Engineering of mucin-type human glycoproteinsin yeast cells
Koh Amano*, Yasunori Chiba*†, Yoshiko Kasahara*, Yukinari Kato*, Mika Kato Kaneko*, Atsushi Kuno*, Hiromi Ito*,
Kazuo Kobayashi‡, Jun Hirabayashi*, Yoshifumi Jigami†§, and Hisashi Narimatsu**Research Center for Medical Glycoscience and §Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology,
1-1-1 Higashi, Tsukuba 305-8566, Japan; and ‡CMC R&D Laboratories, Kirin Pharma Co., Ltd., Hagiwara-cho, Takasaki 370-0013, Japan
Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved January 7, 2008 (received for review November 1, 2007)

Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, wereport a genetic engineered yeast strain capable of producing mucintype sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc
4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAcT1, and Drosophila melanogaster core1 !1–3 GalT were introduced
into Saccharomyces cerevisiae. The engineered yeast was able toproduce a MUC1a peptide containing O-glycan and also a mucin-like
glycoprotein, human podoplanin (hPod; also known as aggrus),
which is a platelet-aggregating factor that requires a sialyl-core1
structure for activity. After in vitro sialylation, hPod from yeast could
induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable inboth hPod proteins on a lectin microarray. Most of O-mannosylation,
a common modification in yeast, to MUC1a was suppressed by theaddition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential
for use in basic research and pharmaceutical applications.
glycosylation engineering ! mucin-type glycan ! podoplaninMucin-type glycosylation is one of the most abundant posttranslational modifications. The modification is initiated by
O-linked N-acetylgalactosamine (GalNAc) to Ser or Thr residues
on a peptide backbone and is involved in a variety of importantbiological processes, such as processing of hormones (1), endocytosis (2), and sorting of apical proteins in the Drosophila embryo (3).
Mucin-type glycans are sometimes clustered, forming the ‘‘mucindomain’’ found on both membrane-bound (4) and secreted mucins(5), which function as a selective molecular barrier at the epithelial
surface (6) and are involved in morphogenetic signal transduction(7). It is also known that changes in expression of mucin and in theirglycosylation state are closely associated with the development ofcancer and cancer-related processes such as cell growth, differentiation, adhesion, invasion, and immune surveillance (8). Immunohistochemical studies have identified several tumor-associated antigens (TAAs) of adenocarcinoma (9), and most TAAs on mucins
were originally found as sialylated mucin-type glycans (10). Podoplanin (also called aggrus) (11, 12), one of mucin-type glycoproteins, acts as a platelet-aggregating factor for cancer cells, a findingthat has attracted recent attention because it is well known that theplatelet-aggregating activity of cancer cells influences tumor metastasis. In fact, the anti-human podoplanin antibody NZ-1 has an
inhibitory effect on lung colonization of human podoplanintransfected cells (13). Additionally, podoplanin is a potential diagnostic marker for many tumors, including testicular tumors, several
squamous cell carcinomas, and brain tumors, and may be associatedwith malignancy (14, 15).
Mucins and mucin-like glycoproteins have the potential to be
novel cancer markers and thus a new technology for production ofa large amount of mucin-like glycoprotein for induction of specificantibodies could prove very useful. Production of a large amount ofrecombinant mucin-like glycoprotein may also help researchers to3232–3237 ! PNAS ! March 4, 2008 ! vol. 105 ! no. 9

