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. 2015 May 4;20(6):521–542. doi: 10.1111/gtc.12246

Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing

Miki Yamamoto-Hino 1,2,†, Hideki Yoshida 2,3,4,†, Tomomi Ichimiya 3, Sho Sakamura 5, Megumi Maeda 5, Yoshinobu Kimura 5, Norihiko Sasaki 3,6, Kiyoko F Aoki-Kinoshita 3, Akiko Kinoshita-Toyoda 2,7, Hidenao Toyoda 2,7, Ryu Ueda 2,8, Shoko Nishihara 2,3,*, Satoshi Goto 1,2,*
PMCID: PMC4682476  PMID: 25940448

Abstract

Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, β1,4-galactosyltransferase I, β1,3-galactosyltransferase II, and β1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

Introduction

A wide variety of glycans play important roles in a diverse range of biological processes, such as organ development (Haltiwanger & Lowe 2004), lymphocyte homing (Carlow et al. 2009), and cancer invasion (Isaji et al. 2010), by regulating protein–protein, lipid–protein, and cell–cell interactions. Glycans are synthesized by sequential reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters (Nishihara 2007; Yamamoto-Hino et al. 2012). Accordingly, different glycan structures are synthesized by different sets of GR proteins. Thus, it is likely that mutation of GR genes involved in synthesis of the same glycans will result in the same phenotype. For example, glycosaminoglycans (GAG) are sequentially synthesized by peptide-O-xylosyltransferase, β1,4-galactosyltransferase I, β1,3-galactosyltransferase II, and β1,3-glucuronyltransferase (Nishihara 2010; Mikami & Kitagawa 2013). Mutations of these GAG synthesizing enzymes principally impair the same developmental pathways, namely those regulated by decapentaplegic, wingless, hedgehog, and fibroblast growth factor in Drosophila (Haltiwanger & Lowe 2004; Nishihara 2010; Yamamoto-Hino et al. 2012). However, because complete sets of GR gene mutants are not available in metazoa, no comprehensive examination has yet been undertaken to determine whether impairment of GR genes involved in synthesis of the same glycans results in the same phenotypes.

It is possible to silence almost all the genes in Drosophila and Caenorhabditis elegans by RNA interference (RNAi) (Yamamoto-Hino & Goto 2013). In particular, spatiotemporally regulated gene silencing is possible in Drosophila when it is implemented using the Gal4/upstream activation sequence (UAS) system (Brand & Perrimon 1993). In this system, the yeast Gal4 transcription factor binds to the UAS and activates expression of the downstream gene; theoretically, the gene downstream of the UAS is not expressed in the absence of Gal4. Consequently, a genetic cross between UAS- and Gal4-fly strains will induce expression of the gene downstream of the UAS. By placing genes expressing hairpin RNAs downstream of a UAS, RNAi is readily induced by genetic crossing. In addition, there are a large number of Gal4 strains in which the Gal4 gene is conditionally expressed, such as in a specific tissue, at a particular developmental stage, or under specific temperature conditions (Hayashi et al. 2002). Therefore, spatiotemporal patterns and levels of expression of hairpin RNAs can be controlled by the Gal4 strains and temperature conditions used.

In this study, we determined 120 Drosophila GR genes and five core proteins by sequence similarity searches and literature mining. Of these GR genes, 72 were silenced in the whole body. Silencing of 56 of these genes resulted in lethality before eclosion. Thus, it was not possible to assess phenotypic clustering of essential GR genes when genes were silenced in the whole organism. To overcome this difficulty, spatiotemporally regulated gene silencing was carried out using several Gal4 driver strains. The induced phenotypes were linked to the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and terminal domains of glycans. Based on this phenotypic clustering, the functionally unknown gene CG33145 was predicted to be involved in the synthesis of terminal domains. Our biochemical analysis provided direct evidence that CG33145 functioned as a novel galactosyltransferase in terminal N-linked glycan synthesis. In summary, phenotypic clustering in this study proved useful for functional prediction of novel GR genes.

Results

Drosophila GR genes

Drosophila GR genes (67) were identified through similarity to human glycosylation genes using the human GlycoGene DataBase (http://jcggdb.jp/rcmg/ggdb/). The Drosophila GR gene set comprised 54 glycosyltransferases, seven glycan-modifying enzymes, and six nucleotide-sugar transporters. In addition, we manually identified Drosophila genes encoding 44 glycosyltransferases, eight glycan-modifying enzymes, one sugar-nucleotide transporter, and five core proteins from literature searches. In total, 98 glycosyltransferases, fifteen glycan-modifying enzymes, seven sugar-nucleotide transporters, and five core protein genes were identified (Table1). Based on biochemical activities that were directly measured or predicted from homologous mammalian genes, 108 of these 125 GR proteins could be assigned to the following categories: formation of sugar linkages, modification of glycans, or core proteins (Fig.1, Table1).

Table 1.

