©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Sialyltransferase Sialylmotif Participates in Binding the Donor Substrate CMP-NeuAc (*)

(Received for publication, November 3, 1994 )

Arun K. Datta James C. Paulson (§)

From the From Cytel Corporation and the Department of Chemistry and Molecular Biology, Scripps Research Institute, San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

All members of the sialyltransferase gene family cloned to date contain a conserved region, the ``sialylmotif,'' consisting of 48-49 amino acids in the center of the coding sequence. To investigate the function of this motif, mutant constructs of the Galbeta1,4GlcNAc alpha2,6-sialyltransferase were designed by site-directed mutagenesis, replacing 11 individual conserved amino acids with alanine. Each of the mutants was expressed in COS-1 cells, and eight of these retained sialyltransferase activity, allowing comparison of their enzymatic properties with that of the wild type enzyme. Kinetic analysis showed that six of eight mutants had a 3-12-fold higher K for the donor substrate CMP-NeuAc relative to the wild type enzyme, while the K values for the acceptor substrate were within 0.5-1.2-fold of the wild type for all eight mutants evaluated. The K of the donor substrate analog CDP was also evaluated for the recombinant sialyltransferase with the Val to Ala mutation at residue 220, which produced a 6-fold increase in K of CMP-NeuAc. A corresponding increase in K of 3.4-fold was observed for CDP, indicating a decreased affinity for the cytidine nucleotide. Taken together, these results suggest that the conserved sialylmotif in the sialyltransferase gene family participates in the binding of the common donor substrate, CMP-NeuAc.


INTRODUCTION

The family of sialyltransferases that transfers sialic acid from CMP-sialic acid to carbohydrate groups of glycolipids and glycoproteins has been predicted to consist of a group of 10-12 enzymes(1, 2) . So far cDNA clones have been obtained for eight distinct members of this family(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . Analysis of their protein sequences has revealed that all of the sialyltransferases have structural features common to all glycosyltransferases, which are relevant to their function as enzymes involved in post-translational glycosylation of glycoproteins through their transit through the Golgi apparatus. They each have a short NH(2) cytoplasmic domain, a hydrophobic signal-anchor sequence that serves as the membrane-spanning domain, a luminal ``stem'' region not required for catalytic activity, and a large luminal catalytic domain. However, apart from their common topological features, there is little sequence homology among the sialyltransferases except for 10-12% of the sequence in the middle of the catalytic domain and a smaller sequence of 23 residues at the carboxyl end of the sequence(17, 20) . The largest conserved region, termed the ``sialylmotif''(12) , consists of 48 or 49 amino acids in the center of each molecule, which exhibits 40-60% identity between any two enzymes and contains 8 invariant residues among all the cDNAs cloned to date.

The sialylmotif is presumed to contribute to a structural feature related to the common function of the sialyltransferases. Each enzyme transfers sialic acid from the common donor substrate CMP-NeuAc to an oligosaccharide acceptor substrate. Thus, the sialylmotif could form part of the binding sites for either the donor or acceptor substrates, or both. Alternatively, it could be a structural nucleus that maintains the conformation and spatial relationship of variable substrate binding pockets in the sialyltransferase family.

To explore the function of the sialylmotif, we prepared single point mutants of the Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) as a model in this study. This enzyme forms the NeuAcalpha2,6Galbeta1,4GlcNAc- sequence common to many Asn-linked oligosaccharides(21, 22) . The cDNA encoding this enzyme was cloned from rat liver(3) , and its kinetic properties have been established (21, 22, 23) . In this study, we constructed 11 recombinant enzymes by site-directed mutagenesis, changing selected conserved amino acids in the sialylmotif to alanine, and evaluated the expressed enzymes for changes in their enzymatic properties. Most of the mutants retained sialyltransferase activity and had increases in the K of CMP-NeuAc without corresponding changes in the K of the acceptor substrate. Thus, the results suggest that the sialylmotif of the Galbeta1,4GlcNAc alpha2,6-sialyltransferase participates in binding the donor substrate, CMP-NeuAc.


