Promoters A–C have been isolated, and functionally characterized using luciferase reporter gene assays in trans-fected porcine endothelial cells PEC-A.. Results indica-ted that more than
Trang 1Regulation of a1,3galactosyltransferase expression in pig
endothelial cells
Implications for xenotransplantation
Dominique Mercier1,2, Beatrice Charreau3, Anne Wierinckx2, Remco Keijser1, Lize Adriaensens1,
Renate van den Berg1and David H Joziasse1
1 Department of Molecular Cell Biology, Research Institute of Immunology and Inflammatory Diseases and
2
Department of Medical Pharmacology, Research Institute Neurosciences VU, VUmc, Amsterdam, the Netherlands;
3 Institut de Transplantation et de Recherche en Transplantation (ITERT), INSERM U437, Nantes, France
The disaccharide galactosea1,3galactose (the aGal epitope)
is the major xenoantigen responsible for the hyperacute
vascular rejection occurring in pig-to-primates organ
trans-plantation The synthesis of the aGal epitope is catalyzed by
the enzyme a1,3-galactosyltransferase (a1,3GalT) To be
able to control porcine a1,3GalT gene expression
specific-ally, we have analyzed the upstream portion of the a1,3GalT
gene, and identified the regulatory sequences
Porcine a1,3GalT transcripts were detected by 5¢ RACE
analysis, and the corresponding genomic sequences were
isolated from a phage library The porcine a1,3GalT gene
consists of at least 10 different exons, four of which contain 5¢
untranslated sequence Four distinct promoters, termed A–
D, drive a1,3GalT gene transcription in porcine cells A
combination of alternative promoter usage and alternative
splicing produces a series of transcripts that differ in their 5¢
portion, but encode the same protein
Promoters A–C have been isolated, and functionally characterized using luciferase reporter gene assays in trans-fected porcine endothelial cells (PEC-A) Promoter prefer-ence in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quanti-tative PCR More than 90% of the a1,3GalT transcripts in PEC-A cells originate from promoter B, which has charac-teristics of a housekeeping gene promoter While promoter preference remains unchanged, a1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor a-activation of PEC-A cells However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface a1,3-galactosylation Keywords: a1,3galactosyltransferase; promoter; pig endo-thelial cells; regulation; xenotransplantation
The growing disparity between the demand for
transplant-able organs and the supply has renewed interest in the
possibilities of transplanting animal organs to humans
(xenotransplantation) [1] Several animal species have been
evaluated for their suitability as organ donor Currently,
pigs are considered as the most suitable donor animal
because pig organs are physiologically similar to human
organs and the potential risk of pathogen transmission is
low when compared with the use of organs from species
closely related to humans [1, 2] But when transplanted into
humans or nonhuman primates, pig organs are rejected
hyperacutely by antibody-mediated complement activation
[3–5] The hyperacute rejection is initiated by the interaction between natural preformed anti-pig Ig (xenoreactive natural antibodies) and carbohydrate epitopes expressed by endo-thelial cells of donor organs This results in the activation of the classical complement pathway with concomitant endo-thelial cell activation, which ultimately induces graft failure [4] A major portion (about 80%) of xenoreactive natural antibodies is directed against a single determinant, the terminal disaccharide structure galactosea1,3galactose (the aGal epitope), present on the surface of pig vascular endothelium [6–8] These anti-Gala1,3Gal Ig, originally identified by Galili et al [9], are also found in apes and Old World monkeys, but not in lower primates (e.g New World monkeys) or nonprimate mammals (including the pig) The latter species express the aGal epitope, whereas humans and higher primates don’t [9–11]
Xenoantigens that contain the Gala1,3Gal structure are synthesized by UDP-Gal:Galb1,4GlcNAc a1,3galactosyl-transferase (a1,3GalT) Genes and cDNAs encoding the a1,3GalT enzyme have been cloned from several species (cow, mouse and pig) [12–16] In humans, one pseudogene (HGT-10) and one retro-processed pseudogene (HGT-2) have been identified, both containing multiple frame-shift mutations and internal stop codons in the protein coding sequence [17–19] The absence of a functional a1,3GalT gene copy in humans, and in apes and Old World monkeys, explains the absence of aGal epitopes in these species In
Correspondence to D Mercier, Department of Medical Pharmacology,
Research Institute Neurosciences, Vrije Universiteit Medical Center,
van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands.
Fax: + 31 20 444 81 00, Tel.: + 31 20 444 80 96,
E-mail: d.mercier.pharm@med.vu.nl
Abbreviations: AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear
translocator; C/EBP, CCAAT/enhancer-binding protein; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; NF-jB, nuclear factor j
beta; pPAEC, primary pig aortic endothelial cell; Q-PCR, real-time
quantitative PCR; TNFa, tumor necrosis factor a; YY1, Ying Yang 1
transcription factor.
