©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
N- and O-Glycosylation and Phosphorylation of the Bar Secretion Leader Derived from the Barrier Protease of Saccharomyces cerevisiae(*)

(Received for publication, May 25, 1995; and in revised form, August 15, 1995)

Mette U. Jars (§) Sherri Osborn John Forstrom Vivian L. MacKay

From the From ZymoGenetics, Inc., Seattle, Washington 98102

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure.


INTRODUCTION

A leader sequence secures secretion of a protein of interest(1) . As the fusion protein proceeds through the secretory pathway, it may undergo extensive post-translational modifications (e.g. glycosylation of specific asparagine (Asn) and serine or threonine (Ser/Thr) residues(2, 3) ). These modifications may be important for efficient secretion of the protein(3) . However, very little is known specifically about how post-translational modifications (especially O-glycosylation) affect the ability of leaders to export proteins. Therefore, the purpose of this work has been to determine if and how cell physiology and growth conditions influence post-translational modifications of a secretion leader derived from the signal peptide and primarily the third domain of the BAR1 gene product of the yeast Saccharomyces cerevisiae. This secretion leader has been used to export heterologous proteins (e.g. platelet-derived growth factor and insulin precursors (4) ) and will be referred to as the bar secretion domain (BSD). (^1)Previous studies have indicated that the BSD contains substantial levels of N- and probably also O-glycosylation(5) .

The most common post-translational modifications in S. cerevisiae are N- and O-glycosylation. Oligosaccharides consisting of Glc(3)Man(9)GlcNAc(2) may be transferred as a unit to specific asparagine residues (Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except Pro; (6) ) in nascent peptides in the lumen of the endoplasmic reticulum. These oligosaccharides are trimmed in the endoplasmic reticulum to generate a Man(8)GlcNAc(2) intermediate, which in yeast is usually elongated in the Golgi to yield species of ManGlcNAc(2)(6, 7) . On secreted glycoproteins from yeast, some of these core oligosaccharides become further elongated by addition of an alpha1,6-linked mannose and its extension to form an alpha1,6-linked backbone and by addition of short alpha1,2-linked side chains, which in turn may get the addition of alpha1,3-linked side chains to form an outer chain of geq50 mannoses (hyperglycosylation)(6) . Additional mannose residues in phosphodiester linkages may also be present in the core and in the outer chain(8, 9) .

Ballou (10) has isolated mnn mutants that are defective in mannan biosynthesis. The mnn1 mutation results in the synthesis of N- and O-linked structures (N- and O-structures) that lack terminal alpha1,3-linked mannose attached to mannose in alpha1,2-linkage, and the mnn9 mutants lack the ability to elongate the alpha1,6-linked backbone of the N-structures(10) . Due to these mutations, the mnn9 strain primarily makes N-structures of ManGlcNAc(2), and the mnn1 mnn9 mutants make N-structures of mainly ManGlcNAc(2) units along with a small amount of ManGlcNAc(2) units(10) . The MNN1 gene encodes the alpha1,3-mannosyltransferase(11, 12) , and the MNN9 gene is believed to encode a protein involved in maintaining a Golgi apparatus with functioning mannosyltransferases (13) .

No consensus sequence for O-glycosylation has been found(2, 14) . The first sugar transfer of mannosyl residues to Ser or Thr is catalyzed in the endoplasmic reticulum and requires dolichol-phosphate-mannose as an intermediate(15) . The additional mannosyl residues (maximally 3-4) are transferred from GDP-Man(15) . It is uncertain whether the second alpha1,2-linked mannose is added in the endoplasmic reticulum or Golgi(15, 16) , but the third alpha1,2-linked mannose is added in the Golgi(17) . An additional one to two alpha1,3-linked mannoses may then be added to the three alpha1,2-linked mannoses in the Golgi. O-Structures with one to five mannoses are expected on proteins expressed in the wild-type and the mnn9 strains(2, 10, 18) . Addition of the first and probably the second alpha1,3-linked mannose requires the MNN1 gene (10) . Therefore, the expected O-structures on proteins expressed in the mnn1 and mnn1 mnn9 strains have one to three alpha1,2-linked mannoses(10) .

The DNA sequence of the secretion leader used in this work has three potential N-linked (Asn) and 54 potential O-linked (Ser/Thr) glycosylation sites (see Fig. 1) and has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Post-translational modifications of the BSD have been analyzed with respect to site selectivity by peptide mapping, sequencing, and size of O-structures by electrospray (ES) liquid chromatography mass spectrometry (LC/MS). An unusual phosphorylated post-translational modification was investigated by ES tandem mass spectrometry (MS/MS) and elementary analysis.


Figure 1: Site selectivity for post-translational modifications of BSDwt-N and BSDmnn1 mnn9. Amino acid sequence of the secretion leader, showing sites for N- and O-glycosylation and the peptides generated by digestion with lysyl endopeptidase. Potential O-linked Ser and Thr are single underlined and potential N-linked Asn residues are double underlined. Sequences with Ser and Thr, which have been investigated on synthetic peptides by Strahl-Bolsinger and Tanner(42) , are indicated in boxes. The results from Edman degradation of fractions from digests of BSDwt-N and BSDmnn1 mnn9 are shown above the sequence. O and N above a residue indicate highly O- and N-glycosylated, respectively. Similarly, (O) and (N) indicate moderately glycosylated, and ((O)) and ((N)) indicate poorly glycosylated. ? indicates that the residue was late in a sequence so the categorization is less accurate. ?? indicates that Edman degradation data of that residue were not available.




