Báo cáo khoa học: Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) ppt

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferase 1 (GalNAc-T1) Mari Tenno 1 , Shinya Toba 1 , Fere ´ nc J Ke ´ zdy 3 ,A ˚ ke P. Elhammer 3 and Akira Kurosaka 1,2 1 Department of Biotechnology Faculty of Engineering, and 2 Institute for Comprehensive Research, Kyoto Sangyo University, Kamigamo-motoyama, Kyoto, Japan; 3 Pharmacia Corporation, Kalamazoo, Michigan, USA Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyl- transferases, which contain several conserved cysteine resi- dues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc. Keywords: cysteine; GalNAc-transferase; mucin; O-glyco- sylation; UDP-GalNAc. Mucin-type O-glycosylation is an important post-transla- tional modification that is widely distributed on many secretory and membrane glycoproteins [1,2]. The initial step of this glycosylation is catalyzed by the UDP-GalNAc:poly- peptide N-acetylgalactosaminyltransferases (GalNAc-trans- ferases; EC 2.4.1.41). These enzymes transfer GalNAc from UDP-GalNAc to serine or threonine residues of proteins [3]. Recent progress in molecular cloning has revealed that the GalNAc-transferases constitute a large gene family, with 10 distinct isozymes identified to date [4–14], and that they are type II membrane proteins with a short N-terminal cytoplasmic tail, a hydrophobic transmembrane anchor, a luminal stem region, and a large luminal putative catalytic domain (Fig. 1). The luminal putative catalytic domain contains two distinct subdomains; a central catalytic domain and a C-terminal lectin-like domain. The central catalytic domain can be further subdivided into two regions. The N-terminal half is represented by a glycosyltransferase 1 (GT1) motif that is conserved among a wide range of glycosyltransferases [15]. The extreme C-terminal end of the GT1 motif contains a so-called DXH motif, which corres- ponds to the DXD sequence common to many glyco- syltransferases [16]. The C-terminal half of the catalytic domain contains a so-called Gal/GalNAc-T motif, a sequence segment where significant homology can be seen between b1,4-galactosyltransferases and GalNAc-transfer- ases [15,17]. A C-terminal lectin-like domain, called the (QXW) 3 repeats, occurs exclusively in the GalNAc-trans- ferases [18,19]. Although recent reports show that the GalNAc-transfer- ases all have common structural features and the conserved motifs described above, the exact role of each domain in catalysis remains largely unknown. Moreover, these enzymes are also characterized by the presence of highly conserved cysteine residues, several of which are positioned in and around the conserved motifs (Fig. 1). In order to obtain more detailed information on the structure–function relationship of the GalNAc-transferases, we investigated the possible role(s) of the conserved cysteine residues in GalNAc-T1. We used site-directed mutagenesis, in Correspondence to A. Kurosaka, Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan. Fax: + 81 75 705 1914, Tel.: +81 75 705 1894, E-mail: kurosaka@cc.kyoto-su.ac.jp Abbreviations: ABD-F, 4-(aminosulfonyl)-7-fluoro-2, 1, 3-benzoxa- diazole; DFP, diisopropyl fluorophosphate; GalNAc, N-acetylgalac- tosamine; GalNAc-transferase, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase; GT1, glycosyltransferase 1; NEM, N-ethylmaleimide; PCMPS, p-chloromercuriphenylsulfonic acid; UBD, UDP-binding domain; TFA, trifluoroacetic acid. Enzymes: UDP-GalNAc:polypeptide N-acetylgalactosaminyl- transferases (GalNAc-transferases; EC 2.4.1.41). (Received 16 May 2002, revised 9 July 2002, accepted 18 July 2002) Eur. J. Biochem. 269, 4308–4316 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03123.x combination with identification of free cysteine residues (defined as cysteine residues not involved in the formation of a disulfide bond), by cysteine-specific labeling, to study the mechanistic involvement of the conserved cysteine residues in the function of GalNAc-T1. Our results demonstrate that Cys212 and Cys214, which are located at the C terminus of the DXH motif, are free cysteine residues that interact with the nucleotide moiety of UDP-GalNAc, possibly through hydrogen bonding. EXPERIMENTAL PROCEDURES Preparation of soluble bovine GalNAc-T1 A soluble form of bovine GalNAc-T1 was expressed in High Five