EXPERIMENTAL PROCEDURES

Construction of Mutants

Single alanine mutations were introduced in spST-2, a construct of expression vector pSVL (Pharmacia Biotech Inc.) containing cDNA for rat Galbeta1,4GlcNAc alpha2,6-sialyltransferase(24) . The mutations in the cDNA for the corresponding nucleotide(s) were introduced by a two-step polymerase chain reaction following the procedure of Sarkar and Sommer (25) with modifications as follows: the cDNA for the Galbeta1,4GlcNAc alpha2,6-sialyltransferase (GenBank accession no. M18769) previously subcloned in pBluescript KS+ plasmid (26) was used as a template. The mutagenic antisense oligonucleotides (the substituted nucleotides are underlined) used for construction of the corresponding cDNAs were (5`-3`): for C181A, AGACGACGGCAGCCCTTTGCCAAGG (nt (^1)553-529); for V184A, GCAGAAGAGGCGACGGCACACCTTT (nt 560-536); for L190A, GGAGTTTTTCGCAGATCCTGCAGAAG (nt 579-554); R207A, CCCATTAAACGCCAGAACTGCATCA ); for D219A, TTTTGAGCCCACAGCCTGTTGGAAGTTGT (nt 669-641); for V220A, GTTTTTGAGCCCGCATCCTGTTGG (nt 671-648); for G221A, GGTAGTTTTTGAGGCCACATCCTGTT (nt 675-651); for S222A, GGTAGTTTTTGCGCCCACATCCTGTTG (nt 675-649); for K223A, CGAATGGTAGTTGCTGAGCCCACATC (nt 680-655); for T224A, CGAATGGTAGCTTTTGAGCCCACAT (nt 680-656); for T225A, TTAGGCGAATGGCAGTTTTTGAGCCC (nt 685-660). In the first step of PCR, the 5`-end oligonucleotide primer, CCCAGGTTGAATTCTCAAACAGC (5`-3`; nt 170-192), and the mutant antisense oligonucleotide for the desired mutation were first used to generate a megamer using Pfu DNA polymerase (Stratagene); the conditions used were: 94 °C, 30 s, 56 °C, 1 min, 73 °C, 2 min, 25 cycles. The gel analysis showed the generation of a single band for about 450 bp depending on the position of the oligomer. This megamer was purified from the PCR mixture using Geneclean II (Bio101). It was then used in the second step of PCR as follows. The tube containing the megamer, the above template, and all the other reagents including Pfu polymerase except the 3`-primer was placed in the thermocycler (Perkin-Elmer 9600), and the reaction was performed for five cycles at 94 °C, 1 min, 73 °C, 3 min. While the tubes were at 73 °C, the tubes were taken out and briefly centrifuged; the 3`-end antisense oligonucleotide primer, CCAGGAGAGGATCCATAAAATGAC (5`-3`; nt 1270-1247) was added, and the mixture was briefly vortexed and centrifuged and then placed in the thermocycler to continue the polymerase chain reaction at 94 °C, 1 min, 68 °C, 1 min, 73 °C, 3 min for 20 cycles. This modification of the conditions was found to be optimum for a good yield of the final products. The products were analyzed by agarose gel electrophoresis that showed the generation of a major single band of 1.1 kilobase. This band was purified by agarose gel electrophoresis followed by Geneclean II (Bio101) to separate from the megamer. For the cDNAs of D219A, V220A, G221A, S222A, K223A, T224A, and T225A, the gel-purified product was digested with two unique restriction enzymes, ApaI (at nt 629) and BstBI (at nt 824), and the smaller 195-bp fragment was purified by the MERmaid kit (Bio101). This fragment was then subcloned into a similarly digested and finally purified larger fragment of spST-2 (prior to this treatment this plasmid DNA was propagated through the dam negative strain GM2163 of Escherichia coli to inhibit the inhibitory methylation at the ApaI site) following standard techniques(27) . Similarly, for C181A, V184A, L190A, and R207A, the gel-purified product was digested with DraIII (at nt 477) and BstBI, and the 350-bp fragment was purified and subcloned into a similarly digested and purified larger fragment of spST-2. The mutation was confirmed by dideoxy double-stranded sequencing (28) of the entire fragment that has been subcloned, including the restriction sites used. Using Pfu polymerase, we found that all the clones obtained have only the desired mutation.

Expression of the Wild Type and Mutant Sialyltransferases

COS-1 cells obtained from ATCC were routinely maintained as described(29) . COS-1 cells (1-2 times 10^6 cells/100-mm dish) were transfected using 2.0 µg of plasmid DNA using LipofectAmine reagent according to the supplier (Life Technologies, Inc.). Expression of transfected proteins was typically allowed to continue for 36-48 h post-transfection before harvesting the cells. After 36-48 h of post-transfection, the cell culture media were collected and concentrated 10-fold by ultrafiltration using microconcentrators (MWCO 10; Amicon Inc., Beverly, MA). The concentrated media containing the soluble expressed sialyltransferase were used directly for analysis of the enzymatic activity as described below. It was observed that the wild type as well as the mutant recombinant sialyltransferases are quite stable at 4 °C for at least 2 weeks. In this study, the enzyme assays were typically performed within 1 week after collecting the media. Following collection of the media, the transfected cells were subsequently used for radiolabeling of the expressed proteins when desired. Transfections of each mutant were done at least three times with plasmid DNAs from different preparations to determine the degree to which variation in expression was transfection-dependent.

