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Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile
Biochemical and Biophysical Research Communications 402 (2010) 680–686
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile Can Peng a, Akira Togayachi b, Yeon-Dae Kwon b, Chunyan Xie a,d, Gongdong Wu a, Xia Zou a, Takashi Sato b, Hiromi Ito b, Kouichi Tachibana b, Tomomi Kubota b, Toshiaki Noce c, Hisashi Narimatsu b, Yan Zhang a,d,⇑ a
Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, Shanghai, China Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan Laboratory of Reproductive Biology, Mitsubishi Kagaku Institute of Life Sciences, 11 Minama-Ooya, Machida-shi, Tokyo, Japan d State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 197 Rui Jin Road II, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 9 October 2010 Available online 25 October 2010 Keywords: Glycosyltransferase ppGalNAc-T O-glycosylation O-glycan Mucin
a b s t r a c t A novel member of the human ppGalNAc-T family, ppGalNAc-T20, was identified and characterized. Amino acid alignment revealed a high sequence identity between ppGalNAc-T20 and -T10. In the GalNAc transfer assay towards mucin-derived peptide substrates, the recombinant ppGalNAc-T20 demonstrated to be a typical glycopeptide GalNAc-transferase that exhibits activity towards mono-GalNAc-glycosylated peptide EA2 derived from rat submandibular gland mucin but no activity towards non-modified EA2. The in vitro catalytic property of ppGalNAc-T20 was compared with that of ppGalNAc-T10 to show different acceptor substrate specificities and kinetic constants. The ppGalNAc-T20 transcript was found exclusively in testis and brain. In situ hybridization further reveals that ppGalNAc-T20 was specifically localized in primary and secondary spermatocytes of the two meiotic periods, suggesting that it may involve in O-glycosylation during mouse spermatogenesis. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Many secreted and membrane proteins are post-translationally modified with a variety of glycans that have significant roles [1,2]. The transfer of a GalNAc residue from the nucleotide sugar, UDPGalNAc, to the hydroxyl group of Ser or Thr in target proteins is the first step in mucin-type O-glycosylation. This reaction is catalyzed by members in a large family of ppGalNAc-Ts (EC 2.4.1.41) located in Golgi apparatus [2,3]. Therefore, the substrate specificity of each isoform determines the number and site of O-glycans in a protein. ppGalNAc-Ts belong to type II membrane proteins [4,5]. Their primary structure contains a short cytoplasmic tail, a single transmembrane domain, a stem region with variable length, a catalytic domain with GT1 and Gal/GalNAc-T motifs which are the putative binding site for donor and acceptor substrate, respectively, and a Abbreviations: FAM, 5-carboxyfluorescein succinimidyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MALDI-TOF, matrix-assisted laser desorption/ ionization time-of-flight; ppGalNAc-T, UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase; TFA, trifluoroacetic acid. ⇑ Corresponding author at: Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China. Fax: +86 21 34206778. E-mail address: [email protected] (Y. Zhang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.084
C-terminal ricin-type lectin domain which is observed only in ppGalNAc-Ts among all glycosyltransferases [6]. Analysis of EST and genome databases indicates that the human ppGalNAc-T family has 19 distinct members, each of which has unique substrate specificity and expression pattern (Table 1), suggesting that they have distinct biological functions [10,22]. Different ppGalNAc-Ts are directly involved in diverse cellular and developmental processes by modifying specific target proteins. ppGalNAc-T1 contributes to the O-glycosylation of P-selectin ligand on the surface of lymphocytes and B cells. Deficiency of ppGalNAc-T1 will not only affect the microvascular circulation and neutrophil recruitment, but also result in the increased apoptosis of B cells [23]. Overexpression of ppGalNAc-T6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of an oncoprotein MUC1 [24]. The O-glycosylation of death receptors DR4 and DR5 by ppGalNAc-T14 could promote the ligand-stimulated clustering of DR4 and DR5 to mediate the recruitment and activation of the apoptosis-initiating protease caspase-8 [25]. However, functions of individual ppGalNAc-T including their in vivo substrate proteins and O-glycosylation sites remain to be elucidated. Rat ppGalNAc-T9, an orthologous gene of human ppGalNAcT10, is considered as a glycopeptide GalNAc-transferase with no detectable activity towards non-glycosylated peptides [26].
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C. Peng et al. / Biochemical and Biophysical Research Communications 402 (2010) 680–686 Table 1 Summary of 20 human ppGalNAc-Ts. GalNAc-transferase activity
ppGalNAc-T isoform
mRNA accession number
Chromosome Exon Coding region (aa)
ppGalNAc-T1/GALNT1 ppGalNAc-T2/GALNT2 ppGalNAc-T3/GALNT3 ppGalNAc-T4/GALNT4 ppGalNAc-T5/GALNT5 ppGalNAc-T6/GALNT6 ppGalNAc-T7/GALNT7 ppGalNAc-T8/GALNT8 ppGalNAc-T9/GALNT9
NM_020474.3 NM_004481.2 NM_004482.3 NM_003774.3 NM_014568.1 NM_007210.3 NM_017423.2 NM_017417.1 NM_001122636.1
18q12.1 1q41–42 2q24–31 12q21.3–22 2q24.2 12q13 4q31.1 12p13.3 12q24.33
11 16 10 1 10 10 12 11 11
559 571 633 578 940 622 657 637 603
+ + + + + + ND -
+ + + + + + + ND ND
ppGalNAc-T10/GALNT10
NM_198321.2
5q33.2
12
603
-
+
ppGalNAc-T11/GALNT11 ppGalNAc-T12/GALNT12 ppGalNAc-T13/GALNT13
NM_022087.2 NM_024642.3 NM_052917.2
7q34–36 9q31.1 2q24.1
11 10 11
608 581 556
+ + +
+ + +
ppGalNAc-T14/GALNT14 ppGalNAc-T15/GALNTL2
NM_024572.2 NM_054110.4
2p23.2 3p25.1
15 10
552 639
+ +
+ +
ppGalNAc-T16/GALNTL1 ppGalNAc-T17/GALNTL3 ppGalNAc-T18/GALNTL4 ppGalNAc-T19/GALNTL5 ppGalNAc-T20/GALNTL6
NM_020692.1 NM_022479.1 NM_198516.1 NM_145292.2 NM_001034845.2
14q24.1 7q11.23 11p15.3 7q36.2 4q34.1
14 11 11 8 12
542 598 607 443 601
ND ND ND -
ND ND ND +
Transcript expression in tissues
Ref.
Ubiquitously Ubiquitously Testis, pancreas, kidney, prostate, intestine, spleen Ubiquitously Sublingual gland, colon, small intestine, stomach Placenta, trachea, brain, pancreas Ubiquitously Ubiquitously Brain (cerebellum, putamen, temporal lobe, frontal lobe, cerebral cortex) Small intestine, stomach, pancreas, ovary, thyroid gland, and spleen Kidney Small intestine, stomach, pancreas, colon, and testis Fetal brain, adult cerebellum, cerebral cortex, whole brain Kidney and fetal kidney Small intestine, placenta, spleen, cerebral cortex, ovary, uterus, mammary gland, stomach, cerebellum, whole brain ND Cerebellum and cerebral cortex ND ND Testis and brain
[7] [7] [8] [9] [10] [11] [12] [13] [14]
Peptide Glycopeptide
[15] [16] [17] [18] [19] [20]
[21] This work
ND, not determined.
