 5 487-492
© 2000 Oxford University Press
Two distinct domains of the ß-subunit of glucosidase II interact with the catalytic 
-subunit
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received on August 19, 1999; revised on November 1, 1999; accepted on December 8, 1999.
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Abstract |
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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
-subunit and a ß-subunit of unknown function, both of which are highly conserved in mammals. Since the ß-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 ß-subunit protein responsible for mediating the association with the -subunit. By screening a panel of recombinant ß-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 ß-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. Key words: ER quality control/glucosidase II/glycoprotein processing/N-glycosylation
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Introduction |
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The biochemical activity of mammalian glucosidase II (GII) has been extensively characterized since its identification in microsome fractions two decades ago (Ugalde et al., 1978
, 1979, 1980; Grinna and Robbins, 1979; Michael and Kornfeld, 1980; Burns and Touster, 1982; Brada and Dubach, 1984; Hino and Rothman, 1985). This processing enzyme of the endoplasmic reticulum (ER) functions after glucosidase I in early glycoprotein biogenesis to catalyze the hydrolysis of the two 1,3-linked glucose residues present on all Asn-linked precursor oligosaccharides. Why this seemingly redundant yet highly conserved pathway of glucose addition followed by removal should be invoked in oligosaccharide maturation was not immediately apparent. More recently, however, a number of elegant studies have revealed an intimate link between glucose processing events in the ER and the coordination of protein folding and assembly. It is clear from these studies that cleavage of the first 1,3-linked glucose by GII is essential for allowing nascent polypeptides to interact with the membrane-bound ER resident calnexin and its luminal homologue, calreticulin (Hammond et al., 1994; Hebert et al., 1995; Ora and Helenius, 1995; Wada et al., 1997). Calnexin and calreticulin are members of a new family of lectins with specificity for monoglucosylated precursor oligosaccharides (Ware et al., 1995; Spiro et al., 1996; Zapun et al., 1997; Vassilakos et al., 1998). The interaction of polypeptides bearing monoglucosylated carbohydrates with calnexin/calreticulin initiates a cycle of protein folding that is distinct from that provided by classical chaperones in that its initiation appears to be solely dependent on the glycosylation status, rather than the folding status, of the substrate protein (Hammond et al., 1994; Rodan et al., 1996; Zapun et al., 1997). Subsequent cycles of folding are initiated when GII cleaves the second 1,3-linked glucose, generating a nonglucosylated oligosaccharide product that transiently dissociates from calnexin/calreticulin (Hebert et al., 1995, 1996; Rodan et al., 1996; Wada et al., 1997). Nonglucosylated glycoproteins that have not yet achieved their proper conformations are reglucosylated by the UDP-glucose:glycoprotein glucosyltransferase (Sousa et al., 1992), allowing for another cycle of folding (Hebert et al., 1995, 1996; Wada et al., 1997). Once the final conformation is achieved, the glycoprotein is recognized by GII but not the glucosyltransferase (Sousa and Parodi, 1995; Zapun et al., 1997), permitting its egress from the ER. Thus, a multiprotein "quality control apparatus" exists in the endoplasmic reticulum in which GII participates in the initiation and propagation of folding cycles via deglucosylation.
Interestingly, many of these insights into the central function of GII in glycoprotein maturation were acquired before the true molecular identity of the protein was known. In a recent attempt to reconcile this, sequence information was obtained on GII purified from rat liver as a heterodimer of two noncovalently linked but strongly associated subunits (GII and GIIß) (Trombetta et al., 1996). In addition, we recently isolated the mouse cDNAs encoding these two proteins following their copurification with the transmembrane protein-tyrosine phosphatase CD45 from T-cell lysates (Arendt and Ostergaard, 1997). Homology searches revealed that mouse GII and a recently cloned pig homologue (Flura et al., 1997) share, respectively, 90% and 92% protein sequence identity to a human cDNA clone in the GenBank database (Nagase et al., 1995). GII lacks a transmembrane segment and a known retention/retrieval motif, however the mouse ß-subunit coding sequence has a C-terminal His-Asp-Glu-Leu (HDEL) sequence (Arendt and Ostergaard, 1997) that is conserved in its highly homologous human counterpart (Sakai et al., 1989). Since this motif has been shown to be sufficient for recognition by the KDEL receptor (Ozawa and Muramatsu, 1993; Wilson et al., 1993), it has been postulated that GIIß functions to couple the -subunit to this ER-retrieval mechanism (Trombetta et al., 1996). Interestingly, GIIß also possesses two putative EF hand domains and a region of negatively charged repeats, features thought to confer Ca2+ binding properties to other ER proteins (Booth and Koch, 1989; Sonnichsen et al., 1994; Weis et al., 1994). As there is evidence for the involvement of a calcium matrix in the retention of resident ER proteins (Booth and Koch, 1989; Sonnichsen et al., 1994; Weis et al., 1994), it is possible that GIIß invokes multiple mechanisms to ensure localization of GII to the endoplasmic reticulum.
