Effect of the glucosidase inhibitor 1-deoxynojirimycin on protein secretion from Saccharomyces cerevisiae

Effect of the glucosidase inhibitor 1-deoxynojirimycin on protein secretion from Saccharomyces cerevisiae Urs F. Greber and Koji Sode Institute o f Biotechnology, E T H Hoenggerberg, CH-8093 Zurich, Switzerland

and Franz Meussdoerffer* Abteilung Biotechnologie, H e n k e l K G a A , Henkelstrasse 67, D-4000 Dusseldorf, F R G

(Received 3 September 1986; revised 29 August 1987)

1-Deoxynojirimycin (dNM), an inhibitor of the trimming enzymes glucosidase I and H, increased secretion of a cloned human peptide hormone, somatomedin C, about fourfold. The stimulation was reversible and directly dependent on dNM concentration. The effect was observed with cells grown in batch or with immobilized steady-state cultures. Most likely, dNM affected processing of N-linked glycans, since it reduced the catalytic activity of external yeast invertase even in low concentrations. This could be attributed to preventing the outer chain glycosylation of the surface exposed glycans.

Keywords: 1-Deoxynojirimycin; somatomedin C; invertase; secretion; yeast

Introduction Recently, the yeast S a c c h a r o m y c e s cerevisiae has become an attractive host for the production of pharmaceuticals. This popularity is in particular due to its absolute nonpathogenicity, its well-understood genetics, an easy genetic manipulation and its capability of secretion. Like in other eucaryotic organisms, protein secretion from S. cerevisiae requires a complex series of compartmentalized enzymatic reactions.~ The first stages of the secretion pathway are in common with the pathway for ER proteins, lysosomal proteins or integral membrane proteins. TM Proteins destined for this pathway have an N-terminal hydrophobic signal peptide that interacts with the signal recognition particle causing arrest of translation. Upon binding to the docking protein on the endoplasmic reticulum (ER), elongation allows the nascent polypeptide chain to be translocated across the ER membrane and the signal peptide to be cleaved off in the lumen of the ER (for review, see reference 5). There, asparagine residues of potential N-glycosylation sites (Asn-X-Ser/Thr) become modified with the oligosaccharide precursor Glc3Man9GlcNAc2.6 The precursor is trimmed in the

* To whom correspondence should be addressed.

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ER and other sugars chains are subsequently added. This process varies with proteins and organisms and might be restricted to surface oligosaccharide chains, which are more accessible to trimming enzymes. 7 In all eucaryotic organisms the trimming reactions are initiated by the release of three terminal glucose residues by glucosidases I and II. Among several inhibitors specific for trimming, 8 the glucose analog 1deoxynojirimycin (dNM) inhibits glucosidases I and II from a great variety of species, including yeast. 9 In mammalian cells, dNM has been shown to affect both glycosylation and secretion in a complex manner.~°-~2 In S. cerevisiae, the elimination of one trimming enzyme in the ER does not result in a missorting of cellular proteins or in impaired secretion or growth (gls 1 mutant. 13) However, it has been assumed that this mutation causes alterations in the sugar moiety of secreted proteins. On the other hand, it was shown by in vitro studies that the trimming reactions of the alpha factor precursor can be efficiently blocked with dNM without affecting the core-oligosaccharides.14 Moreover, kinetic measurements have shown that the glycosylated oligosaccharide precursor is transferred about 20 times faster to the nascent protein chain than the unglucosylated analog. ~5Therefore, we reasoned that dNM could affect glycosylation and cause a faster transfer of polypeptides from the ER to © 1988 Butterworth Publishers