(Received 28 September 2001, revised 7 January 2002, accepted 16
January 2002)
Trang 2mouse and pig, the coding region of the a1,3GalT gene is
distributed over six exons that span 24 kb of genomic DNA
(18 kb in mouse) [13, 20, 21] In addition, the gene contains
several 5¢ noncoding exons
A suppression of the a1,3-galactosylation of the donor
organ will possibly overcome hyperacute rejection, and thus
facilitate xenotransplantation Therefore, efforts have been
directed at targeting the porcine a1,3GalT gene [22–24], but
thus far, no viable knockout pigs have been produced The
design of more subtle methods to down-regulate a1,3GalT
expression in a time- and tissue-dependent fashion requires
an understanding of the regulation of a1,3GalT gene
transcription Therefore we have analyzed a1,3GalT
regu-lation in pig endothelial cells We have set out to isolate the
sequences upstream of the 5¢ untranslated exons Three of
the four putative promoter regions were isolated from a pig
genomic library, and functionally characterized using gene
reporter assays When transiently transfected into pig aortic
endothelial cells, all three putative promoter regions were
able to drive luciferase transcription Relative importance of
the different promoters was determined in resting and
tumor necrosis factor a (TNFa) stimulated endothelial cells
using real-time quantitative PCR (Q-PCR) Results
indica-ted that more than 90% of the a1,3GalT gene expression in
pig endothelial cells was associated with only one of the four
putative promoters (promoter B) The modest effect of
TNFa treatment on a1,3GalT transcription suggests that
the various promoters are only weakly sensitive to
inflam-matory conditions
M A T E R I A L S A N D M E T H O D S
Cells and cell lines
COS7 cells were obtained from the Netherlands Cancer
Institute (Amsterdam, the Netherlands) The pig kidney
cells (PK15) were obtained from A Roos (Department of
Nephrology, Leiden University Medical Center, the
Neth-erlands) Pig aortic endothelial cells (PEC-A [25]), and pig
primary aortic endothelial cells (pPAECs) were provided by
J Holgersson (Karolinska Institute, Huddinge, Sweden) and B Charreau (Institut de Transplantation et de Recher-che en Transplantation, Nantes, France), respectively All cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum and 100 units
of penicillin/streptomycin (Life Technologies)
Probe preparation Pig genomic DNA was prepared from PK15 cells (2–3· 106 cells) using proteinase K (Boehringer-Mann-heim) treatment [26] and phenol/chloroform extraction PCR using pig genomic DNA (100 ng) and primers p1 and p2 (Table 1) hybridizing to 5¢ untranslated exons 2 and 3, respectively, was carried out under the following conditions: one cycle of 2 min at 94°C, 15 s at 68 °C and 5 min at
72°C; 35 cycles of 10 s at 94 °C, 15 s at 65 °C, and 1 min at
72°C incremented with 1 s at each cycle, followed by a final extension at 72°C for 7 min The amplified fragment (538 bp) was purified from a 2% agarose gel (Nucleotrap nucleic acid purification kit, Clontech), cloned into the vector pGEM-T easy (pGEM-T easy vector system, Promega) and sequenced (T7 sequencing kit, USB) using primers (SP6 and T7) flanking the cloning site This fragment was used as a probe to screen for phage clones containing the A/C region A second probe was generated from PK15 cDNA using primers p3 and p4 (Table 1) under the conditions described previously and was used to screen for phage clones containing the putative promoter B region Isolation of promoter regions B and A/C
A pig genomic library (Clontech) was screened by a standard plaque hybridization method, according to the manufac-turer’s recommendations Two probes were used in these experiments The first one, designed to isolate the promoter
C region, contained the pig a1,3GalT exon 2/intron 2/exon 3 (generated as described above) and the second one, suitable for the cloning of promoter B region, contained exon 1 After plating (2.5–3· 105 plaques, 5· 104 plaques per
Table 1 Sequences of oligonucleotides used.
Trang 3plate), the plaques were transferred to Hybond N+
(Amersham) membranes (two membranes per plate) and
hybridized in 40 mM Na2HPO4, 7% SDS, 1 mM EDTA
with the labeled probe (106c.p.m per membrane) labeled
with [a-32P]dCTP (3000 CiÆmmol)1, Amersham) using the
Prime-a-gene labeling system (Promega) Double-positive
clones were purified by secondary and tertiary rounds of
screening and genomic DNA inserts were purified using
poly(ethylene glycol) precipitation [26] The phage inserts
were mapped by restriction digestion and Southern blot
analysis Hybridizing DNA fragments were then subcloned
in pBluescript SK + (pBS, Stratagene) and sequenced
Generation of a1,3galactosyltransferase promoter
luciferase constructs
All the promoter luciferase constructs were subcloned in the
reporter plasmid Enhancer (Promega)
pGL3-Enhancer contains SV40 enhancer sequences, and is used
rather than pGL3-Basic in view of the relatively low
transcriptional activity of the a1,3GalT promoter
Clone A represents vector pGEM-T-easy containing the
whole PCR fragment (nucleotides 1249–1786)
correspond-ing to the putative promoter A region (see above) For
construct A.1 (promoter A, construct 1), the PCR fragment
(nucleotides 1249–1786) was excised from clone A with
EcoRI Construct A.2 (nucleotides 1388–1786) was