analyze the relationship between mucin-type glycans and theirfunctions. Thus, we have attempted to create a yeast strain capableof producing mucin-type O-glycan. We first created a system for invivo production of MUC1a peptides containing O-linked GalNAc
and core1 structure (Gal!1–3GalNAc"1-O-Ser/Thr). We thenengineered yeast capable of producing functional podoplanin andshowed that yeast-produced podoplanins possess platelet aggregation activity after in vitro sialylation. A combination of glycosyltransferases introduced into yeast cells allowed us to analyze thestructure–function relationship of the O-glycans on podoplanin.
Results
Construction of Yeast Strains Capable of Producing Mucin-Type Glycopeptides. To engineer yeast capable of producing mucin-typesugar chains, three enzymatic steps should be established. Namely,
these are synthesis of UDP-GalNAc and UDP-Gal in the cytosol,
transport of UDP-sugars from the cytosol to the Golgi lumen, andtransfer of the sugars from the UDP-sugars to an acceptor (Fig. 1a).
S. cerevisiae produces intrinsic UDP-GlcNAc, UDP-Glc, and GDPMan, but UDP-Gal is produced only when galactose is used as acarbon source. Thus, we first introduced a UDP-galactose 4-epimerase encoded by the Bacillus subtilis GalE gene (16), which canbring about 4-epimerization of both UDP-GlcNAc and UDP-Glc,
forming UDP-GalNAc and UDP-Gal, respectively. After transformation expressing B. subtilis GalE, yeast cells grown in yeast extract
peptone adenine dextrose (YPAD) medium were analyzed byreverse-phase HPLC to detect the presence of UDP-sugars. Two
new peaks not present in the parental strain appeared in the yeast
strain with GalE and corresponded to authentic UDP-GalNAc and
UDP-Gal [see supporting information (SI) Fig. 5]. Next, we introduced human UDP-Gal transporter, which is encoded by UGT2 totransport both UDP-GalNAc and UDP-Gal from cytosol to theGolgi lumen (17). Finally, we introduced either the human polypeptide:N-acetylgalactosaminyltransferase (ppGalNAc-T) 1 (18), 2(18), or 3 (19) gene. ppGalNAc-Ts transfer GalNAc from UDPGalNAc to serine or threonine residues in polypeptide, forming analpha anomeric linkage. Human ppGalNAc-T family proteinscomprise 20 members (20), and each member appears to have a
different substrate specificity. For core1 structure (Gal!1–
3GalNAc"1-O-Ser/Thr) synthesis, we chose Drosophila melanogaster core1 !1–3 galactosyltransferase (core1 !1–3GalT) (21),because unlike human core1 !1–3GalT (22), the Drosophila core1!1–3GalT does not require chaperones (21). The transmembrane
domain of S. cerevisiae MMN9 (23) (mannosyltransferase), which
Author contributions: Y.C., J.H., Y.J., and H.N. designed research; K.A., Y. Kasahara, Y. Kato,
M.K.K., A.K., H.I., and K.K. performed research; K.A., Y. Kasahara, Y. Kato, and A.K.
analyzed data; and K.A., Y.C., and Y. Kato wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
†To whom correspondence may be addressed. E-mail: y-chiba@aist.go.jp or jigami.yoshi@

aist.go.jp.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0710412105/DC1.
© 2008 by The National Academy of Sciences of the USA

www.pnas.org"cgi"doi"10.1073"pnas.0710412105

UDP

O
NHCOCH3

UDP-GlcNAc

OH

O

UDP

O
NHCOCH3

UDP-GalUDP-GalNAcb

UDP-Gal and UDP-GalNActransporter (UGT2)

MUC1a/T-1

MUC1a/TN-1

polypeptide
MUC1a/N-1

ppGalNAc-Ts

core 1 β1-3 GalT

core1 structurebhuman UGT2

Gal
GalNAc3 X myc gene3 X HA gene

Bacillus GalE

LEU2

URA3

Sc MNN9 TMD 3 X FLAG gene human ppGalNAc-Ts gene
Sc MNN9 TMD 3 X FLAG gene D. melanogaster core 1 β1-3 GalT gene

α-facor prepro

MUC1a gene

6 X His

TRP1

HIS3

Fig. 1. Engineering of a mucin-type glycosylation pathway in yeast. (a) Introduction of a mucin-type glycosylation pathway in yeast. UDP-Gal and UDPGalNAc are synthesized from UDP-Glc and UDP-GlcNAc, respectively, by GalE
protein in the cytosol. UGT2 protein transports UDP-Gal and UDP-GalNAc fromthe cytosol to the Golgi lumen. Next, ppGalNAc-Ts and core1 !1–3 GalT transfer
GalNAc and Gal to polypeptides. (b) Construction of each gene integrated into
the yeast genome. UGT2 protein was C-terminally 3" HA-tagged, and GalE
protein had an N-terminal 3" myc tag. The glycosyltransferases were fused to thetransmembrane domain of yeast MNN9, and a 3" FLAG-tag was inserted between the transmembrane domain and the catalytic domains of the transferases.
MUC1a was fused to the "-factor prepro sequence for secretion into the medium,and a hexa-histidine tag was added to the C terminus.

encodes a component of a Golgi resident mannosyltransferase
complex, was fused to each of the glycosyltransferases (Fig. 1b) toensure that they are localized to the correct cellular compartment.
The yeast strains used in this study are listed in SI Table 2.
Expression of Mucin-Type Glycopeptides in Yeast. Our first attempt
was to produce a mucin-type glycopeptide in yeast. The MUC1a
Amano et al.

