Cloning of a Human UDP-galactose:2-Acetamido-2-deoxy-D-glucose 3beta -Galactosyltransferase Catalyzing the Formation of Type 1 Chains*

Frank Kolbinger, Markus B. Streiff, and Andreas G. KatopodisDagger

From Novartis Pharma AG, Transplantation Preclinical Research, CH 4002 Basel, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human beta 3-galactosyltransferase distinct from the melanoma enzyme. The new beta 3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(beta 1-3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Two types of carbohydrate chains are known to exist in the lacto-series of oligosaccharides, type 1 chains that contain the Gal(beta 1-3)GlcNAc linkage and type 2 chains containing the topoisomer Gal(beta 1-4)GlcNAc. Both types of carbohydrate structures are present in soluble oligosaccharides of human milk (1), are also found on glycoproteins (2) and glycolipids (3), and are important precursors of blood group antigens (4). The differences in function between type 1 and type 2 chains are not well understood. For example, during both embryogenesis (5) and carcinogenesis (6), the ratio of type 1 to type 2 chains produced by the cell changes. Furthermore, both type 1 and type 2 chains can be ligands for the selectin family of leukocyte extravasation receptors (7). The physiological significance of these observations is not yet known (8).

The biosynthesis of type 1 and type 2 structures is catalyzed by specific galactosyltransferases, which transfer galactose to GlcNAc terminating chains. The galactosyltransferase responsible for type 2 chain biosynthesis (beta 4-Gal-T)1 has been cloned and well characterized and was shown to be expressed in various tissues and cell types (reviewed in Refs. 9 and 10). This enzyme requires Mn2+ for activity and is regulated by alpha -lactalbumin to change the kinetics of transfer to glucose, thus favoring the synthesis of lactose. Relatively little information is known about the type 1 elongating enzyme, UDP-galactose:2-acetamido-2-deoxy-D-glucose 3beta -galactosyltransferase (beta 3-Gal-T). This enzyme is clearly different from the beta 4-Gal-T and is expected to have a more restricted tissue distribution. The type 1 elongating enzyme is thought to be distinct from another beta 3-Gal-T activity detected in various sources and transferring to lactose or LacNAc (11-13), although this has not been molecularly established.

A beta 3-Gal-T enzyme catalyzing the synthesis of Gal(beta 1-3)GlcNAc has been purified from pig trachea and shown to require Mn2+, not to be influenced by lactalbumin, and to have an acceptor specificity consistent with its role of being responsible for elongation of oligosaccharide chains on both mucins and glycolipids (14, 15). Another beta 3-Gal-T enzyme capable of forming type 1 chains has been detected in colon carcinoma cell lines (16), as well as normal colonic mucosa (17). Moreover, DNA from COLO 205 cells when transfected into mammalian cells produced cell lines de novo synthesizing type 1 chains (18). No molecular information is available for any of these enzymes, and it is therefore difficult to judge their similarity. A beta 3-Gal-T was recently cloned from the human melanoma WM266-4 cell line using an expression cloning strategy that relied on lectin resistance to identify clones (19). This enzyme, which has no homology to known glycosyltransferases, transfers galactose in vitro to produce Gal(beta 1-3)GlcNAc(beta 1-3)Gal(beta 1-4)Glc and directs the synthesis of sLea in transfected cells.

We report here the cloning and functional expression of a new beta 3-Gal-T from human brain distinct from the enzyme present in melanoma cells. The new enzyme is homologous to the melanoma cell enzyme in the putative catalytic domain, and is mainly expressed in brain and heart. When transfected in mammalian cells, the new gene directs the de novo synthesis of type 1 chains. A soluble version of the enzyme expressed in insect cells transfers galactose to GlcNAc to produce Gal(beta 1-3)GlcNAc. The recombinant enzyme also transfers galactose to Gal, LacNAc, and lactose, at distinctly lower rates. To distinguish the two beta 3-Gal-T enzymes, we propose to name the melanoma enzyme beta 3GalT1 and the human brain enzyme beta 3GalT2.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- All cell culture media, sera, and antibiotics were from Life Technologies, Inc. CHO K1 cells (ATCC CCL-61) were maintained in alpha -minimal essential medium, 5% fetal bovine serum. Sf9 insect cells were grown in Sf-900 II SFM medium. GlcNAc, Gal, Gal(beta 1-3)GlcNAc, p-nitrophenyl beta -D-lactopyranoside, GalNAcalpha -benzyl, and UDP-Gal were purchased from Sigma. Antibody CSLEX was purchased from Becton Dickinson, and GSLA1 was a gift from Dr. J. Magnani (GlycoTech Corp.). The cDNA for Fuc-T III in the expression vector pcDM7 was a gift from Dr. J. B. Lowe (Howard Hughes Medical Institute, Ann Arbor, MI).