Drosophila GR genes

Family of proteins/protein name Protein/gene name CG No. References Glycan structure Mammalian orthologue
Glycosyltransferase
N-acetylgalactosaminyltransferase
 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase pgant1/GalNAc-T1 CG8182 Ten Hagen et al. 2003 Mucin-type O-glycan
pgant2 CG3254 Ten Hagen et al. 2003 Mucin-type O-glycan GALNT2
pgant3 CG4445 Ten Hagen et al. 2003 Mucin-type O-glycan
pgant4 CG31956 Ten Hagen et al. 2003 Mucin-type O-glycan
pgant5 CG31651 Ten Hagen et al. 2003 Mucin-type O-glycan GALNT5
pgant6 CG2103 Ten Hagen et al. 2003 Mucin-type O-glycan GALNT1
pgant7/GalNAc-T2 CG6394 Schwientek et al. 2002; Ten Hagen et al. 2003 Mucin-type O-glycan GALNT7
pgant8 CG7297 Ten Hagen et al. 2003 Mucin-type O-glycan
pgant35A CG7480 Schwientek et al. 2002; Ten Hagen et al. 2003 Mucin-type O-glycan GALNT11
dppGalNAcT9 CG30463 ND Mucin-type O-glycan GALNT3
dppGalNAcT10 CG10000 ND Mucin-type O-glycan
CG31776 ND Mucin-type O-glycan
dppGalNAcT11 CG7304 ND Mucin-type O-glycan
dppGalNAcT12 CG7579 ND Mucin-type O-glycan
 α1,4-N-acetylgalactosaminyltransferase α4GT1 CG17223 Mucha et al. 2004 Glycolipid A4GALT
α4GT2 CG5878 Chen et al. 2007 Glycolipid
 β1,4-N-acetylgalactosaminyltransferase β4GalNAcTA CG8536 Haines & Irvine 2005; Chen et al. 2007; Sasaki et al. 2007 Glycolipid, N-glycan B4GALT2
β4GalNAcTB CG14517 Haines & Irvine 2005; Chen et al. 2007 Glycolipid B4GALT3
N-acetylglucosaminyltransferase
 UDP-GlcNAc:polypeptide O-β-N-acetylglucosaminyltransferase dO-GnT/Sxc CG10392 Sinclair et al. 2009* O-GlcNAc OGT
 α3-D-mannoside-β1,2-N-acetylglucosaminyltransferase dMGAT1/Mgat1 CG13431 Sarkar & Schachter 2001; Ichimiya et al. 2004 N-glycan MGAT1
 α6-D-mannoside-β1,2-N-acetylglucosaminyltransferase dMGAT2/Mgat2 CG7921 Ichimiya et al. 2004 N-glycan MGAT2
 β4-D-mannoside-β1,4-N-acetylglucosaminyltransferase dMGAT3 CG31849 ND N-glycan MGAT3
 α3-D-mannoside-β1,4-N-acetylglucosaminyltransferase dMGAT4-1 CG9384 ND N-glycan MGAT4A
dMGAT4-2 CG17173 ND N-glycan MGAT4B
 i-β1,3-N-acetylglucosaminyltransferase diβ3GnT1 CG3253 ND Unknown
diβ3GnT2 CG9171 ND Unknown
diβ3GnT3 CG15483 ND Unknown
diβ3GnT4 CG11149 ND Unknown
diβ3GnT5 CG9996 ND Unknown
diβ3GnT6 CG11388 ND Unknown
 β1,3-N-acetylglucosaminyltransferase Brn CG4934 Muller et al. 2002 Glycolipid
Fng CG10580 Bruckner et al. 2000; Moloney et al. 2000 Notch O-glycan RFNG
 β1,3-N-acetylglucosaminyltransferase or dβ3GnT or GalT1 CG33145 this study N-glycan
 β1,3-galactosyltransferase* dβ3GnT or GalT2 CG11357 ND Unknown
dβ3GnT or GalT3 CG3038 ND Unknown
dβ3GnT or GalT4 CG8668 ND Unknown
dβ3GnT or GalT5 CG8673 ND Unknown
 Dolichyl phosphate N-acetylglucosaminyltransferase dAlg14 CG6308 ND N-glycan ALG14
dAlg7 CG5287 ND N-glycan DPAGT1/ALG7
dAlg13 CG14512 ND N-glycan GLT28D1/ALG13
Chondroitin synthase
 Chondroitin synthase dCHSY CG9220 ND GAG (CS) CHSY1
 Chondroitin polymerization factor dCHPF CG43313 ND GAG (CS) CHPF
 Chondroitin N-acetylgalactosaminyltransferase dCSGalNAcT1 CG12913 ND GAG (CS) ChGn
 Chitin synthase Chitin Syn1/Kkv CG2666 ND Chitin
Chitin Syn2 CG7464 ND Chitin
Fucosyltransferase
 α1,3/1,4-fucosyltransferase or FucTA CG6869 Fabini et al. 2001 N-glycan
 α1,3-fucosyltransferase* FucTB CG4435 ND Unknown FUT1
FucTD CG9169 ND Unknown
FucTC CG40305 ND Unknown
 α1,6-fucosyltransferase dα6Fut/FucT6 CG2448 Paschinger et al. 2005 N-glycan FUT8
 Protein O-fucosyltransferase OFut1 CG12366 Okajima & Irvine 2002 Notch POFUT1
OFut2 CG14789 Luo et al. 2006 Thrombospondin POFUT2
Galactosyltransferase
 GAGβ1,4-galactosy-ltransferase I dGAGβ4GalTI/β4GalT7 CG11780 Nakamura et al. 2002; Vadaie et al. 2002; Takemae et al. 2003 GAG (common) B4GALT7
 GAGβ1,3-galactosyltransferase II dGAGβ3GalTII CG8734 Ueyama et al. 2008 GAG (common) B3GALT6
 core1β1,3-galactosyltransferase dC1GalT1/C1GalTA CG9520 Muller et al. 2005; Yoshida et al. 2008* Mucin-type O-glycan C1GALT1
dC1GalT2 CG8708 Muller et al. 2005 Mucin-type O-glycan
dC1GalT3 CG18558 ND Mucin-type O-glycan
dC1GalT4 CG2975 Muller et al. 2005 Mucin-type O-glycan
dC1GalT5/Tgy CG7440 ND Mucin-type O-glycan
dC1GalT6 CG34056 Muller et al. 2005 Mucin-type O-glycan
CG34057 Muller et al. 2005 Mucin-type O-glycan
dC1GalT7 CG3119 ND Mucin-type O-glycan
dC1GalT8 CG2983 ND Mucin-type O-glycan
dC1GalT9 CG9109 ND Mucin-type O-glycan
Glucosyltransferase
 Dolichyl phosphate glucosyltransferase dAlg5/Wol CG7870 ND N-glycan ALG5
 Dolichyl pyrophosphate glucosyltransferase dAlg6/Gny CG5091 ND N-glycan ALG6
dAlg8 CG4542 ND N-glycan ALG8
dAlg10 CG32076 ND N-glycan ALG10
 Glucosylceramide synthase dGlcCerT/GlcT-1 CG6437 Kohyama-Koganeya et al. 2004 Glycolipid UGCG
 Protein O-glucosyltransferase Rumi CG31152 Acar et al. 2008 Notch
Ugt CG6850 Parker et al. 1995 N-glycan UGCGL1
Glucuronyltransferase
 GAG glucuronyltransferase I dGlcAT-I CG32775 Kim et al. 2003 GAG (common) B3GAT1
 β1,3-glucuronyltransferase dGlcAT-BSI/GlcAT-S CG3881 Kim et al. 2003 GAG (common), other glycan ?
dGlcAT-BSII/GlcAT-P CG6207 Kim et al. 2003 GAG (common), other glycan ?
CG30438 ND glucuronidation CGT
 Hereditary multiple exostoses (EXT) protein dExt1/Ttv CG10117 ND GAG (HS) EXT1
dExt2/Sotv CG8433 ND GAG (HS)
dExt3/Botv CG15110 Kim et al. 2002 GAG (HS) EXTL3
Mannosyltransferase
 β1,4-mannosyltransferase β1,4ManT/Egh CG9659 Wandall et al. 2003 Glycolipid
 Dolichyl pyrophosphate mannosyltransferase dAlg1 CG18012 ND N-glycan ALG1
dAlg2 CG1291 ND N-glycan ALG2
dAlg11 CG11306 ND N-glycan ALG11
dAlg3/l(2)not CG4084 ND N-glycan ALG3
dAlg9 CG11851 ND N-glycan ALG9
dAlg12 CG8412 ND N-glycan ALG12
dDPM CG10166 ND N-glycan DPM1
 Protein O-mannosyltransferase dPomt1/Rt CG6097 Ichimiya et al. 2004 Dystroglycan POMT1
dPomt2/Tw CG12311 Ichimiya et al. 2004 Dystroglycan POMT2
Sialyltransferase
 Galactoside α2,6-sialyltransferase dST6Gal I CG4871 Koles et al. 2004 N-glycan ST6GAL2
Xylosyltransferase
 Peptide-O-xylosyltransferase dXylT/Oxt CG32300 Wilson 2002 GAG (common) XYLT1
Oligosaccharyltransferase
 Oligosaccharyltransferase OST CG33303 N-glycan
CG9022 N-glycan
CG7830 N-glycan
CG6370 N-glycan
CG13393 N-glycan
STT CG1518 N-glycan
STT CG7748 N-glycan
 Fukutin-related protein CG15651 ND Dystroglycan FKRP
Sulfotransferase
 Chondroitin 4-O-sulfotransferase dC4ST CG31743 ND GAG (CS) CHST13
 N-acetylgalactosamine-4-O-sulfotransferase d4ST1 CG14024 ND GAG (CS), N-glycan ? CHST11
d4ST2 CG13937 ND GAG (CS), N-glycan ?
 N-acetylgalactosamine/N-acetylglucosamine/galactose d6ST1 CG31637 ND GAG (CS), N-glycan ?
 6-O-sulfotransferase d6ST2 CG9550 ND GAG (CS), N-glycan ?
 Heparan sufate sulfotransferase Pipe CG9614 Zhu et al. 2005*; Xu et al. 2007 GAG (HS)
 Heparan N-deacetylase/N-sulfotransferase Sfl CG8339 ND GAG (HS) NDST2
 Heparan sulfate 2-O-sulfotransferase HS2ST CG10234 Kamimura et al. 2006*; Xu et al. 2007 GAG (HS) HS2ST1
 Heparan sulfate 6-O-sulfotransferase dHS6ST CG4451 Kamimura et al. 2001 GAG (HS) HS6ST1
 Heparan sulfate d-glucosaminyl 3-O-sulfotransferase dHS3OSTA CG33147 ND GAG (HS) HS3ST5
dHS3OSTB CG7890 Kamimura et al. 2004 GAG (HS) HS3ST3A1
 C5 epimerase Heparan sulfate C5-epimerase CG3194 ND GAG (HS)
Sugar-nucleotide transporter
 GDP-Fuc transporter (Golgi) Gfr/Nac CG9620 Luhn et al. 2004; Ishikawa et al. 2005; Geisler et al. 2012*
 GDP-Fuc/UDP-GlcNAc/UDP-Xyl transporter (ER) Efr CG3774 Ishikawa et al. 2010 SLC35B4
 UDP-Gal/UDP-GalNAc transporter Csat CG2675 Segawa et al. 2002 SLC35A2
 UDP-sugar transporter Frc CG3874 Goto et al. 2001; Selva et al. 2001 Notch, GAG SLC35D1
 Sugar-nucleotide transporter Meigo CG5802 ND SLC35B1
 PAPS transporter Sll CG7623 Kamiyama et al. 2003; Luders et al. 2003 GAG SLC35B2
dPAPST2 CG7853 Goda et al. 2006 GAG SLC35B3
Core protein
 Glypican Dally CG4974 GAG (HS)
 Glypican Dlp CG32146 GAG (HS)
 Dystroglycan αDystroglycan CG18250 O-Man
 Syndecan dSdc CG10497 GAG (HS)
 Perlecan dPerlecan/Trol CG33950 GAG (HS)
Glycosidase
 α-mannosidase I α-Man-I CG42275 ND N-Glycan
α-Man-II CG18802 Cao et al. 2011 N-Glycan
 β-N-acetylglucosaminidase Fdl CG8824 Leonard et al. 2006 N-Glycan
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*

