©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Folding of Tissue-type Plasminogen Activator
EFFECTS OF DISULFIDE BOND FORMATION ON N-LINKED GLYCOSYLATION AND SECRETION (*)

(Received for publication, August 26, 1994; and in revised form, December 5, 1994)

Simon Allen (1) Hassan Y. Naim (2) Neil J. Bulleid (1)(§)

From the  (1)University of Manchester, School of Biological Sciences, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom and the (2)Institute für Mikrobiologie, Heinrich Heine Universität Düsseldorf, Universitätsstrasse 1, Gebaude 26.12, D-4000 Düsseldorf, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded, and was secreted. We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.


INTRODUCTION

Many of the membrane and secretory proteins synthesized by eukaryotic cells undergo modification during their synthesis by the N-linked addition of oligosaccharides to specific asparagine residues(1) . This modification plays an important role in regulating the activity, stability, and antigenicity of the mature protein(2) . Aside from the physiological roles of N-linked oligosaccharides, a picture is now emerging where N-linked oligosaccharides are central in facilitating the folding, transport, cell surface expression, and secretion of glycoproteins(3, 4) .

N-Linked glycosylation occurs co-translationally on translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum (ER)(^1)(5) . The initial step, core glycosylation, is catalyzed by the enzyme oligosaccharyltransferase, which is associated with the lumenal surface of the ER membrane(1, 6) . Core glycosylation determines the number of individual oligosaccharides attached to a given polypeptide, and involves the transfer of a presynthesized Glc(3)Man(9)GlcNAc(2) unit from a membrane associated donor, dolicholphosphate(7, 8) , to asparagine residues in the tripeptide acceptor sequon Asn-Xaa-Ser/Thr(9) .

Although the sequon is essential for core glycosylation, numerous examples of glycoproteins have been observed containing either unglycosylated sequons or sequons that are glycosylated to a variable extent(10, 11, 12) . The selectivity of sequon utilization suggests that additional regulatory mechanisms operate during core glycosylation to determine the extent of glycosylation of a given protein. The frequency of sequon utilization may be dependent upon the relative lumenal concentrations of oligosaccharyltransferase, dolichol donor and acceptor protein(13, 14) . These factors are reflected in cell culture where the relative amounts of glycoforms synthesized for a constitutive or recombinant glycoprotein depend upon both the expression system used and the culture conditions employed(12, 15) . Previous studies in vitro using membrane extracts as source of oligosaccharyltransferase to glycosylate polypeptide substrates identified the requirement of short range interactions within the sequon, necessary for reactivity between asparagine and the glycosyl donor(16, 17) . Similar experiments demonstrated that unglycosylated or variably glycosylated sequons in native substrates could undergo complete glycosylation if denatured prior to glycosylation(18) .

In this present study we use t-PA as a model secretory glycoprotein to test whether disulfide bond formation within the ER of living cells can influence the frequency of sequon utilization. Human t-PA is a 70-kDa serine protease that contains 17 disulfide bonds and is secreted from human and recombinant mammalian cell lines as a mixture of two glycoforms that differ in their extent of core glycosylation. Type I t-PA undergoes core glycosylation at Asn-117, Asn-184, and Asn-448; type II t-PA is identical except for lacking core glycosylation at Asn-184(11) . Cell-free synthesis of t-PA under conditions favoring disulfide bond formation reduced the likelihood of core glycosylation at Asn-184(19) . It is likely that during co-translational translocation into the lumen of the ER, the folding intermediate that satisfies both the accessibility of the sequon to oligosaccharyltransferase and the glycosyl donor may only be transient resulting in inefficient core glycosylation of Asn-184.

Recently several groups have demonstrated that co-translational disulfide bond formation, folding, and oligomerization of proteins within the ER can be reversibly inhibited by the addition of the reducing agent dithiothreitol (DTT) to the culture medium of living cells(20, 21, 22, 23, 24, 25) . Disulfide bond formation, folding, and oligomerization were shown to recommence post-translationally on removal of DTT. Such treatments were shown to have no deleterious effects upon translation, translocation, and N-linked glycosylation. A similar approach was adopted in this study to achieve the temporal separation of N-linked glycosylation from the disulfide bond formation of nascent t-PA in the ER of CHO cells. A pulse-chase approach in conjunction with immunoprecipitation and reducing and non-reducing SDS-PAGE was used to monitor core glycosylation, disulfide bond formation and secretion of t-PA. Our results demonstrate that co-translational disulfide bond formation of t-PA can be inhibited in vivo with mild concentrations of DTT. Synthesis of t-PA under these conditions resulted in complete glycosylation of the otherwise variably glycosylated Asn-184. On removal of DTT from the cell culture medium, this overglycosylated t-PA could fold post-translationally and undergo normal secretion. Finally, secreted t-PA was shown to be less susceptible to reduction than t-PA present within cells unless pretreated with a denaturing reagent.