gener-ated by PCR (primers p6 and p2, Table 1), in which clone A
DNA was the template In a similar way, construct A.3
(nucleotides 1483–1786) was also made by PCR, using
primers p5 and p2 (Table 1) For construct A.4, the 5¢ part
(nucleotides 1249–1611) of the insert was deleted from the
clone A by HincII digestion For construct A.5, a
StyI-internal fragment was deleted from the clone A (Dnt1484–
1590) Construct A.6 was made using a blunted
StyI-fragment of clone A (nucleotides 1484–1590)
Construct A.7 consisted of a HincII fragment (nucleotides
1249–1611) of clone A
For promoter B sequences, a BamHI fragment was used
that originated from the hybridization-positive phage clone
2.3.1, isolated from the genomic library This 2.7-kb DNA
fragment was ligated with plasmid pBS Part of the intronic
sequence (intron 1, nucleotides 1385–2695) was deleted by
SmaI digestion and re-closure, and the clone thus obtained
(pBS clone B) was used for further constructs Construct B.1
contained a BglII/HindIII fragment of 1.2 kb (nucleotides
175–1385) An antisense construct (B.2, nucleotides 1385–
175) was also made using a BglII/SmaI digestion The two
other constructs (B.3 and B.4) contained SacI fragment
nucleotides 29–1099 cloned in sense (B.3) or antisense (B.4)
orientations
Constructs for the promoter C were made using the
1.8-kb BamHI fragment which was isolated from
hybrid-ization-positive phage clone 2.1.3, and subcloned in pBS
Constructs C.4 and C.5 consisted of a BglII fragment
(nucleotides 712–1219) and a SacI/BglII fragment
(nucleo-tides 1–711), respectively Construct C.1 (nucleo(nucleo-tides
1–1219) was made from construct C.5 linearized with BglII
by insertion of the BglII fragment from C.4 (nucleotides
712–1219) Taking advantage of unique restriction sites in
the insert of C.1 and in the pGL3-Enhancer plasmid, NdeI
(Dnt1–164) and PvuII (Dnt1–475) fragments were also
deleted, yielding constructs C.2 and C.3, respectively
The presence of the insert and its orientation in the plasmids was checked using restriction enzyme mapping and sequencing For the position of the relevant restriction sites see below
Transient transfections and luciferase assays COS7 and PEC-A cells were grown in complete medium as described above (105cells per well plated in 24-well plates) The DNA transfection complex was prepared by mixing 0.5 lg of luciferase construct and 0.25 lg of pCH110 plasmid diluted in 30 lL of serum-free medium and 3 lL of SuperFect reagent (Qiagen) per well After 5–10 min of incubation at room temperature, the mixture was diluted with 170 lL of Dulbecco’s modified Eagle’s medium containing 10% of fetal bovine serum and added to the cells After 3 h of incubation at 37°C, the mixture was replaced by 400 lL of complete medium The b-galactosi-dase plasmid pCH110 (Amersham), containing the SV40 early promoter, served as an internal control for transfection efficiency pGL3-Control (i.e pGL3-Enhancer plasmid containing the early promoter of SV40, Promega) was used
as a positive control, and empty pGL3-Enhancer plasmid as the negative control All the constructs were tested in two or three independent experiments, each performed in triplicate Luciferase reporter and b-galactosidase assays for cell extracts were performed 48 h after the start of the transfec-tion Luciferase activity was measured using the Luciferase assay system (Promega) and 5 lL (out of 60 lL) of cell extract in a BioOrbit-1250 luminometer (BioOrbit) b-Galactosidase activity was assayed using ortho-nitrophe-nyl-b-D-galactopyranoside as the substrate, and the amount
of reaction product was determined from the absorbance at
420 nm
TNFa activation of pig endothelial cells and cDNA synthesis
pPAEC and PEC-A cells were cultured as described above and were stimulated with 100 UÆmL)1 of recombinant human TNFa (hTNFa; CLB, the Netherlands) added to the medium for different periods of time (1, 2, 4, 8, 12, 24, 48 and 72 h) After activation, cells were washed in phosphate buffered saline (NaCl/Pi), lysed in SV RNA lysis buffer (175 lL per well), and total RNA was extracted using the
SV total RNA isolation system (Promega) according to manufacturer’s recommendations One microgram of total RNA was reverse transcribed using the Reverse transcrip-tion system (Promega)
Transcript quantification cDNAs were quantified using Q-PCR Samples were run on the ABI PRISM 7700 sequence detector system using SYBR green PCR core reagents (PE Applied Biosystems) Q-PCR was carried out in a volume of 25 lL containing 12.5 ng of cDNA, 2.5 mMMgCl2, 0.2 mM dATP, dCTP, dGTP and dTTP, 0.35 U Ampli-Taq Gold DNA and 0.14 U AmpErase uracil-N-glycosylase The PCR condi-tions were as follows: 40 cycles of 15 s at 95°C and 1 min at
60°C each
Primers (Table 1) for pig glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (p7 and p8), E-selectin (p9 and
Trang 4p10), a1,3GalT transcripts 5¢-A (p11 and p12), 5¢-B (p13
and p14), 5¢-C (p15 and p16), and total amount of pig
a1,3GalT mRNA (p17 and p18) were used with cDNA
from pPAECs or PEC-A cells (either activated or not with
human TNFa) as the template GAPDH was used to
normalize the quantity of cDNA used for each assay, and
background due to primer dimerization was checked with
nontemplate controls (reaction without cDNA) The
activ-ation efficiency of the endothelial cells was tested by
quantification of E-selectin transcripts (GenBank accession