In the present study, we have utilized a panel of GIIß fusion proteins to identify functional domains that mediate binding of this protein to the catalytic subunit. The results of our analysis indicate the presence of two distinct interaction domains, ID1 and ID2, in GIIß. We hypothesize that synergy in the binding activity of these two sites accounts for the highly stable intermolecular association of the - and ß-subunits of the GII heterodimer.
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Results |
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Two distinct domains of GIIß are capable of associating with GII
activityA schematic of primary sequence features of the ß-subunit of mouse GII is presented in Figure 1. This protein is synthesized with an amino-terminal leader peptide which is absent in the mature form of the polypeptide (Arendt and Ostergaard, 1997
). Cysteine residues are present at high concentrations near the N- and C-termini, but not in the intervening region. The central region of the molecule contains two closely apposed putative EF-hand domains. There is also an acidic stretch of glutamic acid repeats and a seven amino acid alternatively-spliced sequence (Arendt et al., 1999) which are flanked by proline-rich elements.
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Since nothing is known about the sites through which GIIß associates with GII
, we sought to map this interaction by making use of a sensitive and quantitative assay for GII activity. In this assay, samples are incubated with the chromogenic substrate p-nitrophenyl -D-glucopyranoside, which is specifically hydrolyzed by GII at neutral pH (Burns and Touster, 1982; Reitman et al., 1982). In preliminary experiments, we used this assay to determine if GII activity can be detected in immunoprecipitates of GIIß from mouse SAKR cells, the T-lymphoma line from which we originally purified and cloned the two subunits of GII (Arendt and Ostergaard, 1997). Immune complexes were captured on Protein A beads, washed, and incubated with the reaction mix. GII activity was quantitated by measuring the optical density of triplicate samples at 405 nm. As illustrated in Figure 2, antisera 80.1 and 80.2, which recognize two distinct regions of GIIß (Figure 1), are capable of coprecipitating the enzymatic activity encoded by GII. Preimmune sera from the two rabbits, however, fail to retrieve GII activity from the cell lysates. The amount of GII activity coimmunoprecipitated by the 80.1 and 80.2 antisera in this experiment is roughly equivalent to 1% of the activity in the total cell lysate (Figure 2). These data support the notion that the - and ß-subunits of GII are stably associated and indicate that neither antiserum blocks the ability of these two subunits to interact.
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To identify regions of GIIß that mediate the association with the catalytic subunit, we expressed segments of mouse GIIß spanning the entire length of the molecule as GST-fusion proteins in E.coli. These purified fusion proteins were incubated with SAKR cell extracts and then captured by addition of glutathione matrix. In vitro association of GII
with the recombinant GIIß proteins was detected by measuring GII enzymatic activity. The results of this initial binding study are presented in Figure 3. Two nonoverlapping constructs, GSTß(N-258) and GSTß(273-C), were capable of precipitating GII activity from SAKR lysates. In contrast, no activity was found to associate with GSTß(112–258), GSTß(112–344), GSTß(437-C), or GST alone. Confirmation that this activity is due to association with GII was obtained when were unable to detect any activity associated with the recombinant fusion proteins incubated with lysates prepared from the GII-negative cell line PHAR2.7 (Reitman et al., 1982; Flura et al., 1997) (data not shown). We therefore conclude that two independent portions of the ß-subunit of GII are capable of associating with the catalytic subunit. The presence of two such binding domains in GIIß may explain the high stability of this putative heterodimeric complex (Trombetta et al., 1996).
