Control of O-Glycan Branch Formation
MOLECULAR CLONING OF HUMAN cDNA ENCODING A NOVEL beta 1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE FORMING CORE 2 AND CORE 4*

Tilo SchwientekDagger , Mitsuharu Nomoto§, Steven B. Levery, Gerard Merkxparallel , Ad Geurts van Kesselparallel , Eric P. BennettDagger , Michael A. Hollingsworth§, and Henrik ClausenDagger **

From the Dagger  School of Dentistry, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark, the § Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, the  University of Georgia, Complex Carbohydrate Research Center, 220 Riverbend Road, Athens, Georgia 30602, and the parallel  Department of Human Genetics, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands

    ABSTRACT
Top
Abstract
Introduction
References

A novel human UDP-GlcNAc:Gal/GlcNAcbeta 1-3GalNAcalpha beta 1,6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-alpha -D-glucosamine:acceptor beta 1,6-N-acetylglucosaminyltransferase (beta 1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a beta 1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in beta 1,6GlcNAc-transferases capable of forming core O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

    INTRODUCTION
Top
Abstract
Introduction
References

Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide GalNAc-transferases that add GalNAc to selected Ser and Thr residues (1). Further assembly of O-glycan chains involves different biosynthetic pathways: (i) formation of simple mucin-type core 1 structures by UDP-Gal:GalNAcalpha beta 1,3Gal-transferase activity; (ii) conversion of core 1 to complex-type core 2 structures by UDP-GlcNAc:Galbeta 1-3GalNAcalpha beta 1,6GlcNAc-transferase1 activities; (iii) direct formation of complex mucin-type core 3 by UDP-GlcNAc:GalNAcalpha beta 1,3GlcNAc-transferase activities; and (iv) conversion of core 3 to core 4 by UDP-GlcNAc:GlcNAcbeta 1-3GalNAcalpha beta 1,6GlcNAc-transferase activity (for an overview see Ref. 2; see also Fig. 1). Elongation and termination of complex oligosaccharide structures involves a large number of glycosyltransferases. Synthesis of the different O-glycan structures is cell- and tissue-specific. Different core structures are produced upon differentiation and malignant transformation (3-7). For example, increased formation of GlcNAcbeta 1-6GalNAc branching in O-glycans has been demonstrated during T-cell activation, during the development of leukemia, and for immunodeficiencies like Wiskott-Aldrich syndrome and AIDS (3, 8, 9). Core 2 branching may play a role in tumor progression and metastasis (10). In contrast, many carcinomas show changes from complex O-glycans found in normal cell types to immaturely processed simple mucin-type O-glycans such as T (Thomsen-Friedenreich antigen; Galbeta 1-3GalNAcalpha 1-R), Tn (GalNAcalpha 1-R), and sialosyl-Tn (NeuAcalpha 2-6GalNAcalpha 1-R) (see Fig. 1) (11). The molecular basis for this has been extensively studied in breast cancer, where it was shown that specific down-regulation of core beta 6GlcNAc-transferase was responsible for the observed lack of complex type O-glycans on the mucin MUC1 (7). Interestingly, the metastatic potential of tumors has been correlated with increased expression of core 2 beta 6GlcNAc-transferase activity (6). The increase in core 2 beta 6GlcNAc-transferase activity was associated with increased levels of poly-N-acetyllactosamine chains carrying sialyl-Lex, which may contribute to tumor metastasis by altering selectin-mediated adhesion (5, 12). The control of O-glycan core assembly is regulated by the expression of key enzyme activities outlined in Fig. 1; however, epigenetic factors including post-translational modification, topology, or competition for substrates may also play a role in this process (13).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Biosynthetic pathways of mucin-type O-glycan core structures. The abbreviations used are: GalNAc-T, polypeptide alpha GalNAc-transferase; ST6GalNAcI, mucin alpha 2,6 sialyltransferase I; C1beta 3Gal-T, core 1 beta 1,3-galactosyltransferase; C2GnT, core 2 beta 1,6GlcNAc-transferase; C2/4GnT, core 2/core 4 beta 1,6GlcNAc-transferase; C3GnT, core 3 beta 1,3GlcNAc-transferase; ST3GalI, mucin alpha 2,3 sialyltransferase I; beta 4Gal-T, beta 1,4-galactosyltransferases; beta 3Gal-T, beta 1,3-galactosyltransferases; beta 3GnT, elongation beta 1,3GlcNAc-transferase.

The kinetic properties and substrate specificities of the enzymes responsible for O-glycan core formation have been extensively studied (14-20), and one of these enzymes, a core 2 beta 6GlcNAc-transferase (C2GnT),2 has been cloned and characterized to date (21). Human C2GnT was identified by a transfection cloning strategy, and it shows similarity to another beta 6GlcNAc-transferase (IGnT) that is responsible for poly-N-acetyllactosamine branching (22). Surprisingly, these two genes were not similar in sequence to GnTV, which is responsible for formation of the beta 6GlcNAc branch in tetraantennary N-linked glycans (23). Studies of the kinetic properties and acceptor substrate specificities of beta 6GlcNAc-transferases involved in O-glycosylation from different cell lines and organs have suggested that multiple beta 6GlcNAc-transferases exist and that one of these would function in a manner that is distinct from the cloned C2GnT enzyme, in being capable of forming core 2, core 4, and possibly the branched I antigen structure (17, 18). The existence of a homologous beta 6GlcNAc-transferase family containing C2GnT and IGnT suggests that additional beta 6GlcNAc-transferase activities may be encoded by homologous genes (22). Recently, similar genes for large families of galactosyltransferases (24-26) have been identified by using the expressed sequence tag (EST) information for similarity searches. This report describes the use of this strategy for identification of a novel member of the beta 6GlcNAc-transferase family that forms core 2 as well as core 4 structures.