1,750

2,000
m/z

2,250

2,500

22

d

23

100-

24

25

Retention time (min)26

27

R3A-1c R3A-1c +806040200-

Mannosylated
GalNAc-peptide

GalNAc-peptide

Fig. 2. Analysis of MUC1a peptide expressed in engineered yeast cells. (a)MALDI-TOF MS spectra of each exogenously expressed MUC1a peptide.
MUC1a produced by N-1, TN-1, and T-1 cells show MUC1a/N-1, MUC1a/TN-1,
and MUC1a/T-1, respectively. The molecular mass of MUC1a is 1,932. (b) Lectin
microarray. BPL, Bauhinia purpurea lectin; ABA, Agaricus bisporus agglutinin;
Jacalin, Artocarpus integrifolia lectin; PNA, peanut agglutinin; WFA, Wisteria
floribunda agglutinin; ACA, Amaranthus caudatus agglutinin; MPA, Maclura
pomifera agglutinin; HPA, Helix pomatia agglutinin; VVA, Vicia villosa agglutinin. GalNAc binders: Jacalin, WFA, MPA, HPA, and VVA; core1 binders: ABA,
PNA, and ACA; core1 and GalNAc binder: BPL. (c) Enzymatic digestion of
MUC1a/T-1. The peak at 24.5 min corresponds to a core1-MUC1a peptide,whereas the peak at 25.1 min corresponds to the GalNAc-MUC1a peptide. (d)Inhibition of yeast O-mannosylation with a chemical reagent. MUC1a wasexpressed in TN-1 strains in the presence or absence of R3A-1c reagent. The
total area of the recombinant MUCla peak on the chromatogram is set to
100%, and error bars show standard deviation of the mean (n # 3).

O-linked GalNAc

Golgi

MUC1a/TN-1

MUC1a/N-1
1,500

peptide (AHGVT5SAPDTR) was selected as a model, and ourconstruct included a hexa-histidine tag at the C terminus (Fig. 1b).
Because at least in vitro a GalNAc residue can be transferred toThr-5 in MUC1a by ppGalNAc-T1 (24), the yeast strain expressing
the ppGalNAc-T1 gene seemed appropriate for production of the
mucin-type glycopeptide form of MUC1a.
To test for expression and modification of the peptide, theMUC1a coding region was introduced into N-1, TN-1, and T-1 cells(see SI Table 2), and the resultant yeast strains were cultivated in
YPAD medium for 2 d at 30°C. MUC1a peptides produced in thesestrains were then analyzed by matrix-assisted laser desorption
ionization/time-of-flight (MALDI-TOF) mass spectrometry (MS)(Fig. 2a). The protonated molecular ion of MUC1a produced by
N-1 cells (MUC1a/N-1) was observed at m/z 1,933, which is identicalto the estimated molecular mass of MUC1a peptide. Moreover, theprotonated molecular ions of MUC1a produced by TN-1 cells andT-1 cells (MUC1a/TN-1 and MUC1a/T-1, respectively) were observed at m/z 2,136 and 2,298, respectively. The m/z value of
MUC1a/TN-1 is identical to the estimated mass of MUC1a modified by addition of a single N-acetylhexosamine molecule (HexNAc), and the m/z value of MUC1a/T-1 is identical to the estimatedmass of MUC1a modified by addition of a HexNAc and a hexose(Hex) molecule (Fig. 2a). More than 85% of MUC1a/TN-1 contained a HexNAc residue, and a total of !100 #g of HexNAcMUC1a was obtained from the supernatant of a 1-liter culture after
purification by HPLC. The transfer efficiency of Hex to HexNAcMUC1a is 70%, and a total of !70 #g of Hex-HexNAc-MUC1a was
PNAS ! March 4, 2008 ! vol. 105 ! no. 9 ! 3233

APPLIED BIOLOGICALSCIENCES

HO

UDP

Relative Area percentage ( % )

O
OH

HPA

O

MUC1a with
β1-3 galactosidase
MUC1a with
β1-4 galactosidase[M+H]+ 1,933=MUC1a

VVA

OH

MUC1a/TN-1

CH 2OHMPA

HO(GalE)