cells using the baculovirus expression system. The molecule was purified to homogeneity by apomucin- Sepharose chromatography as described previously [20]. Construction of soluble rat recombinant GalNAc-T1 and expression in COS7 cells Rat GalNAc-T1 cDNA was obtained as outlined by Hagen et al. [21]. For the construction of soluble GalNAc-T1, rat GalNAc-T1 full-length cDNA was subcloned into pcDNA4 to create the vector, prT1. prT1 was linearized with BamHI, andthendigestedwithBal31 nuclease. The Bal31 digest was blunt-ended and digested with NotI. The resulting digest was ligated into the EcoRV and NotIsitesofpcDNA4, obtaining pDN42 that encodes GalNAc-T1 with 42 N-terminal amino acid residues, including a cytoplasmic tail and a transmembrane domain, deleted. A NheI-SmaI fragment of the plasmid pGIR201protA (a gift from H. Kitagawa, Kobe Pharmaceutical University) [22,23], containing a cDNA encoding the insulin signal sequence and the Protein A-IgG binding domain, was inserted into NheI-HindIII digested pcDNA3.1, producing the vector, pInsProA. pDN42 was digested with BamHI and NotI, and was subcloned into pInsProA, generating the plasmid pInsProADN42 containing truncated rat GalNAc-T1 fused with IgG-binding domain of Protein A (P-DN42). Site-directed mutagenesis Site-directed mutagenesis was performed on pInsProADN42 using the LA PCR TM in vitro mutagenesis kit, using the primers listed. Nucleotides shown in italic are nucleotides mutated to convert conserved cysteine residues into either alanine or serine residues. C106a, 5¢-TAGAGGGGGCTA AAACAAAA-3¢; c212a, 5¢-ACTGTGCACTCGGCGTG AGC-3¢; c214a, 5¢-ACTGTGGCCTCGCAGTGAGC-3¢; c235a, 5¢-TAGGAGCCACCACTGTCCTC-3¢; c330a, 5¢-CAGAGTCCCTCCAGCCTGCC-3¢; c339a, 5¢-GGAG GCCGTCACTATTTCCA-3¢; c408a, 5¢-GAAAGGCTTG GCCTGTAGTT-3¢;c212s,5¢-GCTCACAGCGAGTGCA CAGT-3¢; c214s, 5¢-GCTCACTGCGAGAGCACAGT-3¢; c212s/c214s, 5¢-GCTCACAGCGAGAGCACAGT-3¢. Expression of P-DN42 and mutant P-DN42 in COS7 cells Expression construct (pInsProADN42 or mutant pInsPro- ADN42) was transfected into COS7 cells using FuGENE TM 6 Transfection Reagent. Three days after the transfection, the culture medium was collected and the Fig. 1. Schematic representation of the domain structure and the position of the cysteine residues in the cloned GalNAc-transferases. Arrows indicate cysteine residues and the numbers indicate residues mutated in this study. The amino acid residue numbering is based on the GalNAc-T1 sequence. Highlyconservedcysteineresiduesarerepresentedbydottedlines.b,r,hT1,bovine,rat,andhumanGalNAc-T1;hT2,humanGalNAc-T2;hT3, human GalNAc-T3; hT4, human GalNAc-T4; rT5, rat GalNAc-T5; hT6, human GalNAc-T6; hT7, human GalNAc-T7; hT8, human GalNAc-T8; hT9, human GalNAc-T9; rppGaNTase-T9, rat ppGaNTase-T9. Ó FEBS 2002 Cys residues in GalNAc-T1 interact with UDP-GalNAc (Eur. J. Biochem. 269) 4309 secreted enzyme was purified on IgG-Sepharose. For analysis by SDS/PAGE, the resins adsorbed with the secreted enzyme were boiled in SDS/PAGE loading buffer. The resulting supernatant was loaded directly on the gel. For Western blotting, the proteins on the membrane were visualized by incubating the blot with an affinity purified, alkaline phosphatase-conjugated, rabbit antibody to mouse IgG, followed by staining with nitrobluetetrazolium and 5-bromo-4-chloro-3-indolylphosphate. The protein bands on the immunoblots was quantified by densitometry scanning and the intensity of each band was determined using the NIH Image software. The enzymatic activity of the P-DN42 and mutant P-DN42 gene products was determined as described below. The activity levels were corrected for enzyme protein concentration in the medium. Assay for GalNAc-transferase activity (PD-10 assay) The enzyme activity was determined in a reaction mixture composed of 50 m M imidazole buffer (pH 7.2), 10 m M MnCl 2 , 0.1% Triton X-100, 6 nmol UDP- 3 H-GalNAc (approximately 10 000 d.p.m.), 150 lg apomucin [24], and an appropriate amount of enzyme. The mixture was incubated for 30 min at 37 °C, and the reaction was stopped by adding 0.25 M EDTA. The reaction mixture was then separated on a PD-10 column. The void fraction containing 3 H-labeled apomucin was recovered and the radioactivity was determined. Modification of GalNAc-T1 with PCMPS and DFP Purified soluble bovine GalNAc-T1 or a recombinant mutant rat GalNAc-T1 was treated