EXPERIMENTAL PROCEDURES

Fermentation

Four different S. cerevisiae strains were used in this study, one wild-type for glycosylation and three with mnn mutations: ZM137 (MATa/MATalpha pep4-3/pep4-3 leu2-3, 112/leu2-3, 112 Deltatpi1::LEU2/Deltatpi1::LEU2), XCY93-1C (MATalpha ade2 leu2-3, 112 ura3-52 Deltapep4::URA3 Deltamnn1::URA3 suc2-Delta9), ZY400 (MATaade2 leu2-3, 112 ura3-52 Deltapep4::URA3 Deltamnn9::URA3 suc2-Delta9), and XCY93-1D (MATaade2 SUPX leu2-3, 112 ura3-52 Deltapep4::URA3 Deltamnn9::URA3 Deltamnn1::URA3 suc2-Delta9). These strains were transformed either with an expression plasmid for the BSD (pZY93 for strain ZM137 and pSW327 for the others) or with a control vector (pRPOT for ZM137 and YEp13 for the others). In both plasmids (constructed by C. Yip and S. Welch, ZymoGenetics), the BSD coding sequence is under the control of the promoter from the TPI1 gene and contains a C-terminal substance P epitope tag for immunological determination(19, 20) .

The transformants were grown in fermentation medium (FM) 1 (7.5 g/liter yeast extract (Difco), 14.0 g/liter (NH(4))(2)SO(4), 2.7 g/liter KH(2)PO(4), 2.0 g/liter MgSO(4)bullet7H(2)O, 125 µg/liter biotin, 2.0 mg/liter thiamine, 2.0 mg/liter pyridoxine, 37.5 mg/liter inositol, 37.5 mg/liter calcium pantothenate, 1.5 mg/liter niacinamide, 250 µg/liter folic acid, 0.5 mg/liter riboflavin, 2.5 mg/liter choline chloride, 6.8 g/liter ZnCl(2), 54 mg/liter FeCl(3)bullet6H(2)O, 19.1 mg/liter MnCl(2)bullet4H(2)O, 2.2 mg/liter CuSO(4).5H(2)O, 2.6 mg/liter CoCl(2), 0.62 mg/liter H(3)BO(3), 21 µg/liter (NH(4))(6)Mo(7)O.4H(2)O, 21 µg/liter KI, 0.1 ml of concentrated HCl, and 2.0% (w/v) D-glucose in tap water). In fermentations of the wild-type, mnn1 mnn9, and mnn9 strains, 0.75 MD-sorbitol was added to the medium. Strains carrying the mnn9 mutation require the presence of D-sorbitol to osmotically stabilize the cells during growth(10) . Tanks of 60 liters of medium were inoculated with 3% (v/v) wild-type or mnn1 mnn9 transformants, and the cells were fermented at 30 °C for 48 h. D-Glucose was fed continuously during the fermentation at a rate of 1.5 g/liter/h, and pH was kept at 4.5 with ammonia. The mnn9 and mnn1 transformants were grown at 30 °C for 48 h in Fernbach flasks of 10 times 1200 ml of FM1 + D-sorbitol and 6 times 1200 ml of FM1, respectively. Additional D-glucose was added to the cultures to a final concentration of 2% (w/v) after 24 h. Wild-type transformants were also grown in 1200 ml of FM2 containing 40 g/liter yeast extract (Difco), 20 g/liter Bacto-peptone (Difco), 0.1 ml/liter pluronic acid (100% v/v), 18 g/liter D-glucose, pH 6.5 at 30 °C. At 90 h, 5% (v/v) glacial acetic acid was added to the medium, and the fermentation was stopped after 96 h.

Purification

BSD from a wild-type strain will be designated BSDwt, a similar nomenclature is used for the other strains. The number 2 in the name (e.g. BSDwt2-N) indicates that the strain has been grown in FM2, and the letter N indicates that the N-structures have been enzymatically removed. For BSDwt and BSDwt-N, the culture supernatant of 60 lilters was filtered through a 0.45 µm^2 polypropylene Gelman filter, concentrated to 1.5 liters on a hollow fiber system (UFP-5-C-55, cut off 5000 Da, A/G Technology Corporations), precipitated by ethanol(20) , and resuspended in 640 ml of 5 mM EDTA. A volume of 40 ml of the resuspended precipitate (BSDwt) containing 10 mM dithiothreitol was fractionated on a Sephacryl column (4.0 times 86 cm S-200, HR; Pharmacia) equilibrated in 0.15 M NaCl and 5 mM EDTA. After addition of 16 ml of 0.5 M sodium acetate, pH 5.2, 80 ml of resuspended ethanol precipitate was deglycosylated by incubation at 37 °C for 2 days with 3 units of endo-beta-N-acetylglucosaminidase H (Boehringer Mannheim). This batch (BSDwt-N) was then fractionated as described for BSDwt. The two batches were individually desalted on a reversed phase HPLC C-4 column (2.2 times 25 cm, Vydac) by elution with a gradient of 5-35% (v/v) acetonitrile (ACN) in H(2)O and 0.1% (v/v) trifluoroacetic acid. The samples were lyophilized and stored at -20 °C. BSDwt2-N was purified from 500 ml of broth after 96 h of fermentation as described for the BSDwt-N, except that BSDwt2-N was concentrated on an Amicon cell (cut off 50 kDa, Amicon) instead of the hollow fiber system.