EST Data Base Searches-- The cDNA sequence of beta 3GalT2 was cloned by its homology to beta 3GalT1 (19). Using the complete 322-amino acid protein sequence of beta 3GalT1, a TBLASTN search was performed (20) on the dbest data base (release: Feb. 4, 1996). The best aligning EST sequences (accession numbers R13867, H14861, and R13064) from infant brain showed sequence identities of about 67% over a stretch of about 68 amino acids to the putative catalytic domain of beta 3GalT1. Thirty-eight amino acids deduced from the cDNA sequence of R13867 were then used to search again the dbest data base (release: Feb. 29, 1996) using TBLASTN. An additional human fetal EST clone D81474 was found, further extending the homology within the catalytic domain.

Screening of lambda gt10 Library-- The sequences of EST clones R13867 and D81474 were artificially combined, and primers were designed, allowing PCR amplification of a 307-bp DNA fragment specific for combined ESTs R13867 and D81474. These primers, designated galtdia2.pcr (ACT CGC CAG TGA TTG AAC ACA AAC) and galtdia3.pcr (TGA AGC CAG ATC TGC CTC CC) were then used to screen a collection of 16 heat-inactivated human lambda  cDNA libraries (QUICK-screenTM, CLONTECH) by PCR for the presence of the 307-bp diagnostic fragment.

A viable form of the human brain lambda gt10 cDNA library (CLONTECH, HL3002) was screened for the full-length cDNA of the putative new beta 3-Gal-T. Screening was done by preparing hierarchical lambda  phage pools and screening them by PCR using primers galtdia2.pcr and galtdia3.pcr, essentially as described previously by D'Esposito et al. (21). In brief, 9.5 × 105 plaque-forming units from the human brain lambda gt10 cDNA library were used to infect Escherichia coli C600 hfl cells, mixed with LB top agar, and plated onto 32 six-well tissue culture plates. After bacterial lysis, lambda  phage were eluted with SM buffer and extracted once with chloroform. The samples were briefly centrifuged and the clear lysates were subsequently stored at 4 °C (top level pool). For each 96-well plate of the top level, column and row pools were prepared by mixing equal volumes of phage lysate from each well to obtain a total of 40 column and row lambda  phage pools. Aliquots (1 µl each) of every well in each column and row were pooled, heat-treated (5 min at 95 °C), and tested by PCR with primer pair galtdia2.pcr and galtdia3.pcr for the presence of the 307-bp DNA fragment. The intersections of each positive column and row pool were identified as a potentially positive wells possibly containing single independent clones and were individually re-tested by PCR. Two positive top level wells were selected and their phage titer determined. Level 1 pools were obtained by replating 13,000 plaque-forming units each from the selected top level wells onto 16 six-well plates and preparation of the corresponding phage lysates. Positive level 1 pools were identified by PCR using the strategy described above. To further reduce phage complexity, 450 plaque-forming units of several positive level 1 wells were subsequently plated onto four 24-well tissue culture plates each. Phage from positive level 2 wells were plated at a density so that single lambda  plaques could be isolated. Forty-eight single phage lysates were prepared and analyzed by PCR. Purity and insert size of each lambda  phage clone was determined by PCR using lambda  DNA specific oligonucleotide primers lgt10-5'.seq (AGC AAG TTC AGC CTG GTT AAG T) and lgt10-3'.rev (TTA TGA GTA TTT CTT CCA GGG). One of the 307-bp positive phage lysates, designated GA4/1, contained the largest insert (3 kb).

Subcloning and DNA Sequencing-- After plaque purification, the insert of clone GA4/1 was subcloned into the single EcoRI site of expression vector pZEOSV (Invitrogen). Isolation of the insert of lambda  clone GA4/1 was done either by PCR amplification using the lambda gt10-specific primers shown above or by excision from purified lambda  DNA using restriction endonuclease EcoRI. The DNA sequences of several clones of GA4/1 in pZEOSV (GA4/1.zeo) were determined. Both strands were sequenced using 21-mer and 22-mer oligonucleotide primers synthesized according to the sequence of the cDNA insert. The DNA sequences were assembled with the CAP program (22) and analyzed using Clone Manager (Scientific & Educational Software) and the sequence analysis software package of the University of Wisconsin Genetics Computer Group (23).

Northern Blot Analysis-- A premade Northern blot of poly(A)+ RNA (multiple tissue Northern blot, CLONTECH) was prehybridized in 5 × SSC, 5 × Denhardt's, and 1% SDS solution at 65 °C for 4 h and then hybridized overnight at 65 °C with a 32P-random prime-labeled (24) probe from the GA4/1 insert, containing the entire coding region of beta 3GalT2. Filters were washed at room temperature in 5 × SSC, 0.2% SDS two times and then in 2 × SSC, 0.2% SDS two times. Final wash was in 0.1 × SSC, 0.2% SDS at room temperature.