    EXPERIMENTAL PROCEDURES

Identification of C2/4GnT-- The BLASTn and tBLASTn were used with the reported coding sequence of human C2GnT (GenBankTM accession number M97347) to search the dbEST data base at The National Center for Biotechnology Information as described previously (24). Human EST and genomic bovine sequences similar but not identical to known members of the beta 6GlcNAc-transferase family were identified.

Cloning and Sequencing of the Gene Encoding C2/4GnT-- EST clone 178656 (5' EST GenBankTM accession number AA307800), derived from a putative homologue to C2GnT, was obtained from the American Type Culture Collection. Sequencing of this clone revealed a partial open reading frame with significant sequence similarity to C2GnT. The coding region of human C2GnT and a bovine homologue was previously found to be organized in one exon (27).3 Because the 5' and 3' sequence available from the C2/4GnT EST was incomplete but likely to be located in a single exon, the missing 5' and 3' portions of the open reading frame were obtained by sequencing genomic P1 clones. P1 clones were obtained from a human foreskin genomic P1 library (DuPont Merck Pharmaceutical Co. Human Foreskin Fibroblast P1 Library) by screening with the primer pair TSHC27 (5'-GGAAGTTCATACAGTTCCCAC-3') and TSHC28 (5'-CCTCCCATTCAACATCTTGAG-3'). Two genomic clones for C2/4GnT, DPMC-HFF#1-1026(E2) and DPMC-HFF#1-1091(F1), were obtained from Genome Systems Inc. DNA from P1 phage was prepared as recommended by Genome Systems Inc. The entire coding sequence of the C2/4GnT gene was represented in both clones and sequenced in full using automated sequencing (ABI377, Perkin-Elmer). Confirmatory sequencing was performed on a cDNA clone obtained by PCR (30 cycles at 95 °C for 15 s, 55 °C for 20 s, and 68 °C for 2 min 30 s) on total cDNA from the human COLO 205 cancer cell line with the sense primer TSHC 54 (5'-GCAGAATTCATGGTTCAATGGAAGAGACTC-3') and the antisense primer TSHC 45 (5'-AGCGAATTCAGCTCAAAGTTCAGTCCCATAG-3'). The composite sequence contained an open reading frame of 1314 base pairs encoding a putative protein of 438 amino acids with type II domain structure predicted by the TMpred-algorithm at the Swiss Institute for Experimental Cancer Research.4 The sequence of the 5' end of C2/4GnT mRNA including the translational start site and 5'-UTR was obtained by 5' rapid amplification of cDNA ends (35 cycles at 94 °C for 20 s, 52 °C for 15 s, and 72 °C for 2 min) using total cDNA from the human COLO 205 cancer cell line with the antisense primer TSHC 48 (5'-GTGGGAACTGTATGAACTTCC-3') (see Fig. 2).

Expression of C2/4GnT and C2GnT in Insect Cells-- An expression construct designed to encode amino acid residues 31-438 of C2/4GnT was prepared by PCR using P1 DNA and the primer pair TSHC55 (5'-CGAGAATTCAGGTTGAAGTGTGACTC-3') and TSHC45 (see Fig. 2). The PCR product was cloned into the EcoRI site of pAcGP67A (PharMingen), and the insert was fully sequenced. An expression construct encoding amino acid residues 40-428 of human C2GnT was prepared by PCR on genomic DNA of a healthy male blood donor using the primer pair EBCT1 (5'-AGCGGATCCTTTGTAAGTGTCAGACACTTGGAG-3') and EBCT2 (5'-AGCGGATCCAAAATTGCCCGTAATGGTCAGTG-3'). The PCR product was cloned into the BamHI site of pAcGP67B (PharMingen), and the sequence was found to be identical to the sequence originally reported by Bierhuizen and Fukuda (21). Plasmids pAcGP67-C2/4GnT-sol and pAcGP67-C2GnT-sol were co-transfected with Baculo-GoldTM DNA (PharMingen) as described previously (24). Recombinant Baculo-virus were obtained after two successive amplifications in Sf9 cells grown in serum-containing medium, and titers of virus were estimated by titration in 24-well plates with monitoring of enzyme activities. Controls included the pAcGP67-GalNAc-T3-sol (28). The kinetic properties were determined with partially purified enzymes expressed in High FiveTM cells. Partial purification was performed by consecutive chromatography on Amberlite IRA-95, DEAE-Sephacryl, and CM-Sepharose essentially as described (29). Protein concentrations were determined using the Bio-Rad reagent with bovine serum albumin as standard protein.