Structural elements underlying the recognition of glycopeptide substrate by ppGalNAc-T10 have been resolved [27]. Substrate specificities of ppGalNAc-T1, -T2, and -T10 revealed that the glycopeptide-specific ppGalNAc-T10 has a unique GalNAc-O-Ser/ Thr-binding site in its catalytic domain, which was not found in ppGalNAc-T1 or -T2 [28]. In this study, we identified a novel member of the human ppGalNAc-T family, ppGalNAc-T20, which has high homology with human ppGalNAc-T10. The in vitro catalytic activity and expression profile of ppGalNAc-T20 were investigated to provide insights into its possible roles in O-glycosylation. 2. Materials and methods 2.1. Cloning of human ppGalNAc-T20 A BLASTN search of human genome database with the ORF of human ppGalNAc-T10 (AJ505950) as a query identified potential exons of a novel ppGalNAc-T gene in sequence NT022792. The potential transmembrane protein contained 601 amino acids. By using the cDNA of human small-cell lung carcinoma cell, Lu130, as the template, the full-length cDNA of this novel polypeptide GalNAc transferase was amplified by PCR (forward primer, 50 -CAC CATGAAGAGGAAACAGAAGAGATTTCTG-30 ; reverse primer, 50 -CTA GGAGTTGGCATGGTGGTTA-30 ) and cloned into pENTR/D-TOPO for sequencing. The obtained ORF was deposited into GenBank with the accession number GU220060.
purified with anti-FLAG M2 affinity gel (Sigma), and analyzed by Western blotting using anti-FLAG antibody (Sigma) and ECL plus reagents (GE Healthcare). Human ppGalNAc-T10 and -T1, containing residues 34–603 and 34–559, respectively, were also prepared by the same method. 2.3. Polypeptide GalNAc-transferase assay Standard polypeptide GalNAc-transferase assay was performed as described previously [18]. In brief, the enzymatic mixture (20 ll) was incubated at 37 °C for various periods and boiled at 95 °C for 3 min to terminate the reaction. FAM-labeled peptide substrates derived from tandem repeats of corresponding mucins [29–32], EA2 (PTTDSTTPAPTTK-5,6FAM), Muc5AC (SAPTTSTTSA PTK-5,6FAM), Muc1a (5,6FAM-AHGVTSAPDTR), and Muc2 (PTTTPI TTTTTVTPTPTPTGTQTK-5,6FAM), were purchased from Sawady Co. Ltd. Reaction products were evaluated by RP-HPLC (Shimadzu) on a C18 analytical column (COSMOSIL 5C18-AR-II, 4.6 250 mm). The isolated reaction products were lyophilized and subjected to MALDI-TOF mass spectrometry (Reflex IV, Bruker Daltonics) in the positive ion mode to determine the number of transferred GalNAc. To identify the position of attached GalNAc, amino acid sequencing was carried out by Edman degradation [18,33]. To determine the Km value for acceptor substrate, the reaction condition was modified to include a saturated concentration of UDPGalNAc (0.5 mM), and a series of concentrations from 0.005 to 0.15 mM of mono-GalNAc-glycosylated EA2. Km and Vmax values were calculated using OriginPro 7.5 with a non-linear curve fitting.
2.2. Production of purified recombinant ppGalNAc-Ts The cDNA fragment encoding the catalytic and lectin domain of ppGalNAc-T20 (aa 33–601) was obtained by PCR (forward primer, 50 -GACGACAAGCTTGAGCAGCAGACATTTCCACTG-30 ; reverse primer, 50 -GCTCTAGAGGATCCAGAGCTGCCAGGAAACAG-30 ) and cloned into pFLAG-CMV3 (Sigma). HEK293T cells were transfected with the reconstructed plasmid. The secreted recombinant protein was
2.4. Quantitative analysis of the ppGalNAc-T20 transcript by real-time PCR in human tissues and tumor cell lines Quantitative real-time PCR was carried out using a TaqMan Universal Master Mix (PE Applied Biosystems). The primer set and probe for ppGalNAc-T20 were as follows: forward, 50 -ACTGC GAGGTCAATGTGAACTG-30 ; reverse, 50 -CACACGATGGTTTTGTGGTT
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TAG-30 ; probe, 50 -CTGCCCCCACTCCTCAACCAAATTG-30 . Standard curves for ppGalNAc-T20 and GAPDH, were generated by serial dilution of pENTR/D-TOPO containing the ppGalNAc-T20 gene and pCR2.1 (Invitrogen) with the GAPDH gene, respectively. Realtime PCR was performed at 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). 2.5. In situ hybridization In situ hybridization was performed as previously described [34,35] in mouse testis. The partial 30 -UTR sequence of mouse ppGalNAc-T20 (nucleotides 1951–2709 in BB641428) was amplified by PCR using mouse brain cDNA as the template. The fragment amplified by 50 -GAGACGTTCCTACATCTGAAGAACTTG-30 and 50 CCTCTCCACTGTCTTCTTTAATAAAAATT-30 was inserted into pCR– Blunt II-TOPO (Invitrogen) to generate anti-sense and sense RNA probes using the RNA digoxigenin-labeling kit (Roche) after linearization. 3. Results 3.1. cDNA cloning of human ppGalNAc-T20 The ppGalNAc-T20 gene was identified as a putative novel ppGalNAc-T by BLASTN search of human genome database using ppGalNAc-T10 as a query. The full-length cDNA of human ppGalNAc-T20 encoded a 601-aa type II membrane protein, which contains a N-terminal 7-aa cytoplasmic region, a transmembrane segment of 21 aa, a stem region, a putative catalytic region, and a C-terminal lectin domain. ppGalNAc-T20 retained the DXH motif (aa 232–234) which was conserved in the catalytic domain of all ppGalNAc-T members and was essential for divalent cation binding. Amino acid alignment of human ppGalNAc-T20 and -T10 (Fig. 1A) revealed their high sequence identity (71%). Three possible N-glycosylation sites were found in ppGalNAc-T20, which were conserved in ppGalNAc-T10 [27]. The genomic structure of ppGal-
NAc-T20 and -T10 was also compared (Fig. 1A). The ppGalNAc-T20 gene was found to have different chromosome location from the ppGalNAc-T10 gene (Table 1). Both genes contained at least 12 exons, the length of which was exactly the same between the two genes, except for exon 1. To better understand the evolutionary relationship of 20 human ppGalNAc-Ts, a neighbor-joining tree is reconstructed by MEGA3.1 (Fig. 1B). ppGalNAc-T20 is positioned with ppGalNAc-T10 in one clade, confirming that ppGalNAc-T10 is the closest homologue to ppGalNAc-T20. 3.2. Different catalytic properties between ppGalNAc-T20 and -T10 To determine the substrate specificity and kinetic property of ppGalNAc-T20, the recombinant enzyme ppGalNAc-T20 fused with the FLAG tag was purified from the medium of transfected HEK293T cells. Recombinant FLAG-fused enzymes ppGalNAc-T1 and -T10 were also produced as control. As shown in Fig. 2A, the ppGalNAc-T1 enzyme (20 ng) generated one product peak (P1) that was shifted from the acceptor substrate peak (S) of EA2 with 100% product conversion rate after incubation for 30 min, and more highly glycosylated products appeared with an extended incubation of 24 h. Similarly, ppGalNAc-T1 (20 ng) generated a major product peak (P4) when incubated with Muc5AC as an acceptor substrate for 30 min and 24 h. However, no GalNAc-transferase activity of up to 600 ng of enzyme ppGalNAc-T20 or -T10 was observed after incubation for 3 h (data not shown) and 24 h, respectively. These results demonstrate that the two highly homologous enzymes, ppGalNAc-T20 and -T10 show no GalNAc-transferase activities to non-GalNAc-modified peptides EA2 and Muc5AC. Similar results were obtained with Muc1a and Muc2 as acceptor substrates (data not shown). To assess whether ppGalNAc-T20 has GalNAc-transferase activity on glycopeptides, we incubated mono-GalNAc-Thr7th-EA2 produced by ppGalNAc-T1 (P1) [26] as the acceptor substrate with 300 ng of ppGalNAc-T20 or -T10. Both enzymes effectively utilized mono-GalNAc-EA2 and yielded one product peak with different catalytic efficiency (Fig. 2B). As shown in Fig. 2C, the molecular
Fig. 1. (A) Amino acid sequence comparison of human ppGalNAc-T20 and -T10. Positions of introns, the putative transmembrane region, the GT1 motif, the Gal/GalNAc-T motif, the lectin domain, and potential N-glycosylation sites are indicated by arrows, square boxes, double lines, dashed lines, single solid lines, and circles, respectively. (*), identical residues; (:), conserved substitutions; (.), semiconserved substitutions. (B) Phylogenetic tree generated from amino acid alignment of 20 human ppGalNAC-Ts.