Construction and Characterization of CHO/Fuc-T III Cell Line-- To create a cell line capable of producing sLea, CHO-K1 cells were transfected with the gene for Fuc-T III (25). Expression vector pcDNA(+).hyg was constructed by inserting into the single NheI restriction endonuclease site present in vector pcDNAI/Amp (Invitrogen) a XbaI-restricted 1.68-kb DNA fragment encompassing the entire expression unit for hygromycin-B resistance. The fragment was obtained from vector pREP7 (Invitrogen) by PCR amplification using primers Hyg1.Xba (GCT CTA GAG CGT TTG CTG GCG GTG TCC) and Hyg2.Xba (GCT CTA GAC CAT GGG TCT GTC TGC TCA GTC CA). In pcDNA(+).hyg both, the hygromycin-B resistance gene and inserted genes/cDNAs are transcribed in the same direction. The complete cDNA for human Fuc-T III was excised from pcDM7 using restriction endonuclease XhoI and ligated into pcDNA(+).hyg cut with the same restriction endonuclease to create vector FTIII.hyg. Correct orientation of the Fuc-T III coding sequence was verified by restriction analysis.

CHO-K1 cells were seeded overnight in six-well plates and transfected with 4 µg/well of FTIII.hyg, using LipofectAMINE according to the manufacturer's instructions. Cells were trypsinized into a T-175 flask, and 0.2 mg/ml hygromycin (Calbiochem) was added to the medium after 24 h. After 3 weeks of selection, surviving cells were sorted by FACS for the surface expression of the sLex epitope by incubation with monoclonal antibody CSLEX-1 and staining with FITC-labeled polyclonal anti-mouse IgM (Jackson ImmunoResearch). The sorted cells were placed in 96-well plates at a density of 0.5 cells/well. Cells from wells containing single colonies were analyzed for surface expression of sLex by FACS with CSLEX, and clones displaying the highest staining were used for further experiments.

Expression of Full-length beta 3GalT2 in CHO/Fuc-T III Cells and FACS Analysis-- A mammalian expression vector was prepared containing the complete open reading frame of beta 3GalT2. Construction was done by PCR amplifying a DNA fragment from plasmid GA4/1.zeo using oligonucleotides NGalTATG.ECO (CGC GAA TTC GCC ACC ATG CTT CAG TGG AGG AGA AGA CAC TGC) and NGalTTAG.ECO (CGC GAA TTC CTA ATG TAG TTT ACG GTG GCG ATA CCT GCC). Oligonucleotide NGalTATG.ECO had a 5' extension containing an EcoRI restriction endonuclease site and consensus sequence for efficient translation (26). Oligonucleotide NGalTTAG.ECO included the putative stop codon of the beta 3GalT2 and an 5' extension containing an EcoRI restriction endonuclease site. The gel-purified, amplified 1.29-kb PCR fragment was directly ligated into plasmid pCR3TM-uni (Invitrogen). Correct orientation of the DNA insert present in plasmid pGA4/1CDS.uni was verified by restriction analysis.

CHO/Fuc-T III cells seeded overnight in a six-well plate were transfected with 4 µg of plasmid pGA4/1CDS.uni using LipofectAMINE. Cells were trypsinized in a T-175 flask, and after 24 h 0.5 mg/ml G418 was added to the culture medium. After 3 weeks of selection, cells were analyzed by FACS for the expression of sLea on their surface. FACS analysis was performed by first incubating the cells with the monoclonal antibody GSLA1 and after washing with a FITC-labeled polyclonal anti-mouse IgG (Jackson ImmunoResearch).