MATERIALS AND METHODS

Reagents

Recombinant Chinese hamster ovary cells (CHO 1-15500; catalog no. CRL 9606) were purchased from American Type Culture Collection (Rockville, MD). Tissue culture reagents were purchased from GIBCO Life Technologies Ltd. (Glasgow, Scotland). Goat anti-human t-PA polyclonal antibody was purchased from American Diagnostica Inc. (Greenwich, CT). EXPRESS [S]methionine and [S]cysteine protein labeling mixture was purchased from DuPont NEN (Dreiech, Germany). Endoglycosidase H was purchased from Boehringer Mannheim (East Sussex, United Kingdom). Human t-PA was purchased from Integrated Genetics (Framingham, MA). All other reagents were purchased from Sigma (Dorset, United Kingdom).

Cell Line, Metabolic Labeling, and Chase Conditions

Chinese hamster ovary (CHO) cells, stably transfected to express t-PA, (American Type Culture Collection), were grown as monolayers in Ham's F-12 medium with 10% fetal calf serum. CHO cells at 90% confluence were washed twice with methionine- and cysteine-free Dulbecco's modified Eagle's medium, then preincubated in the same medium for 20 min at 37 °C. Each monolayer was pulse-labeled with 100 µCi of EXPRESS label in 5 ml of above prewarmed medium/100-mm dish for 10 min at 37 °C. DTT was included in the pulse medium, where indicated, to give a final concentration of 5 mM. The pulse was ended by adding 5 ml of prewarmed Dulbecco's modified Eagle's medium with 5 mM each of unlabeled methionine and cysteine. Cycloheximide (500 µM) was included in the chase medium to block completion of labeled nascent chains. DTT (5 mM) was included in the chase medium where indicated. After chase times up to 3 h, the cells were transferred to ice and washed twice with ice-cold phosphate-buffered saline containing 20 mMN-ethylmaleimide (NEM) to prevent formation or rearrangement of disulfide bonds in the protein. The cells were lysed with 750 µl of ice-cold lysis buffer/dish (buffer: 25 mM Tris-HCl, pH 7.4, containing 0.5% Triton X-100, 50 mM NaCl, 2 mM EDTA, 20 mM NEM, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each antipain, chymostatin, leupeptin, and pepstatin, and 10 µg/ml soybean trypsin inhibitor). Lysates were adjusted to 1.5 ml with lysis buffer, then spun for 15 min at 12,000 times g to pellet nuclei and cell debris.

Immunoprecipitation

Cell lysates (300 µl) and culture medium (750 µl) were made up to a final volume of 1 ml with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 0.02% (w/v) sodium azide). Samples were preincubated with 50 µl of protein A-Sepharose (10% (w/v) in phosphate-buffered saline) for 1 h at 4 °C to preclear the samples of protein A binding components. Precleared samples were each incubated with 50 µl of protein A-Sepharose and 5 µg of goat anti-human t-PA polyclonal antibody for 15 h at 4 °C. The complexes were washed three times with fresh immunoprecipitation buffer, and then prepared for analysis by SDS-PAGE with or without prior endoglycosidase treatment as described below.

Endoglycosidase H Digestion

For endoglycosidase H digestion, after washing the immunoprecipitates, the final pellet was resuspended in 15 µl of dissolution buffer (50 mM Tris-HCl, pH 8.0, containing 1% (w/v) SDS), and boiled for 5 min. An equal volume of endoglycosidase H digestion buffer (150 mM sodium citrate, pH 5.5, containing 0.5 mM phenylmethylsulfonyl fluoride and 0.02% (w/v) sodium azide), was added to each sample then incubated for various times with 1 milliunit of endoglycosidase H at 37 °C. The reaction was terminated by adding an equal volume of SDS-PAGE sample buffer containing 50 mM DTT.

Unfolding of t-PA in Vitro

Samples of purified t-PA (8 µg) were incubated in the absence of urea and in the presence of 0-100 mM DTT for 10 min at 37 °C. Alternatively, samples were incubated in the presence of 0-6 M urea for 10 min at 37 °C and for another 10 min in the presence of 5 mM DTT. NEM was added to each sample to a final concentration of 200 mM to quench the DTT and alkyate any free thiols. Finally, an equal volume of SDS-PAGE sample buffer was added to each sample.