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Fig. 2. (A) HPLC analysis of in vitro O-glycosylation on FAM-labeled EA2 and Muc5AC by recombinant ppGalNAc-T20, -T10, and -T1. S represents the original non-glycosylated peptide peak, while P1 and P4 respectively represent the product peaks of ppGalNAc-T1 when using EA2 and Muc5AC as the acceptor substrate. (B) Time-course of the secondary activity of ppGalNAc-T20 and -T10 towards mono-GalNAc-EA2 produced by ppGalNAc-T1 (P1) as the acceptor substrate. (C) MALDI-TOF mass measurement of reaction products derived from EA2 and mono-GalNAc-EA2 by ppGalNAc-Ts as shown in (B). (D) Mono-GalNAc-Muc5AC produced by ppGalNAc-T1(P4) was used as the peptide substrate to compare the glycopeptide GalNAc-transferase activity of ppGalNAc-T20 and -T10. Reaction products were eluted with a flow rate of 1 mL/min and the following buffer B (100% acetonitrile, 0.05% TFA) gradient: 0–30 min, 20–40%, 30–35 min, 40–80%.
masses of P2 (m/z 2080.91) and P3 (m/z 2080.85) matched with that of di-GalNAc-EA2. The glycosylation sites of P2 and P3 were determined by Edman sequencing. Both products P2 and P3 were glycosylated at Thr6th and Thr7th of peptide EA2 (data not shown). Hence, ppGalNAc-T20 and -T10 transferred one GalNAc to Thr6th of mono-GalNAc-Thr7th-EA2. But when 300 ng of ppGalNAc-T20 or T10 was incubated with mono-GalNAc-Muc5AC (P4) produced by ppGalNAc-T1 as the acceptor substrate, only ppGalNAc-T10 showed GalNAc-transferase activity (Fig. 2D). Therefore, ppGalNAc-T20 demonstrated to be a novel typical glycopeptide GalNAc-transferase that shows different substrate specificities from ppGalNAc-T10 in vitro. The kinetic constants of ppGalNAc-T20 and -T10 for mono-glycosylated EA2 were then determined to investigate their different catalytic properties. As shown in Table 2, ppGalNAc-T20 and -T10 exhibited a Km of 0.06 and 0.025 mM, respectively, indicating a lower substrate affinity of ppGalNAcT20 than that of ppGalNAc-T10, and the catalytic efficiency (Vmax/Km) of ppGalNAc-T20 is 40-fold lower than that of ppGalNAc-T10. 3.3. Distribution of the ppGalNAc-T20 transcript Expression levels of the ppGalNAc-T20 transcript in human tissues and tumor cell lines were determined by real-time PCR as shown in Fig. 3. ppGalNAc-T20 was restrictively expressed in testis and brain (Fig. 3A), with low or negligible expression observed in other tissues, and it had relatively high expression in lung small cell carcinoma cell lines (Fig. 3B). As the ppGalNAc-T10 transcript was ubiquitously expressed in human tissues [15], the differential
expression pattern between ppGalNAc-T20 and -T10 suggests that they may control distinct O-glycosylation process in vivo. To investigate the location of ppGalNAc-T20 during testis development, the expression of mouse ppGalNAc-T20 (mT20) transcript was examined by in situ hybridization. As shown in Fig. 4, mT20 is strongly expressed in primary spermatocytes of the first meiosis (stage I–V) and secondary spermatocytes of the second meiosis (stage IX–XII), with no signals observed in spermatogonia and late spermatids. This result suggests that ppGalNAc-T20 might have certain functions in spermatogenesis. 4. Discussion In this work, we describe the identification of the 20th member of human ppGalNAc-T family, ppGalNAc-T20, which shares high sequence homology to ppGalNAc-T10. ppGalNAc-T20, the same as ppGalNAc-T10, exhibited significant glycopeptide GalNActransferase activity towards mono-glycosylated EA2, but only ppGalNAc-T10 showed activity to mono-glycosylated Muc5AC, revealing unique acceptor substrate specificities of the two enzymes. As EA2 has seven potential glycosylation sites and
Table 2 Kinetic constants of recombinant ppGalNAC-T20 and -T10 for acceptor substrate.
ppGalNAc-T20 ppGalNAc-T10
Km (mM)
Vmax (nmol/min/lg)
Vmax/Km
0.06 0.025
0.064 1.080
1.067 43.2
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Fig. 3. Quantitative analysis of the ppGalNAc-T20 transcript by real-time PCR in human tissues (A) and tumor cell lines (B). The expression level of ppGalNAc-T20 was normalized to that of GAPDH. Values are shown as copy numbers of the target mRNA per lg of total RNA. Data were obtained from triplicate experiments and indicated as means ± SD.
