Note: Descriptions are shown in the official language in which they were submitted.
~ WO 94/26873 21~1~ 7 3 PCT/SE94~00421
I
YEAST STRAIN AND METHODS FOR EXPRESSING
HETEROLOGOUS PROTEINS IN YEAST.
Technical field:
The invention concerns a novel yeast (Sd~ a,~ ces
cerevisiae) strain that performs O-glycosylation of at least one
serine and/or at least one Ll,r~oll Ie residue at a reduced level. The
inventio~ also provides novel methods for the production of
heterologous proteins, primarily eukaryotic such as Illdllllll '- )
proteins, in yeast cells.
Technical background:
In fungi O-glycosylation is initiated in the endopldsllldLic
reticulum and consists of the transfer of a single mannose residue
from dolichol~ llo~,ul)dLe-mannose (Dol-P-Man) to the nascently
secreted protein (Haselbeck and Tanner, FEBS lett. 158 (1983) 335-
338). ~rl the yeast S. cerevisiae further additions of 3-4 mannose
residues using GDP-Man as donor can occur in the Golgi system
leading to maximal O-glycosyl chain lengths of 5 mannose residues
(Sharma et al., Eur. J. Biochem. 46 (1974) 35-41). Linkages between
mannose residues at positions 1, 2 and 3 are 1-2, while c~l-3
linkages e%ist between mannose residues at positions 3, 4 and 5.
While the in vivo dLLd-,lllllent sites for O-glycosyl chains on
yeast proteins have not been defined, sites on some heterologous
proteins secreted by S. cerevisiae are known. Human granulocyte-
llla~lupllage colony-stimulating factor (hGM-CSF) is partially 0-
glycosylated in yeast (Ernst et al., Eur. J. Biochem. 203 (1992)
663-667). O-glycosylated hGM-CSF species appear to carry either
an extended chain of up to 5 I--an-loses on serine-9, or single
mannose residues simultaneously on serine-9 and threonine-l 0.
Thus, serine-9 is the principal O-glycosyl d~LdCh~ llL site in hGM-
CSF. In yeast-secreted human insulin-like growth factor (hlGF-I)
about 50% of the secreted protein carries a di,lldllnosyl chain on
~I,r~oll;,le-29 (Gellerfors et al., J. Biol. Chem. 19 (1989) 1144~49).
Serine and threonine dlLd~ llL sites have also been identified in
nL~ in vitro using synthetic peptides as substrates for
21~1 ~7~ .
WO 94/26873 PCT/SE94100421 0
yeast mannosyl~,d"~rt:,dses (Bause and Leh!e, Eur. J. Biochem. 101
(1979) 531-40; Strahl-Bolsinger and Tanner, Eur. J. Biochem. 196
(1991 ) 185-90; Lorenz et al., Eur. J. Biochem. 205 (1992) 1163-67).
Like in hGM-CSF and hlGF-I the O-glycosylated serine or ~ o"i"e
5 residues in synthetic peptides may be directly flanked at their N-
terminal by a proline residue. The glycosylation patterns in
~lldlllllldlidl~ systems and in S. cerevisiae differ Siyl,iricd"~ly,
particularly with regard to O-glycosylation dLlacl""~"L sites
(serine and ll"eO";I~e) and types of carbohydrates involved.
Recently, Strahl-Bohlsinger et al (Yeast 8 (1992) S489, the
1 6th Illl~llldlioridl Conference on Yeast Genetics and Molecular
Biology, Vienna (Austria), August 15-21, 1992) described a gene in
S. cerev~siae, whose mutation reduces the in vivo O-glycosylation
by about 50%, while the in vitro O-glycosylation in the mutant
extract is absent. The mnt1 mutation leads to sl,u,le,1ed O-glycosyl
chains col1si~tli"g of only 2 mannose residues; in this mutant a
specific 1-2 mannosyltransferase is defecl:ive (Hausler et al.,
Proc. Natl. Acad. Sci. USA 89 (1992) 6846-50). The mnt7 mutation
is also known as kre2 and renders S. cerevisiae resistant to the
action of K1 killer toxin (Hausler et al., Proc. Natl. Acad. Sci. USA
89 (1992) 6846-50). In mnnl mutants, a defective ~ 1-3
mannosyltransferase does not add the terminal 1-3 linked mannose
residues in O-, and N-glycosyl chains (Ballou et al., Proc. Natl.