SDS-Polyacrylamide Gel Electrophoresis

Immunoprecipitation pellets were resuspended in 30 µl of SDS-PAGE sample buffer (0.25 M Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 20% (v/v) glycerol, 0.004% (w/v) bromphenol blue). DTT (50 mM) was added to reduced samples where indicated. All samples, reduced and non-reduced, were boiled for 5 min prior to electrophoresis. Cooled samples were loaded into the wells of a 10% SDS-polyacrylamide gel for electrophoresis. Gels were stained, fixed, dried under vacuum, and visualized by either autoradiography using Kodak XAR-5 film or imaged using a Fujix Bas2000 phosphorimager.


RESULTS

N-Linked Glycosylation of t-PA Reduced in the Endoplasmic Reticulum

Human t-PA is secreted from human and recombinant cell lines as a mixture of two glycoforms that differ in their extent of core N-linked glycosylation. A previous study using a cell-free translation system supplemented with canine pancreatic microsomes suggested that this variability of core glycosylation was dependent upon the ability of t-PA to fold and form disulfide bonds (19) . To investigate whether disulfide bond formation and protein folding actually determine the extent of core N-linked glycosylation in living cells, we applied a pulse-chase approach to cultured CHO cells stably transfected with the cDNA encoding human t-PA.

CHO cells were pulsed with [S]methionine and -cysteine at 37 °C for 10 min in the absence or presence of varying concentrations of DTT in the labeling medium. The cells were cooled immediately and then treated with the membrane-permeable alkylating agent NEM to block free thiols and prevent further rearrangement of disulfide bonds. The cells were lysed and the post-nuclear supernatant immunoprecipitated with a goat polyclonal anti-tPA. The immunoprecipitates were subjected to both reducing and non-reducing SDS-PAGE (Fig. 1). The t-PA synthesized during the 10-min labeling period in the absence of DTT migrated as a doublet band when reduced prior to electrophoresis (Fig. 1, lane1), indicating that variable glycosylation of Asn-184 had occurred. When subjected to non-reducing SDS-PAGE (Fig. 1, lane1) t-PA migrated with a greater mobility and with a more diffuse banding pattern than on reducing gels, indicating that during the 10-min labeling period t-PA had formed intramolecular disulfide bonds. As the concentration of DTT present during the labeling period was increased from 0 to 5 mM, the doublet pattern progressively disappeared until only a single band was present at concentrations of DTT above 1 mM (lanes2-10). This change in banding pattern was coincident with a progressive reduction in the formation of disulfide bonds. Thus, at 1 mM DTT no disulfide bonds were formed as no faster migrating bands were present when the sample was run under non-reducing conditions (Fig. 1, lowerpanel).


Figure 1: N-Linked glycosylation of t-PA synthesized in the presence of varying concentrations of DTT. CHO cells were labeled with [S]methionine and -cysteine for 10 min in the absence (lane1) or presence (lanes2-10) of varying concentrations of DTT. Cells were lysed in the presence of 20 mM NEM and the lysates immunoprecipitated with goat polyclonal anti-t-PA. Immunoprecipitates were separated by SDS-PAGE under reducing (upperpanel) and non-reducing conditions (lowerpanel).



To ascertain the number of oligosaccharide side chains present on the t-PA polypeptide synthesized either in the absence or presence of DTT, we carried out a limited endoglycosidase H digestion. As t-PA contains three glycosylation sites that are known to be glycosylated (11) , we would expect to see four products after limited digestion corresponding to tri-, di-, mono-, and unglycosylated t-PA. This was indeed the case for both t-PA synthesized in the absence of DTT (Fig. 2a) and in the presence of DTT (Fig. 2b). This demonstrates conclusively that t-PA synthesized in the presence of DTT was exclusively the triglycosylated type 1 t-PA.


Figure 2: Limited deglycosylation of t-PA with endoglycosidase H. CHO cells were labeled with [S]methionine and -cysteine for 10 min in the absence (a) or presence (b) of 5 mM DTT. Cell lysates were immunoprecipitated with goat polyclonal anti-t-PA. Immunoprecipitates were digested with endoglycosidase H for various times as indicated. Samples were separated by SDS-PAGE under reducing conditions. Bars indicate the position of tri-, di-, mono-, and unglycosylated t-PA.