ppGalNAc-T20 and -T10 incorporated only one GalNAc into monoGalNAc-EA2, the two bulky GalNAc residues may cause steric hindrance to interfere with the subsequent O-glycosylation. Moreover, the fact that ppGalNAc-T1 can transfer more than two GalNAc residues to the acceptor substrate EA2 with extended incubation (Fig. 2A) further indicates diversified catalytic mechanisms among human ppGalNAc-T family enzymes. Although ppGalNAc-T20 and -T10 both exhibit GalNAc-transferase activity towards monoGalNAc-EA2, the 40-fold higher efficiency of ppGalNAc-T10 suggests that the glycosylated EA2 derived from rat submandibular gland mucin may be a more appropriate substrate for ppGalNAcT10. Besides, the lower reactivity of ppGalNAc-T20 towards EA2 is reasonable, as it has no expression in salivary grand. Mucins specifically expressed in testis or brain may be endogenous substrates for this enzyme. The different catalytic property and expression pattern of ppGalNAc-T20 and -T10 suggest that the two enzymes may have distinct biological functions. The GalNAc-transferase activity of 20 human ppGalNAc-Ts characterized to date is summarized in Table 1. Many ppGalNAcT isoforms can transfer GalNAc to both non-glycosylated and
glycopeptides, while ppGalNAc-T20, as well as two other glycopeptide-specific ppGalNAc-T7 [12] and -T10, require the prior addition of GalNAc to peptides before they transfer additional GalNAc residues. Each ppGalNAc-T isoform has unique enzymatic activity and there may have synergistic, competitive, or even inhibitory effects among them. The 20 human ppGalNAc-Ts provide us a systematic model to investigate the roles of O-glycosylation under certain physiological and pathological conditions. The ppGalNAc-T20 transcript was exclusively expressed in testis and brain, suggesting that it may involve in cell maturation and differentiation of these two organs. Most ppGalNAc-Ts were distributed in peripheral blood cells, digestive and lymphatic organs that produce various O-glycosylated proteins, while ppGalNAcT9, -T13, and -T17 specifically expressed in brain, ppGalNAc-T11 and -T14 restrictively distributed in kidney (Table 1). It was recently reported that normal human testis showed a very restricted expression pattern of simple mucin-type O-glycans and polypeptide GalNAc-transferases [36]. To date, only ppGalNAc-T3 has been detected in maturing postmeiotic germ cells including sperm [11]. Hence, ppGalNAc-T20 is the second ppGalNAc-T isoform found in
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Fig. 4. In situ hybridization of mT20 during testis spermatogenic stages I–XII. Mouse testis sections were hybridized with mT20 30 region anti-sense probe. Signals can be seen over primary spermatocytes of the first meiosis (stage I–V) and secondary spermatocytes of the second meiosis (stage IX–XII), whereas hybridization with sense construct did not result in significant staining.
germ cells of normal testis. As changes in the profile of simple mucin-type O-glycans and ppGalNAc-Ts in human testis and testicular neoplasms are associated with germ cell maturation and tumor differentiation [36], lack of ppGalNAc-T3 or -T20 might impair maturation of spermatozoa and sperm-egg binding. Because ppGalNAc-T3 has enzymatic activity on both non-glycosylated peptides and glycopeptides [37], there may have coordination and competition between ppGalNAc-T3 and -T20 to ensure appropriate glycosylation of substrate proteins in testis. Transcript expression of mT20 was observed in primary spermatocytes of the first meiosis (stage I–V) and secondary spermatocytes of the second meiosis (stage IX–XII), while the mouse ppGalNAc-T3 transcript was expressed in spermatogenic stages I–VII and IX–XII (data not shown), suggesting that they might play distinct functions. The substrate profiling for individual ppGalNAc-T in testis remains largely unknown, and we hope to find the specific substrates for ppGalNAc-T20 with more research progress in characterizing glycoproteins in testis. In conclusion, the present study provides evidence that ppGalNAc-T20 is a new glycopeptide GalNAc-transferase member of the human ppGalNAc-T family. The significant high level of ppGalNAc-T20 transcript in testis strongly suggests that ppGalNAc-T20 is involved in O-glycosylation in spermatogenesis. Further study
is necessary for the identification of natural substrates, which are also expressed in testis as ppGalNAc-T20. Acknowledgments This work was sponsored by grants from the National Natural Science Foundation (30770482), the Pujian project (07pj14057), and the National Key Scientific Program (2007CB914703) of China. This work was also performed as a part of the National Institute of Advanced Industrial Science and Technology (AIST) work supported by the New Energy and Industrial Technology Development Organization (NEDO) of Japan. References [1] R.S. Haltiwanger, J.B. Lowe, Role of glycosylation in development, Annu. Rev. Biochem. 73 (2004) 491–537. [2] E. Tian, K.G. Ten Hagen, Recent insights into the biological roles of mucin-type O- glycosylation, Glycoconj. J. 26 (2009) 325–334. [3] K.G. Ten Hagen, T.A. Fritz, L.A. Tabak, All in the family: the UDP-GalNAc: polypeptideN-acetylgalactosaminyltransferases, Glycobiology 13 (2003) 1R– 16R. [4] J. Roth, Y. Wang, A.E. Eckhardt, R.L. Hill, Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptideN-acetylgalactosaminyltransferasemediated O-glycosylation reaction in the submaxillary gland, Proc. Natl. Acad. Sci. USA 91 (1994) 8935–8939.
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[21] N. Nakamura, S. Toba, M. Hirai, S. Morishita, T. Mikami, M. Konishi, N. Itoh, A. Kurosaka, Cloning and expression of a brain-specific putative UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase gene, Biol. Pharm. Bull 28 (2005) 429–433. [22] F.K. Hagen, K.G. Ten Hagen, T.M. Beres, M.M. Balys, B.C. Van Wuyckhuyse, L.A. Tabak, cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase, J. Biol. Chem. 272 (1997) 13843–13848. [23] M. Tenno, K. Ohtsubo, F.K. Hagen, D. Ditto, A. Zarbock, P. Schaerli, U.H. von Andrian, K. Ley, D. Le, L.A. Tabak, J.D. Marth, Initiation of protein O glycosylation by the polypeptide GalNAcT-1 in vascular biology and humoral immunity, Mol. Cell Biol. 27 (2007) 8783–8796. [24] J.H. Park, T. Nishidate, K. Kijima, T. Ohashi, K. Takegawa, T. Fujikane, K. Hirata, Y. Nakamura, T. Katagiri, Critical roles of mucin 1 glycosylation by transactivated polypeptide N-acetylgalactosaminyltransferase 6 in mammary carcinogenesis, Cancer Res. 70 (2010) 2759–2769. [25] K.W. Wagner, E.A. Punnoose, T. Januario, D.A. Lawrence, R.M. Pitti, K. Lancaster, D. Lee, M. von Goetz, S.F. Yee, K. Totpal, L. Huw, V. Katta, G. Cavet, S.G. Hymowitz, L. Amler, A. Ashkenazi, Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL, Nat. Med. 13 (2007) 1070–1077. [26] K.G. Ten Hagen, G.S. Bedi, D. Tetaert, P.D. Kingsley, F.K. Hagen, M.M. Balys, T.M. Beres, P. Degand, L.A. Tabak, Cloning and characterization of a ninth member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family ppGaNTase-T9, J. Biol. Chem. 276 (2001) 17395–17404. [27] T. Kubota, T. Shiba, S. Sugioka, S. Furukawa, H. Sawaki, R. Kato, S. Wakatsuki, H. Narimatsu, Structural basis of carbohydrate transfer