Enzymatic Assays and Product Characterization-- Standard assays were performed using culture supernatant from infected cells in 50-µl reaction mixtures containing 100 mM MES (pH 8.0 for C2/4GnT and pH 7.0 for C2GnT), 10 mM (C2/4GnT) or 5 mM EDTA (C2GnT), 10 mM 2-acetamido-2-deoxy-D-glucono-1,5-lacton, 180 µM UDP-[14C]-GlcNAc (6,000 cpm/nmol) (Amersham Pharmacia Biotech), and the indicated concentrations of acceptor substrates (Sigma and Toronto Research Laboratories Ltd.; see Table I for structures). Semi-purified C2GnT and C2/4GnT were assayed in 50-µl reaction mixtures containing 100 mM MES (pH 7), 5 mM (C2/4GnT) or 2 mM EDTA (C2GnT), 1 mM dithiothreitol (C2GnT), 90 µM UDP-[14C]-GlcNAc (3,050 cpm/nmol) (Amersham Pharmacia Biotech), and the indicated concentrations of acceptor substrates. Reaction products were quantified by chromatography on Dowex AG1-X8. Complete glycosylation of core3-pNph was performed in a reaction mixture consisting of 6.9 milliunits C2/4GnT (specific activity determined with core1-pNph), 2 mg core3-pNph, 100 mM MES (pH 7.0), 5 mM EDTA, 4.6 µmol UDP-GlcNAc, and 100 milliunits alkaline phosphatase in a final volume of 200 µl. The glycosylation of core3-pNph was monitored by thin layer chromatography and run for 8 h until completed. The reaction product was purified on an octadecyl-silica cartridge (Bakerbond; J. T. Baker), deuterium exchanged by repeated lyophilization from D2O and then dissolved in 0.5 ml of D2O for NMR analysis. One-dimensional 1H NMR, two-dimensional 1H-1H TOCSY (30, 31) and ROESY (32, 33), 1H-detected, 13C-decoupled, phase sensitive, gradient (34) 13C-1H HSQC (35), and HMBC (36, 37) experiments were performed at 298 K on a Varian Unity Inova 600 MHz spectrometer using standard acquisition software available in the Varian VNMR software package. A 2-mg sample of core3-pNph was prepared in similar fashion and analyzed under identical conditions for comparison. Chemical shifts are referenced to internal acetone (2.225 and 29.92 ppm for 1H and 13C, respectively).

Northern Analysis-- Total RNA was isolated from human colon and pancreatic adenocarcinoma cell lines AsPC-1, BxPC-3, Capan-1, Capan-2, COLO 357, HT-29, and PANC-1 essentially as described (38). 25 µg of total RNA was subjected to electrophoresis on a 1% denaturing agarose gel and transferred to nitrocellulose as described previously (38). The cDNA fragment of soluble C2/4GnT was used as a probe for hybridization. The probe was random primer-labeled using [alpha 32P]dCTP and an oligonucleotide labeling kit (Amersham Pharmacia Biotech). The membrane was probed overnight at 42 °C as described previously (28) and washed twice for 30 min each at 42 °C with 2 × SSC, 0.1% SDS and twice for 30 min each at 52 °C with 0.1 × SSC, 0.1% SDS. Human multiple tissue Northern blots, MTN I and MTN II (), were probed as described above and washed twice for 10 min each at room temperature with 2 × SSC, 0.1% SDS; twice for 10 min each at 55 °C with 1 × SSC, 0.1% SDS; and once for 10 min with 0.1 × SSC, 0.1% SDS at 55 °C.

Chromosomal Localization of C2/4GnT: in Situ Hybridization to Metaphase Chromosomes-- Fluorescence in situ hybridization was performed on normal human lymphocyte metaphase chromosomes using procedures described previously (24). For evaluation of the chromosomal slides a Zeiss epifluorescence microscope equipped with appropriate filters for visualization of fluorescein isothiocyanate was used. Hybridization signals and 4,6-diamidino-2-phenylindole-counterstained chromosomes were transformed into pseudo-colored images using image analysis software. For precise localization and chromosome identification 4,6-diamidino-2-phenylindole-converted banding patterns were generated using the BDS-imageTM software package (ONCOR).

    RESULTS

Isolation and Characterization of Human C2/4GnT-- Analysis of the GenBankTM and dbEST data bases suggested the existence of additional members of the beta 6GlcNAc-transferase family. EST clones encoding the putative enzymes were obtained, and the full coding sequence of a novel gene with significant sequence similarity to human C2GnT (21) and blood group IGnT (22) was obtained by 5'-rapid amplification of cDNA ends and analysis of P1 DNA. The sequence encoded a type II transmembrane protein of 438 amino acids with an N-terminal cytoplasmic domain of 9 residues, a transmembrane segment of 18 residues, a stem region, and catalytic domain of 411 residues with two potential N-linked glycosylation sites (Fig. 2). A Kyte and Doolittle (39) hydropathy plot of C2/4GnT showed similarity to C2GnT and indicated that the putative stem region was hydrophilic, similar to other Golgi-localized glycosyltransferases (data not shown). The cloned cDNA included a single initiation codon according to the Kozak rule (40). The 3'-UTR of 510 base pairs was confirmed in additional EST clones and contains a polyadenylation signal at base pair 1749 (+435) (Fig. 2).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 2.   Nucleotide sequence and predicted amino acid sequence of human C2/4GnT. The amino acid sequence is shown in single-letter codes. The hydrophobic segment representing the putative transmembrane domain is underlined with a double line (Kyte and Doolittle (39), window of 8). Two consensus motifs for N-glycosylation are indicated by asterisks. The location of the primers used for preparation of the expression constructs are underlined with a single line. A potential polyadenylation signal is indicated in boldface underlined type.

A multiple sequence alignment (ClustalW) of three human beta 6GlcNAc-transferases is shown in Fig. 3. C2/C4GnT shows a higher overall amino acid sequence identity to human C2GnT (52%) than to human IGnT (41%). Sequence similarities among the three human proteins are found predominantly in the putative catalytic domains, and no significant sequence similarities were detected in the N-terminal regions. Eight cysteine residues are conserved in all three beta 6GlcNAc-transferases; a ninth cysteine residue in the N-terminal region of C2GnT and C2/C4GnT was not found in IGnT (Fig. 3). N-linked glycosylation consensus sequence sites are not generally conserved in homologous as well as orthologous glycosyltransferases, although one site is conserved in the central region of four beta 3Gal-transferases (25) and one site is conserved in the C-terminal region of five of six beta 4Gal-transferases (41). Interestingly, a single potential N-linked site located in the stem region of C2/4GnT, C2GnT and IGnT is conserved (Fig. 3). The two N-glycosylation sites found in C2GnT are utilized, and the N-Glycan in the conserved position was shown to be essential for function of the recombinant enzyme (42).