Construction of Soluble beta 3-Gal-T2 Fused with Protein A-- A basic mammalian expression vector for the expression of Staphylococcus aureus protein A fusions was prepared as follows. First an expression vector was constructed containing 20 amino acids of the human gamma -interferon signal sequence and the first 6 amino acids of mature human gamma -interferon (27) plus suitable cloning sites for the cDNA insertion. Oligonucleotides IFNG-NEW1 (GTG GCA AAG CTT TCT AGA GGC GCG CCA CCA CCA TGA AAT ATA CAA GTT ATA TCT TGG CTT TTC AGC TCT GCA TCG TTT TGG GTT CTC TTG) and IFNG-NEW3 (GGA CTA GTT CTA GAA CCG GTT TAC TAC TCG AGG GAT CCG TCG ACG GGG TCC TGG CAG TAA CAG CCA AGA GAA CCC AAA ACG AT) were annealed, the single-stranded regions were filled in with the Klenow fragment of DNA polymerase I, and the HindIII/SpeI-restricted DNA fragment was subcloned into vector pcDNAI/neo (Invitrogen) restricted with HindIII and XbaI, resulting in plasmid IFNG-new.neoI. A DNA fragment encoding for the Ig-binding domains of protein A was amplified from plasmid pRIT2T (Pharmacia Biotech Inc.) by PCR using oligonucleotides SPANEW1.SAL (GGT ACG GTC GAC TGG GAT CAA CGC AAT GGT TTT ATC) and SPANEW3.XHO (GGT GCA CTC GAG ATT TGT TAT CTG CAG ATC GAC). This DNA fragment was then cut with restriction endonucleases SalI and XhoI and subcloned into plasmid IFNG-new.neoI restricted with the same enzymes. At the carboxyl-terminal end of protein A were added XhoI and AgeI cloning sites for in-frame insertion of cDNAs. The resulting plasmid, designated sPROTA2.neoI, is capable of directing the expression of secreted protein A or protein A fusion proteins under control of the human cytomegalovirus promoter (data not shown).

A DNA fragment encoding for amino acids 125-422 (Fig. 1) of beta 3GalT2 was inserted into the XhoI and AgeI sites of vector sPROTA2.neoI. Construction was done by PCR amplification of the corresponding DNA fragment from GA4/1.zeo using oligonucleotides NGALTS1.XHO (GGT GCA CTC GAG AAA GGT ACT GGA CAT CCA AAT TCT TAC) and NGALTEND.AGE (GGT GCA ACC GGT TAC TAA TGT AGT TTA CGG TGG CGA TAC C). The amplified 0.92-kb PCR fragments was restricted with AgeI and XhoI and subcloned into sPROTA2.neoI, cut with the same enzymes. The presence of the beta 3GalT2 insert in the resulting protein A fusion vector SPA2GATS.neoI was verified by sequencing. The complete expression cassette for the protein A fusion of beta 3GalT2 of vector SPA2GATS.neoI was then transferred into an insect cell expression system to be able to purify larger quantities of the fusion protein for functional studies. This was achieved by transferring a 1.8-kb XbaI fragment from SPA2GATS.neoI into the unique XbaI site of donor plasmid pFastBac1 (Life Technologies, Inc.). Correct orientation of the DNA insert present in the resulting donor plasmid SPA2GATS.bac1 was verified by restriction analysis.


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Fig. 1.   Nucleotide and deduced amino acids sequence of human beta 1-3- or beta 3-galactosyltransferase-2. The double underlined amino acids correspond to a putative transmembrane domain. The asterisks indicate potential N-glycosylation sites (Asn-X-(Ser/Thr)).

Expression and Purification of Protein A Fusion of beta 3GalT2 in Insect Cells-- Donor plasmid SPA2GATS.bac1 was used to create in E. coli recombinant SPA2GATS bacmid DNA containing all the genetic elements for the production of recombinant virus particles, by utilizing Tn7-mediated transposition according to protocols given in the instruction manual of the BAC-TO-BAC baculovirus expression system (Life Technologies, Inc.). The recombinant bacmid DNA was then used to transfect Sf9 insect cells, and isolated virus was then used to obtain a high titer virus stock.

For large scale production of the beta 3GalT2/protein A fusion, Sf9 cells grown at 28 °C in flasks to a density of 2 × 106 were infected with virus at a multiplicity of infection of 5, and culture supernatants were harvested 72 h post-infection. After centrifugation, filtration and concentration, supernatants were made 25 mM in sodium cacodylate, pH 6.5, and 100 mM in NaCl and loaded onto a SP-Sepharose column (Pharmacia). The column was washed with 25 mM sodium cacodylate, pH 6.5, 200 mM NaCl, and the beta 3GalT2/protein A fusion protein was eluted with 25 mM sodium cacodylate, pH 6.5, 500 mM NaCl. The eluted fractions were dialyzed against 40 mM sodium cacodylate, pH 6.5, 100 mM NaCl, 20 mM MnCl2 and loaded onto a UDP-hexanolamine-Sepharose column, equilibrated in the same buffer. The column was washed with loading buffer, followed by washing with loading buffer without MnCl2. Elution of the enzyme was accomplished with buffer containing 50 mM sodium cacodylate, pH 6.5, 100 mM NaCl, 10 mM EDTA, 1 mg/ml UDP (Fluka). The enzyme was stored at 4 °C.

