The UDP-glucose:Glycoprotein Glucosyltransferase Is Organized in at Least Two Tightly Bound Domains from Yeast to Mammals*

Marcelo Guerin and Armando J. Parodi {ddagger}

From the Institute for Biotechnological Research, University of San Martin, 1650 San Martin, Argentina

Received for publication, January 27, 2003 , and in revised form, March 18, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The endoplasmic reticulum UDP-Glc:glycoprotein glucosyltransferase (GT) exclusively glucosylates nonnative glycoprotein conformers. GT sequence analysis suggests that it is composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins and was proposed to be involved in the recognition of non-native conformers and the C-terminal or catalytic domain, which displays a similar size and significant similarity to members of glycosyltransferase family 8. Here, we show that N- and C-terminal domains from Rattus norvegicus and Schizosaccharomyces pombe GTs remained tightly but not covalently bound upon a mild proteolytic treatment and could not be separated without loss of enzymatic activity. The notion of a two-domain protein was reinforced by the synthesis of an active enzyme upon transfection of S. pombe GT null mutants with two expression vectors, each of them encoding one of both domains. Transfection with the C-terminal domain-encoding vector alone yielded an inactive, rapidly degraded protein, thus indicating that the N-terminal domain is required for proper folding of the C-terminal catalytic portion. If, indeed, the N-terminal domain is, as proposed, also involved in glycoprotein conformation recognition, the tight association between N- and C-terminal domains may explain why only N-glycans in close proximity to protein structural perturbations are glucosylated by the enzyme. Although S. pombe and Drosophila melanogaster GT N-terminal domains display an extremely poor similarity (16.3%), chimeras containing either yeast N-terminal and fly C-terminal domains or the inverse construction were enzymatically and functionally active in vivo, thus indicating that the N-terminal domains of both GTs shared three-dimensional features.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Most proteins following the secretory pathway in eukaryotic cells are N-glycosylated in the endoplasmic reticulum (ER).1 A glycan (Glc3-Man9-GlcNAc2) is transferred to Asn in growing polypeptides. Glucoses are then trimmed by the action of glucosidase I, which removes the external Glc unit, followed by glucosidase II, which excises both Glc residues remaining in the glycan (1). Monoglucosylated N-glycans may be formed by partial deglucosylation of the transferred oligosaccharide or by reglucosylation of Glc-free glycans by the UDP-Glc:glycoprotein glucosyltransferase (GT) (2). This enzyme is a sensor of glycoprotein conformations, as it exclusively glucosylates N-glycans in not properly folded conformers. One or two ER-resident {alpha}-mannosidases may degrade Man9-GlcNAc2 to Man8-GlcNAc2 and Man7-GlcNAc2, which may also be reglucosylated by GT. Folding glycoproteins oscillate then between monoglucosylated and unglucosylated forms catalyzed by the opposing activities of GT and glucosidase II. The monoglucosylated forms are recognized by two ER-resident lectins, calnexin and calreticulin. Upon reaching the proper tertiary structures, glycoproteins become substrates of glucosidase II, but not of GT. Properly folded molecules thus liberated from the lectins are then free to pursue their transit to the Golgi. Proteins that fail to properly fold are retained in the ER and are eventually transported to the cytosol, where they are degraded in the proteasomes. Interaction of monoglucosylated glycans with ER lectins not only retains misfolded glycoproteins in the ER, but also facilitates glycoprotein folding by preventing aggregation. GT is therefore the key constituent of the ER quality control of glycoprotein folding, as it is the only element in such a process that discriminates between glycoprotein conformers.

In vitro and in vivo assays have shown that, under both conditions, GT preferentially glucosylates glycoproteins not in extended conformations, but at more advanced folding, molten globule-like stages, when glycoprotein substrates already display secondary structures and some long-range interactions (3, 4, 5). Solvent-accessible hydrophobic amino acid patches have been identified as the structural elements recognized by GT in non-native conformers, as they are the only structural features exclusive of molten globule-like folding intermediates (5). Moreover, in vitro assays have shown that GT preferentially glucosylates N-glycans in the close vicinity of protein structural perturbations (6, 7).

Sequence analysis (BLAST search using a BLOSUM62 matrix) of mammalian, insect, and yeast GTs has suggested that they are composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins, and has been suggested to be involved in non-native conformer recognition; and the C-terminal domain, which binds 5-azido-[{beta}-32P]UDP-glucose and displays a similar size and significant similarity to members of glycosyltransferase family 8 (8, 9, 10, 11, 12). Members of this family conserve the anomeric configuration of the monosaccharide transferred from a sugar nucleotide, presumably by forming a monosaccharide-enzyme intermediate. All GT C-terminal domains from different species share a significant similarity (65–70%), but no such similarity may occur between N-terminal domains. For instance, Rattus norvegicus GT (Gen-BankTM/EBI accession number AAF67072 [GenBank] ) and Drosophila melanogaster GT (accession number Q09332 [GenBank] ) N-terminal domains share a 32.6% similarity, but they show only 15.5 and 16.3% similarities, respectively, to the same portion of Schizosaccharomyces pombe GT (accession number S63669 [GenBank] ) (8, 9, 10). Although there is both structural and experimental evidence supporting the idea that the C-terminal domain is the catalytic portion of the enzyme, the notion that the N-terminal domain is responsible for recognition of non-native conformers has not been experimentally confirmed yet.

