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J. Biol. Chem., Vol. 276, Issue 24, 21555-21561, June 15, 2001

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Substrate Recognition by UDP-galactose and CMP-sialic Acid Transporters

DIFFERENT SETS OF TRANSMEMBRANE HELICES ARE UTILIZED FOR THE SPECIFIC RECOGNITION OF UDP-GALACTOSE AND CMP-SIALIC ACID^*

Kazuhisa Aoki, Nobuhiro Ishida, and Masao Kawakita

From the Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan

Received for publication, February 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi membrane proteins with 10 transmembrane helices. We have constructed chimeras between these proteins in order to identify submolecular regions responsible for the determination of substrate specificity. To assess the UGT and CST activities, chimeric cDNAs were transiently expressed in either UGT-deficient mutant Lec8 cells or CST-deficient mutant Lec2 cells, and the binding of plant lectins, GS-II or PNA, respectively, to these cells was examined. During the course of analysis of various chimeric transporters, we found that chimeras whose submolecular regions contained helices 1, 8, 9, and 10, and helices 2, 3, and 7 derived from hUGT1 and hCST sequences, respectively, exhibited both UGT and CST activities. The dual substrate specificity for UDP-galactose and CMP-sialic acid of one such representative chimera was directly confirmed by in vitro measurement of the nucleotide sugar transport activity using a heterologous expression system in the yeast Saccharomyces cerevisiae. These findings indicated that the regions which are critical for determining the substrate specificity of UGT and CST resided in different submolecular sites in the two transporters, and that these different determinants could be present within one protein without interfering with each other's function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nucleotide sugar transporters are a family of related proteins located in the endoplasmic reticulum and the Golgi membranes (for recent reviews, see Refs. 1 and 2). Their substrates, nucleotide sugars, are synthesized in either the cytoplasm or the nucleus. These nucleotide sugars have to be delivered into the lumen of the endoplasmic reticulum or the Golgi apparatus by appropriate transporters before they are utilized by glycosyltransferases as substrates for glycoconjugate biosynthesis. The transporters are thus indispensable for this process. In fact, nucleotide sugar transporter deficiency leads to a pleiotropic aberration in cellular glycoproteins and glycolipids (3, 4), and may be responsible for a congenital disorder, leukocyte adhesion deficiency type II (5). These transporters may also be involved in controlling the spectrum of glycoconjugates synthesized by a cell, by regulating the amounts of nucleotide sugars delivered into the Golgi lumen. We need to learn much more about the molecular basis of nucleotide sugar transporter function and its regulation to better understand the mechanisms and significance of transporter-mediated glycoconjugate control.

Several cDNAs encoding nucleotide sugar transporters have been cloned so far, including cDNAs for UDP-galactose (UDP-Gal) transporter (UGT)¹ (6-9), CMP-sialic acid (CMP-Sia) transporter (CST) (7, 10, 11), UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (12-14), and GDP-mannose (GDP-Man) transporter (15-18), each from a few species. These transporters constitute a family of related membrane proteins that are localized in the Golgi apparatus and have similar hydropathy profiles. Regarding their structure, Eckhardt et al. (19) recently proposed a structure for murine CST in which there are 10 membrane-spanning segments (19). We used an eight-transmembrane model for hUGT1 and hCST predicted by computer analysis in our previous reports (6, 20). However, in this report we have adopted the 10-transmembrane model for both hUGT1 and hCST because of the high similarity between them and murine CST. This model provides a basic framework for discussions of structure-function relationships, but the structural basis of the specificity and mechanisms of nucleotide sugar transport remains largely obscure.