with p-chloromercuri- phenylsulfonic acid (PCMPS) in 40 m M imidazole buffer (pH 7.2) for 90 min at room temperature, or with diiso- propyl fluorophosphate (DFP) in 40 m M imidazole buffer (pH7.2)for30minat37°C. Following treatment, the reaction mixture was dialyzed against 25 m M imidazole buffer (pH 7.2) containing 300 m M NaCl, 10% glycerol, and 0.1% taurodeoxycholate. The enzymatic activity of the samples was determined using the PD-10 assay (see above). To study the influence of UDP-GalNAc or UDP on the effect of PCMPS, the treatment was carried out in 0.1 m M PCMPS in the presence of UDP-GalNAc or UDP. Identification of free cysteine residues Labeling of bovine GalNAc-T1 with ABD-F and fraction- ation of the labeled peptides were carried out as described [25,26]. Briefly, GalNAc-T1 was first labeled with ABD-F, followed by reduction with tributylphosphine and S-carbo- xymethylation with iodoacetic acid. The alkylated protein was digested with endoproteinase Lys-C. The digest was then fractionated by HPLC on a C 18 HPLC column. The fluorescent peptides were purified by rechromatography on aC 8 column and sequenced with an automated Edman sequencer. Kinetic analysis K m for UDP-GalNAc was obtained by varying the concentration of UDP-GalNAc from 1.5 to 43.5 l M in the presence of 1.88 mgÆmL )1 apomucin. To determine the K m for apomucin, GalNAc-transferase activity was assayed in the presence of 7.5 l M UDP-GalNAc and 0.625–8.75 mgÆmL )1 apomucin. Calculation of kinetic parameters was done from double reciprocal plots (1/v vs. 1/[S]), using standard procedures. RESULTS Involvement of the free cysteine residues of GalNAc-T1 in catalysis To investigate the functional role of the cysteine residues (Fig. 1), we first modified GalNAc-T1 with a cysteine- specific reagent, PCMPS. We then examined the influence of the modification on the GalNAc-transferase activity. A purified bovine GalNAc-T1, expressed as a secreted protein in High Five cells, was used for this experiment [20]. As shown in Fig. 2, PCMPS caused a marked, concentration dependent decrease in enzyme activity, with a K i of 0.03 m M . This suggests that free cysteine residues, possibly located at the catalytic site of GalNAc-T1, might be involved in the catalytic function of the enzyme. To investigate whether the cysteine residues modified by PCMPS are involved in the binding of UDP-GalNAc, we treated recombinant GalNAc-T1 with PCMPS in the presence of either UDP-GalNAc or UDP. To increase the sensitivity in this experiment, the cysteine modification was performed with the minimal PCMPS concentration (0.1 m M ) required for complete inhibition of GalNAc-T1 (Fig. 2). Fig. 3 shows that GalNAc-T1 retained enzymatic activity in the presence of either UDP or UDP-GalNAc. This suggests that the sulfhydryl groups of free cysteine residues modified by PCMPS may interact with UDP- GalNAc, or at least be located in the UDP-GalNAc binding cleft. Furthermore, the data suggest that the cysteine residues predominantly interact with a UDP moiety of UDP-GalNAc, as UDP and UDP-GalNAc were equally effective at protecting the enzyme from inactivation. Fig. 2. Inhibition of GalNAc-T1 with PCMPS. Purified bovine GalNAc-T1 was incubated with increasing concentrations of PCMPS for 90 min at room temperature. Following incubation, the treated enzyme was dialyzed to remove excess PCMPS, and assayed for activity as described in Experimental procedures. 4310 M. Tenno et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Mutagenesis of the cysteine residues in and around the GT1 and Gal/GalNAc-T motifs To investigate which cysteine residues are involved in the catalytic function of GalNAc-T1, site-directed mutagenesis was carried out on the conserved cysteine residues in the catalytic domain. A rat GalNAc-T1 cDNA, cloned by PCR as outlined by Hagen et al. [21], was used for this experiment. Rat GalNAc-T1 is 98% identical to the bovine ortholog and all of the cysteine residues are conserved between the two enzymes (Fig. 1). For ease of purification and detection, 42 N-terminal amino acid residues containing the cytoplasmic tail and the transmembrane region were deleted from the rat isozyme, and an insulin signal sequence and a Protein A-IgG binding domain were fused to the resulting N terminus of the sequence. The recombinant truncated GalNAc-T1 was