Acad. Sci. USA 88 (1991 ) 3209-12). Other mutations defective in
the synthesis of glycosylation precursors, mannose, GDP-Man and
Dol-P-Man, also affect both O-or N-glycosylation. Mutations of this
type include defects in SEC59, DPM1, SEC53 and PMI genes encoding
dolichol kinase, dolichol pl~ospl1dle mannose synthase,
phospl)~" ,a,~"o" ,utase and phosphomannose is~" ,e, ase, respectively.
At present it is not clear, if O-glycosylation irl yeast is essential
for viability. Only mutations abolishing O- and N-glycosylation
completely (SEC59, DPM1, SEC53 and PMI) lead to lethality (Heller
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 7013-16; Orlean et al.,
J. Biol. Chem. 163 (1988) 17499-507; Kepes and S~l,enk",d", J. Biol. t
Chem. 163 (1988) 9155-61; Smith et al., Mol. Cell. Biol. 12 (1992)
2924-30).
~ WO 94/26873 2161~7 ~ PCT/SE94/00421
An Article in J. Protein Chem. 9:95-104 (1990) by Elliot et al.
describes the purification and p, up~, lies of yeast-produced hlGF-
1.The hlGF-1 protein has been mutated to reduce O-glycosylation.
Site-directed mutagenesis was used to convert Thr 29 to Asn29
5 which reduced but not c' "i"d~ed IGF-I glycosylation. It thus
describes dlLeld~ions in the IGF-I protein itself, and not the host
cell.
EP 314096 concerns yeast mutants that are defective in the
addition of outer N-glycosyl chains. EP 276846 discloses the
10 biological activities of various forms of GM-CSF lacking sites for
N- and/or O-glycosylation. This patent describes mutants of the
~x,ul~s~ed protein GM-CSF, and not the host mutants.
P,.h'~ ~.,solved bythe i,.~nLi~n.
The unique glycosylation patterns of yeast strains have in
many cases a negative impact on the yield obtained of the desired
form of heterologously produced proteins. Moreover extra
precautions during purification must be applied in order to remove
undesired forms of the produced protein.
Figure 1 A and B shows reversed phase HPLC cl"un,dloy,d",~.
OL;e th/c~ of the i"~nLion:
One objective of the invention is to provide S. cerevisiae
25 c~ ed proteins, in particular he~,ul~gous proteins, that are
minimally O-glycosylated at one or more particular serine and/or
threonine attachment site(s). An addiliol1dl objective is to provide
a production method for these proteins and a means (a mutated
S.cerevisiae strain) for the method. In addition the invention will
30 provide S. cerevisiae homologous proteins that are minimally 0-
glycosylated at a serine or threonine site.
The invention:
The S. cerevisiae strain of the invention is cl)d,d~eri~ed in
35 that it is defective in initiating O-mannosylation of the hydroxyl
group of at least one residue selected from serine and ll"t:oni"e in
a protein ~ ssed by the strain. This is probably due to a defect
_
2~6~973
WO 94/26873 PCT/SE94/00421 0
in the ~ Og,li~iol~ of serine, or threonine acceptor sites, or in the
mannos~,lLldll:,rt:rdse activity required to glycosylate these sites.
A serine and/or threonine specific O-mannosyl~,dll~r~rd~e may be
missing or altered in activity. Other aspects of glycosylation are
5 similar to wild-type glycosylation, e.g glycosylation p~i rul Ill~d by
strain YE-465. Thus, further dLLd~ lL of saccl~a,ide units to a
ll-ol~or,-dl~,~osylated serine or threonine residue and/or N-
glycosylation and/or in vivo synthesis of necessary carbohydrate
i"L~rll,edidLes are in most cases normal as deL~ ed by the
10 procedures given in the ~,-peri"le"Ldl section.