number L39076) as a control Ct values, corresponding to
the cycle number required for fluorescence intensity to
exceed an arbitrary threshold in the exponential phase of the
amplification (0.3 arbitrary units), were determined for all
the samples and the gene to be analyzed In addition, to
quantify the mRNA copy numbers standard curves were
generated Plasmids containing exon 2-intron 2-exon 3
(pGEM-T-easy clone A), exon 1 (pBS clone B), exons 8
and 9 (PCR product obtained with primers p17 and p18,
Table 1, subcloned in pGEM-T-easy) or pig E-selectin
cDNA, corresponding to transcripts 5¢-A and -C, 5¢-B
and -E, a1,3GalT coding sequence and E-selectin,
respect-ively, were selected Various amounts of these different
plasmids (from 103to 106copies per reaction) were used in
Q-PCR assays, and data obtained for each concentration
(2Ct) were plotted against the amounts
R E S U L T S
The organization of the pig a1,3GalT gene
Using 5¢ RACE analysis, we confirmed the occurrence
in porcine endothelial cells of four transcripts 5¢-A, -B, -C
and -E, described earlier by Katayama et al [20] In order to
complete the model of the pig a1,3GalT gene organization,
we have compared the structure of the various a1,3GalT
transcripts with partial maps of the gene organization as
established by Koike et al [21] and Katayama et al [20]
Transcripts A–E encode the same protein, but differ in the
structure of their 5¢ ends by the presence or absence of one
or more untranslated exons These exons were mapped onto
genomic sequences using long-distance PCR, which allowed
us to establish the order of the exons, and also to estimate
intron sizes A model of the a1,3GalT genomic structure,
showing how the various transcripts are formed by a
combination of alternative start site usage and alternative
splicing, is presented in Fig 1 By 5¢ RACE analysis of
primary porcine endothelial cell (pPAEC) cDNA we have
identified an additional, sixth transcript, termed 5¢-F This
transcript contains untranslated exons 0, 1 and 3, which
confirms that exon 0 previously identified by Katayama
et al [20] is indeed authentic In addition, PCR analysis of
porcine genomic DNA showed that the start sites that give
rise to transcripts 5¢-A and -C are closely spaced An intron
of only 427 bp separates exon 2 from exon 3 In fact,
transcript A starts within this intron 2, 94 bp upstream of
the start of exon 3 Similarly, sequences upstream of exon 1
either serve as intron (intron 0), or are retained in the
processed mRNA, depending on the start site used More
recently, Koike et al [21] detected two more transcripts
starting in intron 0 (cf Fig 1)
Based on RT-PCR, pig a1,3GalT is expressed in lung and
in all cell types investigated so far (kidney PK15 cells,
hepatocytes, endothelial cells) Transcripts 5¢-B and/or -E have been detected in all samples, and 5¢-A in most of them with the exception of hepatocytes The 5¢-C and -F transcripts are present in pPAECs Transcript 5¢-D was not detected in any of the samples studied here
Cloning and sequence analysis of pig a1,3GalT promoter regions
The available a1,3GalT cDNA sequences (this paper and [20]) were used to generate DNA probes by PCR, with the aim to isolate relevant 5¢ flanking sequences of the gene from a genomic library As start sites A and C had been found to be closely spaced, a single probe was sufficient to screen the library for their individual regulatory sequences
To isolate the genomic region upstream of the 5¢-A transcript, we performed PCR on pig genomic DNA using primers p1 and p2 that hybridize with exons 2 and 3, respectively A fragment of 538 bp was obtained (clone A), which contains exon2-intron2-exon3 sequences that overlap with the putative promoter A region (Fig 2B, GenBank accession number: AF415202) This fragment was used as a probe to screen the pig genomic library A single hybrid-ization-positive clone (phage 2.1.3) containing an insert of
14 kb was isolated Southern blot analysis of the phage DNA confirmed that a major portion of the probe sequence
is included in a 1.6-kb BamHI fragment (Fig 2A) This DNA fragment contains1.2 kb of sequence upstream of start site C (Fig 2B, GenBank accession number: AF415202), as well as 430 bp of clone A Sequences thus obtained were scanned for putative transcription factor binding sites using TRANSFAC Exon 3 is preceded by a well-conserved pyrimidine-rich acceptor splice site which is functional in all transcripts 5¢-B, -C and -F The 538-bp sequence of clone A does not contain TATA or CAAT-boxes, but multiple putative transcription factor binding
ATG
5'-A
5'-B 5'-E
5'-C
5'-D 5'-F
16 kb 0.5 kb
n.d.
n.d.
Koike et al [21]
Fig 1 Schematic representation of the a1,3galT gene and transcript structures Exon numbers are indicated above the corresponding exons
on the gene structure The sizes of introns 2 and 3, determined using long-PCR, are indicated below the gene structure The length of introns 0 and 1 could not be determined (n.d.) Untranslated exonic sequence is indicated by white or gray boxes, coding sequence is indicated in black Gray boxes are flanked on both sides by consensus splice sites Structures of the 5¢ end of the different transcripts identified
in this paper (5¢-A, -B, -E and -F) or by Katayama et al (5¢-A to -E [20]), and/or Koike et al [21] are drawn below the gene structure.