The work reported here provides experimental evidence for the two-domain GT structure and shows that both domains are tightly bound. If the N-terminal domain is indeed responsible for conformation recognition, this finding provides a molecular rationale for the close proximity between protein structural perturbations and N-glycans required for glucosylation. Moreover, the S. pombe GT N-terminal domain linked to the D. melanogaster GT C-terminal portion or the inverse construction formed enzymatically and functionally active enzymes in vivo. We therefore concluded that N-terminal domains from different GTs share three-dimensional features despite showing an extremely poor similarity in their primary sequences. In addition to the suggested role in the recognition of misfolded conformers, the results presented here indicate that the N-terminal domains are required for proper folding of the catalytic C-terminal portions, as the latter were enzymatically and functionally inactive and rapidly degraded in vivo when expressed in the absence of the former.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains, Plasmids, Expression Vectors, and Yeast Growth Media—The following S. pombe strains were used: wild-type, Sp95 (h90, ura4-D18, ade6-M216, leu1-32), Sp61G4 (h, ade6-M210, leu1-32, ura4-D18, gpt1::ura4-D1684), and Sp61G4A (h, ade6-M210, ade1, leu1-32, ura4-D18, gpt1::ura4-D1684, alg6::ura4+) (13). Plasmid pBluescript SKgpt1+33A and expression vector pREP3Xgpt1+, which encode S. pombe GT, were generated in our laboratory (13). Plasmid pOT2gpt1+ (D. melanogaster GT) was a generous gift from Dr. S. E. Trombetta (Department of Cell Biology, Yale University Medical School). Expression vectors pREP2 and pREP3X encode the S. pombe ura4+ gene and the Saccharomyces cerevisiae LEU2 gene, respectively. The latter complements the S. pombe leu1 mutation. Rich medium contained 3% glucose (Sigma) and 0.5% yeast extract (Difco). Minimal medium was as described by Alfa et al. (14). The final concentrations of adenine, uracil, and leucine supplements were 75, 75, and 250 µg/ml, respectively.

Materials—[14C]Glc (300 Ci/mol) was from New England Nuclear. UDP-[14C]Glc (300 Ci/mol) was prepared according to Wright and Robbins (15) with slight modifications. N-Methyldeoxynojirimycin and endo-{beta}-N-acetylglucosaminidase H were from Sigma. Anti-c-Myc monoclonal antibodies were a gift from Dr. S. E. Trombetta.

Methods—GT was assayed using UDP-[14C]Glc as a sugar donor and denatured thyroglobulin as a glucosyl acceptor as described (16). Proteins were microsequenced at the Department of Biochemistry and Molecular Biology of the University of Nebraska (Omaha, NE). In constructs described below, the accuracy of all DNA junctions generated was checked by DNA sequencing. S. pombe cells were labeled with [14C]Glc, and the N-glycans were analyzed as described (17), but with 2.5 mM N-methyldeoxynojirimycin. Paper chromatography was performed with Whatman No. 1 papers using solvent system A (1-propanol/nitromethane/water (3:2:1)) and solvent system B (1-butanol/pyridine/water (10:3:3)). Similarities of N- and C-terminal domains were obtained by BLAST analysis using a BLOSUM62 matrix.

Enzyme Purification and Related Procedures—Microsomes were prepared from R. norvegicus liver and S. pombe cells, and GTs were purified from them to homogeneity as described (17, 18). Assays used for attempting to separate cleaved GT N- and C-terminal domains were as described in the indicated purification procedures (17, 18).

Limited R. norvegicus GT Proteolysis—Purified enzyme (40 µg) was incubated in a total volume of 250 µl containing 10 mM imidazole buffer (pH 7.0), 5% sucrose, and 1 µg of endoproteinase Glu-C (protease V8, Sigma) at 37 °C. Aliquots of 5 µl were used for GT assays after addition of 3,4-dichloroisocoumarin (Sigma) at a final concentration of 1 mM. Aliquots of 10 µl were used for 10% SDS-PAGE analysis.

Electrophoresis—Nonreducing standard 10% polyacrylamide gels were employed. Purified GT (2 µg) was resuspended in 10 µl of solution containing 10% glycerol, 2% SDS, and 50 mM Tris-HCl (pH 7.6). N-Ethylmaleimide was added to nonreduced samples at a final concentration of 25 mM. Dithiothreitol was added to reduced samples at a final concentration of 10 mM. Both reduced and nonreduced samples were heated at 95 °C for 3 min, and N-ethylmaleimide (25 mM) was added to the reduced samples to allow direct comparison of both type of samples. Reduced and nonreduced samples were run side by side on the same gels.