Particularly illli~oi LdllL modes of the invention are
Lldll~r~.lll,ed S. cerevisiae strains c~""~ i"~ an e,~ sion vector
co~lL..;,I 19 a gene for a heterologous, in particular Illdlllll -'' 1,
protein, such as hGM-CSF or hlGF-I. It follows that in these modes
of the invention, the mutation causing decreased O-glycosylation is
in a yeast cl)lull,,aso,llal gene.
A second main aspect of the inventiorl is a method for
producing a protein, often a heterologous, in particular II,d,l,,,,alidi~,
protein c~""~ i-.y culturing the inventive S. cerevisiae strain and
recovering the protein from the r~lllellLdLion medium or from the
cells. It is conceivable that conventional S. cerevisiae culturing
methods and conventional recovering methods may be used.
Best Mode at the priority date:
The greatest advantage based on today's kll~ clge for the
inventive S. cerevisiae strains and production method is obtained
for proteins co"i .9 one or more serine or threonine residue(s)
that are recognized as O-glycosylation sites by wild-type strains
of S.cerevis;ae (co..L~;. -9 a vector ~ i"g such a protein) but
30 not by Illdllllll 'ian systems such as in humans. In other words the
most preferred S. cerevisiae strains of the invention contain an
e~-,u~s~iull vector (e.g. plasmid) for a mammalian, in particular
human, protein that is not O-glycosylated by its original species
but O-glycosylated by for instance YE-449 c~"; ,g the
35 appropriate t~ s~k~ll plasmid for the protein. The most preferred
strain has the same mutation as the Ml 95 strain (see below). The
most preferred heterologous protein is hlGF-I.
~ WO 94/26873 21~ 1~ 7 3 PCT/SE94/0(~4ZI
FXPERIMENTAL PROCEDURES
Strains and growth conJi~io,'~. The starting strain for
muld~e~e~i~ was S. cerevisiae YE-465 which consists of host
strain rE-449 (Biogen) (MATct leuZ ura3-52 prb~-7 122 pep4-3 cir)
carrying plasmid p539/12 (Gellerfors et al., J. Biol. Chem. Z64
(1989) 11444-49). p539/12 is an eA~,les~idn plasmid for hlGF-I
that leads to the secretion of b;c!~v lly active hlGF-I into the
growt~ medium of yeast transformants.
Muld~enesis. Ethyl methane sulfonate (EMS) was used to
mutagenize strain YE-465. Treatment of a log-phase culture for
120 min and 180 min with 2.5% EMS yielded killing rates of 81%
a~d 91%, respectively. The 180 min culture was plated for single
colonies on minimal medium. Single colonies were inoculated with
a ~eedle into 2 ml production medium cor l Ig 4% casamino acids
in reagent tubes and grown for 3 days at 30C. One ml of the
culture su~uellldldlll was analysed by concanavalin A blotting, or
imml"lobl~llillg using an anti-hlGF-I antibody.
To lose the e~ ,ion plasmid in putative mutants they were
grown non-selectively in YPD medium (Sherman et al., Methods in
Yeast Genetics (Cold Spring Harbor Lab.) Cold Harbor, N.Y. (1986)
163), followed by the analysis of single isolates for the Ura-
phenotype. While mutants are named "M", cured mutants were given
the de~iy,ldli~ll "CM". Cured mutants (and YE-449 as control) were
l~ll dll~ UI Illed with p539/12, or with a secretion vector for hGM-
CSF, pER545/4. pER545/4 is a derivative of pER562 (Ernst et al.,
Bio/Te~ oluyy 5 (1987) 831-34). Tldl~rUlllldlll~ were first grown
in selective minimal medium (Sherman et al., Methods in Yeast
Genetics (Cold Spring Harbor Lab.) Cold Spring Harbor N.Y. (1986)
164); this culture was used to inoculate production medium
containing 4% casamino acids (Ernst et al., Bio/Technology 5
(1987) 831-34) and the culture was grown for 2-3 days at 30C to
an OD60onm=l 0. Cells were removed by centrifugation; for hlGF-I
~,ul~ssillg lldll~rUlllldlll~ the culture Sl~,UellldLdllL was
concel.LldL~d 25-fold by Trichloro acetic acid (TCA) ~ ,iLaLion
2~g7~
WO 94/26873 PCT/SE94/00421 0
prior to ele~,upllor~is followed by concanavalin A and
immu,~oLl~LLil,g procedures.