We have recently started to construct and analyze a series of chimeras between human UGT1 (hUGT1) and human CST (hCST) proteins in order to define the structural basis of the specificity and function of nucleotide sugar transporters (20). These two transporters show 43% identity in amino acid sequence, and have similar topological characteristics, although they transport quite different substrates. The replacement of the N-terminal or C-terminal cytoplasmic region of hUGT1 by the corresponding region of hCST did not destroy the UDP-Gal transporting activity (20). Putative helices 9 and 10 of hUGT1 could also be replaced by those of hCST without loss of the UDP-Gal transporting activity. The above results indicated that the regions substituted in these chimeras were not critically involved in the determination of substrate specificity of hUGT1. However, further analysis of functionally important regions has been hampered because the stability of hUGT1 protein was markedly reduced when longer N- and C-terminal stretches were replaced by the corresponding hCST stretches. Moreover, our previous experimental procedures did not allow the assessment of the CST activity of chimeric transporters.

In the present study, we first devised a convenient method for assessment of the CST activity, and then used this method to analyze the properties of a number of chimeric transporters. In the course of this analysis, we noticed that the hCST protein is more tolerant to replacements of stretches of the N or C terminus than hUGT1, and also found that hUGT1 is tolerant to replacements of internal segments by hCST counterparts. Analyses based on these findings allowed us to identify distinct submolecular segments that are critical for specific recognition of UDP-Gal and CMP-Sia, respectively. Chimeras containing both of these critical segments were expressed in the Golgi membranes, and transported both UDP-Gal and CMP-Sia.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

UDP-[6-³H]Gal (60Ci/mmol) and CMP-[9-³H]Sia (15Ci/mmol) were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO).

Construction of Chimeric cDNAs

Amino acid sequence alignment between hUGT1 and hCST is shown in Fig. 1. Crossover sites in chimeras, which are specified by alphabetical symbols a-j, are also indicated in the figure. All the chimeric constructs were sequenced to rule out PCR-induced mutations.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Amino acid sequences of hUGT1 and hCST. Amino acid sequences of hUGT1 and hCST are aligned, and identical amino acids are indicated by short vertical ties. Thick lines below the sequences (numbered 1 to 10) show the positions of putative transmembrane helices. Thin vertical lines labeled with letters (a to j) represent the positions of crossover sites used to construct chimeric proteins. The accession numbers of GenBankTM for hUGT1 and hCST are D84454 and D87969, respectively.

(i) Chimeras Containing One Crossover Site-- Chimeras A1, A2, B1, B2, C1-C3, and D1-D3 (see Fig. 3) with a single crossing-over at a, b, c, d, j, i, h, j, h, and g (Fig. 1), respectively, were constructed as follows: chimeric cDNAs A1, C1, and C3, previously designated CU-0, UC-7, and UC-8, respectively, were constructed as described before (20), except that HA-tag was not attached to the chimeras. Other chimeras in the A- to D-series were constructed in a similar manner, using appropriately designed PCR primer sets. Briefly, N- and C-terminal hUGT1 and hCST segments with a terminal overlap at a desired internal crossover site were separately amplified by PCR using appropriate sets of primers. Two primers, one from each set, were complementary to each other to create the overlap at the crossover site. The PCR-amplified fragments were mixed to serve as the templates for a second PCR to amplify the desired full-length chimeric cDNA, using the two outside primers from the primer sets used in the first PCR. Chimeric cDNA from the second PCR was inserted into the mammalian expression vector pMKIT-neo as described before (20).

(ii) Chimeras Containing Two Crossover Sites-- Chimeras E1-E3 and F1 (see Fig. 5) with two crossing-overs at c and h, c and g, c and f, and e and g (Fig. 1), respectively, were constructed as follows. E-series chimeras were constructed by ligating two restriction fragments derived from chimera B1 and appropriate D-series chimeras. For instance, chimera E1 was constructed from chimeras B1 and D2. B1 and D2 were cut with BstEII (at nucleotide number 354-360 of hCST) and NotI (at the 3' terminus of the inserts), and the smaller fragment obtained from D2, containing the C-terminal half of the desired new chimera, was inserted into the larger fragment obtained from B1, containing the N-terminal half of the desired new chimera linked to the vector DNA. Chimera E2 was constructed in the same way, using chimera D3 instead of D2, and E3 was constructed using chimera D4 (1-194hCST/220-393hUGT1, not shown in the figure) instead of D2. Chimera F1 was constructed by the two-step PCR method outlined above in (i), using hUGT1 and chimera D3 as templates.