Sialyltransferase Assay

Enzyme assays were performed in duplicate as described previously(21) . Unless otherwise stated, 10 µl of concentrated COS-1 cell medium containing expressed recombinant sialyltransferases was assayed in a 60-µl reaction mixture containing 9 nmol of CMP-[^14C]NeuAc (DuPont NEN; 6600 cpm/nmol) as donor, 50 µg of asialo alpha(1)-acid glycoprotein (alpha(1)-acid glycoprotein from Sigma was desialylated by mild acid hydrolysis following the procedure of Schmid (30) ), 50 µg of bovine serum albumin, 50 mM sodium cacodylate (pH 6.0) with 0.5% Triton CF-54. After incubation at 37 °C for 1 h, the radiolabeled product was isolated and quantitated as described previously (22, 31) employing chromatography on Sephadex G-50 (Sigma). The assays were repeated in duplicate at least three times from different transfection experiments.

Pulse-Chase Labeling of Transfected COS-1 Cells and Analysis of the Transiently Expressed Proteins

Metabolic labeling of cells using TransS-Express protein label (DuPont NEN; 100 µCi/ml) and immunoprecipitation of expressed proteins with rabbit anti-rat alpha2,6-sialyltransferase were performed using media from cells of 36-48 h post-transfection essentially as described earlier(32) .

Western Blot

The protein samples from the concentrated media were boiled for 5 min in 1times Laemmli gel sample buffer containing 10% beta-mercaptoethanol. These were run using 10% SDS-polyacrylamide gel electrophoresis in Tris/glycine-SDS buffer. The electrophoresed proteins were transferred to nitrocellulose membrane following the usual procedure(33) . The blot was developed by adsorption of the rabbit anti-rat Galbeta1,4GlcNAc alpha2,6-sialyltransferase antibody (1:500) followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000; Amersham Corp.). The protein bands were finally visualized by following the chemiluminescence (ECL) technique as suggested by the supplier of the reagents (Amersham Corp.).

Protein Determination

The concentration of proteins in the media was determined using the bicinchoninic acid protein assay reagent kit (Pierce) with bovine serum albumin as standard.

Enzyme Assay for Kinetic Analysis

For kinetic measurements, initial rate data using asialo alpha(1)-acid glycoprotein as acceptor were obtained. All sialyltransferase assays were done in duplicate. To determine K(m) of the donor substrate, a fixed concentration of asialo alpha(1)-acid glycoprotein (50 µg) and five concentrations of unlabeled CMP-NeuAc (Sigma) were added to assay mixtures containing a fixed amount of CMP-[^14C]NeuAc (DuPont NEN; 315 µCi/µmol; 58,500 cpm/assay) to give a final concentration of 20-200 µM (unless otherwise specified). Similarly, to determine K(m) of asialo alpha(1)-acid glycoprotein, assays containing six concentrations were examined ranging from 40 to 400 µM based on galactose content (1 µmol of asialo alpha(1)-acid glycoprotein or 40 µg is equivalent to 16.6 µmol of Gal acceptor unit), using a fixed concentration of CMP-[^14C]NeuAC (6433 cpm/mmol). Kinetic constants were obtained from double-reciprocal plots (for details see (34) ) by linear regression analysis of Lineweaver-Burk plots(35) . In all cases consumption of substrates was kept below 20% to ensure accurate initial rates by adjusting the amount of enzyme added if necessary.


RESULTS

Expression of the Sialylmotif Mutants and Comparison of Their Enzyme Activity

The plasmid cDNAs used to examine the sialyltransferase activity of selected mutations in the sialylmotif were constructed as shown in Fig. 1. The mutants were derived from an expression vector of the wild type enzyme in which the N-terminal cytoplasmic tail, signal anchor domain, and the stem regions (first 71 amino acids) were replaced with a cleavable signal peptide of dog pancreas insulin(32) . Thus, this vector results in the expression of the Galbeta1,4GlcNAc alpha2,6-sialyltransferase protein in the media as a soluble form. The nomenclature for the mutant sialyltransferases reflects the residue number for which alanine was substituted (e.g. for D219A, aspartic acid 219 is replaced by alanine, etc.).


