From the 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
Department of
Human Genetics, University Hospital Nijmegen,
6500 HB Nijmegen, The Netherlands
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ABSTRACT |
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A novel human
UDP-GlcNAc:Gal/GlcNAc 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:GalNAc1-3GalNAc
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-
-D-glucosamine:acceptor
1,6-N-acetylglucosaminyltransferase (
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
1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in
1,6GlcNAc-transferases capable of forming core 2 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
1,3Gal-transferase activity; (ii) conversion of
core 1 to complex-type core 2 structures by
UDP-GlcNAc:Gal
1-3GalNAc
1,6GlcNAc-transferase1
activities; (iii) direct formation of complex mucin-type core 3 by
UDP-GlcNAc:GalNAc
1,3GlcNAc-transferase activities; and (iv)
conversion of core 3 to core 4 by UDP-GlcNAc:GlcNAc
1-3GalNAc
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 GlcNAc
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;
Gal
1-3GalNAc
1-R), Tn (GalNAc
1-R), and sialosyl-Tn
(NeuAc
2-6GalNAc
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 2
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
6GlcNAc-transferase activity (6). The increase in core 2
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):
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Fig. 1.
Biosynthetic pathways of mucin-type
O-glycan core structures. The abbreviations used
are: GalNAc-T, polypeptide GalNAc-transferase;
ST6GalNAcI, mucin
2,6 sialyltransferase I;
C1
3Gal-T, core 1
1,3-galactosyltransferase;
C2GnT, core 2
1,6GlcNAc-transferase; C2/4GnT,
core 2/core 4
1,6GlcNAc-transferase; C3GnT, core 3
1,3GlcNAc-transferase; ST3GalI, mucin
2,3
sialyltransferase I;
4Gal-T,
1,4-galactosyltransferases;
3Gal-T,
1,3-galactosyltransferases;
3GnT, elongation
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 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
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
6GlcNAc branch in tetraantennary
N-linked glycans (23). Studies of the kinetic properties and
acceptor substrate specificities of
6GlcNAc-transferases involved in
O-glycosylation from different cell lines and organs have
suggested that multiple
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
6GlcNAc-transferase family containing C2GnT and IGnT suggests that
additional
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
6GlcNAc-transferase family that forms core 2 as well
as core 4 structures.
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EXPERIMENTAL PROCEDURES |
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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 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
[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).
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RESULTS |
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Isolation and Characterization of Human C2/4GnT--
Analysis of
the GenBankTM and dbEST data bases suggested the existence
of additional members of the 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).
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A multiple sequence alignment (ClustalW) of three human
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
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
3Gal-transferases (25) and
one site is conserved in the C-terminal region of five of six
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).
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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).
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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 (Gal1-3GalNAc
1-R) and core 3 structures
(GlcNAc
1-3GalNAc
1-R). In contrast, no activity was found with
lacto-N-neo-tetraose as well as
GlcNAc
1-3Gal-methyl as acceptor substrates, indicating that C2/4GnT
has no IGnT-activity. Additionally, no activity could be detected wih
-D-GalNAc-1-para-nitrophenyl indicating that
C2/4GnT does not form core 6 (GlcNAc
1-6GalNAc
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).
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Characterization of Core 4 Product by 1H and
13C NMR Spectroscopy--
The product derived from
reaction of the putative Core 4 6GlcNAc-transferase with
-D-GlcNAc-(1-3)-
-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
-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
GlcNAc
1
6GalNAc
linkage in the product is clearly
demonstrated by strong cross-peaks correlating the
-GlcNAc H-1
at 4.454 ppm with
-GalNAc C6 and the corresponding
-GlcNAc
C-1 at 100.93 ppm with both
-GalNAc H-6 resonances. Consistent with
this, rotating frame Overhauser enhancements were observed between
-GlcNAc H-1 and
-GalNAc H-6 in a ROESY spectrum (data not
shown).
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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).
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DISCUSSION |
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Additional members of a human 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
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 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
6GlcNAc-transferase purified to apparent homogeneity with a
molecular weight of 69,000 (specific activity, 70 units/mg with
Gal
1-3GalNAc
-benzyl) utilized both core 1 and 3 acceptors. In
addition, activity was detected with GlcNAc
1-3Gal
1-R for I
antigen biosynthesis. This
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 Gal
1-3GalNAc
-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 6GlcNAc-transferase
activities may exist. Sekine et al. (49) reported that a
purified mouse kidney
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 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
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
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 6GlcNAc-transferase
gene family may be predicted. Leppänen et al. (43) provided evidence for an additional I
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
6GlcNAc branching either to the
Gal found in Ref. 59 or
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 2- and
3/4-fucosyltransferase families have members clustered at one locus
(63). Interestingly, the three members of the
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
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
1-3 linkage to
GalNAc
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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are:
6GlcNAc-transferase, UDP-N-acetyl-
-D-glucosamine:acceptor
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 ).
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REFERENCES |
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