then expressed in COS7 cells and the secreted fusion protein was purified from the culture medium on IgG-Sepharose. The purified recombinant, truncated rat GalNAc-T1, designated P-DN42, retained full enzymatic activity and had kinetic properties almost identi- cal to those of soluble bovine GalNAc-T1 [27]. Hence, it was used for the following site-directed mutagenesis studies. The amount of fusion protein secreted into the medium was quantified by Western blotting in combination with densi- tometric scanning of the bands on the blotting membrane. The enzymatic activities were correlated with the concen- tration of recombinant proteins in the media. This was done to evaluate the effects of the mutations on both the specific activity and the absolute levels of the secreted mutant enzymes. In a first experiment, we mutated Cys106, Cys212, Cys214, and Cys235 in P-DN42, individually, to alanine. These residues are located in (Cys212 and Cys214), and around (Cys106 and Cys235) the GT1 motif. Mutation of C106A, C212A, and C214A, resulted in a considerable decrease in secretion of the mutant proteins (Fig. 4), indicating that the replacement of cysteine with alanine significantly affected enzyme stability and/or efficiency of secretion. Apomucin was used as the acceptor when comparing the activity of the secreted mutants. As shown in Fig. 4, C106A was completely inactive. The relative activities of C212A and C214A were drastically decreased, to 6% and 17% of that of P-DN42, respectively. These results indicate that the conserved Cys106, Cys212, and Cys214 residues are essential for efficient enzyme function. In contrast, the C235A mutant retained almost full activity, as well as a high level of secretion into the culture medium. Hence, Cys235 appears not to be required for GalNAc-T1 activity or secretion. The Gal/GalNAc-T motif contains one conserved cysteine residue, Cys330. In addition, there are two conserved cysteine residues (Cys339 and Cys408) at the C-terminal side of this motif. Each of these cysteine residues was also mutated to alanine. Secretion of the mutants, especially C339A, decreased significantly. Moreover, there was a complete loss of activity in all three mutant enzymes (Fig. 4). These results demonstrate that the cysteine residues in positions 330, 339 and 408 are important for both secretion and function of GalNAc-T1. Identification of free cysteine residues in GalNAc-T1 The inactivation observed for several of the GalNAc-T1 mutants may result either from conformational changes caused by the disruption of disulfide bridges or from mutational effects of cysteine residues involved in enzyme function. However, the results from modification of GalNAc-T1 with PCMPS (Fig. 2) strongly suggest the presence of essential, free cysteine residues. To identify the free cysteine residues in the native enzyme, we labeled soluble bovine GalNAc-T1 with a cysteine specific Fig. 3. Protection of GalNAc-T1 from PCMPS inactivation. GalNAc- T1wastreatedwith0.1m M PCMPS in the presence of increasing concentrations of UDP-GalNAc (d)andUDP(s). Following incu- bation, the enzyme activity was determined as described in Fig. 2. Fig. 4. Enzyme activity of GalNAc-T1 with mutated cysteine residues. Each mutant was expressed in COS7 cells and the secreted recom- binant protein was recovered from the culture medium. The amount of the secreted protein was determined by Western blotting followed by the densitometric scanning (lower panel). The enzymatic activity secreted in the medium was corrected for the amount of mutant proteins in the medium and expressed as activity relative to that of the wild-type, P-DN42. Solid bars represent percent enzyme activity relative to that of P-DN42 (hatched bars). Ó FEBS 2002 Cys residues in GalNAc-T1 interact with UDP-GalNAc (Eur. J. Biochem. 