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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-[-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 (6570%), 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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--N-acetylglucosaminidase H were from Sigma. Anti-c-Myc
monoclonal antibodies were a gift from Dr. S. E. Trombetta.
MethodsGT 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 ProceduresMicrosomes 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 ProteolysisPurified 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.
ElectrophoresisNonreducing 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
EpitopeMicrosomes prepared from yeast cells grown to
A595 nm = 0.51.0 were resuspended in 2% SDS, 5
mM EDTA, 50 mM Tris-HCl, 10 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM
N-p-tosyl-L-lysyl chloromethyl
ketone, 1 mM
N
-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 DomainA 519-bp fragment of S. pombe GT (bases 36584176) 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 12201392 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 DomainsTwo 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).
|
Construct with the D. melanogaster GT N-terminal and S. pombe GT
C-terminal DomainsFour 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 ConstructsThe 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 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 TerminiFor 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Noncovalent Linkages Tightly Bind Both DomainsThe 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--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.
|
Noncovalently Bound N- and C-terminal Domains Are Enzymatically Active
and Able to Discriminate between Native and Non-native
ConformersWe 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
-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).
|
N- and C-terminal Domains May Be Expressed Separately, Yielding an Active EnzymeThe 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).
|
|
D. melanogaster and S. pombe GT N- and C-terminal Domains Are Mutually InterchangeableAs 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 (62.374.0%), much lower values occur for the N-terminal portions (32.6% for the R. norvegicus and D. melanogaster GTs and 15.516.3% 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--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,
AD. Labeled glucose residues were present only in
substances derived from cells transformed by the expression vector encoding
full-length GTs (Fig. 7,
EH).
|
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, AC, 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.)
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-[-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 |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|