Determined by mutant phenotype.

ND: not determined.

Figure 1.

Figure 1

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Drosophila GR genes assigned to linkage formation and modification of N-linked glycan (A), glycosaminoglycans (B), mucin-type glycans (C), Notch-related glycans (D), Dystroglycan-related glycan (E), and arthro-series of glycolipid (F). Core proteins are also assigned (B, D, and E).

There are structural variants of N-linked glycans. Aoki and colleagues determined the number of N-linked glycan variants in Drosophila embryo using mass spectrometry (Aoki et al. 2007). The authors detected GlcNAc structures that were synthesized by Mgat1, Mgat2, and Mgat4, and also observed extended forms such as Galβ-3GlcNAc and SAα2-6Galβ-3GlcNAc. However, no terminal GlcNAc structures synthesized by Mgat3, Mgat5, or Mgat6 were detected. Accordingly, sequence comparisons showed the absence of Mgat5 and Mgat6 in Drosophila, and expression of Mgat3 was very low (Flybase). In addition, a small amount of N-linked glycans was capped by LacdiNAc (GalNAc-GlcNAc) or GlcA in the Drosophila embryo (Aoki & Tiemeyer 2010). LacdiNAc was also found in arthro-series glycosphingolipids in embryo. LacdiNAc structures on glycoproteins and glycosphingolipids were synthesized by Drosophila β4GalNAcTA (Sasaki et al. 2007).