269) 4311 fluorescent reagent, 4-(aminosulfonyl)-7-fluoro-2, 1, 3-ben- zoxadiazole (ABD-F), in the absence of reducing agent [25,26]. ABD-F is nonfluorescent until it reacts with thiols. Therefore, free cysteine residues can be identified as carrying a fluorescent label following ABD-F treatment of a protein. Purified bovine GalNAc-T1 was first labeled with ABD-F, followed by reduction and S-carboxymethylation. The labeled, reduced enzyme was then cleaved with endopro- teinase Lys-C, and the resulting peptide fragments were fractionated by HPLC on a C 18 column. As shown in Fig. 5, a number of peptide peaks absorbing at 220 nm, were detected but only a few were fluorescent. All of the major fluorescent peaks (indicated by arrows) were collected and re-fractionated by HPLC using a C 8 column. This revealed that peaks 1, 2, and 3 were artifacts as all of them separated into several small peaks on the C 8 column and none of these (secondary peaks) contained any polypeptide sequence detectable by sequence analysis (data not shown). By contrast, peak 4 produced a single peak on the C 8 column. Edman analysis showed that this peak contained an ABD-F labeled peptide. The peptide contained the N-terminal sequence G202QVITFL DAHC212EC214TV. The sequence includes the GalNAc-T1 DXH motif (underlined above), a region believed to be involved in coordination of a divalent cation or the binding of UDP-GalNAc [16,28,29]. The two cysteine residues in the sequence, Cys212 and Cys214, were both labeled by ABD-F (Fig. 6), as demonstrated by the presence of a peak corresponding to fluorescent cysteine and the complete absence of a peak corresponding to carbo- xymethylated cysteine, in the sequence analysis of the peptide. Consequently, both Cys212 and Cys214 can be considered free cysteine residues that probably are exposed on the surface of the UDP-binding pocket. The other essential cysteine residues, which were identified by muta- tional analysis but not labeled by ABD-F, may form intramolecular disulfide bonds required for proper folding of the enzyme. Cys235, on the other hand, does not seem to be involved in formation of a disulfide bond, as mutation at this site did not affect the activity of the enzyme (Fig. 4). Why this residue was not labeled with ABD-F is not clear. Fig. 5. HPLC fractionation of endoproteinase Lys-C digest of ABD-F labeled GalNAc-T1. Soluble bovine GalNAc-T1 was labeled with ABD-F, reduced with tributylphosphine, alkylatedwithiodoaceticacid,andthen digested with endoproteinase Lys-C. The digest was loaded onto a C 18 column (4.6 · 250 mm) equilibrated with 0.1% trifluoroacetic acid. Elution was carried out with a linear gradient from 0 to 50% acetonitrile in 0.1% trifluoroacetic acid, at flow rate of 1 mLÆmin )1 . The chromatograms were monitored by (A) relative absorbance at 220 nm and (B) by relative fluorescence (excitation at 385 nm, emission at 520 nm). The peaks labeled 1–4 were pooled and re-fractionated by C 8 column chromatogra- phy for subsequent amino acid sequence analysis. Fig. 6. Amino acid sequence analysis of the ABD-F labeled peptide. The amino acid sequence of the fluorescent peptide (peak 4 in Fig. 5) was determined with an automated Edman sequencer. The solid and the open arrows indicate the elution position of ABD-F labeled cysteine, and S-carboxymethylated cysteine, respectively. HPLC profiles of (A) cycle11(Cys212)and(B)13(Cys214)areshown. 4312 M. Tenno et al. (Eur. J. Biochem. 269) Ó FEBS 2002 As the fluorescent labeling was performed without dena- turing reagents, it is possible that only free cysteine residues exposed to the solvent were labeled by the ABD-F. Expression and kinetic studies of cysteine-to-serine mutant GalNAc-T1 enzymes The findings that the activities of the C212A and C214A mutants were drastically decreased and that UDP or UDP- GalNAc prevented PCMPS inactivation of GalNAc-T1 suggest that electrostatic interactions through the polar sulfhydryl groups of these cysteine residues may be involved in the interaction(s) between the nucleotide moiety of UDP-GalNAc and GalNAc-T1. To examine this hypothesis, we generated two single point mutants, C212S and C214S, and one double point mutant, C212S/ C214S. In these mutants, the cysteine residues (212 and 214) were replaced by serine residues, thereby generating proteins with hydroxyl, instead of sulfhydryl, side chains at positions 212 and 214. This should allow retained hydro- gen bonding capacity at these positions and at least theoretically, if this capacity is an essential function of these residues, result in functional enzyme. The results shown in Fig. 7 suggest that this is indeed the case. C212S and C214S retained approximately 60% and 80% of parent enzyme activity, respectively. This is significantly higher than the activity of the corresponding alanine mutants (6% and 17%, respectively) (Fig. 4). The activity