CH 2OH

2,136=MUC1a+HexNAc[M+H]+

A210

Cytosol

UDP-Gal 4-epimerase

MUC1a/T-1
no treatment

MUC1a/T-1

WFA

UDP-Glc

ACA

HO

c

[M+H]+ 2,298=MUC1a+HexNAc+Hex

PNA

UDP

O

ABA

OH

Jacalin

O
OH

HO

a

CH 2OHO

BPL

CH 2OHOH

IntensityaInhibition of Yeast O-Mannosylation by a Chemical Reagent. Al-

though most of the MUC1a/TN-1 in yeast cells contained amucin-type glycan, the peptide was also partially modified byO-mannosylation ($8%). In eukaryotic cells, protein Omannosylation occurs by transfer of a mannose from Dol-P-Man toa serine or threonine residue of polypeptide. The reaction can becatalyzed by at least six members of the protein O-mannosyltransferase (Pmt) family in yeast (28). Both Pmt and ppGalNAc-T
recognize Ser and Thr residues as an acceptor substrate; therefore,
Pmt may compete with ppGalNAc-T in yeast cells. Moreover,terminal modification of O-linked sugar chains by "-1,3mannosyltransferase encoded by MNN1 sometimes evokes animmune response in human (29). Thus, suppression of yeast Omannosylation is important to produce mucin-type glycoproteins as
a therapeutic agent. We first constructed six pmt single disruptants(pmt1–pmt6) to inhibit O-mannosylation of MUC1a. However,
MUC1a was still partially O-mannosylated in cells carrying these
disruptions (data not shown). Next, we tried to suppress Omannosylation via a rhodanine-3-acetic acid (R3A) derivative thatwas developed as an antifungal compound. The compound is
known to act against C. albicans, and more specifically, it inhibits C.
albicans Pmt1p activity (30). We also chose to use 5-(3,4-bisphenylmethoxyphenylmethylene)-4-oxo-2-thioxo-3-thiazolidinacetic acid (R3A-1c) in the study because the compound is mucheasier to synthesize than other inhibitors of Pmt (30).
TN-1 cells with MUC1a were cultivated in YPAD medium for 3 d
at 30°C with 10 #M R3A-1c. Cell growth and expression of the
MUC1a peptide in TN-1 cells did not change in the presence orabsence of 10 #M R3A-1c (data not shown). However, the peak ofmannosylated GalNAc-MUC1a visible on the HPLC chromatogram all but disappeared after addition of R3A-1c (data notshown). Additionally, the ratio of GalNAc-MUC1a increased from
85% to 93%, suggesting that R3A-1c is effective in suppression ofO-mannosylation in MUC1a (Fig. 2d).
ppGalNAc-T1, but Not ppGalNAc-T2 or -T3, Recognizes and Modifies
the PLAG Domain of Podoplanin. The mucin-type glycoprotein po-

doplanin (11) acts as a platelet-aggregating factor on cancer cells.
Podoplanin has a platelet aggregation-stimulating (PLAG) domain,
and we previously reported that a sialylated core1 structure at
Thr-52 in the PLAG domain is essential for induction of platelet
aggregation (12). However, it is unclear which ppGalNAc-T is3234 ! www.pnas.org"cgi"doi"10.1073"pnas.0710412105

a

GST

PLAG domain

6 X HisGSSRARGEDDTETTGLEGGVAMPGAEDDVV TPG
Thrombine recognition sequenceb(M.W. 4056)[M+H]+ 4057

PLAG/N-1[M+H]+ 4260

intensity

obtained from the supernatant of a 1-liter culture after HPLC.
The results of protein sequencing revealed that MUC1a/TN-1
contains a HexNAc at Thr-5 (see SI Fig. 6), indicating that
ppGalNAc-T1 has the same substrate specificity in yeast as it
does in in vitro analysis (24).
We next analyzed the glycan of the MUC1a peptides from eachstrain on a lectin microarray (Fig. 2b) (25). MUC1a/N-1 gave nosignal, whereas MUC1a/TN-1 resulted in positive signals on Gal/
GalNAc binders (Jacalin, BPL, and MPA) and GalNAc binders(WFA, HPA, and VVA). In contrast, MUC1a/T-1 resulted inpositive signals on core1 binders (ABA, PNA, and ACA) but not
on GalNAc binders. These results indicate that MUC1a/TN-1 andMUC1a/T-1 were successfully modified by addition of O-GalNAc
and a core1 structure, respectively.
MUC1a/T-1 was further analyzed by exo-glycosidase digestion toconfirm the sugar chain linkage. To do this, MUC1a/T-1 wastreated with !1–4 galactosidase from Jack bean (26) or !1–3
galactosidase from Xanthomonas sp. (27). Untreated MUC1a/T-1
eluted at 24.5 min on reverse-phase HPLC (Fig. 2c); however, after!1–3 galactosidase digestion, the peak was shifted to 25.1 min,which is identical with what is observed for MUC1a/TN-1. The shift
was not observed, however, after !1–4 galactosidase digestion.
These results clearly indicate that the MUC1a/T-1 produced in theengineered yeast contains the core1 structure (Gal!1–3GalNAc"1O-Ser/Thr).