Protein secretion from Saccharomyces cerevisiae: U. F. Greber et al. the Golgi vesicles. This step is known to be rate limiting in secretion from higher eucaryotic cells.16 Moreover, dNM prevents an outer chain extension of the core-oligosaccharides and thereby prevents the attachment of polymannosides, which are known to elicit a high antigenic response in mammals.~7 Here we describe that dNM enhances the secretion of the recombinant human peptide hormone, somatomedin C, from yeast. Somatomedins or insulin-like growth factors are potent cell growth stimulators in vivo and in vitro. ~8 The coding sequence for the mature hormone comprises 70 amino acids, but no potential N-glycosylation site.18 It was fused to the yeast mating type alpha 1 sequence so that the SMC was preceded by the secretion signal peptide and the N-glycosylated propeptide but not the coding sequences of the alpha factor pheromone. The secreted SMC exhibits high biological activity but is not glycosylated (J. Ernst, personal communication). To discuss possible mechanisms of dNM on the secretion of SMC from yeast, we further studied the dNM dependent glycosylation and secretion of external invertase, a well-characterized secretory yeast enzyme.

Materials and methods

Reagents Three g of dNM were a gift of Bayer AG, Leverkusen, FRG. A rabbit anti-GAPDH antiserum was provided by H. Riezman, Lausanne, Switzerland. All other chemicals were of the best grade available.

immunoblots, proteins from SDS gels were electrophoretically transferred to nitrocellulose and subsequently stained with convenient rabbit antibodies using the protein A-HRP detection method (Bio-Rad kit).

Immunotechniques Anti-invertase antibodies were raised in rabbits by two subcutaneous injections of denatured, Endo H deglycosylated invertase. Booster injections were given with purified commercial invertase from Boehringer, FRG. 23 After bleeding, IgG fractions were enriched by chromatography on DEAE Affi-Gel (Bio-Rad), and polymannose specific antibodies were removed on an affinity column with immobilized glycopeptides of pronase digested invertase. 24 For immunoprecipitations, cells (7 A600 per 2 ml) were labeled with 14C leucine (100 uCi per ml) for 3 h with sucrose as carbon source. Immunoprecipitations were performed according to the method of Roberts and Roberts 25 from 100 000g supernatants of cells disintegrated with glass beads.

Assays SMC was detected with a commercial radioimmunoassay (Nichols Inst. Diagnostics, San Juan Capistrano, US). Periplasmic invertase activity was determined from whole cells according to the method of Riezman 26 using incubations without sucrose as a blank. Native invertase was deglycosylated according to the method of Trimble et al. 7 with Endo H purified as described by Tarentino and Maley. 27 Protein was determined according to the method of Bradford. z8

Yeast strains and culture conditions Wild type yeast $288C (MAT alpha), a kind gift from G. R. Fink, and FY 171 (leu2, ura 3, trpl, MAT alpha) were used in a synthetic medium according to the method of Sherman, ~9 with the carbon source as indicated. SMC was produced from a constitutive yeast actin promoter on a 2u, L E U 2 plasmid vector. The SMC coding sequence TM was fused to the pre-pro sequence of the yeast mating type alpha 1 sequence (a gift of J. Ernst, Biogen, Geneva). Immobilizations and continuous cultures of yeast cells were performed as described earlier. 2° External invertase was derepressed in either 0.1% glucose or 4% sucrose for the times indicated.

Electrophoresis and immunoblotting Yeast cells were disintegrated with glass beads and cell debris removed by centrifugation. Aliquots of the supernatants were applied either to 4.5% continuous polyacrylamide gels and stained for invertase activity2~ or to SDS gradient gels (7-25%) according to the method of Laemmli. 22 Molecular weight markers were: catalase (232 000), lactate dehydrogenase (140 000), fl-galactosidase (116 000), phosphorylase b (94 000), bovine serum albumin (67 000), ovalbumin (45 000), carbonic anhydrase (29 000), soybean trypsin inhibitor (20 000) and alpha lactalbumin (14 000). For