A clear difference in mobility of type 1 t-PA synthesized in the absence or presence of 5 mM DTT (Fig. 1, lanes1 and 10) was observed. Thus, t-PA synthesized in the presence of DTT had a reduced mobility, which could result either from alkylation of the 34 free thiol groups or an effect of DTT on oligosaccharide processing. To investigate the cause of this increase in mobility, we repeated the experiment in the absence of alkylating agents and fully deglycosylated the t-PA with endoglycosidase H (Fig. 3). When cells were lysed in the absence of NEM, the t-PA synthesized in the presence of DTT had a greater mobility than when cells were lysed in the presence of NEM (compare lanes3 and 4). These differences in mobility were still evident after deglycosylation (lanes7 and 8), demonstrating that this effect is due to differential alkylation of disulfide-bonded t-PA relative to non-disulfide-bonded t-PA.


Figure 3: Effect of alkylation and glycosylation on the electrophoretic mobility of t-PA. CHO cells were labeled with [S]methionine and -cysteine for 10 min in the absence (lanes 1, 2, 5, and 6) or presence (lanes3, 4, 7, and 8) of 5 mM DTT. Cells were lysed in the absence (lanes1, 3, 5, 7) or presence (lanes2, 4, 6, and 8) of 20 mM NEM. Cell lysates were immunoprecipitated with goat polyclonal anti-tPA. Immunoprecipitates were digested with endoglycosidase H (lanes 5-8) for 5 h. Samples were separated by SDS-PAGE under reducing conditions.



Disulfide Bond Formation of t-PA in the ER

It was clear that when disulfide bond formation and folding of t-PA was prevented in the ER, t-PA was synthesized exclusively as the type I glycoform. To investigate whether reduced and overglycosylated t-PA synthesized under these conditions could form a native complement of disulfide bonds within the ER, we chased the reduced t-PA in the absence of DTT. Cycloheximide was also included in the chase medium to inhibit protein synthesis. Cells were chased for up to 120 min, treated with NEM, lysed, and t-PA precipitated using the polyclonal antibody. A time course for intracellular disulfide bond formation in non-treated and DTT-treated cells was constructed by separating the immunoprecipitates by SDS-PAGE under non-reducing and reducing conditions (Fig. 4, a and b).


Figure 4: Disulfide bond formation in t-PA synthesized in the absence or presence of DTT. CHO cells were labeled for 10 min with [S]methionine and -cysteine in the absence (a) or presence (b) of 5 mM DTT, followed by a 120-min chase with unlabeled medium in the absence of DTT. Chase times were terminated by cell lysis in the presence of 20 mM NEM. Cell lysates were immunoprecipitated with goat polyclonal anti-t-PA. Immunoprecipitates were separated by SDS-PAGE under non-reducing (a and b, upper panels) and reducing conditions (a and b, lowerpanels).



When CHO cells were labeled for 10 min in the absence of 5 mM DTT, non-reducing SDS-PAGE (Fig. 4a, upperpanel) revealed a diffuse band at 0 min into the chase, indicating that disulfide bond formation in t-PA was near completion 10 min into synthesis. When CHO cells were pulse labeled for 10 min in the presence of 5 mM DTT, non-reducing SDS-PAGE (Fig. 4b, upperpanel) revealed a sharp band at 0 min into the chase, indicating that no disulfide bond formation had occurred. After 5 min of the chase, the band became more diffuse and downshifted, indicating partial disulfide bond formation. By 10 min of the chase, there was an additional increase in mobility, which was also observed when the samples were reduced prior to electrophoresis (Fig. 4b, lowerpanel). As described above, this was due to differential alkylation of partially oxidized t-PA. No further increase in mobility was observed after 45 min of chase. These results indicate that disulfide bond formation was complete between 30 and 45 min into the chase. At later time points, there was a decrease in the intensity of t-PA immunoprecipitated, suggesting that t-PA was either being transported out the cell or degraded.