BSDmnn1 mnn9 was purified as the BSDwt sample, but the broth was concentrated on a 1.5-liter Amberchrom CG71-md column (TosoHaas). After loading the sample, the column was washed with 20 liters of 0.1 M sodium acetate, 2 mM EDTA, and NaCl (to 15 millisiemens/cm), pH 4.5, followed by 8 liters of 2 mM EDTA. The sample was eluted with 1050 ml of 40% (v/v) ethanol in 2 mM EDTA and ethanol precipitated(20) . BSDmnn1, BSDmnn1-N, and BSDmnn9 were purified as described for BSDwt-N, but after the HPLC C-4 column, the samples were individually separated on a reversed phase HPLC C-18 column (0.46 times 25 cm, Vydac) eluted with a gradient from 2-41% (v/v) ACN in H(2)O and 0.05% (v/v) trifluoroacetic acid. BSDmnn9 was further separated on a divinyl benzene column (0.46 times 50 cm, PLRP-S 1000 Å, Polymer Laboratories) with a similar gradient. BSDmnn1-N and BSDmnn9 were then individually purified by gel filtration on a TSK SW3000 column (0.75 times 30 cm, Hewlett Packard) by elution with 0.15 M NaCl and 5 mM dithiothreitol, concentrated on an HPLC C-18 column, vacuum rotor evaporated, and stored at -20 °C. The BSD batches were analyzed by SDS-polyacrylamide gel electrophoresis (21, 22) and immunoblotted with antibodies against substance P(19, 20, 23) .

Amino Acid Sequence Analysis

N-terminal sequences were determined by automated Edman degradation for the BSD batches and several peptides from peptide mapping of the BSD using an Applied Biosystems model 475 or model 476A(24) .

Amino Acid Analysis

All samples, except BSDmnn1, BSDmnn1-N, and BSDmnn9, were analyzed by amino acid analysis through hydrolysis of known volumes containing 0.5-0.8 nmol of protein with 6 M HCl/2% phenol for 22 h at 112 °C(25) ; results were not corrected for loss during hydrolysis.

Peptide Mapping

Approximately 0.2 amidase unit of lysyl endopeptidase from Achromobacter protease I (lysyl endopeptidase, Wako Chemicals U. S. A., Inc.) was added per 1.0 mg of BSD glycoprotein dissolved in 0.9 ml of 50 mM Tris-HCl and 6.5 mM dithiothreitol, pH 8.9. The sample was left at 4 °C for 92-144 h, and then the digestion was stopped with 50 µl of glacial acetic acid/ml sample. The glycopeptides were chromatographed on reversed phase HPLC C-18 columns (0.46 times 25 cm or 0.21 times 25 cm, Vydac) eluted in gradients from 2-80% (v/v) ACN in H(2)O and 0.05% (v/v) trifluoroacetic acid.

ES MS

The analysis was performed using an API III liquid chromatography triple quadrupole mass spectrometer fitted with an articulated ion spray plenum and an atmospheric pressure ionization source (Perkin Elmer Sciex) and a scanning range of m/z 0-2400 Da(26) . The instrument was tuned and calibrated with the singly charged ammonium adduct ions of polypropylene glycols under unit resolution. The orifice value was usually held at 65 V to avoid O-glycosidic cleavage of the glycopeptides. In ES MS analysis, the samples were introduced at a flow rate of 5 µl/min and analyzed in the mass range of m/z 600-2400 Da at a step size of 0.1-0.2 atomic mass unit and a dwell time of 0.5-0.75 ms. In comparative peptide mapping by ES LC/MS, 40-200 µg of BSD digestion mixtures were separated on a reversed phase HPLC C-18 column (0.21 times 25 cm, Vydac), typically with a flow rate of 0.2 ml/min (split to 30 µl/min flow and infused onto the ES MS). The following gradient was used: t = 0 min, ACN in H(2)O = 2% (v/v); t = 80 min, ACN = 17.6% (v/v); t = 110, ACN = 41% (v/v); t = 120, ACN = 80% (v/v). In ES MS/MS analysis, the fractions from a peptide digest of the BSDwt-N were introduced at a flow rate of 5 µl/min and analyzed in the mass range of m/z 0-1800 Da at a step size of 0.1-0.2 atomic mass unit and a dwell time of 0.5-0.75 ms. The collision gas was a mixture of argon and nitrogen (90:10).

Matrix-assisted Laser Desorption (MALDI) MS

Intact BSDmnn1 mnn9 and BSDwt-N as well as glycopeptides of BSDwt-N collected from a peptide digest were analyzed by Charles Evans & Associates on a reflector Vision 2000 MALDI MS time of flight mass analyzer (Finnigan)(27) . The mass range was 500-500,000 Da. A total of 5 pmol BSDmnn1 mnn9, dissolved in 0.1% (v/v) trifluoroacetic acid to l0 pmol/µl and mixed 1:1 with the 2,5-dihydroxybenzoic acid matrix, was analyzed with 5 pmol of apomyoglobin as an external calibrant. BSDwt-N was analyzed the same way. Lyophilized fractions from a peptide digest of 1.0 mg of BSDwt-N were redissolved in 0.1% (v/v) trifluoroacetic acid, mixed 1:2 with the above matrix, and analyzed with renin substrate and human insulin as internal standards.