Trang 5sites that could be important for promoter activity in pig
cells, such as GATA- and GC-boxes, AP-1, Inr and YY1
are present (Fig 2B)
Analysis of the sequences upstream of the exon 2
transcriptional start site revealed the presence of four
putative NF-jB binding sites (Fig 2B) located at
nucleo-tides 86, 167, 371 and 552, respectively Additional potential
transcription factor binding sites such as Oct-1, AP-1,
GATA- and GC-boxes are distributed all along the
promoter C sequence, and a TATA-box is present 16 bp
downstream of the transcriptional start site of exon 2
(Fig 2B)
In order to clone the putative promoter B region, the genomic library was screened with a probe corresponding to exon 1 Phage clone 2.3.1 thus isolated contained an 11-kb insert; BamHI digestion of the DNA produced a 2.7-kb fragment that hybridized with the probe (Fig 3A, GenBank accession number AF415201) This fragment contains 1.18 kb of sequence upstream of exon 1, exon 1 itself, and 1.36 kb of intron 1 The GC content of the whole fragment
is about 60%, and in the 1.6-kb region between nucleotides
724 and nucleotide 2335 (Fig 3B) it reaches 68% Associ-ated with the high GC content of this region, 12 putative Sp1 binding sites are present In addition, the promoter B region contains numerous putative transcription factor binding sites including GATA-boxes, Oct-1, ets-1, AP-1, NF-jB and C/EBP sites (Fig 3B)
Unfortunately, out of the 6· 105plaques screened with a probe corresponding to exon 0, no positive genomic clones containing exon 0 upstream sequences (promoter D region) were isolated The strong homology of exon 0 sequences with a portion of the porcine invariant chain gene [21] could
be responsible of the isolation of the false positive clones from the library
To search for preferred transcriptional start sites, a RNA polymerase II context analysis was performed on promoter regions A, B and C using PROMOTERINSPECTOR software (Genomatix) Results indicated that promoter region B contains two putative RNA polymerase II binding regions located at nucleotides 677–1296 (upstream of exon 1) and nucleotides 2201–2392 (within intron 1), respectively, whereas promoters A and C do not seem to contain such regions
Functional characterization of the porcine a1,3GalT promoters
Luciferase reporter gene assays were performed to test the ability of the cloned sequences to drive transcription To characterize the promoters in more detail, a deletion analysis was carried out The various test fragments were placed upstream of the luciferase gene, and the resulting plasmid constructs were transiently transfected into cultured cells In view of the relatively low efficiency of transfection
of primary endothelial cells, these experiments were per-formed using the established cell line PEC-A [25]
Construct A.1 that contains the entire 538-bp promoter A fragment is able to drive luciferase gene transcription in PEC-A pig endothelial cells (Fig 4A, open bars) Deletion
of the 140 bp 5¢ portion of the fragment to give A.2 did not affect activity, but deletion of an additional 96 bp (construct A.3) resulted in a fivefold lower luciferase activity (Fig 4A)
A segment of 1.2-kb containing putative promoter C regulatory regions was also analyzed The full 1.2-kb sequence (construct C.1, containing nucleotides 1–1219) was able to drive transcription in PEC-A cells (Fig 4B, open bars) Deletion of a 164-bp (C.2) or 719-bp (C.4) 5¢ fragment resulted in a fourfold and twofold reduction in luciferase activity, respectively
For promoter B, a fragment of1.2 kb (nucleotides 175–
1385 in Fig 3B) containing most of the GC-rich region was studied When transiently transfected into PEC-A cells, construct B.1 produced a luciferase activity ninefold greater than negative control (Fig 4C) Deletion of the 3¢ 286-bp portion (construct B.3) did not change the activity, which
5'-C [20]
A
1 2 3 4
6
3
1.5
0.5
Size in kb
AP-1 Oct-1 GATA NF-κB
BamH I
NF-κB NF-κB
ets-1
Bgl II
C/EBP
Sp1 AP-1 TATA
Bgl II
Ap-1
p1
ets-1 GATA YY1 GATA
Sty I exon 2
NF-κB
GATA
Sp1
Sty I Hinc II BamH I
5'-A [20]
GATA
Ap-1 Inr GATA
p2
Sp1
exon 3
B
Fig 2 Schematic representation of the a1,3GalT promoter A and C
regions (A) Southern blot analysis DNA prepared from phage 2.1.3
isolated from the pig genomic library was digested with various
restriction enzymes, and hybridized with the clone A DNA fragment
(see Materials and methods) Sizes of the different bands of the DNA
marker are indicated on the left of the figure Lane 1: PstI, lane 2:
HindIII, lane 3: EcoRI, lane 4: BamHI (B) Schematic representation
of the a1,3GalT promoter A and C regions Positions of primers used
to generate the 538-bp fragment (Table 1) are positioned under the
sequence and are indicated by horizontal arrows Restriction enzyme
sites used to generate the different reporter gene constructs are
indi-cated by vertical lines above the sequence Exonic sequences (exon 2
and 3) are indicated by gray boxes The start sites of transcript 5¢-A
and -C are indicated [20] Putative transcription factor binding sites
detected using TRANSFAC are indicated above the sequence and are
represented by horizontal lines.
Trang 6underlines the importance of the 5¢ part of the fragment.