Blotting Drocedures: Gl~op~uL~ in the culture medium were
identified by concanavalin A blotting (Clegg Anal. Biochem. 127
5 (1982) 389-94). For this purpose 20 ul of the TCA-concer,L,dled
medium was sepa,dLt:d by SDS-PAGE (17,5% gel), l~d~r~ d to
r,i~ lose and stained as des~ ed. For immu,,oblul~i,,y~
proteins were ~,dn~r~:"~:d to ,,,e,,ll,,diles (Immobilon-P, Millipore)
and reacted with the ",oll~ llal anti-hlGF-I antibody 5B3 diluted
10 1:1000 followedby~l~d~ withpe,u~;.ld~e-coupledgoatanti-
mouse IgG antibody diluted 1:2000 (Jackson Immuno Research,
USA). For the dete~iù,~ of hGM-CSF a rabbit polyclonal anti-hGM-
CSF antibody (kindly supplied by Gla%o Institute for Molecular
Biology, Geneva) was used as first antibody (diluted 1:100)
15 followed by alkaline pllO~,.)l,d~dse-coupled goat anti-rabbit IgG
antibody (Dianova, Germany) diluted 1:5000.
Chr~llldluyld~Jhy Drocedures: Reversed pllase high pel~u,ll,dllce
liquid clllullldloyldpll.y (HPLC) was pe~ru,l"ed with a diphenyl silica
analytical column equipped with a C4-silica precolumn. The
20 ",ollon,e,i~ hlGF-I forms were eluted with an ac~ù"i~ trifluoro
acetic acid (TFA) gradient co"i , ,9 0.1% (v/v) TFA. The hlGF-I
forms were eluted at around Z5 % (v/v) act:~ù"i~,ile.
Other procedures: Standard procedures were used for genetic
analyses and crosses of mutants to laboratory strains S1 50-2B
25 (MAT~ leu2-3, 112 ura3-52 trp 1-289 his3- 7) and BJ 19 9 1 (MATc~
ura3-SZ leu2 trp7 prb7-7 722 pep4-3 gal2) (Sherman et al.
Methods in Yeast Genetics (Cold Spring Harbor Lab.) Cold Spring
Harbor N.Y. (1986) 17-27). Hygromycin B-sensitivity was analyzed
on antibiotic gradient plates (gradient from 0 to 200 l~g/ml).
30 Resistance/sensitivity to killer toxin K1 was assayed as
previously described (Sherman et al., Methods in Yeast Genetics
(Cold Spring Harbor Lab.) Cold Spring Harbor N.Y. (1986) 57-60)
using strain RC1777 (MATc~ ade his4C ¢KlL-k1)).
~ WO94/26873 ~ 7~ PCI/SE:941aO421
RESULTS
Screening for 0-glycosviation mutants: Out of 600
m~ldgerli~èd isolates 8 putative mutants were isolated. To exclude
the possibility that the m~L~yenesi~ had affected the hlGF-I gene
5 or other genetic material present on the e~,ure~ plasmid we
obtained derivatives of the mutants that had lost their plasmid
during non-selective growth. When these cured mutants were
~,d,lsrurllled with the e~Jle~ n plasmid, an identical phenotype
as in the original mutant was obtained. Two of the identified
10 mutants, M195 and M38, were cl,ald~Le,i~ed in more detail as
described below.