Gene silencing in the whole Drosophila body

To examine the phenotypes caused by silencing of GR genes, we established RNAi-inducible fly strains for 72 Drosophila GR genes. RNAi could not be established for the remaining 53 genes. The established UAS-IR strains bore transgenes containing IR sequences of the target genes under the control of the UAS. First, we calculated off-target probability scores (OTPS) for each UAS-IR strain using the dsCheck website (http://dscheck.rnai.jp/, Table2). Our previous research showed that UAS-IR strains with OTPS <3 were most likely to silence on-target genes (Yamamoto-Hino et al. 2010). Therefore, UAS-IR strains with OTPS >2 were not analyzed further.

Table 2.

Off-target probability score (OTPS) and phenotypes caused by whole-body gene silencing

Family of proteins/protein name Protein/gene name CG No. OTPS Act5C
Glycosyltransferase
N-acetylgalactosaminyltransferase
 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase pgant1/GalNAc-T1 CG8182 1 Lethal
pgant2 CG3254 0 Lethal
pgant3 CG4445 2 Viable
pgant4 CG31956 0 Lethal
pgant5 CG31651 0 Lethal
pgant6 CG2103 0 Lethal
pgant7/GalNAc-T2 CG6394 1 Lethal
pgant8 CG7297 0 Lethal
pgant35A CG7480 0 Lethal
dppGalNAcT9 CG30463 2 Lethal
dppGalNAcT10 CG10000 1 N.T.
CG31776 no line N.T.
dppGalNAcT11 CG7304 no line N.T.
dppGalNAcT12 CG7579 1 N.T.
 α1,4-N-acetylgalactosaminyltransferase α4GT1 CG17223 0 Viable
α4GT2 CG5878 0 N.T.
 β1,4-N-acetylgalactosaminyltransferase β4GalNAcTA CG8536 0 N.T.
β4GalNAcTB CG14517 1 Viable
N-acetylglucosaminyltransferase
 UDP-GlcNAc:polypeptide O-β-N-acetylglucosaminyltransferase dO-GnT/Sxc CG10392 0 Lethal
 α3-D-mannoside-β1,2-N-acetylglucosaminyltransferase dMGAT1/Mgat1 CG13431 2 Lethal
 α6-D-mannoside-β1,2-N-acetylglucosaminyltransferase dMGAT2/Mgat2 CG7921 1 Lethal
 β4-D-mannoside-β1,4-N-acetylglucosaminyltransferase dMGAT3 CG31849 1 Lethal
 α3-D-mannoside-β1,4-N-acetylglucosaminyltransferase dMGAT4-1 CG9384 2 Viable
dMGAT4-2 CG17173 0 Lethal
 i-β1,3-N-acetylglucosaminyltransferase diβ3GnT1 CG3253 1 Viable
diβ3GnT2 CG9171 38 N.T.
diβ3GnT3 CG15483 0 Viable
diβ3GnT4 CG11149 0 Lethal
diβ3GnT5 CG9996 0 Lethal
diβ3GnT6 CG11388 0 Lethal
 β1,3-N-acetylglucosaminyltransferase Brn CG4934 0 Lethal
Fng CG10580 0 Lethal
 β1,3-N-acetylglucosaminyltransferase or dβ3GnT or GalT1 CG33145 0 Lethal
 β1,3-galactosyltransferase dβ3GnT or GalT2 CG11357 2 Lethal
dβ3GnT or GalT3 CG3038 0 Viable
dβ3GnT or GalT4 CG8668 0 Lethal
dβ3GnT or GalT5 CG8673 11 N.T.
 Dolichyl phosphate N-acetylglucosaminyltransferase dAlg14 CG6308 no line N.t.
dAlg7 CG5287 0 Lethal
dAlg13 CG14512 0 Lethal
Chondroitin synthase
 Chondroitin synthase dCHSY CG9220 2 n.t.
 Chondroitin polymerization factor dCHPF CG43313 0 Lethal
 Chondroitin N-acetylgalactosaminyltransferase dCSGalNAcT1 CG12913 2 Viable
 Chitin synthase Chitin Syn1/Kkv CG2666 0 Lethal
Chitin Syn2 CG7464 0 Lethal
Fucosyltransferase
 α1,3/1,4-fucosyltransferase or FucTA CG6869 6 N.T.
 α1,3-fucosyltransferase FucTB CG4435 0 Lethal
FucTD CG9169 1 Lethal
FucTC CG40305 no line N.T.
 α1,6-fucosyltransferase dα6Fut/FucT6 CG2448 1 Lethal
 Protein O-fucosyltransferase OFut1 CG12366 0 Lethal
OFut2 CG14789 0 Viable
Galactosyltransferase
 GAGβ1,4-galactosyltransferase I dGAGβ4GalTI/β4GalT7 CG11780 0 Lethal
 GAGβ1,3-galactosyltransferase II dGAGβ3GalTII CG8734 1 Lethal
 core1β1,3-galactosyltransferase dC1GalT1/C1GalTA CG9520 0 Viable
dC1GalT2 CG8708 1 Lethal
dC1GalT3 CG18558 0 N.T.
dC1GalT4 CG2975 8 N.T.
dC1GalT5/Tgy CG7440 0 Lethal
dC1GalT6 CG34056 8 N.T.
CG34057 8 N.T.
dC1GalT7 CG3119 2 N.T.
dC1GalT8 CG2983 2 Viable
dC1GalT9 CG9109 1 Lethal
Glucosyltransferase
 Dolichyl phosphate glucosyltransferase dAlg5/Wol CG7870 2 N.T.
 Dolichyl pyrophosphate glucosyltransferase dAlg6/Gny CG5091 0 N.T.
dAlg8 CG4542 2 N.T.
dAlg10 CG32076 1 N.T.
 Glucosylceramide synthase dGlcCerT/GlcT-1 CG6437 1 Lethal
 Protein O-glucosyltransferase Rumi CG31152 no line N.T.
Ugt CG6850 no line N.T.
Glucuronyltransferase
 GAG glucuronyltransferase I dGlcAT-I CG32775 0 Lethal
 β1,3-glucuronyltransferase dGlcAT-BSI/GlcAT-S CG3881 0 Viable
dGlcAT-BSII/GlcAT-P CG6207 24 N.T.
CG30438 0 Viable
 Hereditary multiple exostoses (EXT) protein dExt1/Ttv CG10117 0 Lethal
dExt2/Sotv CG8433 0 Lethal
dExt3/Botv CG15110 ? Lethal
Mannosyltransferase
 β1,4-mannosyltransferase β1,4ManT/Egh CG9659 0 N.T.
 Dolichyl pyrophosphate mannosyltransferase dAlg1 CG18012 0 Lethal
dAlg2 CG1291 2 N.T.
dAlg11 CG11306 0 N.T.
dAlg3/l(2)not CG4084 0 N.T.
dAlg9 CG11851 no line N.T.
dAlg12 CG8412 0 N.T.
dDPM CG10166 0 N.T.
 Protein O-mannosyltransferase dPomt1/Rt CG6097 0 Lethal
dPomt2/Tw CG12311 0 Lethal
Sialyltransferase
 Galactoside α2,6-sialyltransferase dST6Gal I CG4871 3 N.T.
Xylosyltransferase
 Peptide-O-xylosyltransferase dXylT/Oxt CG32300 0 Lethal
Oligosaccharyltransferase
 Oligosaccharyltransferase OST CG33303 0 N.T
CG9022 no line N.T
CG7830 0 N.T
CG6370 no line N.T
CG13393 no line N.T
STT CG1518 0 N.T
STT CG7748 no line N.T
 Fukutin-related protein CG15651 0 Lethal
Sulfotransferase
 Chondroitin 4-O-sulfotransferase dC4ST CG31743 0 Viable
 N-acetylgalactosamine-4-O-sulfotransferase d4ST1 CG14024 6 N.T.
d4ST2 CG13937 0 Viable
 N-acetylgalactosamine/N-acetylglucosamine/galactose d6ST1 CG31637 0 N.T.
 6-O-sulfotransferase d6ST2 CG9550 0 Lethal
 Heparan sufate sulfotransferase Pipe CG9614 1 Lethal
 Heparan N-deacetylase/N-sulfotransferase Sfl CG8339 1 N.T.
 Heparan sulfate 2-O-sulfotransferase HS2ST CG10234 0 Viable
 Heparan sulfate 6-O-sulfotransferase dHS6ST CG4451 0 N.T.
 Heparan sulfate d-glucosaminyl 3-O-sulfotransferase dHS3OSTA CG33147 2 N.T.
dHS3OSTB CG7890 3 N.T.
 C5 epimerase Heparan sulfate C5-epimerase CG3194 0 lethal
Sugar-nucleotide transporter
 GDP-Fuc transporter (Golgi) Gfr/Nac CG9620 0 N.T.
 GDP-Fuc/UDP-GlcNAc/UDP-Xyl transporter (ER) Efr CG3774 no line N.T.
 UDP-Gal/UDP-GalNAc transporter Csat CG2675 0 Lethal
 UDP-sugar transporter Frc CG3874 2 Lethal
 Sugar-nucleotide transporter Meigo CG5802 0 Lethal
 PAPS transporter Sll CG7623 0 Lethal
dPAPST2 CG7853 2 Lethal
Core protein
 Glypican Dally CG4974 4 N.T.
 Glypican Dlp CG32146 2 Lethal
 Dystroglycan αDystroglycan CG18250 1 N.T.
 Syndecan dSdc CG10497 3 N.T.
 Perlecan dPerlecan/Trol CG33950 0 N.T.
Glycosidase
 α-mannosidase I α-Man-I CG42275 no line N.T.
α-Man-II CG18802 0 Lethal
 β-N-acetylglucosaminidase Fdl CG8824 no line N.T.
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no line: no UAS-IR line; N.T.: not tested.