Western Blot Analysis of Proteins Bearing the c-Myc Epitope—Microsomes prepared from yeast cells grown to A595 nm = 0.5–1.0 were resuspended in 2% SDS, 5 mM EDTA, 50 mM Tris-HCl, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM N{alpha}-p-tosyl-L-lysyl chloromethyl ketone, 1 mM N{alpha}-p-tosyl-L-phenylalanyl chloromethyl ketone, 1 µM E-64, 1 µM pepstatin, and 10 µM leupeptin. Samples were separated by 10% SDS-PAGE, electrotransferred to nitrocellulose filters, and developed with anti-c-Myc antiserum according to standard procedures.

Antibodies against the S. pombe GT C-terminal Domain—A 519-bp fragment of S. pombe GT (bases 3658–4176) was amplified using Pwo DNA polymerase (Roche Applied Bioscience), pBluescript SKgpt1+33A as template, and the following oligonucleotides as primers: 5'-CCGGAATTCGTACAATTGGCCACACTGGC-3' (sense) and 5'-ATAAGAATGCGGCCGCTAAATCGATTGTTTTGG-3' (antisense). The fragment released by EcoRI and NotI treatment (the primers employed generate cleavage sites for these two enzymes) was ligated to the pET22b+ expression vector (Novagen) previously treated with the same enzymes, and the construct was amplified in Escherichia coli NovaBlue cells (Novagen). A protein comprising amino acids 1220–1392 of S. pombe GT fused to a polyhistidine tail was synthesized by transforming E. coli BL26(DE3) cells (Novagen) with above-described construct. The protein in inclusion bodies was washed; dissolved in 6 M urea, 25 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 5 mM imidazole buffer; and purified by affinity chromatography on Ni2+-iminodiacetic acid-Sepharose (Amersham Biosciences). A single 20-kDa protein appeared upon 10% SDS-PAGE. Antibodies raised in rabbits reacted with the recombinant protein at least at a 1:1000 serum dilution.

Construct with the S. pombe GT N-terminal and D. melanogaster GT C-terminal Domains—Two synonymous mutations (A3461G and A3464C) were introduced into the gpt1+ sequence encoding the N- and C-terminal domain junction in expression vector pREP3Xgpt1+, thus creating a StuI site (cleavage with this enzyme generates a blunt end). For this purpose, the mutagenic oligonucleotide 5'-TTTCAAACGTAAAGAGGCCTCTATAAATATTTTTTCTGTTGCC-3' and the Altered Sites II in vitro mutagenesis system (Promega) were employed. The resulting vector (pREP3Xgpt1StuI) (Fig. 1A) coded for Glu1154 and Ala1155, the same as the parental one. The fragment encoding the D. melanogaster GT C-terminal domain was synthesized using oligonucleotide primers 5'-CATCTATCAACATTTTCTCTGTGGC-3' (sense; generates a blunt end) and 5'-TGTACATCCGGACGGGGCTCATGAGAAGGCGTC-3' (antisense; generates a BspEI site), plasmid pOTgpt1+ as template, and Pwo DNA polymerase. The PCR product was digested with BspEI and ligated to pREP3Xgpt1StuI previously digested with StuI and BspEI. The latter cuts S. pombe gpt1+ just before the ER retrieval sequence. The expression vector generated was termed pREP3Xgpt1CTDm (Fig. 1A).



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FIG. 1.
Constructs employed. A, constructs encoding the S. pombe GT N-terminal and D. melanogaster GT C-terminal domains (pREP3Xgpt1CTDm-c-myc). SP, signal peptide; RS, retrieval sequence. c-myc indicates the location of insertion of the c-Myc-encoding sequence, and the dotted line indicates the S. pombe GT C-terminal fragment against which antiserum was raised. The locations of restriction sites are also indicated. The black boxes on the left of the constructs indicate the presence of the signal peptide-encoding sequence. The approximate lengths of DNA fragments are indicated. B, constructs encoding the D. melanogaster GT N-terminal and S. pombe GT C-terminal domains (pREP3Xgpt1NTDm-c-myc). C, constructs expressing full-length S. pombe GT (pREP3Xgpt1+-c-myc) and the S. pombe GT N-terminal (pREP2gpt1NT-c-myc) and C-terminal (pREP3Xgpt1CT-c-myc) domains.

