From the Department of Molecular Physiology and
Biophysics, Vanderbilt University Medical School, Nashville, Tennessee
37232, ¶ Barbara Davis Center for Childhood Diabetes, University
of Colorado Health Sciences Center, Denver, Colorado 80262, and
Institut de Biologie-CNRS 8090, Institut Pasteur de
Lille, Lille Cedex, France
Received for publication, February 19, 2001, and in revised form, March 27, 2001
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ABSTRACT |
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Islet-specific glucose-6-phosphatase (G6Pase)
catalytic subunit-related protein (IGRP) is a homolog of the catalytic
subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been
defined. Since IGRP gene expression is restricted to
islets, this suggests a possible role in the regulation of islet
metabolism and, hence, insulin secretion induced by metabolites. We
report here a comparative analysis of the human, mouse, and rat
IGRP genes. These studies aimed to identify conserved
sequences that may be critical for IGRP function and that specify its
restricted tissue distribution. The single copy human IGRP
gene has five exons of similar length and coding sequence to the mouse
IGRP gene and is located on human chromosome 2q28-32
adjacent to the myosin heavy chain 1B gene. In contrast, the rat
IGRP gene does not appear to encode a protein as a result
of a series of deletions and insertions in the coding sequence.
Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is
not expressed in islets or islet-derived cell lines, an observation
that was traced by fusion gene analysis to a mutation of the TATA box
motif in the mouse/human IGRP promoters to TGTA in the rat sequence.
The results provide a framework for the further analysis of the
molecular basis for the tissue-restricted expression of the
IGRP gene and the identification of key amino acid
sequences that determine its biological activity.
Glucose-6-phosphatase
(G6Pase)1 is located in the
endoplasmic reticulum (ER) and catalyzes the terminal step of the
gluconeogenic pathway in liver and kidney. The enzyme is thought to be
a multi-subunit complex; however, the exact number of subunits, their
stoichiometry, and topological relationships are unclear (1, 2). The
36-kDa G6Pase catalytic subunit spans the membrane multiple times and appears, based on studies using microsomes, to have its catalytic site
oriented toward the lumen (1, 2). A model has therefore been proposed
in which the G6Pase catalytic subunit is postulated to be associated
functionally with a 46-kDa glucose 6-phosphate (G6P) transporter (3)
and hypothetical transporters for inorganic phosphate and glucose,
which serve to deliver cytosolically generated G6P to the active site
and shuttle the reaction products back to the cytosol (1, 2). Rapid
kinetic data, on the other hand, favor an alternative model that places
the catalytic site within the membrane and ascribes both a transport
function and catalytic activity to the 36-kDa catalytic subunit (1, 2). Mutations within the G6Pase catalytic subunit cause glycogen storage disease type 1a (4), which is characterized by severe hypoglycemia in
the post-absorptive state, hepatomegaly associated with excessive glycogen deposition, growth retardation, and renal failure (4). In
glycogen storage disease type 1b mutations in the G6P transporter give
rise to a similar phenotype and additional complications possibly
related to independent functions of this molecule in other tissues
(4).
Hepatic G6Pase activity is increased in poorly controlled human type 1 and 2 diabetics (5, 6) and in experimental rodent diabetic models
(7-11). Along with the elevated activity of other gluconeogenic
enzymes, it contributes to an increase in hepatic glucose production
and the hyperglycemia that characterizes the disease (5, 6, 12, 13).
The change in G6Pase activity has been attributed to changes in
expression of the genes encoding both the G6Pase catalytic subunit and
the G6P transporter. The former probably reflects the combined
stimulatory effect of glucose (10, 14, 15) and the loss of the
inhibitory action of insulin (16, 17). Less is known about the factors
that regulate expression of the G6P transporter (18). However,
experimental overexpression of either the G6Pase catalytic subunit (19)
or the G6P transporter (20) in hepatocytes using recombinant adenovirus
leads to enhanced rates of G6P hydrolysis as well as changes in
glycogen metabolism.
Most studies show that islets also contain a hydrolytic
activity that is specific for G6P but that is present at a lower
specific activity than liver (2, 21-25). G6Pase activity is elevated in islets isolated from ob/ob mice resulting in increased glucose substrate cycling (26, 27). The question of whether islet G6Pase
activity is catalyzed by the same G6Pase catalytic subunit as in liver,
however, has proven controversial (2, 21). Thus, the G6P hydrolytic
activity in islets displays distinct kinetic behavior and inhibitor
profiles compared with that in hepatic extracts (21).