(iii) Chimeras Containing Four Crossover Sites-- Chimera G1 was constructed in three steps. First, chimera CU-2 (1-120hCST/145-393hUGT1) (20) and chimera B1 were cut with EcoRI (at the 5' terminus of the inserts) and BstEII, and the smaller fragment from CU-2 was inserted into the larger fragment from B1, to yield chimera G1-a (1-68hUGT1/46-120hCST/145-393hUGT1). Chimera G1-b (1-219hUGT1/195-237hCST/263-393hUGT1) was then constructed by the two-step PCR method using hUGT1 and chimera D3 as templates. Finally, G1-a and G1-b were cut with PstI (at nucleotide number 499-504 of hUGT1) and NotI, and the smaller fragment from G1-b was inserted into the larger fragment from G1-a, to yield chimera G1.

(iv) Constructs for Expression in Yeast-- The cDNAs encoding hUGT1, hCST, and chimera G1 were inserted into yeast inducible expression vector pYEX-BX (CLONTECH Laboratories, Inc., Palo Alto, CA) (14).

Cell Cultures

Chinese hamster ovary cell (CHO) strain K1 and its derivatives, Lec2 (a CST-deficient mutant) (21) and Lec8 (a UGT-deficient mutant) (22), were cultured in minimum essential medium  supplemented with 10% fetal calf serum (23, 24).

Assessment of the UGT and CST Activities

The UGT activity of chimeric molecules was assessed essentially as described previously with slight modifications (20). UGT-deficient Lec8 cells were transfected with chimeric cDNAs using LipofectAMINE reagent (Life Technologies, Inc., Rockville, MD). The cells were then cultured for 1 day, transferred onto a chamber slide (Nalge Nunc International, Corp., Rochester, NY), incubated overnight, and then fixed with methanol at 20 °C for 6 min. The fixed cells were incubated with an appropriate primary antibody in phosphate-buffered saline containing 3% bovine serum albumin for 1 h at room temperature, washed twice with phosphate-buffered saline for 10 min, and then incubated with a fluorescent secondary antibody for 1 h at room temperature. The cells were next washed twice with phosphate-buffered saline and once with water, and then mounted with Permafluor (IMMUNOTECH, a Coulter Company, Marseilles, France). Staining with 20 µg/ml fluorescein isothiocyanate (FITC)-conjugated GS-II (Griffonia simplicifolia lectin II; EY Laboratories, Inc., San Mateo, CA) was carried out either before the incubation of cells with a primary antibody, or simultaneously with the incubation with a secondary antibody. Fluorescence labeling was visualized under a Carl Zeiss laser-scanning confocal microscope (LSM510). The CST activity of chimeras was assessed by a similar method in which CST-deficient Lec2 cells and FITC-conjugated PNA (peanut agglutinin; EY Laboratories, Inc.) were used instead of Lec8 cells and GS-II, respectively.

Subcellular Fractionation of Yeast and in Vitro Transport Assay

Saccharomyces cerevisiae strain YPH500 (MAT/ura3-52/lys2-801/ade2-101/trp1-63/his3-200/leu2-1) was cultured in a synthetic medium containing 0.67% (w/v) Bacto-yeast nitrogen base without amino acids and 2% (w/v) glucose (YNBD) supplemented with L-leucine, L-histidine, L-tryptophan, L-lysine, and adenine. Uracil was omitted for selection of transformants. Transformations were performed using the lithium acetate method (25).