Elementary Analysis

Phosphorus and sulfur were analyzed by inductively coupled MS on an Elan 5000 A (Perkin Elmer Sciex) in collaboration with Thomas Chapin (University of Washington). The instrument was blanked against 1% (v/v) HNO(3) in H(2)O and calibrated with solutions of (NH(4))(2)SO(4) and KH(2)PO(4) in 1% (v/v) HNO(3) in H(2)O. The samples were analyzed in positive ion peak hop mode where the mass analyzer dwells at m/z 31 for 1000 ms and then 34 m/z for 1000 ms, etc. The samples were introduced with a continuous flow nebulizer at a flow rate of 1 ml/min(28) . Intact BSDwt-N, BSDmnn1 mnn9, and fractions from a peptide digest of BSDwt-N were analyzed.

Peptide Synthesis

A peptide (LVNIQTDGSISGAK) corresponding to lysyl endopeptidase peptide 3 of the BSD was synthesized on an Applied Biosystems model 431A peptide synthesizer using Fmoc (9-fluorenylmethoxycarbonyl) chemistry and standard cycles for 1-hydroxybenzotriazole activation(29, 30, 31) . The product was purified by reversed phase HPLC, and the identity was confirmed by ES MS analysis and Edman degradation.


RESULTS

Fermentation and Purification

BSD preparations purified from each of the four yeast transformant strains were contaminated with the heat shock protein Hsp150p(32) . Based on amino acid analysis and the yield of the first amino acid in Edman degradation (data not shown), the following yields (mg) and purities (%) were determined for the different preparations: BSDmnn1 mnn9 (0.4 mg/liter, 89%), BSDwt-N (51.0 mg/liter, 93%), BSDwt (6.2 mg/liter, 83%), and BSDwt2-N (67.3 mg/liter, 81%). The yields and purities of the remaining batches were as follows: BSDmnn1 (0.5 mg/liter, 28%), BSDmnn1-N (0.04 mg/liter, 89%), and BSDmnn9 (0.006 mg/liter, 52%), assuming that 1 mg/ml is equal to 1.0 on the UV absorbance at 280 nm. Hsp150p was more difficult to remove from the BSDmnn1, BSDmnn1-N, and BSDmnn9 batches.

Characterization of the BSD Batches

The proposed signal peptide of the bar secretion leader contains 24 amino acid residues(20) . Amino acid sequence analysis of purified BSD confirmed that the signal peptide in all batches is processed between amino acids Ala^0 and Leu^1 (Fig. 1) as expected from von Heijne's rule(33) . The polypeptide has a calculated molecular mass of 16,766 Da. However, based on MALDI MS the molecular mass range of the BSDmnn1 mnn9 was 35,000-48,000 Da peaking at 44,317 Da, and the range of the BSDwt-N was 30,000-40,000 Da peaking at 38,949 Da. On an immunoblot, the molecular masses were as follows: BSDwt = BSDmnn1 (127,000-208,500 Da) > BSDmnn9 (73,4000-91,900 Da) > BSDmnn1 mnn9 (61,000-70,400 Da) > BSDwt-N = BSDwt2-N = BSDmnn1-N = BSDmnn9-N = BSDmnn1 mnn9-N (40,500-54,000 Da).

Site Selectivity of Post-translational Modifications by Peptide Mapping of BSDwt-N and BSDmnn1 mnn9

Theoretically, lysyl endopeptidase should cleave the BSD (referred to as Leu^1 to Met) into 8 peptides (P1-P8; Fig. 1). Peptide 2 consists of Lys only; the rest were glycopeptides and will be referred to as (GP1-GP8). Separation of the digestion mixtures on a reversed phase HPLC C-18 column gave reproducible but complex UV-215 absorbance traces with broad overlapping peaks, as shown for BSDwt-N in Fig. 2. The cleavage site between Lys and Pro was not always cleaved. In general, GPm,n indicates that glycopeptide GPm and GPn are not cleaved, so in this case the above glycopeptide is designated as GP6,7. Additionally, an unexpected cleavage site between Leu and Ser was observed in BSDwt-N, giving rise to GP1`` ( Fig. 1and Fig. 2).


Figure 2: Elution of glycopeptides for BSDwt-N after peptide mapping with lysyl endopeptidase. The indicated C-18 fractions were analyzed by Edman degradation, ES MS, or MALDI MS. The gradient in the percentage of ACN is depicted.