 

Construct with the D. melanogaster GT N-terminal and S. pombe GT C-terminal Domains—Four mutations (C56G, A57C, A58G, and G59G) were introduced into the gpt1+ sequence encoding the junction between the S. pombe GT signal sequence and the N-terminal domain to create a BssHII site. These mutations resulted in the point mutation K20R at the amino acid level. Mutations were generated by conducting three PCRs. The first one used primers 5'-CGCGGATCCCCCGGGCTGCAGG-3' (sense; hybridizes with a noncoding region ~100 bp upstream of the ATG codon and generates a BamHI site) and 5'-CTTGACATCTAAAGGGCGCGCGGCATAGCAAATCG-3' (antisense and mutagenic; hybridizes with the sequence encoding the signal peptide/N-terminal domain junction). The second PCR used primers 5'-CGATTTGCTATGCCGCGCGCCCTTTAGATGTCAAG-3' (sense and mutagenic; hybridizes with the sequence encoding the signal peptide/N-terminal domain junction) and 5'-CAACCACTGCGTACGGAATGCC-3' (antisense; hybridizes ~500 bp downstream from the ATG codon and generates a BsiWI site), the expression vector pREP3Xgpt1+ as template, and Pwo DNA polymerase. The products of both PCRs were purified, mixed, and used as template for a third PCR, in which the sense and antisense primers were those employed in the first and second reactions, respectively. The 606-bp fragment obtained was digested with BamHI and BsiWI and ligated to expression vector pREP3Xgpt1StuI (Fig. 1A) previously treated with the same enzymes, thus generating expression vector pREP3Xgpt1StuI-BssHII (Fig. 1B). The fragment encoding the N-terminal domain of D. melanogaster GT was synthesized using primers 5'-TTGGCGCGCGAATCCAGTCAGAGCTATCC-3' (sense; creates a BssHII site at the 5'-end) and 5'-CCGTATCCTCATCAGAGGC-3' (antisense; generates a blunt end), plasmid pOTgpt1+ as template, and Pwo DNA polymerase. The PCR product was digested with BssHII and ligated to pREP3Xgpt1StuI-BssHII previously digested with StuI and BssHII. The expression vector generated was termed pREP3X gpt1NTDm (Fig. 1B).

Truncated Protein Constructs—The expression vector encoding the S. pombe GT signal peptide followed by the N-terminal domain and the ER retrieval sequence was synthesized by first performing inverse PCR using pBluescript SKgpt1+33A (which encodes full-length GT) as template and oligonucleotide primers 5'-CCGGACGAACTTTGAAAC-3' (sense) and 5'-AAAGAAGCTGAGAGACTT-3' (antisense). The first primer encodes the retrieval plus stop sequences, and the second one encodes the N- and C-terminal domain junction (centered in base 3444 from the ATG codon). The fragment was purified, phosphorylated, religated, and amplified in E. coli DH5{alpha} cells (Invitrogen). The resulting plasmid (pBluescript SKgpt1NT) was digested with SnaBI and BspEI. The 555-bp fragment generated was ligated to expression vector pREP2gpt1+ previously treated with the same restriction enzymes. The resulting expression vector was pREP2gpt1NT (Fig. 1C). The expression vector encoding the GT signal peptide followed by the C-terminal domain and the ER retrieval sequence was constructed in a similar way, but with the following primers: 5'-AATTTCAAACGTAAAGAAGC-3' (sense) and 5'-GGCGGCATAGCAAATCG-3' (antisense). The first primer encodes the N- and C-terminal domain junction, and the second one starts at the sequence coding for the junction between the GT signal sequence and the mature protein sequence. The resulting plasmid (pBluescript SKgpt1CT) was digested with BamHI and NruI. The resulting 590-bp fragment was ligated to the expression vector pREP3Xgpt1+ previously treated with the same restriction enzymes. The resulting expression vector was pREP3Xgpt1CT (Fig. 1C).

Insertion of the c-Myc Epitope at the C Termini—For inserting the c-myc epitope (EQKLISEEDLN) into chimeric and truncated proteins before the ER retrieval sequence, oligonucleotide primers 5'-CCGGAACAAAAACTCATCTCAGAAGAGGATCTGAAT-3' (sense) and 5'-CCGGATTCAGATCCTCTTCTGAGATGAGTTTTTGTT-3' (antisense) were annealed and ligated to the above-described constructs previously digested with BspEI. The sequence coded for the epitope and left, at both ends, sequences able to bind DNA previously digested with the restriction enzyme. All constructs had an additional Pro residue before the c-Myc epitope. The resulting expression vector designations include "c-myc" to indicate insertion of the epitope-encoding sequence.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Linkage between the N- and C-terminal Domains Is Extremely Sensitive to Endoproteolysis—Fig. 2A shows that almost all pure S. pombe GT preparations yielded three bands when subjected to 10% SDS-PAGE. The same result was obtained also with some R. norvegicus enzyme preparations (Fig. 2B). The sizes of the fragments were 161, 130, and 35 kDa for yeast GT and 172, 135, and 38 kDa for the mammalian enzyme. The results obtained from microsequencing the N termini are depicted in Fig. 2 (A and B). They reveal that, in both cases, the smaller fragments had been produced by endoproteolytic breakage of the full-length species at the junction between the N- and C-terminal domains (amino acids 1149 and 1220 from the initial Met residue for the yeast and mammalian GTs, respectively) (Figs. 2, A and B, and 3). As expected, the largest and smallest (but not the middle) fragments of S. pombe GT reacted with polyclonal antibodies directed against a C-terminal 172-amino acid fragment of the molecule (Figs. 1A and 2C).