The same fragments in antisense orientation did not differ
significantly from the negative control (cf B.2 and B.4)
To determine whether the constructs mediated cell-type
specific expression, they were also transfected into African
green monkey COS7 cells Each of the constructs A.1, B.1
and C.1 was able to drive transcription, and, generally,
activities observed in COS7 cells were higher than those in
PEC-A cells Deletion of the 140-bp 5¢ portion of A.1
(construct A.2) resulted in a 10-fold increased luciferase
activity (Fig 4A) Upon deletion of an additional 96-bp
(construct A.3) activity decreased to a value close to that of
A.1 Deletion constructs A.4 to A.7, even when containing
fragment nucleotides 1388–1483, were inactive (Fig 4A) For promoter C, activity in COS7 cells seems to be associated with the 0.5-kb 3¢ segment of C.1, as the 0.7-kb 5¢ portion by itself (C.5 in Fig 4B) is 10 times less active than the full-length fragment C.1 Construct B.1 produced a luciferase activity four times higher than in PEC-A cells The
2
BamH I Nde I Pvu II Bgl II Bgl II
0 50 100 150 200 1000 6000
5136 48
pGL3-Control
pGL3-Enhancer 5
5
Normalized Luciferase Activity (mV)
B
0 50 100 150 200 1000 6000
1
Bgl II
48 pGL3-Enhancer 5
Normalized Luciferase Activity (mV)
C
5136
Sty I Sty I
Hinc II
0 50 100 150 200 1000 6000
pGL3-Enhancer 5
pGL3-Control
5 48
1170
Normalized Luciferase Activity (mV)
A
Fig 4 Transcriptional activity of a1,3GalT promoter constructs in COS7 and PEC-A cells The left part of the figure shows the structure
of constructs made for promoter A (A), C (B) and B (C), and their relative positions in the a1,3GalT gene Exonic (gray boxes), and intronic (solid lines) sequences and restriction enzyme sites are indicated
as well as sequences derived from the plasmid pBS (vertical lines) For each construct, the segment of genomic sequence tested in luciferase assay is indicated by horizontal gray bars The right part of each panel shows the results of transfection experiments for each construct; values (in mV) are the means of three or four separate experiments, per-formed in triplicate, ± SEM Luciferase activities are normalized on b-galactosidase activity from a cotransfected vector (see Materials and methods) Solid and open bars correspond to COS7 and PEC-A transfection, respectively Constructs A.4 to A.7, C.3 and C.5 were only tested in COS7.
A
6
3
1.5
0.5
Size in kb 1 2 3 4 5
B
GATA GATA GATA
Bgl II
Koike et al [21]
Ap-1 GATA
BamH I
Oct-1
Koike et al [21]
Sp1 Sp1 Sp1 Sp1
Sp1 Sp1 Sp1 AhR/Arnt Sp1
Sac I
5'-E [20]
GATA
exon 1
Sp1
5'-B [20]
GATA
Sma I
Sp1
GATA GATA Sp1 ets-1 GATA CREB Arnt
Oct-1 GATA Sp1 ets-1 NF- κB GATA
C/EBP C/EBP
BamH I
GATA
GATA
Sac I
Fig 3 Schematic representation of the a1,3GalT promoter B region.
(A) Southern blot analysis DNA of phage 2.3.1, isolated from the pig
genomic library, was digested with various restriction enzymes, and
hybridized with a probe corresponding to exon 1 (see Materials and
methods) Sizes of the different bands of the DNA marker are
indi-cated on the left of the figure Lane 1: SacI, lane 2: PstI, lane 3:
HindIII, lane 4: EcoRI, lane 5: BamHI (B) Schematic representation
of the promoter B region Restriction enzyme sites used to generate the
different constructs are indicated by vertical lines above the sequence.
Exon 1 sequences are indicated by a gray box The main
transcrip-tional start site used in transcripts produced from promoter B is
indicated [20] Putative transcription factor binding sequences are
indicated above the sequence and are represented by horizontal lines.
The GC-rich region of promoter B is indicated by a thick line.
Trang 7same fragment inserted in pGL3-enhancer in antisense
orientation was 3.5-fold less active (Fig 4C) Values
obtained for the 3¢ truncated fragments B.3 and B.4
followed the same pattern as those observed for PEC-A
cells (Fig 4C), in that the ability to drive transcription is
orientation dependent, and that 3¢ deletion of 286 bp did
not significantly alter activity
Relative levels of a1,3GalT transcripts in pig
endothelial cells
Levels of a1,3GalT transcripts in pPAEC and in a PEC-A
were measured using Q-PCR in order to establish the relative
importance, within the context of the full gene, of the three
different promoters identified above Taking advantage of
the sequence differences between the 5¢ regions of the
promoter specific transcripts, and assuming that the
differ-ences observed in terms of transcript levels are proportional
to promoter activity, specific primers (Table 1) were
designed to follow variations of a1,3GalT gene expression
in resting and TNFa-stimulated pig endothelial cells The
strong homology of exon 0 to a portion of the porcine
invariant chain gene did not allow to design primers specific
of exon 0 and to quantify the 5¢-D/5¢-F transcripts
Additional primers were designed to determine expression
of GAPDH (normalization of cDNA quantities used in
Q-PCR) and E-selectin (control of TNFa stimulation) genes
Before activation of pPAEC, E-selectin transcripts were
present at 2 · 105 copies per lg of total RNA, and
a1,3GalT transcripts at 1.1· 106copiesÆlg)1 Amounts of
104, 106 and 2· 104copiesÆlg)1 of total RNA were
measured for 5¢-A, 5¢-B and 5¢-C transcripts, respectively
During TNFa-induced activation, E-selectin transcript
levels rapidly increased, reaching a maximum of about
60· 106 copiesÆlg)1 after only 2 h of TNFa-treatment
(340-fold increase, Fig 5A) A second peak was observed
after 24 h of induction (21· 106 copiesÆlg)1, Fig 5A),
clearly indicating that the cells were properly activated in
this experiment Total mRNA levels of a1,3GalT were also
checked during the time course of TNFa induction, as well
as the levels of 5¢-A, 5¢-B and 5¢-C transcripts Total amount
of a1,3GalT transcripts started to rise 2 h after the addition
of TNFa, to reach a plateau after 4 h of activation (55%
increase, to 1.8· 106copies per lg of total RNA, or 35
copies per cell) After 12 h the amount began to decrease to