In Edman degradation data, the repetitive yield for each sequence was calculated based on a regression analysis of the more stable amino acids in each sequence (Ala, Glu, Gly, Ile, Leu, Lys, Phe, and Val), and the average repetitive yield was 91% for all the sequences from the BSD. The post-translationally modified amino acids were identified as missing PTH-derivatives in Edman degradation. The obtained yield (Y(n)) in percentage of the expected yield (Y(e)) (based on the regression analysis) was calculated for every PTH-Ser, PTH-Thr, and PTH-Asn in each sequence. PTH-Ser and PTH-Thr are unstable. Additionally, the position of Ser, Thr, and Asn in a sequence and the amount of material used in Edman degradation both affect the yields of these amino acids. These effects were tested with a synthetic lysyl endopeptidase Peptide 3, where the Y(n) in percentages of Y(e) were 62, 26, and 29% of PTH-Thr, PTH-Ser, and PTH-Ser, respectively (Fig. 1).

Each glycopeptide was analyzed several times due to overlapping peaks. A Ser or Thr was categorized as highly O-glycosylated if the Y(n) of that residue in all analyses was less than 10% and the increase in yield (Y(n) - Y) was less than 5% of Y(e), moderately O-glycosylated if Y(n)<25% of Y(e), and poorly O-glycosylated if 25% < Y(n) < 70% of Y(e). Potential N-glycosylation sites were categorized similarly. The results of Edman degradation for both BSDmnn1 mnn9 and BSDwt-N are shown in Fig. 1. Because not every fraction was analyzed, the summary in the figure is an indication of the glycosylation rather than its absolute quantitation. The BSDmnn1 mnn9 had 30 highly, 13 moderately, and 10 poorly O-glycosylated sites of the total 54 Ser and Thr, respectively. Asn and Asn residues were moderately N-glycosylated, and Asn were poorly N-glycosylated. The BSDwt-N had 35 highly, 8 moderately, and 11 poorly O-glycosylated sites, respectively. Asn and Asn residues seemed to be highly N-glycosylated, and Asn seemed to be moderately N-glycosylated. Differences in O-glycosylation sites on BSD expressed in the mnn1 mnn9 strain and the wild-type strain were apparent on 22 sites (Fig. 1), with the most pronounced differences seen at Ser, Thr, and Ser. However, there was no clear pattern in the extent of O-glycosylation on any of the 22 sites. If there was a pattern as to the extent of N-glycosylation, the BSDwt-N had a higher amount of Asn involved in N-glycosyl linkages than the BSDmnn1 mnn9.

ES MS analysis of different fractions from the peptide maps showed that they contained the expected heterogeneous glycopeptides. For example, fraction 27 from BSDwt-N contained GP5 with 1 N-acetylglucosamine and 29-33 mannoses (referred to as P5+1GlcNAc+(29-33)Man) and GP5 with 1 N-acetylglucosamine and 28-33 mannoses and an additional 80 mass units. This additional mass could represent a sulfate group (S) or phosphate group (P) (34) (referred to as P5+1GlcNAc+(28-33)Man+P/S), as discussed in the next section. As the heterogeneity of a glycopeptide varied with respect to the number of N-acetylglucosamines, mannoses, and a phosphate group (or sulfate group), in the following section, a distribution will refer to a given glycopeptide that has a particular number of GlcNAcs and a phosphate group (or sulfate group) across a range of mannoses (e.g. P5+1GlcNAc+(28-33)Man+P/S). Each of the ``members'' within the distribution will be referred to as a variant of that distribution (e.g. P5+1GlcNAc+28Man+P/S). Each glycopeptide variant eluted with decreasing mass as a function of time, as expected (35) . All the glycopeptides were observed in distributions with and without an addition of 80 mass units. Some of the glycopeptides with different numbers of mannoses, N-acetylglucosamine, phosphate, and/or sulfate may have the same masses (e.g. GP5,6 and GP5) and cannot be distinguished by ES MS analysis. In this case it was determined by Edman degradation that only GP5 was present. However, Edman degradation could not solve this problem with GP4 and GP8, because they coeluted.

By using orifice values between 45-70 V, the heterogeneity observed by ES MS seemed real and not generated during the analysis by bond breakage between sugar units on the glycopeptide in the declustering region between the orifice and the first reference-only quadrupole. In agreement with the literature(35) , orifice values between 55-65 V seemed to be optimal to avoid fragmentation and to obtain good sensitivity. Generally, the same distributions and relative intensities of the variants were observed by MALDI MS and ES MS, showing that the heterogeneity was in fact real. However, the ES MS data gave the best accuracy.

An Unusual Post-translational Modification

Because no phosphate or sulfate was added during the purification procedures of the BSD batches and the last purification step was a reversed phase C-4 column, it was assumed that phosphorus and/or sulfur found in these samples would be covalently attached to the glycoprotein. The sulfur concentration in the BSD batches was expected to be 7 sulfurs/molecule of protein, because the DNA sequence has 3 Cys and 4 Met. In elementary analysis the sulfur concentration was 6.3 and 7.8 sulfurs/molecule in the BSDwt-N and BSDmnn1 mnn9, respectively, values which were within 2-3 times the standard deviation of the expected value. Therefore, there is no evidence for post-translational sulfate addition. In contrast, the BSDwt-N and BSDmnn1 mnn9 contained approximately 6.9 and 3.6 mol of phosphorus/mol of protein, respectively, all of which must be added post-translationally. On the basis of these results, it was assumed that the unexpected variants with an additional 80 mass units could be associated with post-translational addition of a P. Analysis of fractions containing GP1, GP3, or GP5 from the BSDwt-N enriched with an additional 80 mass units showed the presence of phosphorus. Treatment with Escherichia coli or bovine alkaline phosphatase of the BSDwt-N did not change the elution time on a reversed phase HPLC C18 column, but treatment in mild acid delayed the elution time of BSDwt-N on the column (data not shown), as expected if a phosphate group was chemically cleaved by the acid treatment(36) .