activity by human UDPGalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAcT10), J. Mol. Biol. 359 (2006) 708–727. [28] C.L. Perrine, A. Ganguli, P. Wu, C.R. Bertozzi, T.A. Fritz, J. Raman, L.A. Tabak, T.A. Gerken, Glycopeptide-preferring polypeptide GalNAc transferase 10 (ppGalNAc T10) involved in mucin-type O-glycosylation has a unique GalNAc-O-Ser/Thr-binding site in its catalytic domain not found in ppGalNAc T1 or T2, J. Biol. Chem. 284 (2009) 20387–20397. [29] J.R. Gum, J.C. Byrd, J.W. Hicks, N.W. Toribara, D.T. Lamport, Y.S. Kim, Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism, J. Biol. Chem. 264 (1989) 6480–6487. [30] S.J. Gendler, C.A. Lancaster, J. Taylor-Papadimitriou, T. Duhig, N. Peat, J. Burchell, L. Pemberton, E.N. Lalani, D. Wilson, Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin, J. Biol. Chem. 265 (1990) 15286–15293. [31] E.F. Albone, F.K. Hagen, B.C. Van Wuyckhuyse, L.A. Tabak, Molecular cloning of a rat submandibular gland apomucin, J. Biol. Chem. 269 (1994) 16845–16852. [32] V. Guyonnet Duperat, J.P. Audie, V. Debailleul, A. Laine, M.P. Buisine, S. Galiegue-Zouitina, P. Pigny, P. Degand, J.P. Aubert, N. Porchet, Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes?, Biochem J. 305 (1995) 211–219. [33] H. Iwasaki, Y. Zhang, K. Tachibana, M. Gotoh, N. Kikuchi, Y.D. Kwon, A. Togayachi, T. Kudo, T. Kubota, H. Narimatsu, Initiation of O-glycan synthesis in IgA1 hinge region is determined by a single enzyme UDP-N-acetyl-alpha-Dgalactosamine: polypeptide N-acetylgalactosaminyltransferase 2, J. Biol. Chem. 278 (2003) 5613–5621. [34] Y. Fujiwara, T. Komiya, H. Kawabata, M. Sato, H. Fujimoto, M. Furusawa, T. Noce, Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage, Proc. Natl. Acad. Sci. USA 91 (1994) 12258–12262. [35] T. Hiruma, A. Togayachi, K. Okamura, T. Sato, N. Kikuchi, Y.D. Kwon, A. Nakamura, K. Fujimura, M. Gotoh, K. Tachibana, Y. Ishizuka, T. Noce, H. Nakanishi, H. Narimatsu, A Novel Human b1, 3-N-Acetylgalactosaminyltransferase That Synthesizes a Unique Carbohydrate Structure, GalNAcb13GlcNAc, J. Biol. Chem. 279 (2004) 14087–14095. [36] E. Rajpert-De Meyts, S.N. Poll, I. Goukasian, C. Jeanneau, A.S. Herlihy, E.P. Bennett, N.E. Skakkebaek, H. Clausen, A. Giwercman, U. Mandel, Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation, Virchows Arch. 451 (2007) 805–814. [37] H.H. Wandall, H. Hassan, E. Mirgorodskaya, A.K. Kristensen, P. Roepstorff, E.P. Bennett, P.A. Nielsen, M.A. Hollingsworth, J. Burchell, J. Taylor-Papadimitriou, H. Clausen, Substrate specificities of three members of the human UDP-Nacetyl-alpha-D-galactosamine: Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3, J. Biol. Chem. 272 (1997) 23503–23514.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile Can Peng a, Akira Togayachi b, Yeon-Dae Kwon b, Chunyan Xie a,d, Gongdong Wu a, Xia Zou a, Takashi Sato b, Hiromi Ito b, Kouichi Tachibana b, Tomomi Kubota b, Toshiaki Noce c, Hisashi Narimatsu b, Yan Zhang a,d,⇑ a
Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, Shanghai, China Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan Laboratory of Reproductive Biology, Mitsubishi Kagaku Institute of Life Sciences, 11 Minama-Ooya, Machida-shi, Tokyo, Japan d State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 197 Rui Jin Road II, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 9 October 2010 Available online 25 October 2010 Keywords: Glycosyltransferase ppGalNAc-T O-glycosylation O-glycan Mucin
a b s t r a c t A novel member of the human ppGalNAc-T family, ppGalNAc-T20, was identified and characterized. Amino acid alignment revealed a high sequence identity between ppGalNAc-T20 and -T10. In the GalNAc transfer assay towards mucin-derived peptide substrates, the recombinant ppGalNAc-T20 demonstrated to be a typical glycopeptide GalNAc-transferase that exhibits activity towards mono-GalNAc-glycosylated peptide EA2 derived from rat submandibular gland mucin but no activity towards non-modified EA2. The in vitro catalytic property of ppGalNAc-T20 was compared with that of ppGalNAc-T10 to show different acceptor substrate specificities and kinetic constants. The ppGalNAc-T20 transcript was found exclusively in testis and brain. In situ hybridization further reveals that ppGalNAc-T20 was specifically localized in primary and secondary spermatocytes of the two meiotic periods, suggesting that it may involve in O-glycosylation during mouse spermatogenesis. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Many secreted and membrane proteins are post-translationally modified with a variety of glycans that have significant roles [1,2]. The transfer of a GalNAc residue from the nucleotide sugar, UDPGalNAc, to the hydroxyl group of Ser or Thr in target proteins is the first step in mucin-type O-glycosylation. This reaction is catalyzed by members in a large family of ppGalNAc-Ts (EC 2.4.1.41) located in Golgi apparatus [2,3]. Therefore, the substrate specificity of each isoform determines the number and site of O-glycans in a protein. ppGalNAc-Ts belong to type II membrane proteins [4,5]. Their primary structure contains a short cytoplasmic tail, a single transmembrane domain, a stem region with variable length, a catalytic domain with GT1 and Gal/GalNAc-T motifs which are the putative binding site for donor and acceptor substrate, respectively, and a Abbreviations: FAM, 5-carboxyfluorescein succinimidyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MALDI-TOF, matrix-assisted laser desorption/ ionization time-of-flight; ppGalNAc-T, UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase; TFA, trifluoroacetic acid. ⇑ Corresponding author at: Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China. Fax: +86 21 34206778. E-mail address: [email protected] (Y. Zhang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.084
C-terminal ricin-type lectin domain which is observed only in ppGalNAc-Ts among all glycosyltransferases [6]. Analysis of EST and genome databases indicates that the human ppGalNAc-T family has 19 distinct members, each of which has unique substrate specificity and expression pattern (Table 1), suggesting that they have distinct biological functions [10,22]. Different ppGalNAc-Ts are directly involved in diverse cellular and developmental processes by modifying specific target proteins. ppGalNAc-T1 contributes to the O-glycosylation of P-selectin ligand on the surface of lymphocytes and B cells. Deficiency of ppGalNAc-T1 will not only affect the microvascular circulation and neutrophil recruitment, but also result in the increased apoptosis of B cells [23]. Overexpression of ppGalNAc-T6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of an oncoprotein MUC1 [24]. The O-glycosylation of death receptors DR4 and DR5 by ppGalNAc-T14 could promote the ligand-stimulated clustering of DR4 and DR5 to mediate the recruitment and activation of the apoptosis-initiating protease caspase-8 [25]. However, functions of individual ppGalNAc-T including their in vivo substrate proteins and O-glycosylation sites remain to be elucidated. Rat ppGalNAc-T9, an orthologous gene of human ppGalNAcT10, is considered as a glycopeptide GalNAc-transferase with no detectable activity towards non-glycosylated peptides [26].