Next, we examined whether RNAi-mediated gene silencing reduced the amounts of corresponding mRNA and glycan in Drosophila. Peptide-O-xylosyltransferase (XylT, CG17772) is required for the formation of the common core region of GAGs such as heparan sulfate (HS) GAG and chondroitin sulfate (CS) GAG, whereas hereditary multiple exostoses protein 3 (DExt3, CG15110) participates in the extension of HS but not CS. Expression of XylT and DExt3 was silenced in whole larval bodies using Act5C-Gal4. XylT and DExt3 mRNA in the silenced larvae were reduced to 15–30% of control levels (Act5C-Gal4) (Fig.2). GAG fractions were extracted from the silenced larvae, treated with heparitinase, and subjected to HPLC for detailed analyses of GAGs. Silencing of XylT resulted in the reduction in both HS and CS, whereas DExt3 silencing caused the specific reduction in HS (Fig.2). These results clearly showed that the RNAi-mediated silencing in the present study resulted in specific reduction in GAGs as well as the mRNA expression levels of each glycosyltransferase.

Figure 2.

Figure 2

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Reduction in mRNA and GAG levels by silencing of CG15110 (Dext3) and CG32300 (dXylT). (A, B) The mRNA levels of CG15110 and CG32300 in Act5C-GAL4/UAS-IR-CG15110 (A), Act5C-GAL4/UAS-IR-CG17772 (B), and Actin5C-GAL4/+ (as control in A and B) were quantified by real-time PCR. (C–E) Typical chromatograms of GAG-derived oligosaccharides in the third instar larvae of Actin5C-GAL4/+ (C), Act5C-GAL4/UAS-IR-CG15110 (D), and Act5C-GAL4/UAS-IR-CG17772 (E). HS, chromatograms of unsaturated disaccharides from heparan sulfate. CS, chromatograms of unsaturated disaccharides from low-sulfated chondroitin 4-sulfate. Peaks: 1, ΔUA-GlcNAc; 2, ΔUA-GlcNS; 3, ΔUA-GlcNAc6S; 4, ΔUA-GlcNS6S; 5, ΔUA2S-GlcNS; 6, ΔUA2S-GlcNS6S; 7, ΔDi-0S; and 8, ΔDi-4S. The peak heights in each chromatogram reflect the amount of oligosaccharides and can be compared between different genotypes.