Subcellular fractionation was performed as described previously with slight modifications (14, 26). Cells were cultured to a density of 0.8 A₆₀₀ and cupric sulfate was added to the medium at a final concentration of 2 mM at 3 h before harvest. Cells were then harvested, washed twice with ice-cold 10 mM NaN₃, resuspended in a spheroplast solution (1.4 M sorbitol, 50 mM potassium phosphate (pH 7.5), 10 mM NaN₃, 40 mM 2-mercaptoethanol) containing 2 mg of zymolyase-100T (SEIKAGAKU Corp., Tokyo, Japan) per g of packed cells, and incubated at 37 °C for 20 min. The spheroplasts were collected by centrifugation at 1,000 × g for 5 min, resuspended in 4 volumes of lysis buffer (0.8 M sorbitol, 10 mM HEPES-Tris (pH 7.4), 1 mM EDTA) containing a protease inhibitor mixture (Roche Molecular Diagnostics, Mannheim, Germany), and homogenized with 10 strokes of mechanical shear using a Potter-type homogenizer. The lysate was centrifuged at 1,500 × g for 10 min to remove unlysed cells and debris. The membrane fraction was collected by centrifugation at 10,000 × g for 10 min and resuspended in lysis buffer. The protein concentrations were determined by using BCA reagent (Pierce Chemical, Rockford, IL).

The in vitro transport assay was performed essentially as described before (26). The reaction was started by addition of the membrane preparation (50 µg of protein) to the reaction mixture (0.8 M sorbitol, 10 mM Tris-HCl (pH 7.0), 1 mM MgCl₂, 0.5 mM dimercaptopropanol, 1 µM UDP-[³H]Gal or CMP-[³H]Sia (6,400 Ci/mol)). The mixture (100 µl) was incubated at 30 °C, diluted with 1 ml of ice-cold stop buffer (0.8 M sorbitol, 10 mM Tris-HCl (pH 7.0), 1 mM MgCl₂, 1 µM nonradioactive UDP-Gal or CMP-Sia) to stop the reaction, and poured onto a nitrocellulose filter (0.45 µm; Millipore Corp., Bedford, MA). The filter was washed 3 times with stop buffer and then dried. The radioactivity trapped on the filter was measured in toluene-based scintillator.

Antibodies

Anti-hUGT1 and anti-hCST antibodies were prepared by immunizing rabbits with synthetic peptides representing the C-terminal amino acid sequences of each protein (³⁷⁷RGDLITEPFLPKSVLVK³⁹³ and ³²¹TSIQQGETASKERVIGV³³⁷ for hUGT1 and hCST, respectively) and affinity purified as described previously (27, 28). An Alexa 546 (a substitute for tetramethylrhodamine)-conjugated goat anti-rabbit IgG antibody (Molecular Probes, Inc., Eugene, OR) was used as the secondary antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Procedure for the Assessment of the CST Activity-- In a previous study, we devised a simple method for analyzing the UGT activity of chimeric transporters (20). Chimeric cDNAs were transiently expressed in UGT-deficient Lec8 cells, and the cells were examined under a microscope to determine whether they bound FITC-conjugated plant lectin GS-II. Since GS-II recognizes terminal GlcNAc residues, UGT-negative cells, which expose GlcNAc residues at the termini of surface glycoconjugates, bind GS-II, while UGT-positive cells, which have Sia residues at the termini of sugar chains, do not. Thus, if a chimeric molecule with UGT activity is expressed, the defect of Lec8 cells will be complemented, and the cells expressing the chimera will not bind GS-II. On the other hand, if a chimeric molecule does not have UGT activity, the cells expressing it will bind GS-II.

A similar procedure was developed in the present study to assess the CST activity of chimeric proteins, utilizing CST-deficient Lec2 cells and FITC-conjugated PNA lectin. PNA recognizes terminal Gal residues that are exposed on the surfaces of CST-deficient cells. In Fig. 2A, we show the lectin-binding properties of parental CHO cells, CST-deficient Lec2 cells, and UGT-deficient Lec8 cells. FITC-conjugated GS-II was bound by Lec8 cells, but not by CHO or Lec2 cells. FITC-conjugated PNA was bound by Lec2 cells, but not by CHO or Lec8 cells. Fig. 2B shows the results of a typical transient expression analysis in which Lec2 cells were transfected with the vector alone or the full-length cDNA encoding hCST. When the vector alone was introduced into the cells, hCST protein was not produced in the cells, and therefore, FITC-conjugated PNA stained the cells. On the other hand, when hCST cDNA was introduced, the cells expressing hCST protein, which were stained by the anti-hCST antibody (about 70% of the total population), were not stained by PNA. Transient expression of an active chimeric transporter would give a similar result to hCST/Lec2 in Fig. 2B, while PNA binding would persist if an expressed chimeric protein had no CST activity.