An increase in intensity of t-PA precipitated during the chase period after labeling in the presence of DTT was observed (Fig. 4b). This could reflect a lack of protein synthesis inhibition by cycloheximide. However, this is unlikely to be the case, as no such increase is observed when DTT was not present in the labeling period (Fig. 4a). We also increased the concentration of cycloheximide in the chase from 0.5 mM to 5 mM with identical results (data not shown). In addition, any protein synthesized during the chase period would be variably glycosylated as the redox conditions within the ER are restored rapidly (23) . An alternative explanation is that the antibody used for immunoprecipitation has a reduced affinity for non-disulfide-bonded alkylated t-PA, and as a consequence more t-PA is immunoprecipitated as the protein folds and forms disulfide bonds during the chase period. To test this we took two equal volumes of cell lysate, one of which was treated with 100 mM NEM while the other sample was reduced with 100 mM DTT and then treated with 100 mM NEM. Both samples were passed through a Sephadex G-25 column equilibrated in immunoprecipitation buffer to remove DTT and NEM from the samples prior to immunoprecipitation. Immunoprecipitates were separated by SDS-PAGE (data not shown) and the amount of t-PA precipitated quantitated by phosphorimage analysis. Only 12.5% of the t-PA precipitated from the non-reduced sample was precipitated from the reduced sample, demonstrating that the antibody used has a reduced affinity for non-disulfide-bonded t-PA. The increase in intensity of the immunoprecipitates from the DTT-treated cells during the chase in the absence of DTT therefore reflects post-translational folding and disulfide bond formation during this period.

Transport and Secretion of t-PA

The next stage of our study was to investigate whether t-PA folded post-translationally had undergone the conformational maturation required for its exit from the ER, transport to the Golgi apparatus, and secretion from the cell. To monitor intracellular transport, we treated samples with endoglycosidase H. This approach takes advantage of the fact that high mannose ER forms of secretory glycoproteins are sensitive to deglycosylation by endoglycosidase H, whereas mannose-trimmed secretory proteins exit the ER and are resistant to deglycosylation. However, no detectable difference in electrophoretic mobility was observed after endoglycosidase H treatment for both t-PA synthesized in the absence or presence of 5 mM DTT during the 180 min of chase (results not shown). This observation may be explained by the fact that t-PA secreted from CHO cells carries high mannose oligosaccharides at Asn-117 (26, 27) and that various cell lines transfected with t-PA cDNA secrete unusually high amounts of core glycosylated t-PA(28) .

The alternative approach to demonstrate transport competency of t-PA after removal of DTT from the cells was to determine whether the t-PA could leave the cell. When t-PA was labeled for 10 min in the absence of 5 mM DTT, a band was observed in the immunoprecipitates from the cell lysates immediately after the pulse (Fig. 5a, upperpanel). Between 75 and 240 min of the chase, there was a decrease in the intensity of t-PA immunoprecipitated from the cellular fraction (Fig. 5a, upperpanel). This was accompanied by the appearance and subsequent increase in intensity of a doublet band immunoprecipitated from the chase medium. By 240 min of the chase time, the bulk of the t-PA immunoprecipitated was present in the medium (Fig. 5a, lowerpanel), although a small amount of t-PA was detected in the cell at 240 min of chase (Fig. 5a, upperpanel). This indicated that from its initial synthesis, the fastest moving t-PA molecules took between 70 and 80 min to reach the cell surface, whereas the slowest moving t-PA molecules took over 240 min. This asynchrony in the transport pattern of t-PA in CHO cells has been observed in other cell lines secreting recombinant t-PA (28) as well as recombinant membrane proteins such as influenza hemagglutinin(29) .


Figure 5: Secretion of t-PA synthesized in the absence or presence of DTT. CHO cells were labeled for 10 min with [S]methionine and -cysteine in the absence (a) or presence (b) of 5 mM DTT, followed by a 240-min chase with unlabeled medium in the absence of DTT. Chase times were terminated by cell lysis in the absence of 20 mM NEM. Cell lysates and medium samples were immunoprecipitated with goat polyclonal anti-t-PA. Immunoprecipitates were separated by SDS-PAGE under reducing conditions.



When t-PA was labeled for 10 min in the presence of 5 mM DTT, a sharp singlet band was observed immediately after the pulse (Fig. 5b, upperpanel), indicating the presence of only type I t-PA in the intracellular fraction. There was a reduction in the intensity of the intracellular t-PA immunoprecipitated between 75 and 240 min of the chase time, until no intracellular t-PA was detectable after 240 min (Fig. 5b, upperpanel). Accompanying the disappearance of t-PA immunoprecipitated from the intracellular fraction between 180 and 240 min of the chase (Fig. 5b, upperpanel), there was the appearance and subsequent increase in the intensity of a singlet band in the immunoprecipitates from the chase medium between 75 and 240 min (Fig. 5b, lowerpanel), indicating that the secretion of t-PA was complete between 180 and 240 min of the chase. Therefore, the fastest moving t-PA molecules took between 80 and 90 min to reach the cell surface, and the slowest took between 180 and 240 min to reach the surface from their initial synthesis within the ER, indicating a recovery time of approximately 10 min after DTT treatment.