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Amino acids 1–118 of GIIß specify an N-terminal binding site for GII
To map more thoroughly the N-terminal binding site of GIIß, we constructed a series of progressively truncated versions of the GSTß(N-258) construct. As revealed by the binding experiment presented in Figure 4, removal of the first 19, 40, or 59 amino acids of GSTß(N-258) ablated the association with GII
. This demonstrates that the N-terminus of GIIß (minus the leader peptide) is essential in mediating the interaction with the catalytic subunit. In contrast, truncating the opposite end of GSTß(N-258) initially resulted in full retention of binding function. In fact, construct GSTß(N-118), deleting 140 amino acids at the C-terminus of GSTß(N-258), displayed robust binding activity. However, truncation of an additional 20 amino acids from this construct resulted in a GST fusion protein incapable of associating with GII, as did further truncations in this direction. Significantly, interaction domain 1 (ID1), as defined by construct GSTß(N-118), displays minimal overlap with GSTß(112–258), the fusion protein against which antiserum 80.1 was generated (Figure 1). This is consistent with our finding that antiserum 80.1, but not construct GSTß(112–258), precipitates GII activity (Figures 2, 3). From these data we conclude that a segment consisting of no more than 118 amino acids at the N-terminus of GIIß defines a domain of interaction with GII.
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A second binding site for GII
is contained within amino acids 273–400 of GIIßThe data presented in Figure 3 revealed a second interaction domain (ID2) within GIIß that binds to GII
. Interestingly, the region encompassed by this domain includes an alternatively spliced 7 amino acid segment that defines two isoforms of GIIß (Figure 1). Our evidence that this portion of the molecule is subject to alternative splicing is derived from examination of multiple cDNA library clones and sequencing of genomic DNA to identify donor and acceptor splice sites (Arendt et al., 1999). To address the possible function of this 7 amino acid segment in influencing the association of the - and ß-subunits of GII, GST fusion proteins were generated that either contained or lacked this segment. As the results of the binding studies presented in Figure 5 demonstrate, this segment does not modulate the association of the two subunits, as detected in our in vitro system. The results of Figure 5 also show that deletion of 114 C-terminal amino acids from GSTß(273-C) to generate construct GSTß(273–400) preserves the ability to precipitate GII activity. However, further truncation of 56 amino acids, yielding construct GSTß(273–344), ablates this binding activity, consistent with the results obtained with construct GSTß(112–344) (Figure 3). The finding that 114 residues at the C-terminus of GIIß are excluded from ID2 is in agreement with the ability of antiserum 80.2, generated against 78 residues at the C-terminus (Figure 1), to coprecipitate GII activity (Figure 2). We thus conclude that residues 273–400 of GIIß contain a second domain sufficient for associating with the catalytic subunit and that a region within segment 345–400 is necessary for this association.
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Since we identified two different regions of GIIß that are capable of binding to GII
, it is possible that ID1 and/or ID2 represent homooligomerization domains through which the GIIß GST-fusion proteins are capable of associating with additional molecules of full-length GIIß present in the cell extracts, thereby indirectly linking the recombinant proteins to GII. This is likely not occurring since we could not detect any full-length, endogenous GIIß bound to any of the recombinant fusion proteins after incubation with cell lysates (data not shown). Taken together, these results fail to provide evidence for homooligomerization of GIIß and support a model whereby ID1 and ID2 interact directly with the catalytic subunit. |
Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Biochemical characterization of glucosidase II has indicated that it exists as a putative heterodimeric complex of two strongly associated, non-covalently-linked soluble subunits (Trombetta et al., 1996
). The -subunit, comprising a 116 kDa protein in mouse, possesses a catalytic consensus sequence (Arendt and Ostergaard, 1997) and appears to represent the functional enzymatic unit of the complex, based on a gene-disruption experiment in S.cerevisiae (Trombetta et al., 1996). The ß-subunit, an alternatively spliced protein (Arendt et al., 1999) that migrates as a doublet at ~80 kDa on polyacrylamide gels, contains a number of potentially interesting primary sequence features, including two putative EF-hand domains, an acidic stretch, proline- and cysteine-rich elements, and a C-terminal HDEL tetrapeptide (Arendt and Ostergaard, 1997). The function of the ß-subunit is at present unclear however a recent study demonstrated that ablation of this subunit in S.pombe results in a loss of GII activity and function (D'Alessio et al., 1999) suggesting an essential role for this subunit.