reach 0.3· 106copiesÆlg)1( 6 copies per cell) after 72 h of
stimulation The 5¢-A and 5¢-B transcript levels varied in
parallel with the total amount of a1,3GalT transcripts,
whereas quantities of 5¢-C transcripts are regulated
differently with two peaks of transcription, a first one
after 2 h (increase of 82%) and a second one at
12 h (119% increase) At any activation time point studied,
5¢-B/E transcripts (which could not be distinguished by the
method as used) were found to correspond to 92–97% of
the total amount of a1,3GalT mRNA
Unlike the pPAECs, PEC-A cells were poorly activated
by recombinant human TNFa (E-selectin increased only
2.5-fold, Fig 5B) Nevertheless, quantities detected for each
of the a1,3GalT transcripts were similar to those observed in
pPAECs The total amount of a1,3GalT increased
signifi-cantly and reached a maximum of 141% after 18 h of TNFa
activation Furthermore, 5¢-B was found to be also the
most expressed transcript in PEC-A cells (80–90%) and
transcripts 5¢-A and 5¢-B followed the same pattern of variation as total a1,3GalT mRNA But in contrast to pPAECs expression, after longer periods of activation no decrease was observed for 5¢-A, 5¢-B and total a1,3GalT mRNAs Both 5¢-A and 5¢-C transcript quantities seem to
be higher in PEC-A cells (3% and 7% of the total amount
of a1,3GalT mRNAs, respectively) than in pPAECs (1% for both) Lastly, the 5¢-C transcript presented a different expression profile with only one peak reached after 4 h of activation followed by a fourfold down-regulation after longer stimulation
D I S C U S S I O N Differences in cell surface glycosylation between human and pigs form a major hurdle in organ transplantation from pig
Fig 5 Kinetics of a1,3GalT mRNA isoforms induction in pPAEC (A) and PEC-A cells during TNFa activation (B) The expression of a1,3GalT after stimulation with TNFa was quantified using Q-PCR Primers specific of the 5¢ region of the different isoforms were used to quantify specifically each transcript species (5¢-A, 5¢-B and 5¢-C) Total amount of a1,3GalT transcripts was estimated using primers binding
to the coding sequence (exons 8 and 9) Effective activation of the cells was verified by amplification of E-selectin mRNA The number of transcripts in the experimental samples was calculated from a calib-ration curve obtained by varying the number of copies of a plasmid containing the fragment to be amplified, and normalized based on GAPDH levels.
Trang 8to man Modification of porcine glycosylation has been
considered as one strategy to facilitate xenotransplantation
In this respect, it will be important to know the mechanism
of regulation of porcine terminal glycosyltransferases
Research focuses on a1,3GalT in particular, as the latter
enzyme produces the Gala1,3Gal structure, the major
porcine xenoantigen with a role both in hyperacute rejection
and in delayed vascular rejection
Here we have assembled the full structure of the 5¢
flanking regions of the porcine a1,3GalT gene, completing
partial structures as reported by Katayama et al [20] and
Koike et al [21] The gene consists of 10 exons, four of
which contain 5¢ untranslated sequence and six coding
sequence The exact structure of the 5¢ flanking regions of
the a1,3GalT gene has been unclear Koike et al [21] have
suggested that exon 0 as detected by Katayama et al [20]
and by themselves (named exon Ii in [21]) in fact is based on
an artifact, and could be the result of an accidental link-up
of two unrelated sequences Independently, we have isolated
from porcine endothelial cells a transcript, 5¢-F, that does
contain exon 0 in conjunction with additional, downstream
a1,3GalT exons 1 and 3–9 The occurrence of this transcript
in porcine cells seems to indicate that exon 0 as described
previously is indeed an authentic portion of the a1,3GalT
gene This would bring the total number of 5¢ noncoding
exons up to four
At least four promoters, here called A–D, are involved in
the initiation of transcription of porcine a1,3GalT
Alter-native start site usage together with alterAlter-native splicing in
the 5¢ region generates at least six different transcripts
Moreover, it is possible that two more transcripts as
described by Koike et al [21] are controlled by still another
regulatory sequence, as they initiate several hundreds of bp
upstream of the start of transcript 5¢-B (Fig 3B)
Alternat-ive splicing of the porcine gene is not limited to the 5¢
flanking sequences, it also occurs in the coding region
Previously, it was reported that the murine a1,3GalT gene is
alternatively spliced in the sequence that encodes the Ôstem
regionÕ of the protein [13] A similar observation has been
made by Vanhove et al [27, 28] for the porcine gene, which
further increases the number of transcripts that can be
obtained from this single gene As yet, it is unclear if the
occurrence of multiple transcript isoforms has a
physiolo-gical relevance Heterogeneity at the 5¢ end of the mRNA
does not affect the protein encoded In contrast, splicing in
the stem region will result in the production of a shortened
enzyme molecule, which may be less sensitive to intracellular
proteolysis, or differ in its ability to transfer galactose to
Galb1,4GlcNAc structures [20]
Various 5¢ flanking regions have been tested for their
ability to drive a1,3GalT gene transcription, and a deletion
analysis has been carried out to identify minimal promoter
regions Each of the promoters A, B, and C was found to be
active in porcine endothelial cells Sequence analysis of
promoter region A, the 479-bp region directly upstream of
exon-3, revealed the presence of several putative
transcrip-tion factor binding sites (Fig 2B) The region contains five
GATA(like) sites One of these, GATA nucleotides 1621–
1624, is in close proximity to an AP-1 motif Cooperative
interactions between AP-1 and GATA were reported to
regulate transcription driven by the human P-selectin
promoter [29, 30] For porcine a1,3GalT, the AP-1/GATA
motif is located just upstream of the start site (at nucleotide