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 3.   Multiple sequence analysis (ClustalW) of human C2/4GnT, C2GnT, and IGnT. Introduced gaps are shown as hyphens, and aligned identical residues are boxed (black for all sequences, and gray for two sequences). The putative transmembrane domains are underlined with a single line. The positions of conserved cysteines are indicated by asterisks. One conserved N-glycosylation site is indicated by an open circle.

Genomic Organization and Chromosomal Localization-- The coding region of the human C2/4GnT gene was found to be organized in a single exon, similar to the genomic structure of the human C2GnT gene (27). The human C2GnT gene was previously found to be localized in a gene cluster with IGnT localized on chromosome 9q21 (22). However, analysis of the Human Gene Map at GenBankTM (The National Center for Biotechnology Information) indicates that the IGnT gene is located on chromosome 6p24 between microsatellite markers D6S1674 and D6S470 (13-17 cM, SHGC-12039). Bierhuizen et al. (22) observed weak binding of a genomic IGnT clone to p23 of chromosome 6 as well as a major hybridization signal at 9q21. This may suggest the existence of an additional highly similar gene or pseudogene at band q21 of chromosome 9. The existence of two IGnT enzyme forms has recently been suggested by Leppänen et al. (43). By fluorescence in situ hybridization of the genomic clone DPMC-HFF#1-1091(F1) to human metaphase chromosomes, the C2/4GnT gene was found to reside on chromosome 15q21.3 (Fig. 4).


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 4.   Fluorescence in situ hybridization of C2/4GnT to metaphase chromosomes. The C2/4GnT probe (C4T, P1 DNA from clone DPMC-HFF#1-1091[F1]) labeled band 15q21.3.

Expression of C2/4GnT-- Transfection of an expression construct encoding soluble C2/4GnT into insect cells resulted in marked increase in GlcNAc-transferase activity compared with uninfected cells or cells infected with a control construct. C2/4GnT showed significant activity with disaccharide derivatives of O-linked core 1 (Galbeta 1-3GalNAcalpha 1-R) and core 3 structures (GlcNAcbeta 1-3GalNAcalpha 1-R). In contrast, no activity was found with lacto-N-neo-tetraose as well as GlcNAcbeta 1-3Gal-methyl as acceptor substrates, indicating that C2/4GnT has no IGnT-activity. Additionally, no activity could be detected wih alpha -D-GalNAc-1-para-nitrophenyl indicating that C2/4GnT does not form core 6 (GlcNAcbeta 1-6GalNAcalpha 1-R) (Table I). No substrate inhibition of enzyme activity was found at high acceptor concentrations up to 20 mM core1-para-nitrophenyl or core3-para-nitrophenyl. C2/4GnT shows strict donor substrate specificity for UDP-GlcNAc; no activity could be detected with UDP-Gal or UDP-GalNAc (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Substrate specificities of C2/4GnT and C2GnT

Characterization of Core 4 Product by 1H and 13C NMR Spectroscopy-- The product derived from reaction of the putative Core 4 beta 6GlcNAc-transferase with beta -D-GlcNAc-(1-3)-alpha -D-GalNAc-1-para-nitrophenyl was characterized by NMR spectroscopy to confirm that the proper linkage was formed between the donor sugar and the acceptor substrate. Comparison of a one-dimensional 1H NMR spectrum of the product (Fig. 5) with that of the substrate (data not shown) clearly showed an additional H-1 resonance (4.454 ppm) from a sugar residue linked in the beta -configuration (3J1,2 = 7-9 Hz). Because we were unable to find NMR data for the para-nitrophenyl glycosides of either the core 3 substrate or the expected core 4 product in the literature or in glycoconjugate NMR data bases and because the substantial anisotropic effects of the para-nitrophenyl group obviate direct comparison of chemical shift data with those of the benzyl-glycosides (44), a de novo sequence analysis of the product was undertaken by consecutive application of two-dimensional 1H-1H TOCSY, 1H-13C HSQC, and 1H-13C HMBC NMR experiments (for a review of this strategy, see Ref. 45). Thus, all 1H and 13C resonances were assigned by the TOCSY and HSQC experiments (Table II; the H-6R and H-6S resonances for each residue were differentiated by comparison of their 3J5,6 coupling constants with those published previously for the corresponding benzyl-glycosides (44)); then the linkages were unambiguously established by observation of interglycosidic H1-C1-O1-Cx and C1-O1-Cx-Hx correlations in the HMBC spectrum. As shown in Fig. 6, the newly formed GlcNAcbeta 1 right-arrow 6GalNAcalpha linkage in the product is clearly demonstrated by strong cross-peaks correlating the beta -GlcNAc H-1 at 4.454 ppm with alpha -GalNAc C6 and the corresponding beta -GlcNAc C-1 at 100.93 ppm with both alpha -GalNAc H-6 resonances. Consistent with this, rotating frame Overhauser enhancements were observed between beta -GlcNAc H-1 and alpha -GalNAc H-6 in a ROESY spectrum (data not shown).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   Sections of a one-dimensional 1H NMR spectrum of the core 4 beta 6GlcNAc-transferase product. GlcNAcbeta 1-3(GlcNAcbeta 1,6)GalNAcalpha 1-1-pNph, showing all nonexchangeable monosaccharide ring methine and exocyclic methylene resonances. Residue designations for GlcNAcbeta 1 right-arrow 3 (beta 3), GlcNAcbeta 1 right-arrow 6 (beta 6), and GalNAcalpha 1 right-arrow 1 (alpha ) are followed by proton designations (1-6). All resonances in this region except for beta 3-5 (3.453 ppm) are marked.