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C. Peng et al. / Biochemical and Biophysical Research Communications 402 (2010) 680–686 Table 1 Summary of 20 human ppGalNAc-Ts. GalNAc-transferase activity
ppGalNAc-T isoform
mRNA accession number
Chromosome Exon Coding region (aa)
ppGalNAc-T1/GALNT1 ppGalNAc-T2/GALNT2 ppGalNAc-T3/GALNT3 ppGalNAc-T4/GALNT4 ppGalNAc-T5/GALNT5 ppGalNAc-T6/GALNT6 ppGalNAc-T7/GALNT7 ppGalNAc-T8/GALNT8 ppGalNAc-T9/GALNT9
NM_020474.3 NM_004481.2 NM_004482.3 NM_003774.3 NM_014568.1 NM_007210.3 NM_017423.2 NM_017417.1 NM_001122636.1
18q12.1 1q41–42 2q24–31 12q21.3–22 2q24.2 12q13 4q31.1 12p13.3 12q24.33
11 16 10 1 10 10 12 11 11
559 571 633 578 940 622 657 637 603
+ + + + + + ND -
+ + + + + + + ND ND
ppGalNAc-T10/GALNT10
NM_198321.2
5q33.2
12
603
-
+
ppGalNAc-T11/GALNT11 ppGalNAc-T12/GALNT12 ppGalNAc-T13/GALNT13
NM_022087.2 NM_024642.3 NM_052917.2
7q34–36 9q31.1 2q24.1
11 10 11
608 581 556
+ + +
+ + +
ppGalNAc-T14/GALNT14 ppGalNAc-T15/GALNTL2
NM_024572.2 NM_054110.4
2p23.2 3p25.1
15 10
552 639
+ +
+ +
ppGalNAc-T16/GALNTL1 ppGalNAc-T17/GALNTL3 ppGalNAc-T18/GALNTL4 ppGalNAc-T19/GALNTL5 ppGalNAc-T20/GALNTL6
NM_020692.1 NM_022479.1 NM_198516.1 NM_145292.2 NM_001034845.2
14q24.1 7q11.23 11p15.3 7q36.2 4q34.1
14 11 11 8 12
542 598 607 443 601
ND ND ND -
ND ND ND +
Transcript expression in tissues
Ref.
Ubiquitously Ubiquitously Testis, pancreas, kidney, prostate, intestine, spleen Ubiquitously Sublingual gland, colon, small intestine, stomach Placenta, trachea, brain, pancreas Ubiquitously Ubiquitously Brain (cerebellum, putamen, temporal lobe, frontal lobe, cerebral cortex) Small intestine, stomach, pancreas, ovary, thyroid gland, and spleen Kidney Small intestine, stomach, pancreas, colon, and testis Fetal brain, adult cerebellum, cerebral cortex, whole brain Kidney and fetal kidney Small intestine, placenta, spleen, cerebral cortex, ovary, uterus, mammary gland, stomach, cerebellum, whole brain ND Cerebellum and cerebral cortex ND ND Testis and brain
[7] [7] [8] [9] [10] [11] [12] [13] [14]
Peptide Glycopeptide
[15] [16] [17] [18] [19] [20]
[21] This work
ND, not determined.
Structural elements underlying the recognition of glycopeptide substrate by ppGalNAc-T10 have been resolved [27]. Substrate specificities of ppGalNAc-T1, -T2, and -T10 revealed that the glycopeptide-specific ppGalNAc-T10 has a unique GalNAc-O-Ser/ Thr-binding site in its catalytic domain, which was not found in ppGalNAc-T1 or -T2 [28]. In this study, we identified a novel member of the human ppGalNAc-T family, ppGalNAc-T20, which has high homology with human ppGalNAc-T10. The in vitro catalytic activity and expression profile of ppGalNAc-T20 were investigated to provide insights into its possible roles in O-glycosylation. 2. Materials and methods 2.1. Cloning of human ppGalNAc-T20 A BLASTN search of human genome database with the ORF of human ppGalNAc-T10 (AJ505950) as a query identified potential exons of a novel ppGalNAc-T gene in sequence NT022792. The potential transmembrane protein contained 601 amino acids. By using the cDNA of human small-cell lung carcinoma cell, Lu130, as the template, the full-length cDNA of this novel polypeptide GalNAc transferase was amplified by PCR (forward primer, 50 -CAC CATGAAGAGGAAACAGAAGAGATTTCTG-30 ; reverse primer, 50 -CTA GGAGTTGGCATGGTGGTTA-30 ) and cloned into pENTR/D-TOPO for sequencing. The obtained ORF was deposited into GenBank with the accession number GU220060.
purified with anti-FLAG M2 affinity gel (Sigma), and analyzed by Western blotting using anti-FLAG antibody (Sigma) and ECL plus reagents (GE Healthcare). Human ppGalNAc-T10 and -T1, containing residues 34–603 and 34–559, respectively, were also prepared by the same method. 2.3. Polypeptide GalNAc-transferase assay Standard polypeptide GalNAc-transferase assay was performed as described previously [18]. In brief, the enzymatic mixture (20 ll) was incubated at 37 °C for various periods and boiled at 95 °C for 3 min to terminate the reaction. FAM-labeled peptide substrates derived from tandem repeats of corresponding mucins [29–32], EA2 (PTTDSTTPAPTTK-5,6FAM), Muc5AC (SAPTTSTTSA PTK-5,6FAM), Muc1a (5,6FAM-AHGVTSAPDTR), and Muc2 (PTTTPI TTTTTVTPTPTPTGTQTK-5,6FAM), were purchased from Sawady Co. Ltd. Reaction products were evaluated by RP-HPLC (Shimadzu) on a C18 analytical column (COSMOSIL 5C18-AR-II, 4.6 250 mm). The isolated reaction products were lyophilized and subjected to MALDI-TOF mass spectrometry (Reflex IV, Bruker Daltonics) in the positive ion mode to determine the number of transferred GalNAc. To identify the position of attached GalNAc, amino acid sequencing was carried out by Edman degradation [18,33]. To determine the Km value for acceptor substrate, the reaction condition was modified to include a saturated concentration of UDPGalNAc (0.5 mM), and a series of concentrations from 0.005 to 0.15 mM of mono-GalNAc-glycosylated EA2. Km and Vmax values were calculated using OriginPro 7.5 with a non-linear curve fitting.