The Effects of DTT on the Folding of t-PA Are More Pronounced in Vivo than in Vitro

Although newly synthesized secretory proteins may be retained within the ER after treatment of cells with mild concentrations of DTT, several groups have established that these effects are only specific to immature disulfide-containing proteins (21, 23, 24, 25) . These studies have shown that the transport and secretion of proteins without disulfide bonds or proteins that are fully oxidized are not affected by subsequent treatment with DTT. As t-PA contains a high number of disulfide bonds in relation to its molecular weight, we investigated the effect of mild concentrations of DTT on disulfide-bonded t-PA present within the cell in comparison with secreted t-PA.

CHO cells were pulsed for 10 min and then chased for 40 min in the absence of DTT. At this time, t-PA should have completed disulfide bond formation but still remains in a pre-Golgi compartment (see Fig. 4). The cells were chased for an additional 180 min in the presence of 5 mM DTT. The cells were lysed at regular intervals during the 180 min in the presence of 20 mM NEM, and t-PA immunoprecipitated and separated by SDS-PAGE under non-reducing conditions (Fig. 6). A t-PA band was observed immediately after the 40-min chase when the immunoprecipitates were reduced prior to electrophoresis (Fig. 6, 0 min), which migrated as a diffuse downshifted band when separated under non-reducing conditions, indicating that disulfide bond formation had occurred. Between 0 and 120 min of the chase, there was an upshift in the mobility of t-PA until a sharp doublet remained, indicating that t-PA became completely reduced between 60 and 120 min after the addition of DTT. There was a decrease in the intensity of t-PA in the cells during the chase until no t-PA was detectable after 180 min. No t-PA was secreted into the medium during this chase period (results not shown). This decrease is probably due to a combination of factors including the reduced affinity of the antibody for non-disulfide-bonded t-PA, nonspecific aggregation, and degradation.


Figure 6: Intracellular reduction of fully disulfide-bonded t-PA. CHO cells were labeled for 10 min with [S]methionine and -cysteine, then chased for 40 min with unlabeled medium. The cells were chased for an additional 180 min with unlabeled medium in the presence of 5 mM DTT. Chase times were terminated by cell lysis in the presence of 20 mM NEM. Cell lysates were immunoprecipitated with goat polyclonal anti-t-PA. Immunoprecipitations were separated by SDS-PAGE under non-reducing and reducing conditions.



The alternative approach involved incubation of purified human t-PA in vitro, in the presence of 0-100 mM DTT and 0-6 M urea (Fig. 7, a and b). When t-PA was incubated for 10 min in the presence of 5 mM DTT, a slight upshift in mobility was observed relative to untreated t-PA when samples were separated under non-reducing conditions (Fig. 7a). No further upshift in mobility was observed even after 3 h (result not shown), which indicated that t-PA was only partially reduced by 5 mM DTT. Complete reduction of t-PA within 10 min required incubation with 100 mM DTT, as indicated by its mobility on non-reducing SDS-PAGE relative to t-PA boiled in DTT sample buffer prior to electrophoresis (Fig. 7a). However, when t-PA was preincubated in the presence of urea, complete reduction of t-PA was observed between 3 and 4 M urea when samples were treated with 5 mM DTT for 10 min (Fig. 7b). We concluded that in vitro t-PA is only partially sensitive to reduction by mild concentrations of DTT that can completely reduce folding intermediates and mature t-PA in vivo. Accessibility of disulfide bonds of t-PA resistant to DTT in vitro therefore required unfolding of the polypeptide with denaturant.


Figure 7: Non-reducing SDS-PAGE analysis of t-PA treated with DTT in vitro in the presence or absence of urea. Samples of purified t-PA (8 µg) were incubated in the absence of urea and in the presence of 0-100 mM DTT for 10 min at 37 °C (a). Alternatively, samples were incubated in the presence of 0-6 M urea for 10 min at 37 °C and for an additional 10 min in the presence of 5 mM DTT (b). NEM was added to each sample to a final concentration of 200 mM to quench the DTT and alkyate any free thiols. t-PA samples were separated by SDS-PAGE under non-reducing and reducing conditions.




DISCUSSION

Although the tripeptide sequon Asn-Xaa-Ser/Thr is an absolute requirement for the attachment of N-linked oligosaccharides to glycoproteins(9) , its presence does not always result in glycosylation. In fact, analysis of protein sequences revealed that approximately one-third of sequons in glycoproteins remain unglycosylated(30, 31) . The current understanding of the temporal and spatial organization of core glycosylation in the ER lumen supports the idea that the conformation of the nascent polypeptide influences the frequency of sequon utilization(1, 3) . A recent study using a cell-free translation system has demonstrated that oligosaccharide transfer only occurs when 12-14 amino acids C-terminal to a sequon have been translocated into the ER lumen(32) . This result suggests that the active site of oligosaccharyltransferase and dolichol oligosaccharide donor, which are tethered to the lumenal surface (6, 33) are projected 30-40 Å into the ER lumen(32) .

