(Received for publication, May 25, 1995; and in revised form, August 15, 1995)
From the
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 1,3-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.
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). ()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 GlcMan
GlcNAc
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
GlcNAc
intermediate, which in yeast is
usually elongated in the Golgi to yield species of
Man
GlcNAc
(6, 7) . On
secreted glycoproteins from yeast, some of these core oligosaccharides
become further elongated by addition of an
1,6-linked mannose and
its extension to form an
1,6-linked backbone and by addition of
short
1,2-linked side chains, which in turn may get the addition
of
1,3-linked side chains to form an outer chain of
50
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 1,3-linked mannose
attached to mannose in
1,2-linkage, and the mnn9 mutants
lack the ability to elongate the
1,6-linked backbone of the N-structures(10) . Due to these mutations, the mnn9 strain primarily makes N-structures of
Man
GlcNAc
, and the mnn1 mnn9 mutants make N-structures of mainly
Man
GlcNAc
units along with a small amount of
Man
GlcNAc
units(10) . The MNN1 gene encodes the
1,3-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 1,2-linked mannose is added in the endoplasmic
reticulum or Golgi(15, 16) , but the third
1,2-linked mannose is added in the Golgi(17) . An
additional one to two
1,3-linked mannoses may then be added to the
three
1,2-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
1,3-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
1,2-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.
The transformants were
grown in fermentation medium (FM) 1 (7.5 g/liter yeast extract (Difco),
14.0 g/liter (NH)
SO
, 2.7 g/liter
KH
PO
, 2.0 g/liter
MgSO
7H
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
, 54 mg/liter
FeCl
6H
O, 19.1 mg/liter
MnCl
4H
O, 2.2 mg/liter
CuSO
.5H
O, 2.6 mg/liter CoCl
, 0.62
mg/liter H
BO
, 21 µg/liter
(NH
)
Mo
O
.4H
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
1200 ml of FM1 + D-sorbitol and 6
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.
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 25 cm, Vydac) eluted with a gradient from
2-41% (v/v) ACN in H
O and 0.05% (v/v) trifluoroacetic
acid. BSDmnn9 was further separated on a divinyl benzene
column (0.46
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
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) .
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) in percentage of the expected
yield (Y
) (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
in percentages of Y
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 of that residue in all analyses was less than
10% and the increase in yield (Y
- Y
) was less than 5% of Y
, moderately O-glycosylated if Y
<25% of Y
, and poorly O-glycosylated if 25% < Y
< 70% of Y
. 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.
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.
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--N-acetylglucosaminidase H. Unexpectedly, GP1 and
GP6,7 were observed with 1 GlcNAc in batches that had not been
deglycosylated with endo-
-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.
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--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
Man
GlcNAc
. Carbohydrate analyses confirmed a
high content of 106-329 mannose residues/molecule in the BSD
batches and the expected size of the N-structures. (
)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-Thr
, 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 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,
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
1,3-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
1,2-linked mannoses or
1,3-linked mannoses.
If the latter is true, then there must be an additional
1,3-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 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
-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.