Cha, d~Le, i~ s of M195: Mutant strain M195 secretes similar
amounts of hlGF-I into the growth medium as co",~.ared to the
parent production strain YE-465 acco" ,9 to SDS-PAGE arld
15 immul,obl~LLilly. In contrast, we col1si:,~elllly detected lower
concanavalin A-reactivity with hlGF-I secreted by M195, than with
hlGF-I secreted by YE-465 ill~i~dlilly reduced 0-glycosylation at
threonine-Z9. This phenotype was not due to mutations in the
expression vector since the cured l~ldll~rulllled mutant (CM195)
20 also showed reduced hlGF-I 0-glycosylation. SDS-PAGE also
showed several minor bands of sor"~-.l,dL larger size than IGF-I.
These bands le~JlejéllL material 0-glycosylated on serine-69,
adding to the evidence that the mutation in strain CM195 affects
0-linked Ll"eon ,e glycosylation but not serine glycosylation.
25 In order to quantify the mutant phenotype we purified and
analyzed hlGF-I secreted by strains M195 and YE-465 by reversed
phase HPLC. HPLC-results for culture media from strain YE-465 and
M195 are le~,,ese"Led in Figure 1 (For legends see after Results).
The ~IIlullldLoy,d,,,~ show four main peaks for r"~l~or"eri~ hlGF-1
30 c~lle~por ,g to 0-glycosylated misfolded hlGF-1, non-
glycosylated misfolded hlGF-1, 0-glycosylated correctly folded
hlGF-I non-glycosylated correctly folded hlGF-I (i.e. the product).
Misfolding of hlGF-1 occurs due to improper disulfide formation
from cysteine (Axelsson et al., Eur. J. Biochem. 206 (1992) 987-
35 994). The results clearly de",on~,dLe that the amount of 0-
glycosylated hlGF-I is reduced and the amount of non-glycosylated
hlGF-I is i"~,~ased for M195 co,,,ua,ed to YE-465.
WO 94/26873 21~ ~ 9 ~ ~ PCT/SE94100421
In order to test if strain M195 is also defective for protein 0-
glycosylation at serine residues, we cured strain M195 of its hlGF-
I e,~ plasmid (resulting in strain CM195) and transformed
an eA~ ioll plasmid for hGM-CSF (pER545/4) into the cured
S strain. No .lirr~:re"ces in the glycosylation pattern was found
between a wild-type L, ~"~ru",.a,-l (YE-449(pER545/4)) and the
mutant L.dl~ru,---d-,L (CM1 95(pER545/4)) as revealed by SDS-PAGE
followed by immu"obluLLi"g. This result suggests that serine
glycosylation is not affected in M195. On the other hand, N-
10 glycosylation of hGM-CSF also does not seem to be affected in
M195, a finding that is confirmed for yeast invertase (see below).
Strain M195 does not show any morphological abi1o"" ' Ly, nor
does it display L~",pe,dL~re sensitivity, or sensitivity to high
os,--uld-iLi~s in the growth medium. M195 is as sensitive as the
15 parent rE-465 for killer toxin K1 and 5 mM vanadate. The only
~e~e~ldble phenotype other than its O-glycosylation defect that
appears a~o~idL~d with strain M195 is an increased serlsitivity
for the aminoglycoside antibiotic hygromycin B. While strain YE-
465 grows well in the presence of 60 ~g/ml hygromycin B, M195 is
20 co...~ ly inhibited. The mnn9 mutation that affects N-
glycosylation also is hygromycin B sensitive but resistant to 5 mM
vanadate (Ballou et al., Proc. Natl. Acad. Sci. USA 88 (1991 ) 3209-
12). To ascertain that the increased hygromycin B sensitivity in
M195 was asso~idl~d with the observed glycosylation defect we
25 crossed M195 to the wild-type strain 51 50-2B and examined the
haploid progeny. Although many of the seg-egd-,l~ of this cross
failed to grow we were able to examine hlGF-I O-glycosylation in
two hygromycin B sensitive haploid seg~d,.L~ by HPLC analysis
and found reduced O-glycosylation in both strains. This finding
30 suggests that reduced O-glycosylation and hygromycin B-
sensitivity are due to defects in the same gene.