We sought to gain insight into the function of GIIß by defining the regions of the molecule that interact with the catalytic subunit of GII. By generating a large panel of native recombinant GST-fusion proteins, we identified two distinct interaction domains, designated ID1 and ID2, that are capable of associating with GII. ID1 is an N-terminal stretch of 118 amino acids that terminates well upstream of the putative EF-hand domain of GIIß. Although ID1 spans the entire N-terminal Cys-rich element of GIIß, disulfide linkages do not mediate its interaction with the -subunit, since the precipitation of GII by GSTß(N-118), as visualized on Coomassie stained gels, is not significantly impaired by addition of 20 mM iodoacetamide to the lysis buffer (data not shown). This is consistent with a previous study that failed to detect covalent intermolecular linkages between the two subunits of GII (Trombetta et al., 1996). In contrast to the N-terminal location of ID1, ID2, as defined by our mapping studies, is contained within residues 273–400 of GIIß. The region encompassed by ID2 contains an acidic stretch flanked by proline-rich elements but excludes the C-terminal Cys-rich element. Although a segment of 7 variably expressed amino acids is located near a 127 amino-acid region of ID2 possessing a critical binding determinant (Figure 5), our binding studies failed to support a role for these 7 residues in modulating the association with the -subunit. However, we cannot exclude the possibility that this segment influences the interaction of the two subunits in vivo in the context of full-length GIIß.
That our experiments implicate not one but two domains in GIIß in the interaction with GII is consistent with biochemical evidence that the - and ß-subunits of GII are highly refractory to dissociation (Trombetta et al., 1996). Analysis of purified GII by sucrose gradient fractionation, gel filtration, and nondenaturing polyacrylamide electrophoresis suggests that the two subunits exist as a 1:1 heterodimeric complex (Trombetta et al., 1996). Together, the available data suggest a model whereby ID1 and ID2 on a single molecule of GIIß, while individually capable of associating with the -subunit, synergize in their binding interaction with a single molecule of GII. While it is unlikely that ID1 and/or ID2 encode homooligomerization domains per se, GII heterodimers may closely interact with one another and other ER proteins as part of a larger protein network (Tatu and Helenius, 1997).
The results of this study derive from observations with multiple GST-fusion proteins encompassing ID1 and ID2 and two antisera directed at other portions of the molecule. Since GII is a soluble enzyme, it should be possible to derive crystals for structural analysis of molecular interactions occurring between the - and ß-subunits. It will be particularly interesting to determine whether ID1 and ID2 are juxtaposed in the native structure of GIIß to form a contiguous binding interface that might interact with a single domain of GII. Interestingly, ID1 superimposes the Cys-rich element at the N-terminus of GIIß, while ID2 is located adjacent to the C-terminal Cys-rich element. Reducing versus nonreducing gel analysis of purified GII has shown that intramolecular disulfide bonds are present in the ß-subunit (Trombetta et al., 1996). It will be important to determine whether such linkages serve to bring the N- and C-termini of GIIß into close apposition.
In summary, we have provided the first biochemical demonstration that GIIß possesses two independent binding domains capable of coupling the molecule to the -subunit of GII. At this point we can only speculate that the role of this tightly associated ß-chain is to maintain GII in the endoplasmic reticulum. Another enticing possibility is that GIIß may also exert a regulatory role upon the physical and functional interplay between GII and other members of the quality control apparatus whose activities are so closely coordinated.
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Materials and methods |
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Cell lines and antibody reagents
SAKRTLS 12.1 (SAKR) and BW5147 (BW) mouse T-lymphoma cells were maintained as described previously (Arendt and Ostergaard, 1995
). The PHAR2.7 cell line, generously provided by Dr. Ian Trowbridge (Salk Institute, La Jolla, CA), is a BW-derived mutant deficient in -glucosidase II activity which was maintained in an identical manner to the parental BW line. Antiserum 80.1, specific for residues 112–258 of the ß-subunit of GII and antiserum 80.2, specific for residues 437–514 (according to the numbering system in Figure 1), have been previously described (Arendt and Ostergaard, 1997). Immunoglobulin was enriched from the 80.2 antiserum by ammonium sulfate precipitation and partially depleted of anti-GST reactivity by two cycles of absorption to immobilized GST.