Northern procedures For mRNA isolation, a preculture of glucose grown cells (5 × 107 ml -~) was harvested and resuspended in sucrose containing minimal medium with or without dNM (I0 raM). Aliquots were removed after times indicated and mRNA was isolated, z9 separated by denaturing agarosegel electrophoresis and blotted to nitrocellulose according to standard procedures. 3° Blots were hybridized with a 32p nicktranslated 2.1 kb HindlII restriction fragment from the suc 2 (invertase) gene 3j or with a plasmid carrying the topo 2 gene from yeast 32 (provided by S. Gasser, Lausanne). Quantification of the bands corresponding to internal and secreted invertase (1.7 and 1.8 kb, 32) or of topo 2 (3.4 kb, 33 was performed by spectrophotometrical analysis of eluted silver grains according to the method of S u i ssa. 33

Results and discussion To evaluate possible effects of dNM on yeast growth or on secretion, wild type strain was grown in synthetic medium in the presence or absence of dNM (10 m~). Only a minimal growth inhibition could be seen in dNM treated cells during a 43 h incubation period (Figure 1A). However, the pattern of proteins secreted from dNM cells differed from that of controls (Figure 1B). After 14 and 23 h of incubation, only a few

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Papers batch culture of yeast transformants was treated with 10 mM dNM, the amounts of SMC in the medium were increased (Figure 2). After 27 h, the SMC level reached a maximum of 1.8 mg 1-1 compared to 1.4 mg 1-1 from the same number of untreated cells. This difference was reproducible and significant, since the detection limit of the assay was below 0.1 mg 1-1 and triple determinations were performed. Although dNM treated cells secreted higher amounts of SMC, their growth rate as well as their glucose consumption differed only slightly from those of control cells

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The stimulation of SMC secretion by dNM could be reversed when the drug was removed from the medium. Yeast cells immobilized in polyacrylamide-alginate beads and cultured in a column reactor for 100 h reached steady-state conditions. Subsequently, dNM (15 mM) was continuously applied for 8 h and the SMC level increased from 0.94 to 1.05 mg 1-1. When the inhibitor was removed, SMC secretion dropped back to the level before the dNM application (Figure 3). It is unlikely that this effect was due to a changed metabolism or to a loss of cells in the reactor, since both parameters remain fairly constant in steady-state reactors. z° Most likely, it reflected a reversible inhibition of the glucosidases I and 11.9 The stimulation of SMC secretion was proportional to dNM concentration. As shown in a series of batch

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Figure I Effect of dNM on the growth (A) and on the secretion of polypeptides (B) from a wild type yeast ($288C) A preculture was grown in glucose medium to a density of 2.6 × 107 m1-1 and resuspended at the same density in fresh glucose medium with or without 15 mM dNM. Cells were counted in a Thoma cell and supernatants (10 ml) were precipitated with TCA and analyzed on a PonceauS stained nitrocellulose filter after SDS gel electrophoresis and electroblotting. At 23 h, a second amount of dNM was added (15 mg m1-1 )

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/ proteins could be detected in both cellular supernatants, while after 43 h, the dNM supernatant contained more proteins than the control. Especially, increased secretion of proteins with molecular weights from 50 000 to 67 000 and of two protein bands of 35 000 and 20 000 was observed, whereas proteins larger than 80 000 were less abundant in the dNM supernatants. This indicates that dNM had an effect on secretion from S. cerevisiae. Control incubations of the same filter with GAPDH antisera were negative in both, dNM and control panels (results not shown). The absence of the major cytoplasmic enzyme GAPDH indicates that the difference in the protein pattern was not due to a different extent in cell lysis of the inhibitor treated and untreated cells. Consequently, we tested the effect of dNM on the secretion of the recombinant peptide hormone, somatomedin C, from an alpha factor plasmid. When a

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about 10-fold higher than those used in in vitro experiments with isolated yeast membranes.14 Since we did not reach the optimum for the SMC secretion at 45 mM dNM, uptake of dNM into the yeast cells could be a limiting factor. To understand the effects of dNM on secretion of polypeptides, we focused further experiments on yeast external invertase. Yeast cells express two forms of invertase, one unglycosylated cytoplasmic enzyme and a highly glycosylated and secreted oligomer. 34 Both forms are encoded by the same gene, suc 2, but only the secreted species is translated from an mRNA, which encodes the N-terminal signal sequence for secretion. 31 The external enzyme is re-

experiments a linear relation was found up to about 45 mM yielding a maximum of about 3.6 mg l -~ SMC (Table 1). This effect persisted for at least 80 h (4th transfer) and indicates that the entrapped cells did not loose their potential to secrete SMC during the semicontinuous culturing. The concentrations of dNM we used (]0-]5 mM) were in the same range as for in vivo studies to inhibit glucosidase I and II in higher eucaryotes. 10They were