of the double mutant, C212S/C214S, was lower but still amounted to 40% of the parent enzyme activity. In a more in-depth evaluation of the function of the three serine mutants, the kinetic properties of the mutant enzymes were compared to those of the parent enzyme, P-DN42. As showninTable1,theK m values of all three mutants, for apomucin, were essentially the same as that of P-DN42, indicating that Cys212 and Cys214 are not involved in the recognition of the acceptor. By contrast, the affinity of the mutant enzymes for UDP-GalNAc was affected quite significantly. When serine was substituted for Cys214, the increase in K m was only slight ( 1.2-fold that of P-DN42). On the other hand, C212S showed a 3.4-fold increase in K m . Furthermore, the effect of mutating the two cysteine residues appear to be cooperative as the double mutant, C212S/C214S, shows an even higher increase in K m (5-fold). These results demonstrate that while Cys212 may be a major site of interaction with UDP-GalNAc, Cys214 is also involved in binding. PCMPS and DFP modification of the serine-mutants of GalNAc-T1 We also examined the sensitivity of the three serine mutants to PCMPS inactivation (shown in Fig. 8). C214S, which contains free Cys212, was inactivated by PCMPS with a K i of 0.03 m M almost identical to that of native GalNAc-T1. On the other hand, C212S was more resistant to the treatment. The K i of  0.65 m M , may be due to a lower reactivity of Cys214 as compared to Cys212. Moreover, no inhibition was observed for C212S/C214S, even in the presence of a large excess of PCMPS (1 m M ), that resulted in the complete inactivation of P-DN42. These results show that both Cys212 and Cys214 are modified by PCMPS, and that, consistent with the kinetic data, Cys212 is the most Fig. 7. Enzymatic activity of cysteine-to-serine mutant GalNAc-T1 enzymes. Enzyme activity measurements and Western blotting of mutant proteins were carried out as described in Fig. 4. Table 1. Comparison of donor and acceptor K m values (apparent) for the parent and mutant enzymes. Values shown are means of three separate determinations. UDP-GalNAc Apomucin K m (l M ) -fold K m (mgÆmL )1 ) -fold P-DN42 5.1 ± 0.8 1.0 4.7 ± 0.1 1.0 C212S 17.4 ± 3.3 3.4 4.6 ± 0.9 1.0 C214S 6.0 ± 1.1 1.2 4.0 ± 0.3 0.9 C212S/C214S 25.0 ± 3.7 5.0 4.0 ± 0.4 0.9 Fig. 8. Inhibition of cysteine-to-serine mutant GalNAc-T1 enzymes with PCMPS. Mutant enzymes expressed in COS7 cells were purified on IgG-Sepharose from the conditioned medium. Proteins adsorbed by the resins were treated with increasing concentrations of PCMPS. Following treatment the resins were washed with buffer and the enzymatic activity of the mutant proteins were determined as described in Experimental procedures. d, C212S; s, C214S; m, C212S/C214S. Ó FEBS 2002 Cys residues in GalNAc-T1 interact with UDP-GalNAc (Eur. J. Biochem. 269) 4313 important site for the interaction with UDP-GalNAc. Complete loss of PCMPS inactivation in the double mutant suggeststhattherearenoPCMPSreactivesitesinnative GalNAc-T1, other than Cys212 and Cys214, and that ABD-F and PCMPS modify the same cysteine residues in GalNAc-T1. We also investigated the function of the serine residues introduced at positions 212 and 214 by modifying the mutant proteins with DFP, a reagent specific for active serine residues. Native bovine GalNAc-T1 is totally insen- sitive to DFP treatment, and thus appears not to contain any serine residues important for enzyme function. By contrast, all three serine mutants were inactivated by DFP to some extent (Fig. 9). The inhibition was more efficient for C212S than C214S, again demonstrating that position 212 is the more important site for substrate interaction. The double mutant, C212S/C214S, was most susceptible to DFP, confirming the cooperative involvement of the two sites observed in the kinetic analysis (Table 1). Taken together, the results from the kinetic, mutational and chemical modification studies presented in this report strongly suggests that the sulfhydryl groups at Cys212 and Cys214, but primarily at Cys212, are involved in substrate binding, possibly as hydrogen bond partners with UDP. DISCUSSION The primary aim of this study is to evaluate the functional role(s) of the conserved cysteine residues found in the GalNAc-transferase family. Using site-directed mutagene- sis, in