Expression and Purification of Protein A Fusion of beta 3GalT1-- A protein A fusion chimera of beta 3GalT1 was cloned and expressed in a manner analogous to that indicated above for beta 3GalT2. A portion of beta 3GalT1 representing the complete stem region and catalytic domains (amino acids 35-326) was amplified by PCR from the genomic DNA of Colo 205 cells (ATCC CCL-222), and recombinant virus were produced in a manner analogous to that described for beta 3GalT2. Cell culture and protein purification was performed as indicated above for beta 3GalT2.

Detection of beta 3GalT2/Protein A Fusion Protein by Enzyme-linked Immunosorbent Assay-- The protein A portion of the beta 3GalT2/protein A fusion protein was used to semi-quantitatively determine the concentration of the soluble form of beta 3GalT2. Microtiter plates were coated overnight at 4 °C with 120 µl of human IgG (5 µg/ml; Sigma) in PBS and blocked with 0.5% BSA in PBS for 60 min at room temperature. Samples as well as a protein A standard (0.5-50 ng/ml recombinant IgG-binding fragment of protein A; Sigma) diluted in 100 µl of PBS containing 0.5% BSA (PBS/BSA) were added to the microtiter plates and incubated for 60 min at room temperature. Wells were washed with PBS containing 0.05% Tween 20 and incubated successively with 100 µl of biotinylated goat anti-protein A antibody (1:100.000; Sigma) and 100 µl of streptavidin-peroxidase conjugate (1:5000; Boehringer) in PBS/BSA, for 60 min at room temperature. Wells were washed six times with PBS plus 0.05% Tween 20 and developed with TMB substrate solution (Bio-Rad), and absorbance at 450 nm was measured after stopping with 50 µl of 1 M H2SO4.

beta 3GalT2 Assays and Product Characterization-- The linkage synthesized by the beta 3GalT2/protein A fusion protein was analyzed by HPAE/PAD as follows: to 28 µl of assay stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/ml BSA, and 0.26 mM UDP-Gal), 2 µl of MnCl2 (500 mM), 1 µl of GlcNAc (500 mM), 14 µl of H2O, and 5 µl of enzyme were added. After incubation at 37 °C for 2 h the reaction was stopped by freezing. An aliquot of 25 µl of the reaction mix was analyzed by HPAE/PAD (Dionex) using the following conditions: 70% H2O, 30% 0.5 M NaOH, at a flow rate of 1 ml/min.

Enzymatic activity of the beta 3GalT2/protein A fusion protein was determined using a radioactive assay similar to the method of Palcic et al. (28) as follows; the appropriate amounts of enzyme were incubated with 14 µl of assay stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/ml BSA, 0.26 mM UDP-Gal, 2 µl of [3H]UDP-Gal (Amersham Corp.)), 1 µl of MnCl2 (500 mM), 2.5 µl of GlcNAc-Lemieux (37 mg/ml in dimethyl sulfoxide) at 37 °C for 60 min. The reaction was quenched by the addition of 1 ml of water and loaded on a C18 Sep-Pack cartridge (Waters), and the column was washed twice with 5 ml of H2O and eluted with 5 ml of methanol. All fractions were counted in a BETAmatic beta  counter (Kontron) after the addition of 10 ml of scintillation fluid. Enzymatic activity was determined by calculating percent UDP-Gal conversion (3H activity in the methanol fraction versus total 3H activity of all fractions).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Cloning and Nucleotide Sequencing of the beta 3GalT2 cDNA-- It is difficult to predict the sequence homology of glycosyltransferases based on their enzymatic activity. Some glycosyltransferases are grouped into families of homologous genes, as for example the alpha 1-3 fucosyltransferases (29), or have characteristic motifs, as is the case for sialyltransferases (30, 31). Many glycosyltransferases, however, have little or no homology even between enzymes that utilize the same activated sugar donor and carbohydrate acceptor. This is the case for galactosyltransferases, which seem to have no common motif except possibly for a hexapeptide (B-D-K-K-N-A, where A is either E or D and B is either R or K) identified by Joziasse et al. (32). TBLASTN (20) data base searches using all possible permutations of this peptide motif revealed no new homologous sequences.