                              
View this table:
[in this window]
[in a new window]
 
Table II
1H, 13C chemical shifts (ppm) and 1H-1H coupling constants J (Hz) for p-nitrophenyl glycoside core 3 substrate and biosynthetic core 4 product


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Section of the 1H-detected 1H-13C HMBC spectrum of the Core 4 beta 6GlcNAc-transferase product, showing interglycosidic H1-C1-O1-Cx and C1-O1-Cx-Hx correlations (cross-peaks marked by ovals). The unmarked cross-peaks are all intra-residue correlations.

Expression Pattern of C2/4GnT-- ESTs from C2/4GnT were derived from colonic and pancreatic cancer tissues as well as germ cell tumors. Northern analysis with mRNA from 16 healthy human adult organs showed expression of C2/4GnT in organs of the gastrointestinal tract with high transcription levels observed in colon and kidney and lower levels in small intestine and pancreas (Fig. 7A). To investigate changes in expression of C2/4GnT in cancer cells derived from tissues normally expressing C2/4GnT, mRNA levels in a panel of human adenocarcinoma cell lines were determined. Analyses of C2/4GnT transcription levels revealed differential expression in pancreatic cell lines; Capan-1 and AsPC-1 expressed the transcript, whereas PANC-1, Capan-2, BxPC-3, and COL0357 did not (Fig. 7B). The colonic cell line HT-29 expressed transcripts of C2/4GnT. The size of the predominant transcript was approximately 2.4 kilobases, which correlates to the transcript size of 2.1 kilobases of the smallest of three transcripts of human C2GnT (21). Additionally, transcripts of approximately 3.4 and 6 kilobases were obtained in mRNA from healthy colonic mucosa (Fig. 7A). The two additional transcripts may resemble the 3.3- and 5.4-kilobase transcripts of C2GnT, which have not yet been characterized. Multiple transcripts of C2GnT have been suggested to be caused by differential usage of polyadenylation signals, which affects the length of the 3'-UTR (21).


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 7.   Northern blot analysis of healthy human tissues and pancreatic and colon tumor cell lines. A, multiple human tissue Northern blots, MTN I and MTN II, from were probed with a 32P-labeled probe corresponding to the soluble expression fragment of C2/4GnT (base pairs 91-1317). B, a Northern blot of total RNA from human colonic and pancreatic adenocarcinoma cell lines was probed as described for A.


    DISCUSSION

Additional members of a human beta 6GlcNAc-transferase gene family were previously predicted from analysis of enzyme activities as well as by low stringency Southern blot analysis (17, 22, 46). An EST cloning strategy produced a novel beta 6GlcNAc-transferase, designated C2/4GnT. The C2/4GnT enzyme is most similar in sequence to C2GnT, and it has the same simple genomic organization of the coding region as C2GnT. The recombinant forms of these enzymes have partly overlapping functions. The IGnT is apparently a more distant member of the family, and it has a different function (22). Three regions of extensive homology have been identified in C2GnT and IGnT: region A in the N-terminal and regions B and C in the C-terminal portion of the catalytic domain. C2/4GnT shows a particularly high degree of homology to C2GnT in region A, supporting the hypothesis that these domains could be directly involved in acceptor binding (22).

Previous analysis of enzyme activities in cell extracts suggested that there exists an enzyme capable of synthesizing core 4 as well as core 2. Brockhausen et al. (17) found that core 2 and core 4 beta 6GlcNAc-transferase activities of colon had similar properties and functioned independently of bivalent cations. Competition experiments with the two substrates yielded no additive effect; it therefore was suggested that in colon one enzyme was responsible for core 2 and core 4 biosynthesis. Ropp et al. (47) found that a bovine tracheal beta 6GlcNAc-transferase purified to apparent homogeneity with a molecular weight of 69,000 (specific activity, 70 units/mg with Galbeta 1-3GalNAcalpha -benzyl) utilized both core 1 and 3 acceptors. In addition, activity was detected with GlcNAcbeta 1-3Galbeta 1-R for I antigen biosynthesis. This beta 6GlcNAc-transferase activity has been designated the M-form for the mucin-secreting tissue form of C2GnT in contrast to the recently cloned leukocyte form or L-form, which is restricted to the formation of core 2 by accepting Galbeta 1-3GalNAcalpha -R substrates exclusively (4, 20, 21). The C2/4GnT enzyme reported here shows broader acceptor substrate specificity in accepting core 1 as well as core 3 but does not resemble the mucin-secreting tissue form because it lacks IGnT activity. C2/4GnT showed approximately 3-4-fold better activity with core 1-para-nitrophenyl than core 3-para-nitrophenyl, which is similar to the differences observed for both the bovine and porcine tracheal enzymes (47, 48).

Species-specific differences in core 2 beta 6GlcNAc-transferase activities may exist. Sekine et al. (49) reported that a purified mouse kidney beta 6GlcNAc-transferase activity utilized the Gal-Gb4 glycosphingolipid as well as core 1 substrates. Subsequently, evidence was presented that the mouse enzyme was encoded by the orthologous gene of human C2GnT (50). Interestingly, mouse kidney cells express a 5'-UTR spliced version of C2GnT that is distinct from other organs. The novel 5'-UTR does not affect the coding region (50). Whereas the normal C2GnT version was ubiquitously expressed at very low levels, a kidney form was highly expressed in Balb/c mice. DBA/2 mice, which lack the branched Gal-Gb4, showed no expression in kidney. To the best of our knowledge, studies have not determined whether human C2GnT or IGnT can use Gal-Gb4 as substrate. This structure was not available for testing in the present study.