We recently identified a novel cDNA from mouse In this paper we report a comparative analysis of the structure of the
IGRP genes from the mouse, human, and rat and an
investigation of their expression at the level of tissue mRNA and
promoter activity. The principal objectives were to identify conserved
amino acids that are potentially critical for IGRP function and
conserved sequences within the IGRP gene promoter that may
specify its restricted tissue distribution. In the process we have
uncovered a major difference in gene structure and expression between
rodent species.
Materials--
[ General Cloning, DNA Isolation, and Sequencing
Procedures--
Plasmid DNA purification, subcloning, and restriction
endonuclease analyses were performed by standard protocols (29). DNA fragments used for subcloning and labeling were isolated from agarose
gels using either the Qiaex II gel extraction kit (Qiagen) or Quantum
Prep spin columns (Bio-Rad). Zeta-probe membranes (Bio-Rad) were used
for DNA hybridization analysis and both alkaline transfer and
hybridization using the standard protocol were performed according to
the manufacturer's instructions. DNA probes were labeled by random
oligonucleotide priming with [ Isolation of Human IGRP Genomic Clones--
An arrayed human
PAC library2 was screened using a
32P-labeled 1028-bp cDNA probe isolated as a
PstI fragment from the pSV.SPORT 1B1 clone, which contained
sequences from all 5 exons of mouse IGRP (21). Thirty PAC library
filters (1,105,920 clones) were incubated overnight in 200 ml of 6×
SSC ((1× SSC = 0.15 M NaCl and 0.015 M
sodium citrate)), 0.5% SDS, 100 µg/ml salmon sperm DNA, and
100 ng of labeled probe (~500,000 dpm/ml), washed five times at
moderate stringency, and exposed for ~2 h at Chromosome Mapping--
Contiguous sequence data spanning ~10
kbp of the cloned human IGRP gene was analyzed by the CENSOR
program (30) to identify and edit out repeated genomic sequences and
used to search the unannotated high throughput-sequencing human genome
data base. Two BACs were identified; one (AC069137) containing the
full-length gene on a 13.3-kbp contig within a 195-kbp insert the other
(AC90045) containing a series of noncontiguous sequences within a
190-kbp insert. The BAC sequences were again edited by CENSOR to remove the repeated sequence and compared with the data base to identify genes
flanking human IGRP.
As an independent approach, two PCR primer sets within exon 2 (forward
5'-ATGTGTGAGAGACCAAGACCTAAG-3' and reverse
5'-TGAAGTTTTAGCATCCTCACTC-3') and exon 5 (forward
5'-AGAACCTCTGTGTCTAATGC-3' and reverse 5'-GGTCTGTGCCTACTCTGTGG-3') were
used to analyze the Stanford radiation hybrid panel G3, which has been
mapped with 1185 markers. Reactions (10 µl) were run for 35 cycles in
PCR buffer (PerkinElmer Life Sciences) containing 3 mM
MgCl2 and 30 ng of template (95 °C × 15 s,
55 °C × 15 s, and 72 °C × 30 s) using
Taq gold polymerase (Applied Biosystems) and a GeneAmp PCR
system 9700 machine (Applied Biosystems).
Isolation of Rat IGRP Genomic Clones--
A fragment of the rat
IGRP gene was generated using rat genomic DNA
(CLONTECH) as the template in a PCR reaction with
the following primers: forward
(5'-CGGAATTCCTCCACAGATGGTCAGCATCACATG-3') and reverse
(5'-CGGAATTCGGGGTCTCCAACATTGGACATAAAATTTAG-3');
EcoRI cloning sites are underlined. The primers were
designed based on conserved sequences in the human and mouse
IGRP genes present in the promoter and exon 1, respectively.
The PCR fragment generated was cloned into the EcoRI site of
pGEM7 (Promega), and the 237-bp IGRP insert was subsequently used as a
labeled probe to screen a rat BAC library (Genome Systems, Inc.
Gene Screening Custom Service). A single rat BAC clone, designated
67/L18, hybridized to the probe. The large scale isolation of 67/L18
BAC plasmid DNA was performed by standard cesium chloride
centrifugation (29).