Figure 1: spST-2 construct and its mutants in relation to the domain structure of sialyltransferase. The cleavable signal anchor domain from dog pancreatic insulin is fused with the catalytic domain of Galbeta1,4GlcNAc alpha2,6-sialyltransferase converting this membrane-bound enzyme into the soluble form(32) . The ``sialylmotif'' in this enzyme spans from amino acid 178 to 225 and contains eight invariant amino acids, which are indicated by boldfaceletters. The underlined amino acid residues were changed to alanine by single point mutagenesis. , cleavable signal sequence; &cjs2113;, sialyl motif; box, catalytic domain.



Expression vectors containing mutant sialyltransferase constructs were transfected into COS-1 cells and evaluated for expression of the recombinant proteins and for expressed sialyltransferase activity. Immunoprecipitation of radiolabeled sialyltransferases using the anti-sialyltransferase antibody, followed by analysis by SDS-polyacrylamide gel electrophoresis, indicated that all mutant sialyltransferases were expressed and exhibited similar molecular weight to the wild type sialyltransferase (not shown). To determine the enzymatic properties of the mutant sialyltransferases, concentrated media from the transfected cells were used as a source of crude enzyme. Media from COS-1 cells ``mock'' transfected using the expression vector (pSVL) only contained low levels of endogenous sialyltransferase, which were typically less than 2% that of media containing the expressed soluble sialyltransferases. All the mutants showed Galbeta1,4GlcNAc alpha2,6-sialyltransferase activities, which varied from 25 to 85% that of the wild type enzyme except for the mutants, C181A, R207A, and S222A, which showed very low activity (5% or less).

By comparing sialyltransferase expression by Western blot to the enzymatic activity observed it was noted that the expression level was apparently similar to the wild type enzyme for V184A, L190A, D219A, V220A, K223A, and T225A, while the enzyme activities obtained were 45, 28, 56, 30, 32, and 27%, respectively. While the mutation of Cys, Arg, and Ser to Ala reduced the enzyme activity to 5% or less, the protein expression appeared to be reduced only 2-3-fold.

Kinetic Analysis of the Mutant Enzymes

Analysis of the kinetics of wild type and each mutant sialyltransferase expressed in COS-1 cells was performed as described under ``Experimental Procedures,'' except for C181A, R207A, and S222A, which had low activity and were not evaluated. The K(m) values for each sialyltransferase were determined for the donor substrate, CMP-NeuAc, and best glycoprotein acceptor substrate of the wild type enzyme, asialo alpha(1)-acid glycoprotein. This glycoprotein acceptor contains five N-linked oligosaccharides, which are predominately tri- and tetra-antennary structures, each branch of which is terminated with the acceptor sequence Galbeta1,4GlcNAc. Data obtained for each of the enzymes produced Lineweaver-Burk plots with correlation coefficients of 0.95 or greater (e.g.Fig. 2).


Figure 2: Double-reciprocal plots of initial rate data with CMP-NeuAc (top) or asialo alpha(1)-acid glycoprotein (bottom) as the varied substrate. Top, rate data with CMP-NeuAc as the donor (26.5-201 µM) were determined at a fixed concentration of the acceptor asialo alpha(1)-acid glycoprotein, 50 µg. Bottom, the concentration of the acceptor asialo alpha(1)-acid glycoprotein was varied (0.04-0.4 mM) at a fixed concentration of the donor, 0.15 mM. The plot was shown only for T225A () in top and V220A () in bottom, as a representative of the mutants and compared with that of the wild type (spST-2) of sialyltransferase (box). The K(apparent) values were determined from the x intercept (-1/K (apparent))(35) .



As summarized in Table 1, the K(m) values obtained for asialo alpha(1)-acid glycoprotein were within 0.5-1.2-fold that of the wild type enzyme for each of the mutant enzymes examined. In contrast, the K(m) values for the CMP-NeuAc were significantly altered, particularly for V184A, L190A, D219A, V220A, K223A, and T225A, with increases in K(m) (apparent) of 3-12-fold observed.



Increase in K of CDP for V220A

Since decreased binding affinity of CMP-NeuAc would be reflected as an increase in the K(m) for this substrate, it seemed reasonable to expect that the amino acids altered in the mutants that exhibited this change participated in the binding of the cytidine or NeuAc moiety of CMP-NeuAc. Previous reports have indicated that the cytidine analog CDP is often a potent inhibitor of sialyltransferases, exhibiting equilibrium dissociation constants similar to that of CMP-NeuAc(34) . To determine if the increase in K(m) for CMP-NeuAc would also be reflected in a decrease in affinity for CDP, CDP was tested as a competitive inhibitor of CMP-NeuAc for mutant V220A (Fig. 3). The K(i) for V220A was determined to be 16.1 µM, representing a 3.4-fold increase over the wild type enzyme (4.7 µM). Thus, the increase in K(i) for CDP observed for V220A is comparable in magnitude with the increase in K(m) for CMP-NeuAc, suggesting that Val participates in binding the common cytidine moiety in these two ligands.