The nascent polypeptide, when translocated into the ER lumen, can undergo co-translational folding and disulfide bond formation(34, 35) . Differential glycosylation has been demonstrated for individual sequons between native or denatured substrates(18, 36) , and between proteins synthesized in vitro under conditions preventing or favoring disulfide bond formation(19) . These experiments have suggested that a folding intermediate that provides both accessibility and the correct orientation of the sequon to the transferase and lipid-linked donor may be transient after the initial translocation of the sequon. Sequon utilization may also be related to the time a protein remains associated with the ER membrane and therefore the glycosylation apparatus. This idea is supported by the fact that when membrane anchors were engineered into rat growth hormone two sequons exhibited increased glycosylation(37) .

The temporal and spatial organization of N-linked glycosylation obviously provides many interconnected regulatory mechanisms. However, its regulation must also depend upon the relative levels of the acceptor protein, dolichol oligosaccharide donor, and oligosaccharyltransferase. This is reflected in cell-free translations were the extent of glycosylation depends upon the source of microsomal vesicles(38) , and in cell culture where core glycosylation differs between cell lines(15) , or cell culture conditions employed(12) . Many studies have demonstrated the importance of the dolichol oligosaccharide pool in regulating N-linked glycosylation. For example, addition of dolichol phosphate to bovine pancreas increased occupancy of a single glycosylation site of ribonuclease from 12% to 90%(13) . Although the relative concentrations of the components of N-linked glycosylation play a direct role in regulating the reaction, the relative concentrations of molecular chaperones and redox enzymes that faciliate protein folding within the ER may differ between expression systems and, therefore, in an indirect fashion influence the extent of glycosylation of proteins.

To date the majority of conformational studies on sequon utilization have used in vitro systems, employing either purified protein and synthetic peptides, or cell-free translation systems. However, such studies have not been possible in vivo, as suitable techniques to study intracellular protein folding have been unavailable until recently. Several groups have demonstrated that co-translational disulfide bond formation can be reversibly inhibited by the addition of DTT to the culture medium of living cells(20, 21, 22, 23, 24, 25) . Such treatments were shown to have no deleterious effects upon translation, translocation, N-linked glycosylation, and the transport of fully oxidized or cysteine-free secretory proteins to the cell surface. Furthermore, when DTT was removed from the cells, all model proteins used to date were shown to fold, oxidize and oligomerize post-translationally, and then move to the cell surface. Using similar techniques in this report, we demonstrated that disulfide bond formation of nascent t-PA in CHO cells can be prevented with exogenously added DTT. Furthermore, t-PA synthesized under these conditions exhibited complete sequon glycosylation at Asn-184, in contrast to variable occupancy of this sequon in untreated cells. Presumably, during co-translational disulfide bond formation Asn-184 becomes buried within the interior of the molecule or between domains. Competition between simultaneous folding and oligosaccharide transfer probably means that the extent of Asn-184 glycosylation depends upon the relative rates of these processes. Inhibition of disulfide bond formation with DTT delays folding, rendering Asn-184 accessible to oligosaccharyltransferase.

We demonstrated that the effects of DTT on the folding of newly synthesized t-PA are reversible. Removal of the reducing agent resulted in disulfide formation and the correct conformational maturation required for t-PA to progress through the secretory pathway to the cell surface. Alongside similar studies, the post-translational folding of t-PA after removal of DTT once more demonstrates the efficiency of the ER in restoring conditions required for protein folding. The ER is specialized as a protein folding compartment, and this is reflected by a high lumenal concentration of molecular chaperones such as BiP, calnexin, Grp94, and redox enzymes such as protein disulfide isomerase and related enzymes. The oxidizing conditions required for disulfide bond formation are buffered by glutathione. An unidentified glutathione transporter is thought to maintain a redox gradient between the ER and cytosol, approximately 1:3-10 GSSG:GSH and 1:100 GSSG:GSH, respectively(39) . It is presumed that the effects of DTT on newly synthesized proteins are due to perturbation of this gradient, and in part by PDI acting as a reductase under reducing conditions(23, 24) . The mechanism by which this redox state is restored when DTT is removed from cells is not understood. However, the data on previously studied secretory proteins and on t-PA in this report indicate that the restoration of oxidizing conditions is extremely rapid, requiring no more than several minutes(23) .