View larger version (74K): [in this window] [in a new window]   Fig. 2.   Lectin binding properties of CHO, Lec2, and Lec8 cells and transient expression analysis to assess the CST activity of cDNA products. A, CHO, Lec2, and Lec8 cells were stained with 20 µg/ml FITC-conjugated GS-II or PNA for 1 h. A fluorescence image and a Nomarski image of the same field were merged. Bar, 20 µm. Typical terminal oligosaccharide structure of each cell line is shown below the images with terminal sugar residues encircled. Target terminal sugar residues of the lectins are depicted to the left of the images. B, Lec2 cells were transfected with the vector DNA or hCST cDNA. Two days after transfection, the cells were double-stained with FITC-conjugated PNA (green) and an anti-hCST antibody. The binding of the antibody was detected with an Alexa 546-conjugated anti-rabbit IgG antibody (red). The two fluorescence images and a Nomarski image of the same field were merged. Bar, 20 µm. The terminal oligosaccharide structure expected to be displayed on the cell surface of transfectants in each experiment is depicted under each image.

Submolecular Regions Critical for Specific Recognition of UDP-Gal and CMP-Sia-- Series of chimeras in which stretches of various lengths from the N terminus or C terminus of hUGT1 or hCST were replaced by the corresponding stretches of hCST or hUGT1, respectively, were constructed (Fig. 3). These chimeras were transiently expressed in Lec8 and Lec2 cells, and their UGT and CST activities as well as their expression and localization were assessed (Fig. 4).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Schematic representation of chimeras A1, A2, B1, B2, C1-C3, and D1-D3 and a summary of their properties. Each protein is represented by a box with its N terminus to the left. The segments derived from hUGT1 are shown as black boxes and those from hCST as white boxes. The numerals in or below the boxes indicate the amino acid residue numbers of hUGT1 or hCST constituting each segment. The letters above the crossover sites correspond to those depicted in Fig. 1. Gray vertical bars (numbered 1 to 10) indicate the positions of putative transmembrane helices. The results of transient expression analyses in terms of UGT activity and CST activity are summarized to the right of each individual chimera.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Transient expression analyses of representative A-D-chimeras. Lec8 and Lec2 cells were transfected with the indicated chimeric cDNAs to assess their UGT and CST activity, respectively. Two days after transfection, the cells were double-stained with FITC-conjugated lectins (GS-II for Lec8 cells and PNA for Lec2 cells; green) and antibodies (an anti-hUGT1 antibody for A- and D-chimeras and an anti-hCST antibody for B- and C-chimeras). The binding of the antibodies was detected with an Alexa 546-conjugated anti-rabbit IgG antibody (red). The two fluorescence images and a Nomarski image of the same field were merged. Bar, 20 µm. Expected terminal oligosaccharide structures on cells with a given phenotype and their reactivity toward lectins are schematically shown below the images.

The UGT activity of a few of these chimeras (A1, C1, and C3) was described in one of our previous reports, but their CST activity was not assessed previously. These chimeras, and also chimeras A2 and C2, were found in this study to be unable to transport CMP-Sia (Fig. 4). Human UGT1 was rather sensitive to replacement of its putative transmembrane regions by their hCST counterparts, with only chimeras C2 and C3 being competent in transporting UDP-Gal among such chimeras. The fact that C3 was active in UDP-Gal transport implies that a contiguous hUGT1 stretch from the N terminus to the eighth helix is sufficient for the specific recognition of UDP-Gal as a transport substrate.