The product ions generated in positive ion mode ES MS/MS analysis of the selected precursor ion m/z 1633.9, representing P3+11Man+P of BSDwt-N, is shown in (Table 1). Note the abundance of ions representative of O-glycosidic cleavage to yield both reducing and nonreducing terminal fragments without any evidence of amide bond cleavage of the peptide backbone, which is typical for a glycopeptide(37) . Adopting the nomenclature proposed by Domon and Costello(38) , the reducing terminal fragments with the alcohol group attached generated from O-glycosidic cleavage are designated Y-type ions. In the high mass-to-charge region is a complete series of doubly charged Y-type ions (m/z 1553.05, 1471.81, 1390.76, 1309.72, 1228.68, 1147.43, 1066.39, 985.34, 904.10, and 823.05) corresponding to consecutive losses of monosaccharide residues with hydrogen rearrangement and charge retention on the reducing end of GP3. The Y-type ions equivalent to losses of 1-10 hexoses from the precursor ion are observed, down to m/z 783.03 representing P3+Man+P. The next mass loss is equivalent to a mannose and a P, although no mass is present that could represent P3+P. At the low mass-to-charge region, three characteristic oxonium ions at m/z 242.93, 405.02, and 567.91 are observed, arising from cleavage at the glycosidic linkage with charge retention on the sugar. These represent Man-P+, ManMan-P+, and ManManMan-P+, respectively. These two observations lead to the conclusion that the phosphate group is attached to the sugar moiety, rather than the backbone of the protein. These oxonium ions were only observed when selecting precursor ions of variants expected to contain a P. As mentioned previously, GP4 and GP8 coeluted and may have the same masses, but based on data from ES MS/MS analysis, GP8 seemed to elute slightly earlier than GP4.



Comparative Peptide Mapping by ES LC/MS

The contour plot from the analysis of BSDwt-N is shown as an example in Fig. 3, with the m/z values for all the masses in the range of m/z 600-2400 for each single scan. The glycopeptides can be observed in the contour plot as negatively sloping ``streaks'' (39) eluting in the same order as observed in Fig. 2. However, the elution times of the glycopeptides are not identical in the two figures due to the two different size columns used. As an example, the sum of all the mass spectra in the scans 327-384 of GP5 in the high mass range is shown in Fig. 4. The data can be summarized as P5+1GlcNAc+(25-36)Man+P and P5+1GlcNAc+(24-37)Man. A summary of the distributions of each glycopeptide eluting in the ES LC/MS analysis of BSDwt-N is shown in Table 2. Extensive O-glycosylation is observed with typically more than two mannoses/O-linked site for the most intense variants of each distribution for every glycopeptide.


Figure 3: Contour plot of the ES LC/MS analysis of BSDwt-N. BSDwt-N (0.2 mg) lysyl endopeptidase digest was separated on a reversed phase C-18 column. The m/z values are shown for each scan. The intensity of the peak is represented by a black-gray scale. The glycopeptide streaks are circled, and the charge states are indicated (e.g. [GP6,7+4H]4+ represents GP6,7 with 4 hydrogens and a charge state of 4+).




Figure 4: Sum of mass spectra of GP5 from ES LC/MS analysis of BSDwt-N. GP5 is eluting in scans 317-384 in Fig. 3. Two distributions are present, one with and one without phosphorylation in charge state 4. All the variants have 1 GlcNAc. The number of mannoses are indicated above the m/z value in parenthesis for the distribution with phosphorylation.





The obtained ES LC/MS data for all the BSD batches have been summarized in Table 3. Only the range of mannoses/O-linked site of each distribution is shown. Obviously, different distributions are observed in batches that have and have not been deglycosylated with endo-beta-N-acetylglucosaminidase H. Unexpectedly, GP1 and GP6,7 were observed with 1 GlcNAc in batches that had not been deglycosylated with endo-beta-N-acetylglucosaminidase H. It cannot be excluded that these distributions with 1 GlcNAc were generated in the ES LC/MS analysis. Such an artifact has previously been reported(40) . GP7 gave weak signals when present due to incomplete cleavage between Lys and Pro. Because BSDmnn9, BSDmnn1, and BSDmnn1-N were analyzed in small amounts of 40-80 µg/sample only, some variants were not observed in these batches (e.g. GP7 and phosphorylated distributions of GP6). In BSDmnn1 and BSDwt, the distributions of glycopeptides with hyperglycosylated N-structures could not be expected to fall inside the mass range of m/z < 2400 Da, and these distributions were not observed. The batches do not show identical distributions, but a great deal of similarity is present, and no clear pattern differences were observed. Differences were primarily noted for the less intense variants (i.e. some phosphorylated distributions of very weak intensities were observed in BSDwt-N but not in BSDwt2-N) (Table 3). The total range of mannoses/O-linked site was as follows: BSDmnn1 mnn9 (1.3-4.0), BSDmnn9 (1.4-4.0), BSDmnn1 (1.4-4.0), BSDmnn1-N (1.2-4.0), BSDwt (0.7-4.3), BSDwt-N (0.0-4.3), and BSDwt2-N (0.7-4.3). The most surprising result was that both BSDmnn1 mnn9 and BSDmnn1-N had variants of GP1 and GP3 with more than 3 mannoses/O-linked site (see Discussion). Data from GP4 and GP8 were not included because the elution of these peptides was uncertain. No distributions of GP5 without GlcNAc were present in any of the batches, indicating that the Asn residue was originally fully N-glycosylated.