The Act5C-Gal4 driver strain was crossed to 72 UAS-IR strains to induce gene silencing in whole bodies during all developmental stages. Progeny from 56 of the crosses (78%) died before developing into third instar larvae, suggesting that these genes were essential for development (Table2). As it was difficult to classify these GR genes from lethality alone, we next carried out spatiotemporally regulated gene silencing using several Gal4 driver strains.

Gene silencing in a spatiotemporally regulated manner

For spatiotemporal RNAi, MS1096/A9-Gal4, scalloped (sd)-Gal4, patched (ptc)-Gal4, and engrailed (en)-Gal4 driver strains were used to induce gene silencing in wing disks, and 69B-Gal4 was used for expression in larval histoblasts and wing disks (Fig.3). Of the 72 strains tested, 20 showed abnormalities in adult wings and abdomens. In wings, extra or thick veins were formed by gene silencing using MS1096/A9-Gal4, scalloped (sd)-Gal4, patched (ptc)-Gal4, and engrailed (en)-Gal4 drivers (Figs4,5, Table3). By contrast, gene silencing using 69B-Gal4 caused abdominal depigmentation (Fig.6, Table3). Formation of extra and thick veins was mainly observed by silencing of genes involved in synthesis of GAGs and Fringe-dependent glycans on Notch, respectively (Figs4,5, Table3). These phenotypes corresponded with those observed for mutant strains (Panin et al. 1997; Goto et al. 2001; Selva et al. 2001; Nybakken & Perrimon 2002). By contrast, abdominal depigmentation has not been observed previously. Depigmentation was caused by silencing of dα6fut/fucT6, gfr/nac, Csat, and CG33145 (Fig.6, Table3). Dα6Fut/FucT6 adds a fucose moiety to the core region of N-linked glycans via α1,6-linkage (Paschinger et al. 2005), whereas Gfr/Nac transports GDP-fucose to the Golgi lumen for fucose addition, including α1,3-fucosylation of the core N-linked glycans (Ishikawa et al. 2010; Geisler et al. 2012). As Gal and GalNAc are often added at or near nonreducing ends of glycans, Csat, a UDP-Gal/UDP-GalNAc transporter (Segawa et al. 2002), may be involved in terminal glycosylation. Therefore, the depigmentation group is possibly involved in synthesis of glycans at or near nonreducing ends, namely terminal domains. We therefore next examined whether CG33145 participated in terminal glycosylation.

Figure 3.

Figure 3

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69B-Gal4 expression in larval histoblasts. The late third instar larva of 69B-Gal4/UAS-GFP expressed GFP in histoblasts.

Figure 4.

Figure 4

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Adult wing phenotypes caused by silencing of GAG genes. Extra veins are indicated by arrows. The combination of UAS-IR and Gal4 strains is indicated under each panel.

Figure 5.

Figure 5

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Adult wing phenotypes caused by silencing of Notch glycosylation genes. Thick veins are indicated by arrows. The combination of UAS-IR and Gal4 strains is indicated in each panel.

Table 3.

Phenotypes caused by spatiotemporally regulated gene silencing

Family of proteins/protein name Protein/gene name CG No. Gal4 driver line Glycans
MS1096/A9 SD en ptc 69B
N-acetylgalactosamine-4-O-sulfotransferase d4ST2 CG13937 Extra vein GAGs
GAG glucuronyltransferase I dGlcAT-I CG32775 Extra vein1
peptide-O-xylosyltransferase dXylT/Oxt CG32300 Extra vein Extra vein
GAGβ1,4-galactosyltransferase I dGAGβ4GalTI/β4GalT7 CG11780 Extra vein Extra vein
C5 epimerase Heparan sulfate C5-epimerase CG3194 Extra vein Extra vein Extra vein2
Hereditary multiple exostoses (EXT) protein dExt1/Ttv CG10117 Extra vein3 Extra vein
Hereditary multiple exostoses (EXT) protein dExt2/Stv CG8433 Extra vein Extra vein Extra vein
Hereditary multiple exostoses (EXT) protein dExt3/Botv CG15110 Extra vein acv deletion
β1,3-glucuronyltransferase dGlcAT-BSII/GlcAT-P CG6207 Extra vein
GAGβ1,3-galactosyltransferase II dGAGβ3GalTII CG8734 Extra vein
PAPS transporter Sll CG7623 Extra vein
Chondroitin 4-O-sulfotransferase dC4ST CG31743 Extra vein
Syndecan dSdc CG10497 Thick vein
Protein O-fucosyltransferase OFut1 CG12366 Thick vein4 Notch
β1,3-N-acetylglucosaminyltransferase Fng CG10580 Thick vein Thick vein Thick vein5
UDP-sugar transporter Frc CG3874 Thick vein
α1,6-fucosyltransferase dα6Fut/FucT6 CG2448 Thick vein Depigmentation6 Terminal
GDP-Fuc transporter (Golgi) Gfr/Nac CG9620 Depigmentation7
UDP-Gal/UDP-GalNAc transporter Csat CG2675 Depigmentation8
β1,3-N-acetylglucosaminyltransferase or β1,3-galactosyltransferase dβ3GnT or GalT1 CG33145 Depigmentation9
Glucosylceramide synthase dGlcCerT/GlcT-1 CG6437 Thick vein Glycolipid
UDP-GlcNAc:polypeptide O-β-N-acetylglucosaminyltransferase dO-GnT/Sxc CG10392 Thick vein pcv deletion O-GlcNAc
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Number of abnormal wings/number of tested wings = 40/76 (1), 46/46 (2), 70/70 (3), 64/64 (4) and 8/8 (5).

Number of depigmented males/number of tested males = 66/79 (6), 5/15 (7), 45/175 (8) and 9/69 (9).

Figure 6.

Figure 6

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Adult abdominal phenotypes caused by silencing of N-glycan genes. Depigmented regions are indicated by arrows and surrounded by dotted lines. The combination of UAS-IR and Gal4 strains is indicated under each panel.