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FIG. 2.
Endoproteolytic cleavage of GTs. A and B, S. pombe and R. norvegicus GTs, respectively, were purified to homogeneity according to standard procedures, subjected to 10% SDS-PAGE, and stained with Coomassie Brilliant Blue. The sizes and N-terminal sequences of proteins are indicated. C, S. pombe GT was submitted to Western blot analysis with an antiserum directed against a fragment of the C-terminal domain.

 


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FIG. 3.
Comparison of S. pombe and R. norvegicus sequences. The MacVector program was used with a window size of 8, a 60% minimum score, a hash value of 2, and a pam250matrix-scoring matrix. The arrow indicates the sites of endoproteolytic cleavages.

 

Noncovalent Linkages Tightly Bind Both Domains—The N- and C-terminal domains of R. norvegicus and S. pombe GTs proved to be tightly bound, as the small fragment yielded by endoproteolytic degradation could not be separated from the middle one by gel filtration on a Bio-Sil SEC 250 column (Fig. 4, A and C), by chromatography on a phenyl-Superose column (Fig. 4, B and D), by ion-exchange chromatography on a Mono Q HR 5/5 column, and by filtration through a Centricon-100 filter (data not shown). Moreover, the linkage between both domains was not mediated by disulfide bonding as revealed by 10% SDS-PAGE performed under reducing and nonreducing conditions (Fig. 4, E and F). The small and middle fragments of both R. norvegicus and yeast GTs were retained on a concanavalin A-Sepharose column despite the fact that the mammalian enzyme C-terminal domain lacks N-glycosylation consensus sequences and that the only one present at such a location in the yeast enzyme was unoccupied as revealed by 10% SDS-PAGE following endo-{beta}-N-acetylglucosaminidase H treatment (data not shown). This result shows that the N- and C-terminal domains could not be separated also by lectin affinity chromatography.



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FIG. 4.
Attempts to separate GT N- and C-terminal domains. S. pombe (A and B) and R. norvegicus (C and D) GTs purified to homogeneity were resubmitted to gel filtration on a Bio-Sil SEC 250 column (A and C) or to chromatography on a hydrophobic phenyl-Superose column (B and D). The results from 10% SDS-PAGE of fractions with maximum activity are shown in the insets. The purified S. pombe (E) and R. norvegicus (F) GT preparations were subjected to 10% SDS-PAGE under reducing (R) and nonreducing (NR) conditions. For further details, see "Experimental Procedures."

 

Noncovalently Bound N- and C-terminal Domains Are Enzymatically Active and Able to Discriminate between Native and Non-native Conformers—We observed that certain S. pombe preparations were enzymatically active despite almost totally lacking the full-length protein component. A slight modification of the purification procedure was then introduced to fully eliminate it: the interaction period of the applied material with the concanavalin A-Sepharose column before elution with {alpha}-methylmannoside was increased from 1 to 12 h. As depicted in Fig. 5 (A and C), although the resulting preparation completely lacked the full-length enzyme, it displayed enzymatic activity. As expected, the largest fragment in the extensively degraded preparation did not react with antibodies directed against the last portion of the C terminus (Fig. 5B). As the same procedure (longer interaction of the applied material with concanavalin A-Sepharose) did not yield an R. norvegicus preparation devoid of the full-length enzyme, the purified GT was incubated with protease V8. As shown in Fig. 5D, the full-length R. norvegicus enzyme disappeared after a 5-min incubation, yielding 135-plus 38-kDa fragments that displayed almost full enzymatic activity. Microsequencing of the latter yielded the sequence SFKWG, indicating that protease V8 cleavage had occurred at amino acid 1211 from the initial Met residue, i.e. 9 amino acids ahead of that produced by endoproteolytic cleavage (see above). The 38-kDa protein was gradually further degraded to smaller fragments, and the enzymatic activity was concomitantly lost (Fig. 5D). Both R. norvegicus and S. pombe enzyme preparations devoid of full-length components glucosylated denatured (but not native) thyroglobulin (data not shown).



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FIG. 5.
Enzymatic activity of noncovalently bound N- and C-terminal domains. A, 10% SDS-PAGE of S. pombe GT maintained for 1 h (lane 1) or 12 h (lane 2) in contact with concanavalin A (ConA)-Sepharose. B, Western blot analysis of the preparations indicated in A using anti-GT C-terminal fragment antiserum. C, enzymatic activities of the preparations indicated in A. D, R. norvegicus GT submitted to protease V8 proteolysis for the times (minutes) indicated at the top. Samples were subjected to 10% SDS-PAGE (upper panel), and enzymatic activities were assayed (lower panel). For further details, see "Experimental Procedures."

