From Novartis Pharma AG, Transplantation Preclinical Research, CH 4002 Basel, Switzerland
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ABSTRACT |
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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 3-galactosyltransferase
distinct from the melanoma enzyme. The new
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(
1-3)GlcNAc. It also has a minor but
distinct transfer activity toward Gal, LacNAc, and lactose, but is
inactive toward GalNAc.
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INTRODUCTION |
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Two types of carbohydrate chains are known to exist in the
lacto-series of oligosaccharides, type 1 chains that contain the Gal(1-3)GlcNAc linkage and type 2 chains containing the topoisomer Gal(
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
(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
-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 3
-galactosyltransferase (
3-Gal-T). This enzyme is clearly
different from the
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
3-Gal-T activity detected in various sources
and transferring to lactose or LacNAc (11-13), although this has not been molecularly established.
A 3-Gal-T enzyme catalyzing the synthesis of Gal(
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
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
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(
1-3)GlcNAc(
1-3)Gal(
1-4)Glc and
directs the synthesis of sLea in transfected cells.
We report here the cloning and functional expression of a new
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(
1-3)GlcNAc. The recombinant enzyme also transfers galactose to Gal, LacNAc, and lactose, at distinctly lower
rates. To distinguish the two
3-Gal-T enzymes, we propose to name
the melanoma enzyme
3GalT1 and the human brain enzyme
3GalT2.
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EXPERIMENTAL PROCEDURES |
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Materials--
All cell culture media, sera, and antibiotics
were from Life Technologies, Inc. CHO K1 cells (ATCC CCL-61) were
maintained in -minimal essential medium, 5% fetal bovine serum.
Sf9 insect cells were grown in Sf-900 II SFM medium. GlcNAc, Gal,
Gal(
1-3)GlcNAc, p-nitrophenyl
-D-lactopyranoside, GalNAc
-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 3GalT2
was cloned by its homology to
3GalT1 (19). Using the complete
322-amino acid protein sequence of
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
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 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
cDNA libraries (QUICK-screenTM,
CLONTECH) by PCR for the presence of the 307-bp
diagnostic fragment.
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 clone GA4/1 was done either by PCR
amplification using the
gt10-specific primers shown above or by
excision from purified
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 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 3GalT2 in CHO/Fuc-T III Cells and
FACS Analysis--
A mammalian expression vector was prepared
containing the complete open reading frame of
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
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.
Construction of Soluble 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
-interferon signal sequence and the first 6 amino
acids of mature human
-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).
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Expression and Purification of Protein A Fusion of 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.
Expression and Purification of Protein A Fusion of
3GalT1--
A protein A fusion chimera of
3GalT1 was cloned and
expressed in a manner analogous to that indicated above for
3GalT2. A portion of
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
3GalT2.
Cell culture and protein purification was performed as indicated above
for
3GalT2.
Detection of 3GalT2/Protein A Fusion Protein by Enzyme-linked
Immunosorbent Assay--
The protein A portion of the
3GalT2/protein A fusion protein was used to semi-quantitatively
determine the concentration of the soluble form of
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.
3GalT2 Assays and Product Characterization--
The linkage
synthesized by the
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.
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RESULTS AND DISCUSSION |
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Cloning and Nucleotide Sequencing of the 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
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.
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Determination of Enzymatic Activity in Cell Culture--
The
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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Production of Soluble 3GalT2/Protein A Fusion Protein and
Enzymatic Assays--
The acceptor specificity of
3GalT2 was also
directly established in enzymatic assays. To purify sufficient amounts
of enzyme for analysis, we constructed and expressed a soluble
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
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(
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
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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Northern Blot Analysis--
The complete open reading frame of
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
1-3- or
3-galactosyltransferase is probably regulated in
a tissue-specific and cell-specific manner. This is in contrast to the
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
3GalT1. Preliminary PCR
experiments using libraries from different tissues and cell types
showed clear differences in the distribution of
3-Gal-T1 and
3GalT2.3
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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: 4-Gal-T,
UDP-galactose:2-acetamido-2-deoxy-D-glucose
4
-galactosyltransferase;
3-Gal-T,
UDP-galactose:2-acetamido-2-deoxy-D-glucose 3
-galactosyltransferase; sLea,
Neu5Ac(
2-3)Gal(
1-3)[Fuc(
1-4)]GlcNAc;
sLex,
Neu5Ac(
2-3)Gal(
1-4)[Fuc(
1-3)]GlcNAc; EST, expressed
sequence tag; FACS, fluorescence-activated cell sorting; UDP-Gal,
uridine diphospho-D-galactose; GlcNAc-Lemieux,
GlcNAc
O-(CH2)8-CO2Me; Gal-Lemieux, Gal
O-(CH2)8-CO2Me;
LacNAc-Lemieux,
Gal(
1-4)GlcNAc
O-(CH2)8-CO2Me; GalNAc
-benzyl, benzyl
2-acetamido-2-deoxy-
-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.
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REFERENCES |
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