The existence of beta 6GlcNAc-transferases with unique functions in either core 2 or blood group I synthesis was predicted from analysis of activities in different organs. The cloning of C2GnT and IGnT confirmed this prediction (21, 22). The C2GnT gene is widely expressed, although the levels may be quite low.3 In contrast, C2/4GnT showed a restricted expression pattern with high expression in colon (Fig. 7A). The mucin-type core 4 structure is less commonly found in mucins than core 1 and 2 (51-53) and has been found predominantly in gastric, respiratory, and colonic mucin preparations (54, 55). However, in some studies colonic mucins have been found to carry exclusively core 3-based structures (56, 57). C2GnT and C2/4GnT may provide redundancy for core 2 synthesis in some cells and tissues. This may be relevant to studies that demonstrated altered expression of core 2 in carcinomas (4, 6, 7). Interestingly, the major core 2 beta 6GlcNAc-transferase activity in normal colonic mucosa may be C2/4GnT, because competition experiments for core 1 and core 3 acceptors revealed no additive effect (17). This is further supported by recent reports showing that the mucin-type core 2 beta 6GlcNAc-transferase activity, which is expressed in healthy colonic mucosa, is replaced by the leukocyte form in colon cancer tissues (4). Further studies of the expression of the two enzymes are required for understanding their individual roles in cellular regulation of core 2 biosynthesis under normal physiological conditions and during progression to cancer (58).

The existence of several additional genes in the beta 6GlcNAc-transferase gene family may be predicted. Leppänen et al. (43) provided evidence for an additional I beta 6GlcNAc-transferase activity and found that this activity differs from the cloned IGnT by acting on the penultimate galactose in poly-N-acetyllactosamine chains and not on galactose residues located centrally in an oligosaccharide chain. Hybrid globo-lactoseries glycosphingolipids may have beta 6GlcNAc branching either to the alpha Gal found in Ref. 59 or beta GalNAc of galactosyl-Gb4 or Gb4 (49).

Originally the IGnT and C2GnT genes were mapped by isotopic in situ hybridization to a gene cluster at 9q21. Genomic DNA clones of C2GnT and IGnT showed a major hybridization signal at 9q21. Additionally weak binding of the IGnT clone to 6p23 was observed (22). C2/4GnT was shown in the present study by in situ hybridization to be located at 15q21.3. The localization of the IGnT gene may require reevaluation, because linkage analysis of 3' ESTs (SHGC-12039) for this gene indicates that it is located at 6p24 (60). Most glycosyltransferase gene families are not arranged in gene clusters (24-26, 61, 62). It is possible that only the alpha 2- and alpha 3/4-fucosyltransferase families have members clustered at one locus (63). Interestingly, the three members of the alpha 3/4-fucosyltransferase family co-localized to chromosome 19 are highly similar in sequence. These appear to have been duplicated very recently, because only one gene for this cluster was found in the cow (64). Two more divergent members of this family are localized differently.

In summary the present data confirm the existence of a beta 6GlcNAc-transferase capable of forming both core 2 and core 4 and establish that core 2 O-glycan branching is controlled by multiple enzymes. This may have important implications for interpreting the molecular events underlying characteristic changes in O-glycan branching during cell development and malignant transformation. The in vitro biosynthesis of O-glycopeptide structures is presently hampered by lack of availability of the key enzymes adding either galactose or N-acetylglucosamine in a beta 1-3 linkage to GalNAcalpha 1-O-Ser/Thr to form core 1 and core 3, because these are required for the enzymes responsible for the build-up of complex type structures (Fig. 1). Most other enzymes required for elongation of branched O-glycans are available, and the core 2/4 enzyme described herein now makes the synthesis of core 4-based structures possible.

    ACKNOWLEDGEMENTS

We thank Dr. Harry Schachter for helpful suggestions and critical reading of the manuscript. We gratefully acknowledge the technical support of Dr. John Glushka (Complex Carbohydrate Research Center).

    FOOTNOTES

* This work was supported by the Danish Cancer Society, the Velux Foundation, the Danish Medical Research Council, National Institutes of Health Grants 1 RO1 CA66234 and 1 RO1 CA66234, funds from the European Union Biotech 4th Framework, National Institutes of Health Resource Center for Biomedical Complex Carbohydrates Grant 5 P41 RR05351, and the Dutch Cancer Society.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) AF038650.

** To whom the correspondence should be addressed: School of Dentistry, Nørre Alle 20, DK-2200 Copenhagen N, Denmark. Tel.: 45-35326835; Fax: 45-35326505; E-mail: henrik.clausen{at}odont.ku.dk.

    ABBREVIATIONS

The abbreviations used are: beta 6GlcNAc-transferase, UDP-N-acetyl-alpha -D-glucosamine:acceptor beta 1,6-N-acetylglucosaminyltransferase; EST, expressed sequence tag; UTR, untranslated region; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; ROESY, rotating frame Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid.

2 C2GnT, IGnT, iGnT, and GnTV represent human GlcNAc-transferases cloned and expressed by Bierhuizen and Fukuda (21), Bierhuizen et al. (22), Sasaki et al. (65), and Shoreibah et al. (23), respectively. Their GenBankTM accession numbers are M97347, Z19550, AF029893, and L14284, respectively.