 

N- and C-terminal Domains May Be Expressed Separately, Yielding an Active Enzyme—The notion of a two-domain GT structure able to display enzymatic activity even if not covalently bound was supported by the synthesis of an active enzyme upon transfection of a GT null mutant (Sp61G4) with two plasmids separately encoding the N and C termini (Table I). Nevertheless, microsomes prepared from cells transformed with both expression vectors displayed a lower specific enzymatic activity than those isolated from cells transformed with a plasmid coding for the full-length enzyme. Constructs coding for either one of both domains had segments encoding the GT signal peptide before the enzymatic domains and a c-Myc epitope (EQKLISEEDLN) and the GT ER retrieval sequence (PDEL) after them (Fig. 1C). Western blots with c-Myc-specific monoclonal antibodies showed that both fragments had been effectively expressed (Fig. 6A). As with wild-type GT proteolytically cleaved at the domain junction, the enzyme formed by the separately expressed domains was also able to discriminate between native and non-native conformers. No enzymatic activity was detected in microsomes isolated from cells transformed with only the C-terminal domain-encoding vector, although, as will be seen below, the fragment was effectively expressed as revealed by Western blot analysis (Table I).


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TABLE I
Activity of separately expressed S. pombe GT N- and C-terminal domains

 


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FIG. 6.
Expression of S. pombe GT N- and C-terminal domains. A, Western blot analysis with anti-c-Myc antiserum performed on microsomes prepared from S. pombe GT null mutant cells (Sp61G4) coexpressing the S. pombe GT N- and C-terminal domains (expression vectors pREP2gpt1NT-c-myc and pREP3Xgpt1CT-c-myc). B, the same Western blot analysis performed on microsomes prepared from Sp61G4A (lanes 1–4) and Sp61G4 (lanes 5 and 6) cells expressing full-length S. pombe GT (expression vector pREP3Xgpt1+-c-myc) (lanes 1 and 2) or only the C-terminal domain (expression vector pREP3Xgpt1CT-c-myc)(lanes 3–6). Cells were incubated with cycloheximide (0.1 mg/ml) for 2 h before microsomal preparation in lanes 2, 4, and 6. Cells were grown at 28 and 37 °C in lanes 1–4 and lanes 5 and 6, respectively. For further details, see "Experimental Procedures."

 

D. melanogaster and S. pombe GT N- and C-terminal Domains Are Mutually Interchangeable—As shown above, in both yeast and mammalian GTs, the N- and C-terminal domains are tightly bound and show enzymatic activity even in the absence of any covalent linkage between them. This was a rather unexpected result because whereas the C-terminal domains of R. norvegicus, D. melanogaster, and S. pombe share a high similarity (62.3–74.0%), much lower values occur for the N-terminal portions (32.6% for the R. norvegicus and D. melanogaster GTs and 15.5–16.3% for the latter enzymes and S. pombe GT). In other words, to bind highly similar C-terminal domains, the tertiary structures of the N-terminal portions of different GTs should be expected to share three-dimensional features. To confirm that the N-terminal domains from different GTs share elements of tertiary structure, two types of chimeras were constructed in expression vector pREP3X. The first one encoded the D. melanogaster GT N-terminal and S. pombe GT C-terminal domains (Fig. 1B). The second construct coded for the yeast GT N-terminal and fly GT C-terminal domains (Fig. 1A). These constructs, together with those encoding either the full-length S. pombe enzyme or only its C-terminal portion, were transfected into S. pombe Sp61G4A mutant cells. All constructs coded for the S. pombe GT signal peptide at the N termini and for the above-mentioned c-Myc epitope before the S. pombe GT ER retrieval sequence (PDEL). Sp61G4A mutants lack GT and transfer to Man9-GlcNAc2 instead of the complete glycan, as they lack the dolichol-P-Glc-dependent glucosyltransferase responsible for Glc1-Man9-GlcNAc2-P-P-dolichol formation. As they synthesize underglycosylated glycoproteins and lack the folding facilitation process mediated by glycoprotein-calnexin interaction, these double mutant cells grow poorly with a round morphology at 28 °C and do not grow at 37 °C. Their transformation with a GT-encoding expression vector rescued the wild-type phenotype, but no rescue was observed when two point mutations that abolished GT activity were introduced at the C-terminal domain (13).

Transformed cells were incubated with 5 mM [14C]Glc in the presence of N-methyldeoxynojirimycin, a glucosidase II inhibitor. Analysis of whole cell N-oligosaccharides released by endo-{beta}-N-acetylglucosaminidase H showed that, in addition to peaks migrating as a Man9-GlcNAc standard, shoulders in the position expected for Glc1-Man9-GlcNAc were formed in cells transfected with full-length S. pombe GT or with both chimeras coding for mixed yeast-fly enzymes (Fig. 7, A, C, and D). On the contrary, the Glc1-Man9-GlcNAc shoulder was absent in cells transformed with the S. pombe GT C-terminal portion (Fig. 7B). Formation of Glc1-Man9-GlcNAc2 in cells transformed with full-length GTs and its absence in mutants transformed with the C-terminal domain were confirmed by strong acid hydrolysis of substances migrating as Glc1-Man9-GlcNAc and Man9-GlcNAc standards in Fig. 7, A–D. Labeled glucose residues were present only in substances derived from cells transformed by the expression vector encoding full-length GTs (Fig. 7, E–H).