combination with identification of free cysteine residues by cysteine-specific labeling, we demonstrated that Cys212 and Cys214, but predominantly Cys212, is involved in the binding of the nucleotide portion of UDP-GalNAc, most probably through hydrogen bonding. This is consis- tent with our previous inhibition study on GalNAc-T1 using various nucleotides and nucleotide sugars [30], showing that the enzyme primarily recognizes the UDP portion of the sugar donor. Recent crystallographic studies on glycosyltransferases indicate that several interactions are involved in binding of the UDP portion of the sugar donors to the enzymes [31–37]. In GalNAc-T1, hydrogen bonding with Cys212 and Cys214 appears to be predominant interactions with UDP. Other interactions may contribute in a more modest fashion, as replacement of Cys212 or Cys214 with alanine resulted in a substantial decrease, but not in a complete loss, of the activity (Fig. 4). We also found that the C212S mutant enzyme works relatively efficiently, with a 3.4-fold difference in the K m value for UDP-GalNAc, as compared to P-DN42, while the activity of the corres- ponding alanine mutant, C212A, was too low for deter- mination of kinetic parameters. This relatively modest difference in efficiency between the wild-type enzyme and the C212S mutant could be a matter of the size of the hydrogen bond forming group: a hydroxyl group (–OH) is smaller than a sulfhydryl group (–SH). If hydrogen bonding is involved in the interaction with the nucleotide sugar, substituting -OH for -SH may increase bond length slightly, thereby making it less efficient and decreasing the affinity. Although we show that the interaction of Cys212 and Cys214 with the sugar donor is important for the GalNAc- T1 activity, it should be noted that some GalNAc-transfer- ases do not have cysteine residues at the corresponding positions. As shown in Fig. 1, GalNAc-T1, -T2, -T3, -T4, and -T6 all contain both cysteine residues, but other GalNAc-transferases contain different amino acids at these positions, such as DSH VEC (GalNAc-T5), DAHCEV (GalNAc-T7), DAH IEV (GalNAc-T8), DAHVEF (GalNAc-T9), and DSHCE A (ppGaNTase-T9). Of these isozymes with variation at the two cysteine sites, GalNAc- T5, -T7 and ppGaNTase-T9 are catalytically active. This indicates that the two cysteine residues C-terminal to the DXH motif may not be crucial for the basic catalytic function of the GalNAc-transferases, but rather are important in defining the catalytic properties of specific isozymes. In fact, the interaction of UDP-GalNAc with GalNAc-T5, which has a valine residue at the Cys212 site, is less efficient than with GalNAc-T1. GalNAc-T5 has a significantly lower affinity for UDP-GalNAc (K m ¼ 55 l M ) [9], than P-DN42 and its serine-mutants (Table 1). The low affinity of UDP-GalNAc for GalNAc-T5 may, at least in part, be ascribed to the substitution with valine at the Cys212 site. The two isozymes GalNAc-T8 and GalNAc-T9 lack cysteine residues at both position 212 and 214. Consequently they may also have a low affinity for UDP-GalNAc and consistent with this, no enzymatic activity has so far been reported for these molecules. It is possible that the activity of these isozymes cannot be measured under the standard assay conditions used for other GalNAc-transferases. Similarly, the importance of four histidine residues for GalNAc-T1 activity has been demonstrated by Wragg et al. [17], using site-directed mutagenesis. Of these residues, His211 and His341 are conserved in all isozymes cloned to date. Some GalNAc- transferases, however, do not contain the other two histidine residues, His125 and His341. His125 and His341 are found in six and eight isozymes out of 10, respectively. Aspartic acid at the DXH motif in GalNAc-T1 is reported to be essential for GalNAc-T1 activity, because the mutation at this site results in inactivation of the enzyme [15]. Contrary to this observation, GalNAc-T4 does not contain the DXH Fig. 9. Inactivation of cysteine-to-serine mutant GalNAc-T1 enzymes with DFP. DFP treatment of mutant proteins was performed as described in Fig. 8. No change in pH was observed during the reaction. This indicates that the inactivation of the mutants was not due to acidification of the incubation but to modification of the active serine residues. d, C212S; s, C214S; m, C212S/C214S. 4314 M. Tenno et al. (Eur. J. Biochem. 