Chdl d~ s of M38: Secretion of hlGF-i by mutant strain
M38 neither shows quantitative, nor qualitative dirr~ "ces
C~ Jdlt:d to the parental strain YE-465; in particular hlGF-I is
35 modified by O-glycosylation to ap~ i"ldlely the same extent in
M38 and YE-465 as revealed by SDS-PAGE followed by
imml"loblollillg. However, ~irr~lel~ces in the gly~op,ul~;., staining
2~ 7~
~ WO 94126873 PCT/SE94100421
pattem by concanavalin A demonstrate that several secreted
glycop,oL~;..s are reduced in size in M38. In particular, a
pl~,.,;,.~..l protein of 35 kDa is missing; instead, a 27 kDa
glycop.uL~;.. of equal intensity is detected.
In a wild-type strain of S. cerevisiae Lldll~rulllled with an
hGM-CSF e~ si"g plasmid immu,.oLluLLi--g on SDS-PAGE of
secreted forms of hGM-CSF give bands at 14.5 kDa
(unglycosylated), 15.5 kDa (0-glycosylated) and 50 kDa (N-
glycosylated) (Ernst et al., Eur. J. Biochem 203 (1992) 663-67). To
assess if glycosylation of hGM-CSF is affected, we isolated a
plasmid-free derivative of M38. This strain, CM38, was
L-d..~ru,...ed with the hGM-CSF e~ ion plasmid pER545/4 and
the hGM-CSF secreted by this Lldl-~ru--lldllL was analyzed by
immu"obl~LLi..g. The unglycosylated 14.5 kDa form of hGM-CSF and
1~ the het~.u~eneous N-glycosylated 50 kDa form of hGM-CSF occur
both in CM38(pER545/4) and control strain YE-449(pER545/4).
However, the 0-glycosylated 15.5 kDa form of hGM-CSF is missing
in CM38(pER545/4). This result suggests that the defect in M38 is
different from the defect in Ml 95, although both defects affect
20 aspects of 0-glycosylation in S. cerevis;ae.
O-glycosylation of chitinase: The 0-glycosylation of a
ho,..ulugous yeast protein, chitinase, which is extensively 0-
glycosylated, was examined in the yeast strains. In YE-465
chitinase appeared on a SDS-PAGE as a band of an d,u,uluAillldL~:
25 molecular weight of 110 kDa. In the mutant strain Ml 95 the
migration of chitinase was ~ d.~ged, i,-~;~dLi..g no dirrt:rt:nce in
the O-glycosyation of this l~o,..ol~gous protein.
N-glycosylation of invertase in mutant strains: To examine if
any of the putative glycosylation mutants is deficient in N-
30 glycosylation, we analyzed the ho",ologous protein invertase,which is esse,.~ lly only N-glycosylated. Extracts of the mutants
were sepdrdLed on a non-denaturing acrylamide gel and invertase
activity was visualised by an activity stain. For two control
strains (mnn9 and mnn1) invertase migrates further than for the
35 strain YE-465 and the mutants. By immu--obl~LLi"g after SDS-PAGE
on the same extracts, the defect in the mnn9 strains is clearly
detected leading to a relatively hor"~geneous protein due to the
WO 94~i873 2161~ 7 3 PCT/SE94/00421
iO
lack of outer glycosyl chains, but the production strain and the
mutants all express invertase as a l1eL~ eneous glycoform. This
result indicates that in the putative mutants including M195 and
M38 N-glycosylation is not affected.
DISCUSSION
The mutant strains M195 and M38 differ from other mutants
that have been shown to be defective in O-glycosylation in S.