2.2. Production of purified recombinant ppGalNAc-Ts The cDNA fragment encoding the catalytic and lectin domain of ppGalNAc-T20 (aa 33–601) was obtained by PCR (forward primer, 50 -GACGACAAGCTTGAGCAGCAGACATTTCCACTG-30 ; reverse primer, 50 -GCTCTAGAGGATCCAGAGCTGCCAGGAAACAG-30 ) and cloned into pFLAG-CMV3 (Sigma). HEK293T cells were transfected with the reconstructed plasmid. The secreted recombinant protein was
2.4. Quantitative analysis of the ppGalNAc-T20 transcript by real-time PCR in human tissues and tumor cell lines Quantitative real-time PCR was carried out using a TaqMan Universal Master Mix (PE Applied Biosystems). The primer set and probe for ppGalNAc-T20 were as follows: forward, 50 -ACTGC GAGGTCAATGTGAACTG-30 ; reverse, 50 -CACACGATGGTTTTGTGGTT
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TAG-30 ; probe, 50 -CTGCCCCCACTCCTCAACCAAATTG-30 . Standard curves for ppGalNAc-T20 and GAPDH, were generated by serial dilution of pENTR/D-TOPO containing the ppGalNAc-T20 gene and pCR2.1 (Invitrogen) with the GAPDH gene, respectively. Realtime PCR was performed at 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). 2.5. In situ hybridization In situ hybridization was performed as previously described [34,35] in mouse testis. The partial 30 -UTR sequence of mouse ppGalNAc-T20 (nucleotides 1951–2709 in BB641428) was amplified by PCR using mouse brain cDNA as the template. The fragment amplified by 50 -GAGACGTTCCTACATCTGAAGAACTTG-30 and 50 CCTCTCCACTGTCTTCTTTAATAAAAATT-30 was inserted into pCR– Blunt II-TOPO (Invitrogen) to generate anti-sense and sense RNA probes using the RNA digoxigenin-labeling kit (Roche) after linearization. 3. Results 3.1. cDNA cloning of human ppGalNAc-T20 The ppGalNAc-T20 gene was identified as a putative novel ppGalNAc-T by BLASTN search of human genome database using ppGalNAc-T10 as a query. The full-length cDNA of human ppGalNAc-T20 encoded a 601-aa type II membrane protein, which contains a N-terminal 7-aa cytoplasmic region, a transmembrane segment of 21 aa, a stem region, a putative catalytic region, and a C-terminal lectin domain. ppGalNAc-T20 retained the DXH motif (aa 232–234) which was conserved in the catalytic domain of all ppGalNAc-T members and was essential for divalent cation binding. Amino acid alignment of human ppGalNAc-T20 and -T10 (Fig. 1A) revealed their high sequence identity (71%). Three possible N-glycosylation sites were found in ppGalNAc-T20, which were conserved in ppGalNAc-T10 [27]. The genomic structure of ppGal-
NAc-T20 and -T10 was also compared (Fig. 1A). The ppGalNAc-T20 gene was found to have different chromosome location from the ppGalNAc-T10 gene (Table 1). Both genes contained at least 12 exons, the length of which was exactly the same between the two genes, except for exon 1. To better understand the evolutionary relationship of 20 human ppGalNAc-Ts, a neighbor-joining tree is reconstructed by MEGA3.1 (Fig. 1B). ppGalNAc-T20 is positioned with ppGalNAc-T10 in one clade, confirming that ppGalNAc-T10 is the closest homologue to ppGalNAc-T20. 3.2. Different catalytic properties between ppGalNAc-T20 and -T10 To determine the substrate specificity and kinetic property of ppGalNAc-T20, the recombinant enzyme ppGalNAc-T20 fused with the FLAG tag was purified from the medium of transfected HEK293T cells. Recombinant FLAG-fused enzymes ppGalNAc-T1 and -T10 were also produced as control. As shown in Fig. 2A, the ppGalNAc-T1 enzyme (20 ng) generated one product peak (P1) that was shifted from the acceptor substrate peak (S) of EA2 with 100% product conversion rate after incubation for 30 min, and more highly glycosylated products appeared with an extended incubation of 24 h. Similarly, ppGalNAc-T1 (20 ng) generated a major product peak (P4) when incubated with Muc5AC as an acceptor substrate for 30 min and 24 h. However, no GalNAc-transferase activity of up to 600 ng of enzyme ppGalNAc-T20 or -T10 was observed after incubation for 3 h (data not shown) and 24 h, respectively. These results demonstrate that the two highly homologous enzymes, ppGalNAc-T20 and -T10 show no GalNAc-transferase activities to non-GalNAc-modified peptides EA2 and Muc5AC. Similar results were obtained with Muc1a and Muc2 as acceptor substrates (data not shown). To assess whether ppGalNAc-T20 has GalNAc-transferase activity on glycopeptides, we incubated mono-GalNAc-Thr7th-EA2 produced by ppGalNAc-T1 (P1) [26] as the acceptor substrate with 300 ng of ppGalNAc-T20 or -T10. Both enzymes effectively utilized mono-GalNAc-EA2 and yielded one product peak with different catalytic efficiency (Fig. 2B). As shown in Fig. 2C, the molecular
Fig. 1. (A) Amino acid sequence comparison of human ppGalNAc-T20 and -T10. Positions of introns, the putative transmembrane region, the GT1 motif, the Gal/GalNAc-T motif, the lectin domain, and potential N-glycosylation sites are indicated by arrows, square boxes, double lines, dashed lines, single solid lines, and circles, respectively. (*), identical residues; (:), conserved substitutions; (.), semiconserved substitutions. (B) Phylogenetic tree generated from amino acid alignment of 20 human ppGalNAC-Ts.
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Fig. 2. (A) HPLC analysis of in vitro O-glycosylation on FAM-labeled EA2 and Muc5AC by recombinant ppGalNAc-T20, -T10, and -T1. S represents the original non-glycosylated peptide peak, while P1 and P4 respectively represent the product peaks of ppGalNAc-T1 when using EA2 and Muc5AC as the acceptor substrate. (B) Time-course of the secondary activity of ppGalNAc-T20 and -T10 towards mono-GalNAc-EA2 produced by ppGalNAc-T1 (P1) as the acceptor substrate. (C) MALDI-TOF mass measurement of reaction products derived from EA2 and mono-GalNAc-EA2 by ppGalNAc-Ts as shown in (B). (D) Mono-GalNAc-Muc5AC produced by ppGalNAc-T1(P4) was used as the peptide substrate to compare the glycopeptide GalNAc-transferase activity of ppGalNAc-T20 and -T10. Reaction products were eluted with a flow rate of 1 mL/min and the following buffer B (100% acetonitrile, 0.05% TFA) gradient: 0–30 min, 20–40%, 30–35 min, 40–80%.
masses of P2 (m/z 2080.91) and P3 (m/z 2080.85) matched with that of di-GalNAc-EA2. The glycosylation sites of P2 and P3 were determined by Edman sequencing. Both products P2 and P3 were glycosylated at Thr6th and Thr7th of peptide EA2 (data not shown). Hence, ppGalNAc-T20 and -T10 transferred one GalNAc to Thr6th of mono-GalNAc-Thr7th-EA2. But when 300 ng of ppGalNAc-T20 or T10 was incubated with mono-GalNAc-Muc5AC (P4) produced by ppGalNAc-T1 as the acceptor substrate, only ppGalNAc-T10 showed GalNAc-transferase activity (Fig. 2D). Therefore, ppGalNAc-T20 demonstrated to be a novel typical glycopeptide GalNAc-transferase that shows different substrate specificities from ppGalNAc-T10 in vitro. The kinetic constants of ppGalNAc-T20 and -T10 for mono-glycosylated EA2 were then determined to investigate their different catalytic properties. As shown in Table 2, ppGalNAc-T20 and -T10 exhibited a Km of 0.06 and 0.025 mM, respectively, indicating a lower substrate affinity of ppGalNAcT20 than that of ppGalNAc-T10, and the catalytic efficiency (Vmax/Km) of ppGalNAc-T20 is 40-fold lower than that of ppGalNAc-T10. 3.3. Distribution of the ppGalNAc-T20 transcript Expression levels of the ppGalNAc-T20 transcript in human tissues and tumor cell lines were determined by real-time PCR as shown in Fig. 3. ppGalNAc-T20 was restrictively expressed in testis and brain (Fig. 3A), with low or negligible expression observed in other tissues, and it had relatively high expression in lung small cell carcinoma cell lines (Fig. 3B). As the ppGalNAc-T10 transcript was ubiquitously expressed in human tissues [15], the differential
expression pattern between ppGalNAc-T20 and -T10 suggests that they may control distinct O-glycosylation process in vivo. To investigate the location of ppGalNAc-T20 during testis development, the expression of mouse ppGalNAc-T20 (mT20) transcript was examined by in situ hybridization. As shown in Fig. 4, mT20 is strongly expressed in primary spermatocytes of the first meiosis (stage I–V) and secondary spermatocytes of the second meiosis (stage IX–XII), with no signals observed in spermatogonia and late spermatids. This result suggests that ppGalNAc-T20 might have certain functions in spermatogenesis. 4. Discussion In this work, we describe the identification of the 20th member of human ppGalNAc-T family, ppGalNAc-T20, which shares high sequence homology to ppGalNAc-T10. ppGalNAc-T20, the same as ppGalNAc-T10, exhibited significant glycopeptide GalNActransferase activity towards mono-glycosylated EA2, but only ppGalNAc-T10 showed activity to mono-glycosylated Muc5AC, revealing unique acceptor substrate specificities of the two enzymes. As EA2 has seven potential glycosylation sites and
Table 2 Kinetic constants of recombinant ppGalNAC-T20 and -T10 for acceptor substrate.