Chimera A2 was constructed to assess the importance of helix 1 of hUGT1. The chimera was expressed in both Lec8 and Lec2 cells, and the products were sorted to the Golgi region, but showed no UGT or CST activity. This indicates that helix 1 of hUGT1 is indispensable for its activity and cannot be replaced by helix 1 of hCST. The importance of helix 8 of hUGT1 in UDP-Gal recognition could not be assessed previously because a chimera in which helices 1-7 and helices 8-10 were contributed by hUGT1 and hCST, respectively, was not sorted to the Golgi region (20). We address this point in experiments described in the next section.

The CST activity was much more tolerant to the replacement of its N- and C-terminal helices by hUGT1 sequences. Thus, helices 1 and 2 could be replaced by the corresponding hUGT1 sequences without loss of the CST activity (chimera B2). Similarly, chimera D3, in which helices 8 to 10 of hCST were substituted by the corresponding hUGT1 sequences, was expressed efficiently and was active in CMP-Sia transport.

These results indicate that helix 1 of hUGT1 is critical for the recognition of UDP-Gal as a transport substrate, while its CST counterpart is not an absolute requirement for the specific recognition of CMP-Sia. This implies that different submolecular regions are involved in the process of specific recognition of UDP-Gal and CMP-Sia. Analysis of chimeras with two crossovers as described below further substantiated this point.

Properties of Chimeras with Internal hCST Stretches-- We constructed chimeras E1 to E3 and F1, in which internal transmembrane helices of hUGT1 were replaced by the corresponding helices of hCST (Fig. 5A), and examined the expression and nucleotide sugar transporting activities of the chimeric proteins. Every chimera was expressed in transfected cells, and definitely showed either or both UGT and CST activities. Chimeras E1 and E2 exhibited CST activity, while chimera E3 did not. These results indicate that the presence of hCST helices 2 to 7 in the hUGT1 context is sufficient for the specific recognition of CMP-Sia, and strongly suggest that helix 7 of hCST is important in this recognition process. The loss of the CST activity of chimera F1 due to the replacement of helices 2 and 3 by their hUGT1 counterparts suggests that these helices are also important for the CST activity.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Structures and properties of chimeras with internal helices of hUGT1 substituted by their hCST counterparts. A, schematic representation of chimeras E1-E3, F1, and G1. The symbols are the same as in Fig. 2. PstI and BstEII sites utilized in construction of these chimeras are also indicated. B, transient expression analyses of chimeras E1-E3, F1, and G1. Lec8 and Lec2 cells were transfected with each chimeric cDNA and double-stained with FITC-conjugated lectins (GS-II for Lec8 cells and PNA for Lec2 cells; green) and an anti-hUGT1 antibody, and the antibody staining was examined with an Alexa 546-conjugated anti-rabbit IgG antibody (red) 2 days after transfection. The two fluorescence images and a Nomarski image of the same field were merged. Bar, 20 µm. Expected terminal oligosaccharide structures on cells with a given phenotype and their reactivity toward lectins are schematically shown below the images.

To show the importance of these helices more clearly, we next constructed chimera G1, in which the segments containing helices 2, 3, and 7 were derived from hCST sequences in the context of hUGT1. This chimera also possessed dual substrate specificity, i.e. it exhibited both UGT and CST activities. Thus, introduction of helices 2, 3, and 7 of hCST into the hUGT1 context was sufficient for the chimeric transporter to recognize CMP-Sia.

In contrast to hCST, which was tolerant of replacement of its N and C termini by hUGT1 sequences, hUGT1 was rather tolerant to substitution of internal helices by their hCST counterparts. Some chimeras with internal hCST stretches in the hUGT1 context, including chimeras E2 and E3, were able to complement the genetic defect of Lec8 cells, and therefore were active in UDP-Gal transport. Only chimera E1 was devoid of UGT activity among the E-series chimeras. Comparison of the structures of E1 and E2 strongly suggested that helix 8 of hUGT1 significantly contributes to the specific recognition of UDP-Gal.

In Vitro Detection of UGT and CST Activities of Chimera G1 Transporter-- To directly demonstrate that chimera G1 transporter is dually specific for UDP-Gal and CMP-Sia, we examined the substrate specificity of the chimeric transporter in vitro, using a yeast expression system.