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FIG. 7.
S. pombe-D. melanogaster full-length GT chimeras (but not the C-terminal domains) are enzymatically active. Sp61G4A cells were transformed with expression vectors pREP3Xgpt1+-c-myc (coding for S. pombe GT) (A), pREP3Xgpt1CT-c-myc (coding for the S. pombe GT C-terminal domain) (B), pREP3Xgpt1NTDm-c-myc (coding for the D. melanogaster GT N-terminal and S. pombe GT C-terminal domain chimera) (C), and pREP3Xgpt1CTDm-c-myc (coding for the S. pombe GT N-terminal and D. melanogaster GT C-terminal domain chimera) (D). Cells were incubated for 30 min with [14C]Glc in the presence of 2.5 mM N-methyldeoxynojirimycin. N-Glycans released from whole cell glycoproteins by endo-{beta}-N-acetylglucosaminidase H treatment were subjected to paper chromatography with solvent system A. Standards were as follows: Glc1-Man9-GlcNAc (arrow 1), Man9-Glc-NAc(arrow 9), and Man8-GlcNAc (arrow 8). In E–H, substances migrating as Glc1-Man9-GlcNAc and Man9-GlcNAc standards in A–D were submitted to strong acid hydrolysis and subjected to paper chromatography with solvent system B. Substances in A generated those in E, substances in B generated those in F, and so on. Standards were as follows: Man (M) and Glc (G).

 

The C-terminal domain was effectively expressed as revealed by Western blot analysis; but the protein was extremely unstable, as no expressed protein was detected after a 2-h incubation of intact cells with cycloheximide, thus strongly suggesting that it had been unable to properly fold in the absence of the N-terminal domain (Fig. 6B, lanes 3 and 4). On the contrary, full-length S. pombe GT continued to be detected after a similar cycloheximide treatment (Fig. 6B, lanes 1 and 2)

To confirm that the chimeric enzymes were not only enzymatically, but also functionally active in vivo, we investigated whether they rescued the non-growth phenotype of Sp61G4A mutants at 37 °C. As shown in Fig. 8, A–C, in all cases, the mutant cells transformed with full-length GT constructs, but not with either the C-terminal domain-encoding expression vector or with the vector itself, grew at 37 °C. As in Sp61G4A double mutant cells grown at 28 °C (Fig. 6B, lanes 3 and 4), Western blot analysis of microsomal proteins isolated from Sp61G4 single mutant cells transformed with the C-terminal domain expression vector showed that the C-terminal domain was expressed at 37 °C, but was unstable, as it disappeared after a 2-h incubation of cells with cycloheximide (Fig. 6B, lanes 5 and 6). (Sp61G4 cells lack GT, but transfer Glc3-Man9-Glc-NAc2 and are able to grow at 37 °C.)



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FIG. 8.
Rescue of the wild-type S. pombe phenotype with GT chimeras. A, S. pombe GT gpt1/alg6 mutant cells (Sp61G4A) transfected with expression vectors pREP3X, pREP3Xgpt1+-c-myc (coding for S. pombe GT), and pREP3Xgpt1CT-c-myc (coding for the S. pombe GT C-terminal domain). B, the same as in A, but in the lower row, cells were transfected with pREP3Xgpt1NTDm-c-myc (coding for the D. melanogaster GT N-terminal and S. pombe GT C-terminal domain chimera). C, the same as in A, but in the lower row, cells were transfected with pREP3Xgpt1CTDm-c-myc (coding for the S. pombe GT N-terminal and D. melanogaster GT C-terminal domain chimera). Cells were grown at the indicated temperatures. For further details, see "Experimental Procedures."

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The results reported herein provide experimental evidence for the two-domain GT structure suggested by the amino acid sequence, as expression of both portions, each of them encoded by different vectors, in an S. pombe GT null mutant resulted in the synthesis of an active enzyme. Expression of the C-terminal or catalytic domain resulted in the synthesis of an inactive, highly unstable protein. Moreover, although linkage between both domains in the wild-type enzyme was extremely sensitive to proteolysis, both portions could not be separated by ion-exchange, size-exclusion, lectin affinity, and hydrophobic interaction chromatographies without loss of enzymatic activity. It is worth remarking that only the N-terminal domain is N-glycosylated and that there is an almost 4-fold difference in size between both domains. Moreover, the cleaved molecules were able to discriminate between native and non-native glycoprotein conformers.

As mentioned above, the C-terminal domains displayed approximately the same size and significant sequence similarity to members of the glycosyltransferase family 8. It has also been reported that this portion of the molecule has the ability to bind 5-azido-[{beta}-32P]UDP-glucose (10). These observations indicate that the C-terminal domain is the catalytic portion of the molecule where formation of the putative glycosyl-enzyme intermediate takes place. Expression of the R. norvegicus GT C-terminal domain in insect cells yielded a protein displaying ~5% of the specific full-length enzymatic activity (10). It was not reported, however, whether the recombinant truncated enzyme discriminated between properly and not properly folded glycoprotein substrates. As shown above and will be further discussed below, we were unable to obtain an enzyme active either in vivo or in vitro upon expressing the S. pombe C-terminal domain in a yeast GT null mutant. It has been suggested that the GT N-terminal domain is responsible for sensing the folding status of glycoprotein substrates. An additional or alternative role (or perhaps the sole one) is, as demonstrated here, to contribute to proper folding of the catalytic or C-terminal domain within the ER luminal environment.