PLAG/TN-1[M+H]+ 4057

PLAG/TN-2[M+H]+ 4057

PLAG/TN-3
3500

4000
m/z

4500

Fig. 3. ppGalNAc-T1 transfers GalNAc to the PLAG peptide. (a) PLAG peptide
was expressed with an N-terminal GST tag and a C-terminal hexa-histidine tag.
The construct contains a thrombin recognition sequence (LVPRGS) between
GST and the PLAG-peptide coding region. MALDI-TOF MS analysis of the PLAG
peptide was done after thrombin digestion. The amino acid sequence of PLAG
peptide after thrombin digestion is GSSRARGEDDTETTGLEGGVAMPGAEDDVVTPG. (b) The molecular mass of the PLAG peptide is 4,056. PLAG/N-1,
PLAG/TN-1, PLAG/TN-2, and PLAG/TN-3 refer to PLAG peptide produced by the
N-1, TN-1, TN-2, and TN-3 yeast strains, respectively.

responsible for the transfer of GalNAc to Thr-52 in mammaliancells. To help address this, we next examined the specificity of
ppGalNAc-Ts by producing the PLAG domain fused to GST in
TN-1, TN-2, and TN-3 yeast cells (Fig. 3a).
In MALDI-TOF MS analysis, the protonated molecular ion of
the PLAG peptide produced by TN-1 cells was m/z 4,260, whereas
those from N-1, TN-2, and TN-3 cells were m/z 4,057 (Fig. 3b). The
former corresponds to the mass of a PLAG peptide modified byaddition of one HexNAc and the latter to the mass of unmodified
PLAG peptide. Thus, the PLAG peptide produced by TN-1 cells
contained GalNAc; consequently, we can conclude that ppGalNAc-T1 is able to transfer GalNAc to the PLAG domain in yeast
cells. By peptide sequence analysis, we detected a peak of Nacetylgalactosaminyl-Thr at Thr-31 of the PLAG peptide, whichcorresponds to Thr-52 of the full-length human podoplanin (SI Fig.
7). The peptide preference of ppGalNAc-T1, which was previously
reported by Gerken et al. (31), is in good agreement with our resultin yeast cells.
Production of Mucin-Type Glycosylated Human Podoplanin in Yeast.

Next, we attempted to produce a mucin-type glycoprotein, human
podoplanin (hPod), which serves as a model glycoprotein (Fig. 4a).
As described above, ppGalNAc-T1 transfers GalNAc to Thr-52 in
PLAG domain in yeast cells. Thus, we used TN-1 and T-1 cells for
production of hPod and TN-3 and T-3 cells for controls. The yeast
strains were cultivated in YPAD medium for 3 d at 20°C, andproduction of hPod was confirmed by immunoblotting with ananti-hPod antibody (NZ-1) (14). The hPods produced in each strain
had different electrophoretic mobility, presumably due to theaddition of different mucin-type glycans (Fig. 4b). We also performed glycan profiling of the hPods produced by yeast strains on
Amano et al.

GST

hPod ∆TMD (22-127)

kDa

6 X His

66.21

GPLGSGASTGQP EDDTETTGLEGGVAMPGAEDDVV TPGTSEDRYKSGLTT

51

LVATSVNSVTGIRIEDLPTSESTVHAQEQSPSATASNVATSHSTEKVDGD

TRY-1

45.031.021.5-

101 TQTTVEKDGLST
Prescission protease recognition sequence100806040-

d hPod/T-1

hP
od
/N
-1

hP
od
/T3

hP
od
/TN
-1

0-

hP
od
/TN
-3

20hP
od
/T1

Pressure rate (%)

csialylated core1 at Thr52

platelet aggregation
PLAG domain

hPod/T-3

no O-glycan at Thr52

platelet aggregation
sialylated core1 structure

Fig. 4. Expression and analysis of PLAG domain and human podoplanin (hPod).
(a) A construct that expresses hPod with an N-terminal GST tag and a C-terminal
hexa-histidine tag. The resultant recombinant protein contains a PreScission
Protease recognition sequence (LEVLFQGP) between GST and hPod. The aminoacid sequence of the TRY-1 trypsin fragment is double-underlined, and the PLAG
domain is in italics. Big T, the essential Thr residue for platelet-aggregationinducing activity. (b) Immunoblot analysis of recombinant hPod. hPod/TN-1,
hPod/T-1, hPod/TN-3, hPod/T-3, and hPod/N-1 shows hPod produced by TN-1 cells,
T-1 cells, TN-3 cells, T-3 cells, and N-1 cells. (c) Platelet aggregation-inducing
activity of recombinant hPod after in vitro sialylation. Before sialylation, no
sample induced platelet aggregation (data not shown). Platelet aggregation wasmeasured using WBA carna with the screen-filtration method. Pressure means avalue of a pressure required for sample to pass through a filter. The pressure ratethat a sample cannot pass through the filter shows 100%. The normalized
mean % SD of three independent experiments is shown. (d) Both hPod/T-1 and
hPod/T-3 are modified with mucin-type O-glycan, although they are glycosylated
at different sites. Thus, hPod/T-1, but not hPod/T-3, contains sialylated mucin-typeO-glycan at Thr-52, and hPod/T-1 can induce platelet aggregation whereas hPod/
T-3 cannot. All O-glycosylation sites of podoplanin except at Thr-52 are estimatedO-glycosylation sites.