Distributions of phosphorylated glycopeptides gave less intense signals in ES MS analysis than those without phosphate. This is as expected due to the electronegative character of the P, which decreases the likelihood of ionization. No distributions with more than one P/peptide were observed in the ES MS analysis; if present, they may not have ionized in the positive ion mode used.

The growth medium tested did not seem to affect the post-translational modifications of the BSD expressed in wild-type S. cerevisiae. The similarities of the glycopeptide distributions between BSDwt-N (fermented in FM1) and BSDwt2-N (fermented in FM2) were high in terms of both the number of mannoses/O-linked site (Table 3) and the abundance of each variant (ES MS data not shown). The only exceptions were some phosphorylated distributions of weak intensities. All this suggests fairly consistent processing in vivo.


DISCUSSION

As previously proposed(5) , the BSD is a heavily glycosylated heterogeneous protein. The N-structures were influenced by the mnn1 and mnn9 mutations as expected, and hyperglycosylation was observed only on the BSD expressed in the mnn1 and wild-type strains(10) . All the BSD had the same molecular mass after endo-beta-N-acetylglucosaminidase H treatment. Therefore, the difference in the peak molecular mass between the BSDwt-N (38,949 Da corresponding to 133 mannose residues/molecule) and BSDmnn1 mnn9 (44,317 Da corresponding to 162 mannose residues/molecule) of 5,368 Da (analyzed by MALDI MS) may be explained by partial occupancy of the 3 potential N-glycosylation sites residues 19, 89, and 124 in the BSDmnn1 mnn9 sample with the expected structure of ManGlcNAc(2). Carbohydrate analyses confirmed a high content of 106-329 mannose residues/molecule in the BSD batches and the expected size of the N-structures. (^2)In all batches, some N-glycosylation was found at Asn and Asn, as determined by Edman degradation and ES LC/MS analysis. Asn seemed to be fully N-glycosylated, based on ES LC/MS experiments, but Edman degradation indicated some unglycosylated material as well. Because an Asn precedes the Asn in the sequence, the result from Edman degradation could be less accurate.

No clear pattern was observed regarding the site selectivity and the extent of O-glycosylation for BSDwt-N and BSDmnn1 mnn9 based on Edman degradation. Almost all the 54 Ser and Thr residues were O-glycosylated to some extent. Clustering of Ser/Thr in O-glycosylated mucins has been described(41) . This was also observed in the BSD, but no clear information that may help predict the amino acid distribution around O-linked sites in S. cerevisiae was established. Strahl-Bolsinger and Tanner (42) observed that a Gly residue immediately preceding Thr (Gly-Thr) and an acidic amino acid in the vicinity of a hydroxy amino acid eliminated the mannosyl acceptor property of the hydroxy amino acid on synthetic peptides in vitro. Their data have been supported by analysis of human IGF-I (43) but not by analysis of the B-chain from platelet-derived growth factor(44) , both expressed in S. cerevisiae. Their conclusions are also not supported in the present study with BSD, at Gly^5-Thr^6, Gly-Thr, Ser-Glu, Glu-Thr, and Glu-Thr, where prevention of the mannosylation of the hydroxy amino acid did not occur (Fig. 1).

Pro enriched in positions -1 and +3 did not seem to be an indicator of O-glycosylation for the 6 Pro in BSD, in contrast to O-linked N-acetylgalactosamine in mucins(41) . This result could suggest, as also proposed by Strahl-Bolsinger and Tanner(42) , that the rules for site selectivity of O-glycosylation are somewhat different in mammalian and yeast cells. Other in vitro studies with the yeast Candida albicans using synthetic peptides indicated that Thr was more frequently O-mannosylated than Ser and that it was unlikely that both Ser and Thr were mannosylated on the same peptide(45) . None of these observations were seen in the BSD.

Surprisingly, no apparent influence on the O-glycosylation by the mnn1 mutation could be detected from the immunoblot. The M(r) after removal of the N-structures indicated substantial amounts of O-glycosylation. In ES LC/MS analysis there were O-structures present with at least 4 mannoses/site (chain) on both the BSD from the mnn1 and mnn1 mnn9 strains. It was confirmed by carbohydrate analysis that all strains produced O-linked chains of 1-5 mannoses,^2 although, it is unknown if these long O-linked chains are present on both Thr and Ser residues. This result was surprising because the mnn1 mnn9 and mnn1 strains were not expected to have O-structures with more than 3 mannoses/site because the mnn1 mutation prevents addition of terminal alpha1,3-linked mannoses(10) . There is no obvious explanation why the O-structures on BSD expressed in the mnn1 and mnn1 mnn9 strains were longer than 3 mannoses/site, and it is unknown how the fourth and fifth mannoses were added, perhaps as alpha1,2-linked mannoses or alpha1,3-linked mannoses. If the latter is true, then there must be an additional alpha1,3-mannosyltransferase present in S. cerevisiae.