Figure 3: Inhibition kinetics of sialyltransferase by CDP. Initial rate data for the wild type enzyme (top panel) were determined in the presence of varied concentrations of the donor substrate CMP-NeuAc (0.025-0.2 mM) and in the absence (box) and presence (⧫) of a fixed concentration of 12.5 µM CDP. Similarly, kinetics for the mutant V220A (bottom panel) were determined in the presence of the varied concentrations of CMP-NeuAc (0.25-2.0 mM) and in the absence (box) or presence (⧫) of a fixed concentration of 16.7 µM CDP. The concentration of the acceptor asialo alpha(1)-acid glycoprotein was kept constant at 50 µg throughout for both enzymes. The K values were extracted from the x intercept in the presence of inhibitor (35) , where the x intercept = -(1/K) (1 + [I]/K) and where K is obtained in the absence of the inhibitor CDP and [I] is the concentration of CDP.




DISCUSSION

The sialylmotif of the sialyltransferase gene family has been identified as a conserved stretch of 48-49 amino acids found roughly in the center of the coding sequence of these enzymes. Based on the premise that this motif was restricted to the sialyltransferase gene family, five sialyltransferase genes have been successfully cloned using a PCR homology approach, bringing the total number of distinct members of sialyltransferases cloned to date to eight(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . Further evidence that the sialylmotif is unique to the sialyltransferase gene family is that no other gene in GenBank, including representative cloned cDNAs from the fucosyltransferase, galactosyltransferase, N-acetylglucosaminyltransferase, or N-acetylgalactosaminyltransferase families, has been found to have a homologous sequence. These facts alone argue that the sialyltransferase genes described to date originate from a common gene and that the sialylmotif participates in a structural feature or function common to all of these enzymes.

The aim of this present study was to evaluate the functional role of the conserved sialylmotif of the sialyltransferase gene family. As described under ``Results,'' the Galbeta1,4GlcNAc alpha2,6-sialyltransferase was used as a representative enzyme in these studies, substituting alanine for conserved amino acid residues in the sialylmotif by site-directed mutagenesis to see if the alteration had an effect on sialyltransferase activity. The 11 mutants reported in this study include 7 of the 8 amino acid residues found to be invariant in all the sialyltransferase genes cloned to date (see Fig. 1). In addition, several other highly conserved amino acids were examined.

The results suggest that the sialylmotif participates in the binding of the donor substrate, CMP-NeuAc, and has little contribution to the binding of the oligosaccharide acceptor substrate. Indeed, of the eight mutant sialyltransferses that exhibited sialyltransferase activity, six exhibited increased K(m) values of 3-12-fold over that of the wild type for the donor substrate, CMP-NeuAc (Table 1). In contrast, all of the mutant enzymes had K(m) values for the acceptor substrate (asialo-alpha(1) acid glycoprotein) that were within 0.5-1.2-fold that of the wild type sialyltransferase. The mutations spanned the length of the sialylmotif (see Fig. 1), and the magnitude of the change in K(m) for CMP-NeuAc did not appear to be dependent on the location of the amino acid in this conserved domain. Further examination of one of the mutants, V220A, revealed that it exhibited a decreased affinity (increased K(i)) for the competitive substrate CDP relative to the wild type enzyme.

The conclusions drawn from these studies are in keeping with observations made previously by others. Wen et al.(10) suggested that the sialylmotif may form a conserved surface feature of the sialyltransferases, consistent with a substrate binding pocket, due to the large number of conserved charged residues found in the sialylmotif. Kurosawa et al.(16) further suggested that the conserved region was less likely to be an acceptor oligosaccharide binding site considering that the cloned enzymes transferred sialic acid to galactose, N-acetylgalactosamine, and sialic acid found in a variety of glycoprotein and glycolipid carbohydrate groups. The fact that the sialylmotif appears to participate in binding the common donor substrate, CMP-NeuAc, suggests that the PCR homology approach to cloning sialyltransferase cDNAs may continue to have utility in defining this diverse gene family.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant GM27904. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 3525 John Hopkins Ct., San Diego, CA 92121. Tel.: 619-552-3020; Fax: 619-552-8801.

(^1)
The abbreviations used are: nt, nucleotide(s); bp, base pair(s); PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. Carl Perez for a suggestion in the mutagenesis experiment by PCR. We also thank Dr. Eric Sjoberg for critical reading of this manuscript and Dorothy Wharry for secretarial assistance.


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