The cloning of beta 3GalT1 by an elegant expression cloning method using lectin resistance for phenotype selection (19) provided the opportunity to search for novel galactosyltransferase genes by sequence similarity. Searches performed on the dbest data base using the TBLASTN algorithm revealed several ESTs with homology to portions of the beta 3GalT1 sequence. From a total of four ESTs identified (all of which were from human brain), a continuous cDNA fragment encoding for about 102 amino acids of a putative new galactosyltransferase gene was assembled. This cDNA fragment allowed the design of primers, which were used to provide a diagnostic PCR signal for the presence of the new putative gene. From the strength of the PCR signal, the new gene was found to exist mainly in libraries from human brain, heart, and cells of the immune system (data not shown). The human brain library gave the strongest PCR signal and was subsequently used as a starting point for the identification of the new gene. To clone the new gene, the method of D'Esposito et al. (21) was used, which relies on successive levels of splits, tests, and expansions of lambda  phage pools to obtain successively enriched fractions of the target gene. Starting with approximately 1 million clones, the first split to 192 wells (level 0) provided 17 wells with a positive PCR signal. This translates into an abundance of the cDNA of at least 1 in 84,000 calculated on the basis of 950,000 independent lambda  phage clones used for the primary infection. Each of the level 0 wells potentially contained a single independent clone from the putative gene. Because the diagnostic PCR amplified signal was from an internal portion of the putative new gene, no information was obtained regarding the length of the various level 0 clones and it was therefore not possible to judge which of the positive wells contained the complete gene. Two level 0 wells were chosen for subsequent expansion solely on the strength and clarity of the PCR signal and were split into 96 separate wells. After testing and splitting again into 96 separate wells, finally single plaques were evaluated for insert size and the largest lambda gt10 clone GA4/1 was determined to contain an insert of ~3 kb.

DNA sequence analysis of lambda gt10 clone GA4/1 revealed that it contained a single, long open reading frame encoding for a protein of 422 amino acids with a predicted Mr of 49,202 (Fig. 1). Hydropathy analysis (Fig. 2) (33), as well as primary amino acid sequence analysis using the SAPS program (34), revealed a hydrophobic, putative transmembrane region at the amino-terminal end of the open reading frame (amino acids 27-43), predicting that this protein has a type II transmembrane topology, typical for all mammalian glycosyltransferases cloned to date.


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Fig. 2.   Hydropathy profile of beta 3GalT2. The hydropathy profile was determined by the program of Kyte and Doolittle (34) with a sliding window of nine amino acids. Positive numbers represent hydrophobic areas; negative numbers indicate hydrophilic areas.

BLAST searches of public data bases using either the deduced amino acids sequence of beta 3GalT2 or the complete cDNA sequence of clone GA4/1 revealed no significant homology with any protein in the Swissprot (release: June 23, 1997) or with any gene in the GenEMBL (release: June 26, 1997) data base. Furthermore, besides the four human brain EST sequences, which were originally used to design the diagnostic PCR primer pair for the cloning of beta 3GalT2, no additional matching entries were found in the EST data base (release: June 23, 1997).

Comparison of the new protein sequence with the 326-amino acid protein sequence of beta 3GalT1 revealed an overall sequence identity of 46%. The highest level of sequence similarity between the two beta 3-Gal-T sequences was found between positions 47 and 326 for beta 3GalT1 and between positions 119 and 405 for beta 3GalT2, respectively. The sequence identity in this region was calculated as 51% (67% similarity), with four 1-3-amino acid insertions found in beta 3GalT2 as compared with beta 3GalT1 (Fig. 3B). Due to the high sequence conservation, we assume that these regions most likely represent the catalytic domains of the two enzymes. Both enzymes contain two conserved potential N-glycosylation sites (N-X-(S/T)) in the putative catalytic domains, with three additional sites present in the stem and putative catalytic domain of beta 3GalT2 (Fig. 3). The major differences between beta 3GalT1 and beta 3GalT2 are a 17-amino acid extension at the carboxyl terminus of beta 3GalT2 and differences in both the lengths and primary sequences of the putative cytoplasmic, transmembrane, and stem regions (Fig. 3, A and B).


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Fig. 3.   Sequence alignment of beta 3GalT1 and beta 3GalT2. A, schematic representations of deduced amino acid sequences of beta 3GalT1 (19) and beta 3GalT2. The number of amino acids contained in each putative region are given above the schematic illustrations; putative N-glycosylation sites are indicated by a Y. The longer cytosolic region, stem region, and carboxyl-terminal extension of beta 3GalT2 are evident. B, amino acid sequence alignment of beta 3GalT1 and beta 3GalT2. The amino acids indicated in bold represent the putative transmembrane domain of the corresponding enzyme.

The high sequence homology within the catalytic domain between the two genes suggested that the new enzyme was also a beta 3-Gal-T. It remained, however, unclear whether the new enzyme transferred galactose to GlcNAc, as would be expected for a type 1 chain extending enzyme, or to other acceptors. Glycosyltransferase stem regions are known to influence enzyme acceptor specificity as has been reported for Fuc-T III and Fuc-T V (35, 36), and cytoplasmic domains are known to influence Golgi localization (37, 38). A distinct possibility therefore existed that beta 3GalT2 would transfer galactose to GalNAc to make the core 1 structure, or to other acceptors. Such an enzyme would be expected to have both different acceptor specificity and Golgi localization compared with beta 3GalT1. To test this possibility, we examined the acceptor specificity of beta 3GalT2 both in cell culture and in enzymatic assays.