1633) of transcript 5¢-A This start site is part of an octanucleotide that is highly similar to the consensus transcriptional initiator sequence [31, 32] The initiator sequence, together with the AP-1/GATA motif, is probably important for the production of transcript 5¢-A in porcine endothelial cells However, additional upstream sequence is essential for transcriptional activity Construct A.2 that contains the nucleotides 1388–1786 region was found to be five times more active in PEC-A cells (luciferase assays, Fig 4A) than construct A.3 (nucleotides 1483–1786) This suggests that a transcriptional activator binds to the region nucleotides 1388–1483 The segment needs to be linked to the transcriptional start site via StyI-fragment nucleotides 1483–1590, as deletion of the latter fragment results in zero activity (Fig 4A) Apart from a single GATA-box, no known transcription factor binding site is present in region nucleotides 1388–1483 (Fig 2B) The GATA box shows only imperfect homology with the consensus sequence Therefore, activation by the nucleotides 1388–1483 segment may result from the binding of a still unknown transcription factor The 538-bp promoter A fragment can also drive transcription in COS7 cells, so does not appear to confer cell type specificity
A second porcine genomic DNA fragment was isolated that contains sequences upstream of exon 2 (nucleotides 1–1219 in Fig 2B) This putative promoter C contains multiple transcription factor binding sites, including five NF-jB sites The latter sites could be important in endothelial cell-specific expression and in the cytokine response to TNFa [33, 34] It has been reported that TNFa can induce the expression of a1,3GalT [27], and NF-jB sites are possibly involved in mediating this effect Other transcription factors such as Sp1, GATA or ets-1 as detected in promoter C (Fig 2B) could also be important for endothelial cell-specific expression [35–38] Reporter gene assays have indicated that regions important for promoter activity are mostly located in the 3¢ portion of the fragment (nucleotides 719–1219) This region contains only
a single NF-jB site The presence of a TATA box at nucleotide 1196 may help to direct initiation specifically to the position nucleotide 1220 An enhancer may be present in the region nucleotides 1–164 of promoter C because deletion
of that region results in a fourfold reduction of luciferase activity in transfected PEC-A cells As shown by reporter luciferase assays, similar activities are observed in both COS7 and PEC-A cells, suggesting that promoter C does not contain species-specific regulatory sequences
The analysis of promoter region B confirmed that the region directly upstream of exon 1 contains a GC-rich sequence of 1.5 kb, as reported earlier by Koike et al [21] Consequently, numerous Sp1 binding sites have been predicted in that region (Fig 3B) The lack of TATA or CAAT-boxes together with the presence of many Sp1 binding sites, as observed for promoter B, is a characteristic
of ÔhousekeepingÕ genes For most of these genes, transcrip-tional start is likely to be imprecise, and indeed a set of transcripts differing in their 5¢ ends is produced from promoter B [20, 21] Several glycosyltransferase promoters present similar structure and characteristics [39–42] For example, the promoter of the long form of b1,4-galactosyl-transferase contains 12 Sp1 binding sites, and is active in a variety of cell types [42, 43] Two putative NF-jB binding sites have been found in promoter B sequence (nucleotides
Trang 9898–907 and nucleotides 2398–2407) suggesting that this
promoter may respond to endothelial cell activation
Interestingly, a1,3GalT transcripts generated from
pro-moter B contain a GC-rich 5¢ untranslated region, which is
predicted to form stable hairpin loops This may interfere
with the efficiency of translation of the mRNA as has been
shown for b1,4-galactosyltransferase [44] In that way,
increased transcription from promoter B could be
compen-sated for by low translation efficiency
We have established which promoter is used
preferen-tially in porcine endothelial cells, and how a1,3GalT gene
transcription is affected by TNFa-activation of endothelial
cells Results obtained for nonactivated pPAEC and PEC-A
cells were similar In both cell types the 5¢-B transcript is the
most highly expressed isoform (Fig 5A,B), and corresponds
to 80–90% of the total amount of a1,3GalT transcript
This indicates that promoter B is the main a1,3GalT
promoter implicated in endothelial cell expression
Prefer-ence for promoter B is not affected by TNFa stimulation
Organ transplantation from pig to man results in an
activation of the donor organ vascular endothelium,
con-comitant with changes in cell surface structures Activation
of endothelial cells by TNFa treatment was reported to
enhance a1,3GalT expression [28] Using Q-PCR we found
that, initially, quantities of a1,3GalT transcripts indeed
increased slightly (25–50%, Fig 5) However, this increase
was followed over time by a strong decrease (fivefold) in
primary endothelial cells, whereas no down-regulation was
observed in PEC-A This indicates that the response of
a1,3GalT to TNFa stimulation in PEC-A is different from
that in primary cells The same holds for E-selectin expression
(about 300-fold increase in pPAECs vs 3-fold in PEC-A)
It remains to be investigated to what extent changes in
endothelial levels of a1,3GalT and other terminal
glycosyl-transferases under inflammatory conditions will affect cell
surface carbohydrate structure, and thus influence the
outcome of organ transplantation The results obtained in
this study will help us to manipulate the expression of
a1,3GalT in porcine cells and tissues in a precise way It is
hoped that, ultimately, this approach will facilitate clinical
xenotransplantation
A C K N O W L E D G E M E N T S
This work was supported by the EU biotechnology project on
xenotransplantation N° BIO4-CT97-2242.
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