269) Ó FEBS 2002 sequence, but contains YXH instead [7]. All these findings indicate that essential amino acid residues for some isozymes are not necessarily conserved in other isozymes. The variations in the primary sequence found in different members of the GalNAc-transferase family could provide each isozyme with distinct kinetic properties, thereby enabling the specific reaction catalyzed by these enzymes in vivo. Sequence variation is also found in the fucosyltransferases. a1,3/4-Fucosyltransferases III, V, and VI are inhibited by the cysteine specific reagent, N-ethyl- maleimide (NEM). The presence of a free cysteine residue has been reported for the NEM sensitive enzymes, whereas those that are insensitive to NEM contain a serine (a1,3/4- fucosyltransferase IV) or a threonine (a1,3/4-fucosyltrans- ferase VII) residue at the corresponding site. Importantly, the NEM-sensitive cysteine residue is reported to be located in or near the binding site for GDP-Fuc [38–41], in analogy to what was found for GalNAc-T1. The known glycosyltransferases have been classified into 52 different families, based both on sequence similarity and substrate/product stereochemistry (inverting or retaining), at the carbohydrate-active enzymes server on world wide web, URL: http://afmb.cnrs-mrs.fr/pedro/CAZY/ db.html [42,43]. The crystal structures of several glyco- syltransferases belonging to different groups have been determined recently [31–37]. Although these proteins have no sequence identity or related functional features, the crystallographic studies show that some of them share a domain structure, called a UBD (UDP-binding domain) [44], also known as a SGC (SpsA N-acetylglucosaminyl- transferase I core) domain [28,32]. The UBD is predicted to consist of alternating a-helices and b-sheets, constituting an a-b-a sandwich [31,44]. The UBD of glycosyltransferases also contains a DXD motif. In all crystallized enzymes, the DXD motifs are located at positions closely related to one anotherandareexpectedtobeindirectcontactwiththe sugar donor or interact with UDP-sugars through binding with a divalent ion [28,29]. The two cysteine residues at positions 212 and 214 in GalNAc-T1, identified in this study as being involved in sugar donor binding, are in the GT1 motif. A DXH motif that precedes Cys212 and Cys214 is located at the C-terminal end of the GT1 motif. The hydrophobic cluster analysis of several glycosyltransferases demonstrates that the DXH motif corresponds to the DXD motif found in most other glycosyltransferases [29]. More- over, it has been reported that the GT1 motif forms a five-stranded parallel b-sheet flanked by four a-helices and that the amino acids essential for enzymatic activity as well as the DXH motif are located near the C-terminal ends of the putative b-strands, lining the face of the predicted active site cleft [15]. These proposed structural features of the GT1 motif in GalNAc-T1 are similar to the UBD, raising the possibility that catalytic mechanisms similar to those described above are conserved in the GalNAc-transferases as well. It therefore appears likely that Cys212 and Cys214 at the C-terminal end of the DXH motif in GalNAc-T1 are located at the active site of the enzyme, and interact with UDP-GalNAc through hydrogen bonding. The results presented in this report offer new insights into the catalytic mechanism of GalNAc-T1. Identification of amino acid residues essential for activity will help us to understand the functions of the different domains in GalNAc-transferases and will allow the development of strategies for engineering new GalNAc-transferases with altered or modified donor and/or acceptor specificities. Together with information on the three-dimensional struc- ture of the GalNAc-transferases, this will allow an under- standing of the catalytic mechanism(s) of these enzymes that in turn can be used for development of isozyme-specific inhibitors and thereby for investigations of the functional roles of mucin carbohydrates. ACKNOWLEDGEMENTS This work was supported in part by the Research Foundation for Pharmaceutical Science, Sasakawa Scientific Research Grant, and the Foundation for Bio-venture Research Center from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 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