ppGalNAc-T20 ppGalNAc-T10
Km (mM)
Vmax (nmol/min/lg)
Vmax/Km
0.06 0.025
0.064 1.080
1.067 43.2
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Fig. 3. Quantitative analysis of the ppGalNAc-T20 transcript by real-time PCR in human tissues (A) and tumor cell lines (B). The expression level of ppGalNAc-T20 was normalized to that of GAPDH. Values are shown as copy numbers of the target mRNA per lg of total RNA. Data were obtained from triplicate experiments and indicated as means ± SD.
ppGalNAc-T20 and -T10 incorporated only one GalNAc into monoGalNAc-EA2, the two bulky GalNAc residues may cause steric hindrance to interfere with the subsequent O-glycosylation. Moreover, the fact that ppGalNAc-T1 can transfer more than two GalNAc residues to the acceptor substrate EA2 with extended incubation (Fig. 2A) further indicates diversified catalytic mechanisms among human ppGalNAc-T family enzymes. Although ppGalNAc-T20 and -T10 both exhibit GalNAc-transferase activity towards monoGalNAc-EA2, the 40-fold higher efficiency of ppGalNAc-T10 suggests that the glycosylated EA2 derived from rat submandibular gland mucin may be a more appropriate substrate for ppGalNAcT10. Besides, the lower reactivity of ppGalNAc-T20 towards EA2 is reasonable, as it has no expression in salivary grand. Mucins specifically expressed in testis or brain may be endogenous substrates for this enzyme. The different catalytic property and expression pattern of ppGalNAc-T20 and -T10 suggest that the two enzymes may have distinct biological functions. The GalNAc-transferase activity of 20 human ppGalNAc-Ts characterized to date is summarized in Table 1. Many ppGalNAcT isoforms can transfer GalNAc to both non-glycosylated and
glycopeptides, while ppGalNAc-T20, as well as two other glycopeptide-specific ppGalNAc-T7 [12] and -T10, require the prior addition of GalNAc to peptides before they transfer additional GalNAc residues. Each ppGalNAc-T isoform has unique enzymatic activity and there may have synergistic, competitive, or even inhibitory effects among them. The 20 human ppGalNAc-Ts provide us a systematic model to investigate the roles of O-glycosylation under certain physiological and pathological conditions. The ppGalNAc-T20 transcript was exclusively expressed in testis and brain, suggesting that it may involve in cell maturation and differentiation of these two organs. Most ppGalNAc-Ts were distributed in peripheral blood cells, digestive and lymphatic organs that produce various O-glycosylated proteins, while ppGalNAcT9, -T13, and -T17 specifically expressed in brain, ppGalNAc-T11 and -T14 restrictively distributed in kidney (Table 1). It was recently reported that normal human testis showed a very restricted expression pattern of simple mucin-type O-glycans and polypeptide GalNAc-transferases [36]. To date, only ppGalNAc-T3 has been detected in maturing postmeiotic germ cells including sperm [11]. Hence, ppGalNAc-T20 is the second ppGalNAc-T isoform found in
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Fig. 4. In situ hybridization of mT20 during testis spermatogenic stages I–XII. Mouse testis sections were hybridized with mT20 30 region anti-sense probe. Signals can be seen over primary spermatocytes of the first meiosis (stage I–V) and secondary spermatocytes of the second meiosis (stage IX–XII), whereas hybridization with sense construct did not result in significant staining.
germ cells of normal testis. As changes in the profile of simple mucin-type O-glycans and ppGalNAc-Ts in human testis and testicular neoplasms are associated with germ cell maturation and tumor differentiation [36], lack of ppGalNAc-T3 or -T20 might impair maturation of spermatozoa and sperm-egg binding. Because ppGalNAc-T3 has enzymatic activity on both non-glycosylated peptides and glycopeptides [37], there may have coordination and competition between ppGalNAc-T3 and -T20 to ensure appropriate glycosylation of substrate proteins in testis. Transcript expression of mT20 was observed in primary spermatocytes of the first meiosis (stage I–V) and secondary spermatocytes of the second meiosis (stage IX–XII), while the mouse ppGalNAc-T3 transcript was expressed in spermatogenic stages I–VII and IX–XII (data not shown), suggesting that they might play distinct functions. The substrate profiling for individual ppGalNAc-T in testis remains largely unknown, and we hope to find the specific substrates for ppGalNAc-T20 with more research progress in characterizing glycoproteins in testis. In conclusion, the present study provides evidence that ppGalNAc-T20 is a new glycopeptide GalNAc-transferase member of the human ppGalNAc-T family. The significant high level of ppGalNAc-T20 transcript in testis strongly suggests that ppGalNAc-T20 is involved in O-glycosylation in spermatogenesis. Further study
is necessary for the identification of natural substrates, which are also expressed in testis as ppGalNAc-T20. Acknowledgments This work was sponsored by grants from the National Natural Science Foundation (30770482), the Pujian project (07pj14057), and the National Key Scientific Program (2007CB914703) of China. This work was also performed as a part of the National Institute of Advanced Industrial Science and Technology (AIST) work supported by the New Energy and Industrial Technology Development Organization (NEDO) of Japan. References [1] R.S. Haltiwanger, J.B. Lowe, Role of glycosylation in development, Annu. Rev. Biochem. 73 (2004) 491–537. [2] E. Tian, K.G. Ten Hagen, Recent insights into the biological roles of mucin-type O- glycosylation, Glycoconj. J. 26 (2009) 325–334. [3] K.G. Ten Hagen, T.A. Fritz, L.A. Tabak, All in the family: the UDP-GalNAc: polypeptideN-acetylgalactosaminyltransferases, Glycobiology 13 (2003) 1R– 16R. [4] J. Roth, Y. Wang, A.E. Eckhardt, R.L. Hill, Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptideN-acetylgalactosaminyltransferasemediated O-glycosylation reaction in the submaxillary gland, Proc. Natl. Acad. Sci. USA 91 (1994) 8935–8939.
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