The glycopeptides were observed with ranges of 0.0-4.3 mannoses/O-linked site in ES LC/MS analysis. It was shown that the same heterogeneity in a given glycopeptide was observed by both MALDI MS and ES MS, but it was not proven that the relative intensities of the variants (peaks) observed in the mass spectra actually reflect the relative quantities of each variant. However, in comparison within (and only within) a given distribution of a glycopeptide that varies only in the number of mannoses, it is assumed that the intensities of the peaks (variants) reflect the actual concentrations of the variants. Then, based solely on the most intense variants in each distribution, the average O-linked chain length/site is greater than 2.0 in all the batches. The total number of mannoses on the 8 glycopeptides from BSDwt-N (attached to the most intense variants in distributions without phosphorylation; Table 2) is 126, equivalent to 2.3 mannoses/O-linked site on average. This hypothetical BSD structure is in agreement with the expected number of 133 mannoses corresponding to 2.5 mannose residues/O-linked site calculated from the M(r) peak of BSDwt-N from MALDI MS.

For BSD expressed in the wild-type strain, no significant differences in post-translational O-linked modification were observed between BSD samples produced in FM1 + D-sorbitol and FM2. In contrast to literature reports(46, 47, 48) , the two media (and pH) did not differently influence the O-glycosylation pattern of the BSD, except for minor differences in a some of the phosphorylated distributions of weak intensities.

ES MS and elementary analysis showed that the BSD was post-translationally modified with phosphate. The phosphate group was attached to the O-linked sugar moiety, a linkage that has not previously been described for S. cerevisiae. A product ion of m/z 783.03, representing [P3+1Man+P+2H]2+, was present in ES MS/MS data of P3+11Man+P, showing that the phosphate may be attached to the first mannose on a Ser or Thr. It is unknown if phosphate was attached to the sites of only Ser, only Thr, or both. Phosphates on the N-linked oligosaccharides in S. cerevisiae are in acid labile diesters with 1-2 mannoses in alpha-glycosidic linkage to the phosphate units that are esterified to position 6 of mannose units in the N-structures(8) . A similar phosphorylated O-structure could explain why treatment of the BSDwt-N with mild acid but not alkaline phosphatases delayed the elution time on a reversed phase HPLC C-18 column(36) .

Yeast glycoproteins with phosphorylation are primarily found in the cell wall, although the role of mannose phosphorylation in S. cerevisiae is unknown(2, 6) . Some prokaryotic and eucaryotic heat shock proteins show a stress-dependent phosphorylation of the polypeptide (e.g. Ser) leading to a modulation of their function(49) . It is possible that the phosphorylation plays a role in the putative function of the BSD as a chaperone, particularly through the cell wall.

The contaminant Hsp150p is a heavily O-glycosylated secretory protein(32) . Other heat shock proteins (e.g. Hsp70p) (50, 51) have been shown to serve important functions as chaperones involved in the maintenance or change of the conformation of other proteins. The Hsp150p and the BSD both have extensive O-glycosylation; therefore, it is most likely that they are nearly fully extended in these regions and have structures resembling a semi-flexible rod, as seen for mucins(52) . Perhaps the shape is important in the proposed role of the BSD as a secretion chaperone for a protein (either the Bar protease or a heterologous protein) in transit through the Golgi and/or cell wall. Also, the O-structures could be important in protecting the heterologous protein from protease attack in transit through the secretory pathway.

A secretion leader derived from a domain of the Barrier protease of S. cerevisiae has been expressed in wild-type, mnn1, mnn9, and mnn1 mnn9 glycosylation strains. These mutations and different growth conditions had limited effect on the post-translational modification of the part of the leader sequence exported to the culture medium (except for the size of the N-structures). It is possible that post-translational modifications are important, especially O-glycosylation, for the leader sequence conformation and thereby perhaps for the proposed function of the sequence as a secretion chaperone.


FOOTNOTES

*
This work was supported in part by grants from the Committee on Industrial Research Fellowship under the Danish Academy of Technical Sciences. 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 correspondence should be addressed: Novo Nordisk A/S, Novo alle, DK-2880 Bagsvaerd, Denmark. Tel.: 45-44-42-75-32; Fax: 45-44-42-12-99.

(^1)
The abbreviations used are: BSD, bar secretion domain(s); N-structure, N-linked structures; O-structure, O-linked structure; FM, fermentation medium; ACN, acetonitrile; PTH, phenylthiohydantoin; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; ES, electrospray; LC, liquid chromatography; MS/MS, tandem mass spectrometry; GP, glycopeptide; Pn, peptide n of the BSD after digestion with Lysyl endopeptidase; HPLC, high pressure liquid chromatography.

(^2)
M. U. Jars, S. Osborn, J. Forstrom, and V. L. MacKay, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Lars Thim, Susanne Jacobsen, and Kenneth A. Walsh for valuable discussion and advice. We are grateful to Thomas Chapin, Collin Lellis, Simon Evans, Mads A. Lausten, James H. Yi, Bob Dedinsky, Kathleen Walker, and Lowell Ericsson for expert technical assistance.


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