a lectin microarray. The results suggest that the hPods weremodified by addition of mucin-type glycans, such as the O-linked
GalNAc and core1 structure, regardless of the type of ppGalNAc-T
that was introduced (SI Fig. 8). Little O-mannosylation of the hPods
was observed on lectin microarray (SI Fig. 8).
Peptide mass fingerprinting (PMF) was done to confirm transfer
of mucin-type glycans to the PLAG domain present in hPod. To dothis, the hPods from each yeast strain were digested with trypsin andthe resultant peptide fragments were analyzed by MALDI-TOF
MS. TRY-1 fragments contain PLAG domain, and those producedby N-1, TN-3, or T-3 cells (hPod/N-1, hPod/TN-3, and hPod/T-3,
respectively) were observed at m/z 4,177 (Table 1), suggesting thatthese hPods did not contain a mucin-type glycan in the PLAG
domain. The presence of a molecular ion at m/z 4,177 and m/z 4,380
Amano et al.

in the TRY-1 fragment of hPod from TN-1 cells (hPod/TN-1)
suggests that in this strain some of the hPod is modified by theaddition of O-linked GalNAc. MS analysis of TRY-1 from hPod
produced by T-1 cells (hPod/T-1) revealed four peaks, at m/z 4,380,
4,542, 4,745, and 4,907. This suggests the presence of four different
types of glycans on TRY-1: (i) one HexNAc, (ii) one HexNAc andone Hex, (iii) two HexNAc and one Hex, and (iv) two HexNAc andtwo Hex, respectively. Thus, we can conclude that the TRY-1
fragments from hPod/T-1 contain both O-linked GalNAc and acore1 structure. With the exception of hPod/N-1, all hPods contained mucin-type glycans; however, it appears that the O-GalNAc
modification of the PLAG domain occurs only in strains that
express ppGalNAc-T1.
Next, we attempted to transfer N-acetylneuraminic acid
(Neu5Ac) to hPods in vitro using human "2–6 sialyltransferase(ST6GalNAc-I) (32) and human "2–3 sialyltransferase (ST3Gal-I)(33), followed by an assay of platelet aggregation-inducing activity.
Purified hPod/TN-1 and hPod/TN-3 were successfully sialylated by
ST6GalNAc-I, and hPod/T-1 and hPod/T-3 were also modified by
ST3Gal-I. The transfer of Neu5Ac to hPod was confirmed on alectin microarray, and the results revealed that after sialylation
hPod/TN-1 has a sialyl-Tn structure (Neu5Ac"2–6GalNAc-O-Ser/Thr) and hPod/T-1 has a "2–3 sialyl core1 structure (Neu5Ac"2–
3Gal!1–3GalNAc-O-Ser/Thr) (SI Fig. 8). Sialylation of hPod produced by TN-3 and T-3 cells was also confirmed on a lectin
microarray (SI Fig. 8).
We investigated whether sialylated hPods were capable of inducing platelet aggregation (Fig. 4c). None of the hPods was able
to induce aggregation before sialylation; however, after sialylation,
hPods produced by T-1 cells but not T-3 cells did induce aggregationof mouse platelets (Fig. 4c). Platelet aggregation was also measured
using human platelets as a substrate. In this assay, hPod producedby T-1 cells induced platelet aggregation, whereas hPod producedby T-3 cells did not (data not shown). The results in a previous
report showed that the presence of a sialylated core1 structure at
Thr-52 is important for the platelet aggregation-inducing activity of
hPod (12). The results in this study also confirm the significance of
having a sialic acid in the nonreducing end of the O-glycan that is
initiated by ppGalNAc-T1, in the PLAG domain of hPod. Moreover, we found that hPod with a sialyl-Tn structure in the PLAG
domain (hPod/TN-1) was able to induce platelet aggregation,although it is not clear whether this type of modification occurs at
Thr-52 in humans cells. We also performed sialylation of PLAG
peptide produced by T-1 cells, but a PLAG peptide with a sialylated
core1 modification alone was not sufficient to induce platelet
aggregation (data not shown). This result suggested that not only
was PLAG domain with sialyl-core1 structure required for induction of platelet-aggregation by podoplanin, but so was the Cterminal domain or a structural conformation of podoplanin.
Discussion
We have generated S. cerevisiae strains capable of producing
mucin-type O-glycans. We confirmed that the sialyl-core1 structureat Thr-52 of podoplanin is essential for the platelet aggregationinducing activity by comparing cases of hPod/T-1 and hPod/T-3(Fig. 4d). Generally, site-directed substitution or deletion of aminoacid sequences has been used for functional analysis of the glycan
on a glycoprotein (34, 35); however, in this work we were able toshow the importance of a glycan by using a newer method. The
biological roles of N-glycosylation are well elucidated at the molecular level; for example, they play roles in both quality control ofglycoproteins (36) and stability of glycoproteins in the blood (37).
By contrast, the biological roles of O-glycosylation have been lesswell understood. It is expected that we are able to control the siteof glycosylation and the number of glycans added to a given
glycoprotein through manipulation of exogenously introduced ppGalNAc-T in the near future. The availability of recombinant
PNAS ! March 4, 2008 ! vol. 105 ! no. 9 ! 3235