CG33145 has β1,3-galactosyltransferase activity for terminal N-glycans

As CG33145 has high sequence similarity to the members of human β1,3-N-acetylglucosaminyltransferase family (60–64%) and those of human β1,3-galactosyltransferase family (61–68%), we searched for glycan structures, including GlcNAc or Gal moiety, via β1,3-linkage in insects including Drosophila. Galβ1,3GalNAc was found in a complex-type N-linked glycan on royal jelly glycoproteins of honeybee: Galβ1,3GalNAcβ1,4GlcNAcβ1,2Manα1,6(Galβ1,3GalNAcβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc (E5, Fig.7A) (Kimura et al. 2006, 2007). Thus, we examined whether CG33145 added Gal to GalNAcβ1,4GlcNAcβ1,2Manα1,6(GalNAcβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc (E2, Fig.7A) via β1,3-linkage. CG33145 protein was expressed in Sf9 cells, and the β1,3galactosyltransferase activity was assessed (Fig.7B,C). An in vitro assay showed that CG33145 protein transferred the Gal moiety to E2 and produced the products Galβ1,3GalNAcβ1,4GlcNAcβ1,2Manα1,6(GalNAcβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc or GalNAcβ1,4GlcNAcβ1,2Manα1,6(Galβ1,3GalNAcβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc (E4), and Galβ1,3GalNAcβ1,4GlcNAcβ1,2Manα1,6(Galβ1,3GalNAcβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc (E5). Digestion of the fractionated E5 product by β1,3galactosidase produced E2 and E4 (Fig.7D), confirming that the linkage between Gal and GalNAc was a β1,3-linkage. These data clearly show that CG33145 protein is a novel β1,3galactosyltransferase of N-glycan.

Figure 7.

Figure 7

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Identification of CG33145 as a novel β1,3galactosyltransferase of N-glycosylation. (A) The structures of an acceptor substrate E2 and its Gal extended forms, E4 and E5, which have one and two terminal Gal moieties, respectively. (B) FLAG-CG33145-PB and FLAG-BAP expressed in insect cells were purified and detected by anti-FLAG antibody. (C) Products of the CG33145-mediated reaction were analyzed by HPLC. CG33145 produced E4 and E5. (D) β1,3galactosidase treatment of reaction product E5. The E5 product peak shifted to peaks corresponding to E4 and E2 after β1,3galactosidase treatment.

Discussion

A wide variety of glycans are involved in diverse biological processes. To date, more than 200 genes in the human genome have been identified as GR candidates. However, biological and biochemical functions of the gene products remain to be studied in detail. In this study, large-scale RNAi silencing was used with Drosophila GR genes. Silencing of genes involved in synthesis of the same glycan resulted in the same phenotypes. Phenotypic clustering was used to identify galactosyltransferase terminal N-glycosylation activity in the previously uncharacterized protein CG33145. This suggests that phenotypic clustering is potentially valuable for the identification of specific glycans synthesized by genes of interest.

Using sequence comparisons, we identified 132 GR gene candidates in the Drosophila genome. Of these, the biochemical and biological functions of 50 genes remain to be studied in detail. However, it is difficult to determine the biochemical properties of GR proteins without predictive information because appropriate substrates and conditions are needed for biochemical assays. For example, a sialyltransferase that adds a sialic acid to a nonreducing end of N-glycans requires both CMP-sialic acid and a part of N-glycans for its biochemical assay. Pgant, a peptidyl-N-acetyl-galactosaminyltransferase that transfers GalNAc to mucin-type proteins, needs both UDP-GalNAc and appropriate peptides. Therefore, to determine the biochemical property of a novel gene, it is advantageous to predict what type of glycosylation is involved. In the present study, we examined the utility of phenotypic clustering for glycosylation prediction.

Silencing of GR genes using several Gal4 drivers resulted in various phenotypes such as formation of extra and thick veins and abdominal depigmentation; however, RNAi abnormalities were less severe than those resulting from classical mutations such as deletion, point mutation, or transposon-insertion. For example, silencing of fringe (fng) and fringe-connection (frc), which play an essential role in Notch glycosylation, produced a thick vein phenotype that was milder than the deleted margin phenotype of their null mutations. This may be due to low efficiency of gene silencing by RNAi and/or unusual persistence of GR proteins. Maternally provided Frc protein and/or mRNA was sufficient for a strong frc mutant to survive to the late third larval stage (Goto et al. 2001).

Knockdown phenotypes also depend on the RNAi library. Mummery-Widmer et al. identified CG12366 (Ofut1), but neither CG10580 (fng) nor CG3874 (frc), as a Notch regulator using Vienna RNAi library (Mummery-Widmer et al. 2009). The reason may be that the different lengths of dsRNAs between Vienna and NIG RNAi libraries. Long dsRNAs (500 bp) in NIG silence target gene expression more effectively than short ones (approximately 300 bp) in Vienna.

RNAi and conventional mutation phenotypes were similar, albeit with milder phenotypes observed with silencing. For example, knockdown of genes involved in GAG synthesis and Notch glycosylation resulted in formation of extra and thick veins, respectively. These phenotypes were also reported in strains with mutations in the corresponding genes. By contrast, the abdominal depigmentation phenotype produced upon knockdown of genes involved in synthesis of terminal domains of glycans has not been observed previously.

Sequence similarity and phenotype-based gene clustering in the present study suggested that CG33145 had a β1,3galactosyltransferase activity in N-glycan synthesis. Biochemical analysis confirmed that the CG33145 protein had β1,3galactosyltransferase activity for N-glycosylation. These results suggest that phenotype-based clustering can be indicative of molecular function. Similarly, Csat (CG2675), which also exhibited the abdominal pigmentation phenotype, may contribute to the synthesis of N-glycan.

The N-glycan gene cluster did not include glycosyltransferases involved in the production of core regions of N-linked glycans. It is possible that core regions of N-linked glycans are essential for protein folding and quality control and that deletion of whole N-linked glycan structures may cause lethal defects. By contrast, nonreducing ends of N-linked glycans play more specific roles such as regulation of ligand-receptor interactions, protein complex formation, and protein trafficking. Thus, defects of the nonreducing ends of N-linked glycans might result in specific, less severe phenotypes such as depigmentation.