Determination of Enzymatic Activity in Cell Culture-- The beta 3GalT2 gene was subcloned in an expression vector and checked for its ability to direct the synthesis of type 1 chains in CHO cells, which do not normally synthesize type 1 chains (39). To readily detect de novo production of type 1 chains, CHO cells were transfected with the gene for Fuc-T III. This enzyme fucosylates both type 1 and type 2 chains so that cells expressing it produce the corresponding fucosylated and also sialylated oligosaccharides (25, 39). CHO/Fuc-T III cells stained brightly with the anti-sLex antibody CSLEX (data not shown) but not with the anti-sLea antibody GSLA1 (Fig. 4, curve A). Transfection of CHO/Fuc-T III cells with a vector containing the newly cloned putative galactosyltransferase produced significant staining of these cells with GSLA1 (Fig. 4, curve B), indicating that the new gene is indeed a type 1 extension enzyme.


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Fig. 4.   Flow cytometry analysis of beta 3GalT2 transfected CHO cells. CHO/Fuc-T III cells transfected with beta 3GalT2 were analyzed by flow cytometry after staining with sLea-specific monoclonal antibody GSLA1 and FITC-labeled anti-mouse IgG antibody as indicated under "Experimental Procedures." Curve A is the histogram obtained from CHO/Fuc-T III control cells, and curve B is that obtained from CHO/Fuc-T III cells transfected with beta 3GalT2.

Production of Soluble beta 3GalT2/Protein A Fusion Protein and Enzymatic Assays-- The acceptor specificity of beta 3GalT2 was also directly established in enzymatic assays. To purify sufficient amounts of enzyme for analysis, we constructed and expressed a soluble beta 3GalT2/protein A fusion protein by removing the putative cytoplasmic, transmembrane, and part of the stem region and replacing them with the IgG binding domain of S. aureus protein A. The beta 3GalT2/protein A fusion protein was expressed in Sf9 cells and purified by ion exchange and affinity chromatography. Enzymatic assays with GlcNAc as the acceptor produced a new peak upon HPAE/PAD analysis, which co-eluted with Gal(beta 1-3)GlcNAc (Fig. 5). Activity assays were also performed using radiolabeled UDP-Gal and measuring the transfer of radioactivity to various sugar acceptors. Using this method, transfer of galactose to GlcNAc-Lemieux was readily observed. Using the radioactive assay with GlcNAc-Lemieux as the acceptor, the final purified beta 3GalT2/protein A fusion protein had a specific activity of about 20 units/mg. This specific activity is typical for several other glycosyltransferase fusion proteins using simple oligosaccharides as acceptors.2


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Fig. 5.   HPAE/PAD analysis of the beta 3GalT2 reaction product. The enzymatic assay was performed using purified protein A/beta 3GalT2 fusion protein with GlcNAc as acceptor substrate and analyzed as indicated under "Experimental Procedures." Curve A is the trace obtained with the authentic standards GlcNAc, LacNAc [Gal(beta 1-4)GlcNAc], and Gal(beta 1-3)GlcNAc as indicated. Curve B is the trace obtained with the complete assay mix at time 0, and curve C is the trace obtained after incubation for 120 min at 37 °C.

Surprisingly, beta 3GalT2 transfers galactose to Gal-terminating acceptors. The results in Table I show Gal to be a relatively good acceptor, with LacNac and lactose being distinctly worse. The relative efficiency of the substrates in Table I, however, must be interpreted with caution since the hydrophobic groups attached on their anomeric sites are different. No transfer was observed to GalNAcalpha -benzyl even when a 10-fold increase of enzyme was used (data not shown). As shown in Table I, beta 3GalT1 also transfers to Gal, but at a much lower relative rate and with more restricted specificity than beta 3GalT2. The reactivity pattern of beta 3GalT2 is partially reminiscent of the human kidney beta 3-galactosyltransferase (11). Although the assays for the kidney enzyme were performed at different conditions (pH 5, 4 mM Cd2+), this enzyme was reported to accept a series of Gal terminating acceptors and to be influenced by the presence of hydrophobic groups at the anomeric site. Unfortunately, no assays with GlcNAc terminating acceptors were reported with the kidney enzyme leaving open the question whether it can synthesize type 1 chains. It is thus not clear if beta 3GalT2 and the kidney beta 3-galactosyltransferase are similar enzymes, although Northern blots show no significant signal for beta 3GalT2 in kidney (Fig. 6). The acceptor specificity of beta 3GalT2 is distinct from those reported for the snail and marsupial beta 3-galactosyltransferases (12, 13), and we expect that these enzymes are also molecularly different. Carbohydrate structures terminating in Gal(beta 1-3)Galbeta - have been reported in both glycoproteins (40, 41) and glycolipids (42). The in vitro specificity of beta 3GalT2 suggests that it is capable of synthesizing such structures.