To confirm the presence of common tertiary structural elements in the N-terminal domains of different GTs, as suggested by the tight association between highly similar C-terminal and highly divergent N-terminal domains, chimeras containing the D. melanogaster GT N-terminal and S. pombe GT C-terminal domains or the yeast GT N-terminal and fly GT C-terminal domains were expressed in S. pombe GT null mutants. Chimeric proteins were enzymatically active in vivo and were able to rescue the wild-type phenotype (growth at 37 °C) of gpt1/alg6 double mutant cells (Sp61G4A). These mutants lack GT and synthesize underglycosylated glycoproteins, as they transfer (inefficiently) Man9-GlcNAc2 instead of Glc3-Man9-GlcNAc2 (13). The severe ER stress to which they are submitted prevents their growth at 37 °C, but the wild-type phenotype could be restored upon transformation with an expression vector coding for an active GT. The underglycosylated glycoprotein(s) that necessarily required GT-mediated interaction with calnexin for proper folding at high temperature was probably somehow involved in cell wall synthesis, as the wild-type phenotype could be rescued also in a hyperosmotic medium (1 M sorbitol) (13). The expressed C-terminal domain was inactive in vivo and was also unable to rescue the wild-type phenotype. As in cells grown at 28 °C, the protein was unstable at 37 °C, probably reflecting its inability to properly fold in the absence of the N-terminal domain.

As protein domains in glycoprotein substrates that determine the exclusive glucosylation of non-native conformers probably do not share identical three-dimensional structures, the corresponding recognition domains in glucosyltransferases from different species might not be expected to necessarily display significant similarity in their tertiary structures. In other words, it may be that recognition of hydrophobic amino acid patches in molten globule conformers could be attained by GTs displaying a variety of different N-terminal domain conformations if those domains were indeed responsible for sensing glycoprotein substrate conformations. In fact, GTs purified from R. norvegicus and S. pombe, which share only a 15.5% similarity in their N-terminal domain primary sequences, were shown to display the same exclusive specificity for non-native conformers in in vitro assays (17, 18). The results presented here, however, show that, despite the extremely poor primary sequence similarity shown by GT N-terminal domains from different species, such portions probably share substantial elements of tertiary structure, as yeast and fly GT N-terminal domains were able to facilitate folding and stabilization of the GT C-terminal portions of both species alike.

A very unique feature of GT is that it glucosylates only N-glycans in the very near proximity of protein structural perturbations. It was recently reported that only the N-glycan attached to the misfolded subunit of an artificial dimer formed by properly folded and misfolded RNase B monomers was glucosylated by GT in in vitro assays (6). It was further demonstrated that the N-glycan was glucosylated only when present in the immediate vicinity of the localized protein structural perturbation introduced into a mutant RNase B monomer by abolishing one of the four disulfide bonds present in the wild-type molecule (7). These recent results agree with previous ones reporting that N-glycans and misfolded protein subunits have to be covalently linked to be glucosylated (19). The close proximity between both structural elements required for efficient glucosylation (N-glycans and structural perturbations) may be best explained by the results reported herein, i.e. by the intimate association between the catalytic (C-terminal) and putative misfolded recognition (N-terminal) domains by noncovalent bonding.

It should be stressed that ascription of the location of the conformation recognition site to the N-terminal domain is merely speculative. It could be that its sole role is, as demonstrated here, to facilitate folding and stabilization of the catalytic C-terminal portion and that the conformation-sensing site resides also in this last part of the molecule. The sole evidence against this possibility is that enzymes belonging to glycosyltransferase family 8, with which GT C-terminal domains share a similar size and significant sequence similarity, probably do not have the capacity to discriminate between glycoprotein conformers, as most of them are not involved in glycoprotein glycosylation. On the other hand, the close proximity between the N-glycan and the protein structural perturbation required for glucosylation could also be explained if both the catalytic and conformation recognition sites reside in the relatively small (~35 kDa) C-terminal domains.


    FOOTNOTES
 
* This work was supported by United States Public Health Service Grant GM 44500, Howard Hughes Medical Institute Grant 55003687, and a grant from the National Agency for the Promotion of Science and Technology (Argentina). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Inst. for Biotechnological Research, University of San Martin, CC30, 1650 San Martin, Argentina. Tel.: 54-11-4580-7255; Fax: 54-11-4752-9639; E-mail: aparodi{at}iib.unsam.edu.ar.

1 The abbreviations used are: ER, endoplasmic reticulum; GT, UDP-Glc:glycoprotein glucosyltransferase. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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