Generation of GST fusion proteins
Flanking primers were designed to introduce an upstream EcoRI restriction site and a downstream in-frame stop codon followed by an XhoI restriction site. Inserts were amplified using the high fidelity Advantage cDNA Polymerase Mix (Clontech) by polymerase chain reaction from GIIß cDNA clones obtained previously (Arendt and Ostergaard, 1997). For most reactions, the thermokinetic parameters were 1 cycle of 1 min at 94°C followed by 25–30 cycles of 30 s at 94°C, 1 min at 54°C, and 50 s at 68°C. Polymerase chain reaction products were cloned into the GST expression vector pGEX-4T-3 (Pharmacia) followed by transformation of E.coli strain JM105. GST-fusion protein expression was induced in log-phase bacterial cultures by addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (Boehringer Mannheim) for 2 h. Bacteria were lysed by sonication in PBS containing 1 mM Pefabloc (Boehringer Mannheim), and GST-fusion proteins were purified by batch elution from glutathione-agarose matrix (Sigma) according to the instructions supplied with the vector. Following extensive dialysis against PBS, protein concentrations were measured by BCA assay (Pierce). Recombinant proteins were adjusted to 0.5 mg/ml and stored at –70°C at for later use. Purity of fusion proteins was assessed by subjecting samples to SDS–PAGE on 10% gels followed by staining with Coomassie brilliant blue dye (Bio-Rad). The DNA sequence fidelity of the following constructs was confirmed by automated sequencing: GSTß(N-118), GSTß(N-98), GSTß(20–258), GSTß(273–400), GSTß*(273–400), and GSTß(273–344).
Binding assays
Cells were lysed at a density of 5 x 107/ml in 0.5% Nonidet P-40/1 mM Pefabloc/phosphate-buffered saline, pH 7.2 and 10 µg of GST-fusion protein or 5 µl of antiserum was added to 1 ml of postnuclear lysate. Following a 30 min incubation on ice, fusion proteins were captured by addition of 50 µl of a 50% slurry of glutathione-agarose while antibodies were captured by addition of the same volume of Protein A beads (Boehringer Mannheim). Samples were placed on a rotator at 4°C for 90 min, after which beads were washed three times with 1 ml of lysis buffer. Beads were then incubated on a rotator for 15 h at room temperature with 125 µl of a reaction mix consisting of 5 mM p-nitrophenyl -D-glucopyranoside (Sigma) in phosphate-buffered saline, pH 7.2. As a positive control, 2.5 µl to 25 µl of the original cell lysates were incubated with the same reaction mix. Color change was quantitated by transferring 100 µl from each tube to a 96-well plate and measuring absorbance at 405 nm. Background absorbance, defined as the average OD405 value obtained when pull-downs were carried out in lysis buffer alone, was subtracted from all values obtained. In all experiments, assays were performed independently in triplicate and mean values were graphed with error bars signifying standard error of the mean.
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Acknowledgments |
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We thank Dr. Ian Trowbridge for providing the PHAR2.7 cell line. This work was supported by a grant from the Medical Research Council of Canada. C. W. A. was supported by a studentship from the Alberta Heritage Foundation for Medical Research. H.L.O. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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ER, endoplasmic reticulum; GII, glucosidase II; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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D. Hu, Y. Kamiya, K. Totani, D. Kamiya, N. Kawasaki, D. Yamaguchi, I. Matsuo, N. Matsumoto, Y. Ito, K. Kato, et al. Sugar-binding activity of the MRH domain in the ER {alpha}-glucosidase II {beta} subunit is important for efficient glucose trimming Glycobiology, October 1, 2009; 19(10): 1127 - 1135. [Abstract] [Full Text] [PDF]
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K. Kato and Y. Kamiya Structural views of glycoprotein-fate determination in cells Glycobiology, October 1, 2007; 17(10): 1031 - 1044. [Abstract] [Full Text] [PDF]
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T. A. Baldwin, M. Gogela-Spehar, and H. L. Ostergaard Specific Isoforms of the Resident Endoplasmic Reticulum Protein Glucosidase II Associate with the CD45 Protein-tyrosine Phosphatase via a Lectin-like Interaction J. Biol. Chem., October 6, 2000; 275(41): 32071 - 32076. [Abstract] [Full Text] [PDF]
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