                              
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Table I
Substrate specificities of beta 3GalT1 and beta 3GalT2
Acceptor substrates were assayed at the indicated concentrations using the radioactivity transfer assay as described under "Experimental Procedures." The same amounts of enzyme were used in all assays. The concentration of acceptor substrate in the assay mix were as indicated. Results are presented as percent conversion with transfer to GlcNAc having been normalized to 100%. ND indicates no detectable transfer under the assay conditions.


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Fig. 6.   Northern blot analysis of beta 3GalT2. Poly(A)+ RNA (2 µg/lane) fractionated on a 1.2% formaldehyde/agarose gel was hybridized under stringent conditions with a 32P-labeled cDNA fragment encoding for the complete open reading frame of beta 3GalT2. RNA was obtained from the indicated human tissues.

Northern Blot Analysis-- The complete open reading frame of beta 3GalT2 was used to probe polyadenylated mRNA from eight different human tissues. Two transcripts were identified by this procedure, with a strongly hybridizing transcript at 3.5 kb and a weaker one of 2.8 kb in size (Fig. 6). Both transcripts were detected only in heart and brain; no transcripts were detectable in placenta, lung, liver, skeletal muscle, kidney and pancreas. Thus, expression of this new beta 1-3- or beta 3-galactosyltransferase is probably regulated in a tissue-specific and cell-specific manner. This is in contrast to the beta 4-Gal-T enzyme, which is transcribed in most tissues (reviewed in Refs. 29 and 43). No information is yet available about the distribution of beta 3GalT1. Preliminary PCR experiments using libraries from different tissues and cell types showed clear differences in the distribution of beta 3-Gal-T1 and beta 3GalT2.3

The beta 3GalT2 described here represents the second type 1 chain extending enzyme cloned. The homology of the two genes in the catalytic domain suggests that they correspond to an evolutionary related family of beta 3-gal-T genes, but the wide divergence both in size and sequence in the cytosolic and stem regions implies different acceptors for the two enzymes and possibly different Golgi compartmentalization. It is plausible that one of the genes is specific for mucin and glycolipid acceptors and the other for N-linked glycoproteins. The restricted tissue specificity of beta 3GalT2 seems to indicate a specific role for this enzyme. At this time, it is not known whether the beta 3-Gal-T family is restricted to these two members or if more genes exist, as is the case for the alpha 3-fucosyltransferases (29). The presence of additional enzymes would imply a diversity of type 1 bearing structures with perhaps varying roles. The availability of the beta 3GalT2 sequence will allow addressing these questions by molecular means and possibly uncovering the physiological functions of type 1 chains.

    ACKNOWLEDGEMENTS

We thank Yves Henriquez and Anja Aenis for excellent technical assistance, Reinhold Öhrlein and Gabi Baisch for providing all the Lemieux substrates, John Lowe for providing the gene for Fuc-T III, and John Magnani for providing GSLA-1.

    FOOTNOTES

* 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) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y15014.

Dagger To whom correspondence should be addressed: Novartis Pharma AG, S386.645, CH-4002 Basel, Switzerland. E-mail: andreas.katopodis{at}pharma.novartis.com.

1 The abbreviations used are: beta 4-Gal-T, UDP-galactose:2-acetamido-2-deoxy-D-glucose 4beta -galactosyltransferase; beta 3-Gal-T, UDP-galactose:2-acetamido-2-deoxy-D-glucose 3beta -galactosyltransferase; sLea, Neu5Ac(alpha 2-3)Gal(beta 1-3)[Fuc(alpha 1-4)]GlcNAc; sLex, Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc; EST, expressed sequence tag; FACS, fluorescence-activated cell sorting; UDP-Gal, uridine diphospho-D-galactose; GlcNAc-Lemieux, GlcNAcbeta O-(CH2)8-CO2Me; Gal-Lemieux, Galbeta O-(CH2)8-CO2Me; LacNAc-Lemieux, Gal(beta 1-4)GlcNAcbeta O-(CH2)8-CO2Me; GalNAcalpha -benzyl, benzyl 2-acetamido-2-deoxy-alpha -D-galactopyranoside; BSA, bovine serum albumin; PCR, polymerase chain reaction; HPAE-PAD, high pH anion exchange chromatography with pulsed amperometric detection; CHO, Chinese hamster ovary; bp, base pair(s); kb, kilobase pair(s); PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; Fuc-T, fucosyltransferase.

2 M. Streiff, unpublished data.

3 F. Kolbinger, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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