3 T. Schwientek and H. Clausen, unpublished observations.

4 The website at the Swiss Institute for Experimental Cancer Research is ).

    REFERENCES
Top
Abstract
Introduction
References

  1. Clausen, H., and Bennett, E. P. (1996) Glycobiology 6, 635-646[Medline] [Order article via Infotrieve]
  2. Brockhausen, I., and Schachter, H. (1997) in Glycosciences (Gabius, H.-J., and Gabius, S., eds), pp. 79-113, Chapman and Hall, Weinheim, Germany
  3. Piller, F., Piller, V., Fox, R. I., and Fukuda, M. (1988) J. Biol. Chem. 263, 15146-15150[Abstract/Free Full Text]
  4. Yang, J. M., Byrd, J. C., Siddiki, B. B., Chung, Y. S., Okuno, M., Sowa, M., Kim, Y. S., Matta, K. L., and Brockhausen, I. (1994) Glycobiology 4, 873-884[Abstract]
  5. Yousefi, S., Higgins, E., Daoling, Z., Pollex-Kruger, A., Hindsgaul, O., and Dennis, J. W. (1991) J. Biol. Chem. 266, 1772-1782[Abstract/Free Full Text]
  6. Fukuda, M. (1996) Cancer Res. 56, 2237-2244[Abstract]
  7. Brockhausen, I., Yang, J. M., Burchell, J., Whitehouse, C., and Taylor-Papadimitriou, J. (1995) Eur. J. Biochem. 233, 607-617[Abstract]
  8. Brockhausen, I., Kuhns, W., Schachter, H., Matta, K. L., Sutherland, D. R., and Baker, M. A. (1991) Cancer Res. 51, 1257-1263[Abstract]
  9. Higgins, E. A., Siminovitch, K. A., Zhuang, D. L., Brockhausen, I., and Dennis, J. W. (1991) J. Biol. Chem. 266, 6280-6290[Abstract/Free Full Text]
  10. Saitoh, O., Piller, F., Fox, R. I., and Fukuda, M. (1991) Blood 77, 1491-1499[Abstract]
  11. Springer, G. F. (1984) Science 224, 1198-1206[Medline] [Order article via Infotrieve]
  12. Kumar, R., Camphausen, R. T., Sullivan, F. X., and Cumming, D. A. (1996) Blood 88, 3872-3879[Abstract/Free Full Text]
  13. Paulson, J. C., and Colley, K. J. (1989) J. Biol. Chem. 264, 17615-17618[Free Full Text]
  14. Schachter, H., McGuire, E. J., and Roseman, S. (1971) J. Biol. Chem. 246, 5321-5328[Abstract/Free Full Text]
  15. Williams, D., Longmore, G., Matta, K. L., and Schachter, H. (1980) J. Biol. Chem. 255, 11253-11261[Abstract/Free Full Text]
  16. Williams, D., and Schachter, H. (1980) J. Biol. Chem. 255, 11247-11252[Abstract/Free Full Text]
  17. Brockhausen, I., Matta, K. L., Orr, J., and Schachter, H. (1985) Biochemistry 24, 1866-1874[Medline] [Order article via Infotrieve]
  18. Brockhausen, I., Matta, K. L., Orr, J., Schachter, H., Koenderman, A. H., and van den Eijnden, D. H. (1986) Eur. J. Biochem. 157, 463-474[Abstract]
  19. Brockhausen, I., Moller, G., Merz, G., Adermann, K., and Paulsen, H. (1990) Biochemistry 29, 10206-10212[Medline] [Order article via Infotrieve]
  20. Kuhns, W., Rutz, V., Paulsen, H., Matta, K. L., Baker, M. A., Barner, M., Granovsky, M., and Brockhausen, I. (1993) Glycoconj. J. 10, 381-394[Medline] [Order article via Infotrieve]
  21. Bierhuizen, M. F., and Fukuda, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9326-9330[Abstract]
  22. Bierhuizen, M. F., Mattei, M. G., and Fukuda, M. (1993) Genes Dev. 7, 468-478[Abstract]
  23. Shoreibah, M., Perng, G. S., Adler, B., Weinstein, J., Basu, R., Cupples, R., Wen, D., Browne, J. K., Buckhaults, P., and Fregien, N. (1993) J. Biol. Chem. 268, 15381-15385[Abstract/Free Full Text]
  24. Almeida, R., Amado, M., David, L., Levery, S. B., Holmes, E. H., Merkx, G., van Kessel, A. G., Rygaard, E., Hassan, H., Bennett, E., and Clausen, H. (1997) J. Biol. Chem. 272, 31979-31991[Abstract/Free Full Text]
  25. Amado, M., Almeida, R., Carneiro, F., Levery, S. B., Holmes, E. H., Nomoto, M., Hollingsworth, M. A., Hassan, H., Schwientek, T., Nielsen, P. A., Bennett, E. P., and Clausen, H. (1998) J. Biol. Chem. 273, 12770-12778[Abstract/Free Full Text]
  26. Lo, N. W., Shaper, J. H., Pevsner, J., and Shaper, N. L. (1998) Glycobiology 8, 517-526[Abstract/Free Full Text]
  27. Bierhuizen, M. F., Maemura, K., Kudo, S., and Fukuda, M. (1995) Glycobiology 5, 417-425[Abstract]
  28. Bennett, E. P., Hassan, H., and Clausen, H. (1996) J. Biol. Chem. 271, 17006-17012[Abstract/Free Full Text]
  29. Wandall, H. H., Hassan, H., Mirgorodskaya, E., Kristensen, A. K., Roepstorff, P., Bennett, E. P., Nielsen, P. A., Hollingsworth, M. A., Burchell, J., Taylor-Papadimitriou, J., and Clausen, H. (1997) J. Biol. Chem. 272, 23503-23514[Abstract/Free Full Text]
  30. Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528
  31. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65, 355-360