The cDNAs encoding hUGT1, hCST, and chimera G1 were inserted into a yeast expression vector. The plasmids were transfected into yeast cells and transformants were obtained. The membrane vesicles were prepared from the transformants and the UDP-[³H]Gal and CMP-[³H]Sia transporting activities were determined.

Fig. 6 shows the time course of uptake of each nucleotide sugar. The UGT activity of chimera G1 was lower than that of hUGT1, but was definitely higher than that of blank membrane vesicles obtained from the vector DNA transformant. The CST activity of chimera G1 was nearly as high as that of hCST. This clearly indicates that the chimeric protein is able to transport both UDP-Gal and CMP-Sia.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Time course of UDP-Gal and CMP-Sia uptake in vitro. Transformation of yeast and subcellular fractionation are described under "Experimental Procedures." The membrane vesicles (50 µg of protein) were incubated at 30 °C with UDP-[3H]Gal (A) or CMP-[3H]Sia (B) for the indicated time periods, and the radioactivity trapped inside the vesicles was measured after filtration and washing. Average values from duplicate experiments are shown. Triangles, membrane vesicles from hUGT1 transformants (A), or hCST transformants (B); circles, vesicles from chimera G1 transformants; squares, vesicles from vector-alone transformants.

It might be argued that chimera G1 may have been rendered simply nonselective for a number of nucleotide sugars, instead of being specific for both UDP-Gal and CMP-Sia. To distinguish between these possibilities, we next examined the substrate specificity of chimera G1 using this in vitro transport system (Fig. 7). CMP-Sia uptake was determined in the presence of excess amounts of various nonradiolabeled nucleotide sugars. If the chimera can transport nucleotide sugars other than CMP-Sia, the additions of those nucleotide sugars would inhibit CMP-Sia uptake by competition.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Substrate specificity of chimera G1. Membrane vesicles from the yeast transformed with vector DNA, hCST, or chimera G1 were incubated with 1 µM CMP-[3H]Sia at 30 °C for 1 min with or without 100 µM nonradiolabeled nucleotide sugars as indicated. The radioactivity trapped inside the vesicles was measured after filtration and washing. Average values from duplicate experiments are shown.

Fig. 7 shows that CMP-Sia uptake of chimera G1 was substantially inhibited by UDP-Gal, but not by addition of nucleotide sugars other than UDP-Gal, such as UDP-GlcNAc, UDP-glucose, UDP-xylose, and GDP-Man. This indicates that chimera G1 is specific for the two nucleotide sugars, UDP-Gal and CMP-Sia, rather than nonselective for a number of nucleotide sugars. It should be noted that the CMP-Sia transporting activity of hCST was not affected at all by UDP-Gal.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our previous studies of modified human and murine UGTs revealed that the N- and C-terminal cytoplasmic loops could be substituted by their CST counterparts, or could even be deleted, without loss of the UDP-Gal transporting activity. We also showed that helices 9 and 10 of hUGT1 could be replaced by the corresponding hCST helices (9, 20). In a further attempt to define the submolecular regions of hUGT1 critical for the specific recognition of UDP-Gal, we have shown in the present study that the substitution of helix 1 of hUGT1 by that of hCST in chimera A2 led to the loss of UGT function without affecting the Golgi localization of the protein. This indicates the indispensability of this helix for the recognition of UDP-Gal. It was also demonstrated that the single substitution of helix 8 of hUGT1 in chimera E1 by its hCST counterpart in E2 abolished the UGT activity of the latter chimera, although the protein was expressed and localized in the Golgi region. These results strongly suggest that both helices 1 and 8 of hUGT1 are necessary for the recognition of UDP-Gal as a transport substrate. On the other hand, assessment of the CST activity of hUGT1/hCST chimeras led to the conclusion that introduction of helices 2, 3, and 7 of hCST into the hUGT1 context was sufficient to elicit CST activity.