APPLIED BIOLOGICALSCIENCES

b

hP
od
/N
-1
hP
od
/TN
-1
hP
od
/T1
hP
od
/TN
-3
hP
od
/T3

a

Table 1. Peptide mass fingerprinting of recombinant hPod from yeast cells
Theoretical m/z[M & H]&

Observed
MALDI-TOF

Expected glycopeptide

TRY-1 from hPod/N-1
TRY-1 from hPod/TN-2
TRY-1 from hPod/TN-3
TRY-1 from hPod/TN-1

4,161.22
4,161.22
4,161.22
4,161.22

TRY-1 from hPod/T-1

4,161.22

4,177*
4,177*
4,177*
4,177*
4,380*
4,380*
4,542*
4,745*
4,907*

TRY-1 with HexNAc
TRY-1 with HexNAc
TRY-1 with one HexNAc and one Hex
TRY-1 with two HexNAc and one Hex
TRY-1 with two HexNAc and two Hex

Peptide name

*TRY-1 possesses one methionine residue, and this residue was oxidized. hPod/TN-1, hPod/T-1, hPod/TN-3,
hPod/T-3, and hPod/N-1 show hPod produced by TN-1 cells, T-1 cells, TN-3 cells, T-3 cells, and N-1 cells.

glycoproteins with different patterns of glycosylation may be usefulfor discovery or validation of novel biological roles for O-glycans.
It is anticipated that in vitro synthesis of mucin-type glycoprotein by sequential glycosyltransferase reactions is expensive
and low-throughput. Hamilton et al. (38) have already reported
in vivo addition of sialic acid to N-glycan in yeast; therefore, it
may be possible to produce sialylated mucin-type glycan in vivo
in the same way, and the addition of sialic acid to mucin-typeglycan in vivo may lead to widespread adoption of our technique.
In our study, however, sialic acid was transferred to podoplanin
by in vitro reaction because the introduction of another six genes
is required to produce a sialylated mucin-type glycan in vivo andthis may reduce the productive efficiency of the sialylated glycan.
Our results show that in yeast the presence of appropriate
ppGalNAc-T(s) for specific acceptor proteins is important for the
production of functional mucin-type glycoproteins. In the previous
work ppGalNAc-T2 had the strongest activity (39) toward the IgA
hinge region, in which alteration of an O-glycan structure is involved
in selective deposition of IgA1 in the renal glomerular mesangium(40). In mammalian cells, a competition among ppGalNAc-Ts will
be influenced by many factors, not only apparent Km but also thedifferent enzymes that may be spatially localized in discrete regionsof the Golgi. Although it is difficult to produce the recombinantglycoprotein that has the same O-glycan structure in the same
position as the mammalian one in yeast cells, for example, theppGalNAc-T2-expressing yeast cell is the first choice to investigate
the function of O-glycans for IgA nephropathy.
O-mannosylation should be eliminated in therapeutic use because yeast O-mannose is highly immunogenic for mammals, andabnormal O-mannosylation reduces the biological activity of an
antibody produced in yeast cells (41). In this study, R3A-1c was used
to suppress O-mannosylation; however, it is hard to inhibit Omannosylation activity completely even if R3A-1c is used because
O-mannosylation is required for the survival of yeast cells. We
assume that the use of other derivatives of R3A in combination with
six pmt single-gene disruptions (pmt1 to pmt6) may be effective forthe further elimination of residual O-mannosylation.
Qualitative and quantitative changes in O-glycosylation are aconsistent feature of cancer cells. Therefore, alternation of glycans
is expected to serve as a tumor-specific epitope, and indeed,tumor-associated carbohydrate antigens such as CA19–9 (42) andCA125 (43) are widely used in serum diagnostic assays. Similarly,
a somatic mutation in Cosmc (core1 !3-Gal-T-specific molecularchaperone) abolished function of core1 !1–3GalT, disrupting core1
synthesis and creating a tumor-specific glycopeptidic neo-epitopeconsisting of O-linked GalNAc and a specific amino acid sequenceof podoplanin (44). A novel anti-mouse podoplanin antibody, 237
mAb, recognizes the aberrant glycoform of podoplanin and is a
specific marker for Ag104A cells, which are a highly aggressive
fibrosarcoma cell type (44). Tumor-specific antigen creation should3236 ! www.pnas.org"cgi"doi"10.1073"pnas.0710412105