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Papers pressed by high concentrations of glucose (e.g., 2%). The two forms (cytoplasmic and external) can be distinguished by the size of their transcripts (1.7 and 1.8

kb, respectively) and by the size and the cellular location of the proteins themselves. 35 Under derepressed growth conditions, the external invertase activity was markedly reduced by dNM already after 1 h and remained low for three more hours until the cells run out of carbon source (Figure 4). In sucrose grown cells, the same effect was most evident after 3 and 4 h, since the derepression occurred slower than in 0.1% glucose medium (Figure 4, cells without dNM). This could be explained by the lag in growth during the first 2 h after transfer (Figure 4), since external invertase is essential for sucrose metabolism and consequently for growth. 36 Most essential, dNM did not affect the cell density (growth) and the activity of the internal invertase. We further investigated whether dNM directly affects the activity of the glycosylated external invertase or whether it interferes with its induction. Therefore, Northern blots of dNM and control cells were probed with the suc 2 gene fragment. At time 0 of derepression, no RNA for external invertase (1.8 kb) could be detected (Figure 5). After 1 h, control cells expressed slightly more of the 1.8 kb RNA than dNM

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cells did, whereas the mRNA level for internal invertase (1.7 kb) was higher in dNM treated cells. After 4 h of derepression, both dNM and control cells, contained about equal amounts of mRNA for external and internal invertases (Figure 5). Thus, it seems that the lag phase observed in dNM treated cells during the first 2 h of derepression is caused by both, a down regulation of external invertase mRNA and a reduced catalytic activity of the enzyme. To evaluate whether dNM interferes with the translation of external invertase, a leucine auxotroph was biosynthetically labeled with 3H leucine in sucrose medium for 3 h in the presence or absence of 15 mM dNM. Immunoprecipitations with invertase antibodies gave about the same amounts of counts (75 290 in the presence of dNM and 72 900 in the absence of dNM). This indicates that dNM did not interfere with the translation of invertase mRNA's. To analyse further the reduced activity of the external invertase in the presence of dNM, we subjected extracts of wild type cells to Western blots with invertase antibodies. After 4 h of derepression, dNM treated cells showed a more heterogeneous pattern of glycosylated invertase than control cells (Figure 6, left

Protein secretion from Saccharomyces cerevisiae: U. F. Greber et al. panel). The protein part of the glycosylated invertase remained unaffected by dNM, as demonstrated after deglycosylation with Endo H (Figure 6, right panel). Three major bands around 67 000 could be detected which most likely represent invertase species that still contain the inner core oligosaccharides. 7 This might indicate that dNM did not affect the formation or the extension of the N-linked glycans within the protein, but affected the surface orientated oligosaccharides. We assume that dNM reduced the amount of catalytically active external invertase by preventing a complete glycosylation. Our experiments indicate that dNM reduces the extent of glycosylation and effects a size reduction of secreted polypeptides from yeast cells. Thus, dNM might interfere with the formation of invertase oligomers, a prerequisite for activity of the enzyme, 37 or it might result in properly folded molecules which are still immunoreactive but less active. Analogous results were obtained for an underglycosylated form of the yeast vacuolar protease, carboxypeptidase y.38 Nomenclature dNM 1-deoxynojirimycin Endo H endo-/3-N-acetylglucosaminidase from

Streptomyces plicatus ER endoplasmic reticulum GAPDH glycerinaldehyde-3-phosphate dehydrogenase HRP horseradish peroxidase SMC somatomedin C TCA trichloroacetic acid topo 2 topoisomerase 2 References 1 2 3

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