  32. Bothner-By, A. A., Stephens, R. L., Lee, J.-M., Warren, C. D., and Jeanloz, R. W. (1984) J. Am. Chem. Soc. 106, 811-813
  33. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 63, 207-213
  34. Davis, A. L., Keeler, J., Laue, E. D., and Moskau, D. (1992) J. Magn. Reson. 98, 207-216
  35. Bodenhausen, G., and Ruben, D. J. (1980) Chem. Phys. Lett. 69, 185-189[CrossRef]
  36. Bax, A., and Summers, M. F. (1986) J. Am. Chem. Soc. 108, 2093-2094
  37. Bax, A., and Marion, D. (1988) J. Magn. Reson. 78, 186-191
  38. Sutherlin, M. E., Nishimori, I., Caffrey, T., Bennett, E. P., Hassan, H., Mandel, U., Mack, D., Iwamura, T., Clausen, H., and Hollingsworth, M. A. (1997) Cancer Res. 57, 4744-4748[Abstract]
  39. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve]
  40. Kozak, M. (1992) Annu. Rev. Cell Biol. 8, 197-225[CrossRef]
  41. Schwientek, T., Almeida, R., Levery, S. B., Holmes, E. H., Bennett, E., and Clausen, H. (1998) J. Biol. Chem. 273, 29331-29340[Abstract/Free Full Text]
  42. Toki, D., Sarkar, M., Yip, B., Reck, F., Joziasse, D., Fukuda, M., Schachter, H., and Brockhausen, I. (1997) Biochem. J. 325, 63-69[Medline] [Order article via Infotrieve]
  43. Leppänen, A., Zhu, Y., Maaheimo, H., Helin, J., Lehtonen, E., and Renkonen, O. (1998) J. Biol. Chem. 273, 17399-17405[Abstract/Free Full Text]
  44. Pollex-Kruger, A., Meyer, B., Stuike-Prill, R., Sinnwell, V., Matta, K. L., and Brockhausen, I. (1993) Glycoconj. J. 10, 365-380[Medline] [Order article via Infotrieve]
  45. Bush, C. A. (1988) Bull. Magn. Reson. 10, 73-95
  46. VanderElst, I. E., and Datti, A. (1998) Glycobiology 8, 731-740[Abstract/Free Full Text]
  47. Ropp, P. A., Little, M. R., and Cheng, P. W. (1991) J. Biol. Chem. 266, 23863-23871[Abstract/Free Full Text]
  48. Sangadala, S., Sivakami, S., and Mendicino, J. (1991) Mol. Cell Biochem. 101, 125-143[Medline] [Order article via Infotrieve]
  49. Sekine, M., Hashimoto, Y., Suzuki, M., Inagaki, F., Takio, K., and Suzuki, A. (1994) J. Biol. Chem. 269, 31143-31148[Abstract/Free Full Text]
  50. Sekine, M., Nara, K., and Suzuki, A. (1997) J. Biol. Chem. 272, 27246-27252[Abstract/Free Full Text]
  51. Hounsell, E. F., Lawson, A. M., Stoll, M. S., Kane, D. P., Cashmore, G. C., Carruthers, R. A., Feeney, J., and Feizi, T. (1989) Eur. J. Biochem. 186, 597-610[Abstract]
  52. Hanisch, F. G., Chai, W., Rosankiewicz, J. R., Lawson, A. M., Stoll, M. S., and Feizi, T. (1993) Eur. J. Biochem. 217, 645-655[Abstract]
  53. Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781[Abstract/Free Full Text]
  54. Karlsson, N. G., Herrmann, A., Karlsson, H., Johansson, M. E., Carlstedt, I., and Hansson, G. C. (1997) J. Biol. Chem. 272, 27025-27034[Abstract/Free Full Text]
  55. Breg, J., Van Halbeek, H., Vliegenthart, J. F., Klein, A., Lamblin, G., and Roussel, P. (1988) Eur. J. Biochem. 171, 643-654[Abstract]
  56. Podolsky, D. K. (1985) J. Biol. Chem. 260, 8262-8271[Abstract/Free Full Text]
  57. Podolsky, D. K. (1985) J. Biol. Chem. 260, 15510-15515[Abstract/Free Full Text]
  58. Brockhausen, I. (1997) Biochem. Soc. Trans. 25, 871-874[Medline] [Order article via Infotrieve]
  59. Natunen, J., Seppo, A., Helin, J., Reinhold, B. B., Rabina, J., Costello, C. E., and Renkonen, O. (1997) Glycobiology 7, 711-718[Abstract]
  60. Olavesen, M. G., Bentley, E., Mason, R. V., Stephens, R. J., and Ragoussis, J. (1997) Genomics 46, 303-306[CrossRef][Medline] [Order article via Infotrieve]
  61. Bennett, E. P., Weghuis, D. O., Merkx, G., van Kessel, A. G., Eiberg, H., and Clausen, H. (1998) Glycobiology. 8, 547-555[Abstract/Free Full Text]
  62. Yoshida, Y., Kurosawa, N., Kanematsu, T., Taguchi, A., Arita, M., Kojima, N., and Tsuji, S. (1996) Glycobiology 6, 573-580[Abstract]
  63. Reguigne-Arnould, I., Couillin, P., Mollicone, R., Faure, S., Fletcher, A., Kelly, R. J., Lowe, J. B., and Oriol, R. (1995) Cytogenet. Cell Genet. 71, 158-162[Medline] [Order article via Infotrieve]
  64. Oulmouden, A., Wierinckx, A., Petit, J. M., Costache, M., Palcic, M. M., Mollicone, R., Oriol, R., and Julien, R. (1997) J. Biol. Chem. 272, 8764-8773[Abstract/Free Full Text]
  65. Sasaki, K., Kurata-Miura, K., Ujita, M., Angata, K., Nakagawa, S., Sekine, S., Nishi, T., and Fukuda, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14294-14299[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.