The fact that chimeras E2 and G1 are competent in transporting both UDP-Gal and CMP-Sia is an important finding. The dual specificity of G1 was confirmed directly by transport measurements using membrane vesicles prepared from yeast cells expressing chimera G1 protein. The dual specificity indicates that different submolecular regions are critically important for specific recognition of the two different substrates, UDP-Gal and CMP-Sia. Moreover, the presence of a region that recognizes one substrate does not disturb the functioning of another region that is specific for the other substrate.

The simplest assumption underlying analyses of chimeras between two related proteins would be that a single set of submolecular regions, occupying corresponding sites in two proteins, is responsible for defining a given property, e.g. substrate specificity, peculiar to each protein. If this assumption is valid, then swapping of the set of submolecular regions would lead to switching of the particular property in question, such as substrate specificity. This was indeed the case with chimeras between two Na⁺-dependent nucleoside transporters, N1 and N2, of rats. In this case, pyrimidine-selective N2 was converted to a purine-selective transporter by replacing its eighth and ninth helices with those of N1 (29).

On the other hand, this assumption was not valid for nucleotide sugar transporters, since swapping of a particular submolecular region between hUGT1 and hCST did not lead to switching of substrate specificity from one nucleotide sugar to the other. Instead, a chimeric UDP-Gal/CMP-Sia transporter was created by the combination of submolecular regions of the two parent transporter molecules. The mechanisms underlying the differential recognition of specific substrates by nucleotide sugar transporters must be more complicated than we assumed at the beginning of the present study. Chimeric transporters such as E2 and G1, which are dually specific for UDP-Gal and CMP-Sia, should be very helpful for achieving an understanding of these mechanisms. Some pertinent questions include: 1) to what extent do the binding sites of UDP-Gal and CMP-Sia overlap in the dually specific chimeric transporters? 2) How many and which helices are actually involved in constructing the whole specific substrate-binding sites and the transport channel? 3) Which nucleotide, UMP or CMP, serves as a countersubstrate in the nucleotide sugar-nucleoside monophosphate antiport reaction? We are currently trying to answer these questions.

Nucleotide sugar transporters have been widely considered to be highly specific for a single nucleotide sugar substrate. This appears true for the transporters so far cloned and characterized, namely UGT, CST, and UDP-GlcNAc transporter (14, 26, 28). However, earlier analyses of the nucleotide sugar transporting activity in the endoplasmic reticulum membranes suggested the possibility that there is a transport system active with multiple nucleotide sugar substrates (30). Construction of a dual specific hUGT1/hCST chimera clearly demonstrated that such a possibility is a real one. Further studies on dual specific chimeras should help us to understand the mode of action and physiological significance of naturally occurring dual specific nucleotide sugar transporters.

	FOOTNOTES

^* This work was supported in part by Grants-in-Aid for Scientific Research 11159220, 11480172, and 11877024 from the Ministry of Education, Science, Sports and Culture of Japan and research grants from Kirin Brewery Co. Ltd., Japan, and Mizutani Foundation for Glycoscience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) for hUGT1 and hCST reported in this paper has been submitted to the GenBank^TM/EBI Data Bank under accession numbers D84454 (6) and D87969 (7), respectively.

Present address and to whom correspondence should be addressed: Department of Applied Chemistry, Kogakuin University, 1-24-2 Nishi-Shinjuku, Shinjuku-ku, Tokyo 163-8677, Japan. Tel.: 81-3-3340-2731; Fax: 81-3-3340-0147; E-mail: bt13004@ns.kogakuin.ac.jp.

Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M101462200

	ABBREVIATIONS

The abbreviations used are: UGT, UDP-galactose transporter; UDP-Gal, UDP-galactose; hUGT1, human UDP-galactose transporter 1; CMP-Sia, CMP-sialic acid; CST, CMP-sialic acid transporter; hCST, human CMP-sialic acid transporter; UDP-GlcNAc, UDP-N-acetylglucosamine; GDP-Man, GDP-mannose; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; GS-II, Griffonia simplicifolia lectin II; PNA, peanut agglutinin; PCR, polymerase chain reaction.

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