In mice, branch positioning near nonreducing ends of N-glycans is required for proper trafficking of Glucose transporter 2, which is essential for glucose-stimulated insulin secretion (Ohtsubo et al. 2005). In Drosophila, the same branch structure and the insulin pathway were shown to be involved in cuticle pigmentation (Shakhmantsir et al. 2014). Therefore, abdominal depigmentation may be caused by impaired trafficking of membrane and/or secretory proteins in the insulin pathway.

Biological functions of some glycans are conserved between Drosophila and humans. For example, POMT1 and POMT2, which transfer a mannose to Dystroglycan via an O-type linkage, are mutated in Walker–Warburg syndrome, a type of muscular dystrophy (Akasaka-Manya et al. 2004; van Reeuwijk et al. 2005). rotated abdomen and twisted, Drosophila mutants of POMT1 (CG6097) and POMT2 (CG12311), respectively, which mediate O-linked mannosylation, also exhibit muscle defects in adults, suggesting a conserved biological function of the O-mannosyl glycan (Martin-Blanco & Garcia-Bellido 1996; Ichimiya et al. 2004; Ueyama et al. 2010). These mutants exhibited the behavioral abnormalities, the shortened lifespan and ultrastructural defects of muscles, as seen in human patients, also indicating that Drosophila POMT mutants are models for human muscular dystrophy. Then enhanced apoptosis was found in muscle progenitor cells of these mutants and provided new insight into the mechanism of WWS development, namely increased numbers of apoptotic myoblasts causing muscle disorganization (Ueyama et al. 2010). Therefore, phenotypic information obtained in Drosophila may shed light on glycan functions in other organisms, including humans.

Experimental procedures

Generation of RNAi fly lines

A 500-bp-long cDNA fragment of the N-terminal region of the ORF of each target gene was amplified by PCR. The fragment was inserted as an inverted repeat (IR) into a modified pUAST transformation vector, pUAST-R57 (GenBank accession: AB233207), which possessed an IR formation site consisting of paired KpnI-CpoI and XbaI-SfiI restriction sites. To enhance the RNAi effect (Kalidas & Smith 2002), pUAST-R57 carries a 282-bp-long genome fragment containing introns 5 and 6 of the Drosophila Ret oncogene between the two IR fragments. The IR was constructed in a head-to-head orientation using a combination of tag sequences in the PCR primers and restriction sites in the vector. Transformation of Drosophila embryos was carried out according to Spradling (Spradling 1986) in the w1118 fly backgrounds. Each line was mated with several of the GAL4 driver lines: Act5C-GAL4 (Bloomington Drosophila Stock Center), GMR-GAL4 (Freeman 1996), ey-GAL4 (Bloomington Drosophila Stock Center), dpp-GAL4 (Staehling-Hampton et al. 1994), en-GAL4 (Johnson et al. 1995), pnr-GAL4 (Heitzler et al. 1996), ptc-GAL4 (Speicher et al. 1994), sd-GAL4 (Milan et al. 1997), A9-GAL4 (Sun & Artavanis-Tsakonas 1997), 29BD-GAL4 (Nakayama et al. 1997), 69B-GAL4 (Brand & Perrimon 1993), and MS1096-GAL4 (Capdevila & Guerrero 1994). F1 progeny were raised at 28°C, and their phenotypes were analyzed. F1 progeny of w1118 crossed with each of the GAL4 driver lines were used as a control, for example, Act5C-GAL4/+, GMR-GAL4/+, etc.

Quantitative analysis of mRNA by real-time PCR

Total RNA was extracted from Act5C-GAL4/UAS-IR-CG4351, CG15110, CG17772, and Act5C-GAL4/+ third instar larvae. First-strand cDNA was synthesized using a SuperScript II first-strand synthesis kit (Invitrogen) according to the manufacturer’s instructions. Quantitation of CG4351, CG15110, and CG17772 mRNA expression was carried out by real-time PCR using the following primers: forward, 5′-ccacgacgtgatcgctttct-3′ (CG4351), 5′-ggagtgcgcggaaatgg-3′ (CG15110), and 5′-gaaatctgcggcggattcta-3′ (CG17772); and reverse, 5′-cagtccctgcggatgtaagag-3′ (CG4351), 5′-tgtttgggcctcagttcacctt-3′ (CG15110), and 5′-agtggtggccgccagtt-3′ (CG17772). The probe, which consisted of 5′-tagtcgggattatgtccaggctcgca-3′ (CG4351), 5′-ccgcccgaaggaaatacctgcttaccta-3′ (CG15110), or 5′-ccatgaacatatacgagaccggaatagccaa-3′ (CG17772), was labeled at the 5′-end with the reporter dye 3FAM and at the 3′-end with the quencher dye TAMRA (Applied Biosystems, Foster City, CA). Real-time PCR was carried out using a TaqMan Universal PCR Master Mix (Applied Biosystems). Relative amounts of CG4351, CG15110, and CG17772 mRNAs were normalized against ribosomal protein L32 (RpL32) mRNA levels from the same cDNA.

Determination of the amount of chondroitin sulfate and heparan sulfate in Drosophila

GAGs were prepared from approximately 20 mg of lyophilized flies. Unsaturated disaccharides were produced by enzymatic digestion and analyzed by fluorometric postcolumn high-performance liquid chromatography, as described previously (Toyoda et al. 2000).

Expression and purification of CG33145 protein

The putative catalytic domain of candidate CG33145 protein (amino acids 92 to 466, CG33145-PB) was cloned using DGC clone RE52041, expressed in insect cells as a secreted protein fused with a FLAG peptide, and purified using Anti-FLAG M1 Affinity gel (Sigma), as described previously (Ueyama et al. 2008).

Galactosyltransferase activity assay

β1,3galactosyltransferase activity was assessed. The acceptor substrate E2 and standards, E4 and E5, were prepared as described previously (Kimura et al. 2006, 2007). Enzymatic reactions, product detection, and product confirmation were also carried out as noted previously (Kimura et al. 2006, 2007).

Acknowledgments

We thank H. Hamamoto, Y. Omae, K. Ohtsu, and W. Awano for technical assistance. This work was supported by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Agency (JST), and MEXT/JSPS KAKENHI.

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