be expanded to include antigens modified by tumor-specific, mucintype glycan. The yeast system described here can be used to produce
glycoproteins containing many types of glycoforms. Some of these
may be specific to cancer cells, and once identified these distinct
glycoforms could serve as tumor-specific antigens, which can beimportant not only for diagnosis but also in the development oftherapeutic agents, such as anti-epitope antibodies. We have already succeeded in creating a strain of the methylotrophic yeast
Ogataea minuta that can produce the mucin-type glycopeptide
GalNAc-MUC1a in this yeast (data not shown). Use of a methylotrophic yeast-based approach makes it easy to produce larger
amounts of recombinant protein. Production of mucin-type glycoproteins in yeast will further the course of basic research andpharmaceutical applications in the near future.
Materials and Methods
Glycan Profiling by Lectin Microarray. Interaction of lectins with glycopeptides
was analyzed by using a lectin microarray as described in ref. 25. Glycopeptides
were purified by HPLC and labeled with Cy3-succimidyl ester in PBST (PBS, 1%
Triton X-100) for 1 h. Cy3-labeled peptides (25 nM) were applied to each well ofa glass slide, and the slides were then incubated at 20°C for 2 h. A fluorescence
image of the array was acquired by using an evanescent-field fluorescencescanner (GTMASScan III, Nippon Laser and Electronics Laboratory) as described inref. 25.
Inhibition of PMT Family Proteins with a Chemical Reagent. The Pmt inhibitor
used in this study—5-(3,4-bis-phenylmethoxyphenylmethylene)-4-oxo-2-thioxo3-thiazolidinacetic acid, designated as R3A-1c—was prepared according to themethod described in ref. 30. The R3A-1c compound was dissolved in 10 mM
dimethyl sulfoxide and added into YPAD to a final concentration of 10 #M. Yeasts
were then grown at 30°C for 2 d. Glycopeptides were purified as described above
and then analyzed by HPLC.
Sialylation of hPod. Each putative catalytic domain of ST3GalI and ST6GalNAcI
was expressed as a secreted protein with a FLAG peptide in human embryonickidney (HEK) 293T cells. To prepare hPod modified by a sialylGalNAc (Neu5Ac"2–
6GalNAc!1-) residue or a sialylcore1 (Neu5Ac"2–3Gal!1–3GalNAc!1-) residue, 25
mM Hepes buffer (pH 7.0) containing the acceptor substrate (25 #g), 10 mM
MnCl2 and an appropriate concentration of CMP-Neu5Ac (Sigma) was used.
Purified ST6GalNAcI and ST3GalI were added to the reaction mixture, respectively, and incubated at 37°C for 20 h. Supernatant from the reaction mixture wassubjected to a platelet aggregation assay after dialysis against PBS.
Platelet Aggregation Assay. Platelet aggregation was measured by WBA Carna(IMI, Saitama, Japan), using the screen filtration pressure method as described inrefs. 12–14. Briefly, 200-#l samples of mouse whole blood were preincubated for
2 min, followed by the addition of 12 #l (1.2 #g) of each partially purified protein.
After 5 min, samples were aspirated to detect aggregation pressure.
ACKNOWLEDGMENTS. We thank Drs. N. Uchiyama and Y. Kubo (ResearchCenter for Medical Glycoscience, National Institute of Advanced Industrial
Science and Technology) for help in preparing the lectin microarray and Dr. T.
Kubota for helpful discussion. This work was supported in part by the New
Energy and Industrial Technology Development Organization.
Amano et al.

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