Additional to the requirement of a compartmentalized redox gradient and a host of folding factors, the intracellular folding and transport of many secretory proteins depend upon N-linked glycosylation. For example in many cases inhibition of core glycosylation with tunicamycin or deletion of glycosylation sites by mutagenesis leads to misfolding, aberrant disulfide bond formation, aggregation, and association with BiP(40, 41, 42) . How oligosaccharides facilitate efficient folding and secretion is poorly understood, especially as the dependence upon glycosylation varies greatly between secretory proteins. Furthermore, the importance of individual oligosaccharides in each protein can also differ greatly(3, 4, 43, 44) . This would suggest an influence on local conformation of polypeptides. In contrast, folding competency of mutant proteins lacking their natural glycosylation sites can be restored by creating new glycosylation sites in distant regions of a polypeptide(45, 46) , implying a holistic influence upon folding(4) . Presumably oligosaccharides act by increasing the solubility of hydrophobic folding intermediates to minimize unproductive aggregation and by modulating the binding of chaperones, in particular calnexin(47) . Tunicamycin treatment and deletion of all three glycosylation sites of t-PA expressed in CHO cells has been shown previously to lead to a decrease in the efficiency of secretion of t-PA and an increase in association of malfolded t-PA with BiP(40) . In the present study, we have not assessed the effects of inhibited disulfide bond formation and overglycosylation of t-PA in the context of binding of reduced or malfolded t-PA to molecular chaperones. However, we have shown that t-PA binds to calnexin during its folding within the ER. (^2)

Although the role of differential sequon utilization and differential processing of t-PA oligosaccharides is at present poorly understood, these aspects of glycosylation do impart vital biochemical properties on t-PA associated with its role in fibrin clot lysis. High mannose oligosaccharides at Asn-117 probably facilitate rapid plasma clearance of t-PA via mannose receptors in the liver(48, 49) . The presence of oligosaccharide at Asn-184 renders t-PA less responsive to fibrin stimulating its plasminogen activation and inhibits the conversion of one-chain t-PA to the more catalytically active two-chain t-PA(27, 50) . Asn-117 and Asn-448 serve to decrease fibrin binding and decrease plasminogen activation in the absence of fibrin. Therefore, the carbohydrate composition of t-PA fulfills a regulatory role in clot lysis so that t-PA is only active in the presence of a fibrin clot (50, 51, 52) .

Previous experiments demonstrated that mild concentrations of DTT did not affect the transport of cysteine free secretory proteins, mature disulfide-bonded proteins, or proteins with disulfides non-essential to a transport-competent conformation(21, 23, 24, 25) . These observations reflected earlier work in vitro showing that disulfide bonds of native proteins are resistant to reduction by DTT, whereas those of denatured proteins are readily reduced(53) . Crystallographic evidence suggests that on average 70% of disulfide bonds reside in the solvent-excluded interior of proteins(54) . We demonstrated that, in the case of t-PA, the fully disulfide-bonded polypeptide was completely reduced by mild concentrations of DTT in vivo. In contrast t-PA in vitro was only partially sensitive to reduction, requiring unfolding in 3 M urea to become fully reduced. These results demonstrate a marked difference between the effects of DTT on the folding of t-PA in vitro and in vivo. Braakman and co-workers (20) have suggested that these enhanced effects of DTT in cells are a likely consequence of the conditions within the ER necessary for protein folding, for example, ``conformational massage'' by chaperones such as BiP, calnexin and Grp94, and the oxidoreductase activity of protein disulfide isomerase. It is likely that fully folded t-PA was no longer a substrate for such molecules prior to the addition of DTT. However, after addition of DTT, t-PA in vivo probably underwent the same degree of unfolding as in vitro with 5 mM DTT due to the presence of solvent exposed disulfide bonds. This would render t-PA transport-incompetent, thus delivering t-PA to the protein folding apparatus once again. Under reducing conditions this would promote further unfolding and protein disulfide isomerase-catalyzed reduction of t-PA.


FOOTNOTES

*
This work was supported by the Wellcome Trust (Grant 35853) and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed. Tel.: 44-61-275-5103; Fax: 44-61-275-5082; neil.bulleid{at}man.ac.uk.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; t-PA, tissue-type plasminogen activator; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; NEM, N-ethylmaleimide.

(^2)
S. Allen and N. J. Bulleid, unpublished results.


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