cerevisiae. Because protein N-glycosylation of hGM-CSF and
10 invertase proceed normally in these mutants their genetic defect
is different from "unspecific" mutations, such as mutations in
SEC59, SEC53, DPM1 or PMI, which affect O-, as well as N-
glycosylation. It has been reported recently that defects in the
mntl gene lead to a specific sl,o, lel ,~ of all O-glycosyl chains to
lS two mannose residues (Hausler et al Proc. Natl. Acad. USA 89
(1992) 6846-50). However, in M195 full-length O-glycosyl chains
are observed in hlGF-I (although at a low frequency) and in hGM-
CSF; in M38 O-glycosylation of hGM-CSF is defective. Thus defects
in the mntl gene appear not to be the reason for the mutant
20 phenotype in M195, or M38, although M38 i~ resistant to the K1
killer toxin, as has been reported previously for mnt7 strains. A
mannos~lL,d"~r~,d~e has recently been purified from S. cerevisiae
(Strahl-Bolsinger et al., Yeast 8 (1992) S489, the 1 6th
I"L~",dLional Co"~ ,)ce on Yeast Genetics and Molecular Biology,
25 Vienna (Austria), August 15-21, 1992) based on in vitro 0-
glycosylation assays using synthetic peptides (Strahl-Bolsinger
and Tanner, Eur. J. Biochem. 196 (1991 ) 185-90). A gene
co"~,ondi"g to the Lldll~rt:ld~e was isolated, the disruption of
which leads to loss of in vitro mannosylLldll~r~ld~e activity and to
30 a reduction of in vivo O-glycosylation to about 50% (Strahl-
Bolsinger et al., Yeast 8 (1992) S489, the 1 6th l~lLt:l ~,dLional
Co"r~,~nce on Yeast Genetics and Molecular 3iology, Vienna
(Austria), August 15-21, 1992). In addition, the disruptant strain
shows normal cytology, but forms multiple adherent clumps of
35 cells. Unlike the phenotype of this disruptant, strains M195 and
M38 do not form clumps during growth; M195 has no morphological
defects, while M38 tends to form rod-like, ekln~dLed cells at high
2~G1~73
~ WO 94n6873 PCT/SE94/00421
temperature. Also, cell extracts of strains Ml 95 and M38 contain
wild-type levels of in vitro O-glycosylation (Strahl-Bolsinger,
u~published results). This evidence indicates that neither gene
known to affect O-glycosylation is mutated in mutants Ml 95 and
5 M38.
The genetic evidence obtained here and in previous studies
strongly suggests that O-glycosylation in S. cerevisiae is a
complex process that requires multiple cellular factors. The
~X~ La by Strahl-Bolsinger et al., (Yeast 8 (1992) S849, the
10 1 6th II~Lt~ dLiol~al Cohr,-,~"ce on Yeast Genetics and Molecular
Biology, Vienna (Austria), August 15-21, 1992) indicate that not
all of the mannosylLId~lar~:~dses that are active in vivo can be
assayed in vitro, a result that clearly d~",or,aL,dL~s the necessity
of a genetic approach. The mutants isolated in the present study
1~ may be defective in the ,~coy,,iLiu,~ of serine, or threonine acceptor
sites, or in the mannosylL, dlla~t:raae activity required to
glycosylate these sites. Thus, Ml 95 may be defective in
mannosylation of ~I" t:o, ,e residues (as in hlGF-I), but not serine
residues (as in hGM-CSF). On the other hand M38 may be defective
20 in mannosylation of serine, but not threonine residues. Further
analyses of the isolated mutants including gene cloning and their in
vivo disruption promise to clarify details of the O-glycosylation
process in S. cerevisiae.
Legends to Figure 1
25 Reversed phase HPLC ~IIlollldLuyld,,,s. (A) culture medium from
M195; (B) culture medium from YE-465. Four main peaks
,es~"Li"g monomeric hlGF-I are visible in each cl"or"d~oy,d""
from left (shorter retention time): O-glycosylated incorrectly
folded hlGF-I ("~is",d~ ed), nonglycosylated ill~ CLly folded
30 hlGF-l ("lia",aL~l,ed), O-glycosylated correctly folded hlGF-I and
nonglycosylated correctly folded hlGF-I (desired product). There is
a ":" , ,g small peak in position (3) for the Ml 95 strain (B). This
peak might l~ ael,L some ~r, ,g O-glycosylation on serine-29
of hlGF-I or possibly a co""~ L~ly different form of hlGF-I with
35 similar retention time.