BAC 67/L18 contained the entire rat IGRP gene. Restriction
enzyme analysis and Southern blotting were performed as with the human
IGRP gene using labeled fragments representing either the 5'
or 3' end of the rat IGRP gene. The fragment representing
the 5' end of the rat gene was the same as that used in the initial library screening (see above, this section). The fragment representing the 3' end of the rat IGRP gene was generated using the
67/L18 BAC plasmid as the template in a PCR reaction with the following primers: forward
(5'-GGAATTCTCACGAGTCCAGCAAAAGGCGTG-3') and reverse (5'-CCCAAGCTTGAGGCCTTTGAACACACTCCAGG-3'); the
EcoRI- and HindIII-cloning sites are
underlined. These primers represent IGRP exon 5 sequences that are
conserved in the human and mouse IGRP genes. The 221-bp rat
IGRP fragment PCR fragment generated was cloned into and subsequently released from the EcoRI-HindIII-digested pGEM7 vector.
Genomic DNA fragments that hybridized to these labeled probes were then
subcloned into the pGEM7 or pSP72 plasmid vectors (Promega) for
sequence analysis. The entire rat IGRP gene was isolated
within two overlapping genomic sub-clones; a ~6-kbp
KpnI-KpnI fragment contained the promoter and
exons 1-3, whereas a ~6-kbp PstI-PstI fragment
contained exons 3 and 5. The identification of the exon/intron
boundaries (Table I) was determined by direct DNA sequencing of both
DNA stands and comparison with the mouse IGRP gene sequence
(28). The sizes of the three introns in the rat IGRP gene
(Fig. 1) were calculated by direct sequencing (introns A and C) or
estimated by PCR (intron B). In the latter case, the size of the intron
was estimated using two separate primer pairs; the difference in the
size of the PCR products was as expected. PCR reactions (100 µl)
contained 100 pmol of each primer, 1× PCR buffer (PerkinElmer Life
Sciences), 0.2 mM each dNTP, 1.5 mM
MgCl2, 200 ng of pGEM7 plasmid DNA containing the ~6-kbp
KpnI-KpnI rat IGRP fragment as the template, and
5 units of AmpliTaq DNA polymerase (PerkinElmer Life Sciences).
Reactions were run for 94 °C for 5 min and then for 30 cycles of
30 s at 94 °C, 30 s at 47 °C, and 2 min at 72 °C
using an MJ Research MiniCycler. Products were analyzed by agarose gel electrophoresis.
Cell Culture--
The pancreatic islet-derived cell lines,
Northern Blotting and RT-PCR Analyses--
Wistar-Furth rats and
Balb-c mice were obtained from Charles River and Jackson Laboratories,
respectively. Animals were fed ad libitum and sacrificed by
CO2 asphyxiation. Islets were isolated by a modification of
the collagenase digestion procedure of Lacy and Kostianovsky
(31) and Guest et al. (32). Viable human islets derived from
cadaveric donors were obtained through the Juvenile Diabetes Foundation
International human islet consortium, shipped at room temperature in
Connaught Medical Research Laboratory medium supplemented with
10% fetal bovine serum and 5.6 mM glucose (Life
Technologies), and maintained in culture for 24-72 h in the same
medium under a 5% CO2 in air atmosphere before harvesting RNA.
Total RNA was prepared from tissues and cell lines using Trizol reagent
(Life Technologies) and quantified in a fluorimetric assay by
DNase-resistant SYBR green binding (RiboGreen kit; Molecular Probes, Eugene, OR). Northern blotting was performed after
electrophoresis of samples (5 µg of total RNA) on denaturing
formaldehyde gels and with commercially available blots prepared with
2-µg samples of poly(A)+ mRNA from various human
tissue sources (multiple tissue northern and multiple tissue
northern 1; CLONTECH, Palo Alto, CA). Blots were
hybridized for 16 h at 42 °C in 50% (v/v) formamide, 5×
saline/sodium phosphate/EDTA, 5× Denhardt's reagent, and 50 µg/ml
salmon testis DNA with a 32P-radiolabeled randomly primed
probe corresponding to the ORFs of human IGRP or rat G6Pase catalytic
subunit (9). Blots were subsequently washed in 2× SSC, 0.05% SDS at
room temperature for 30 min, then in 0.2× SSC, 0.1% SDS at 42 °C
and visualized by PhosphorImager (Molecular Dynamics, Palo Alto, CA).
Reverse transcription of human, mouse, and rat tissue total RNA (300 ng) was performed with oligo(dT18) or random nonamers as
primers using Maloney murine leukemia virus reverse transcriptase at
42 °C with reagents provided in the Stratagene high fidelity RT kit.
Comparative analysis of the expression of IGRP in various tissues of
the mouse, human, and rat was performed using a conserved primer pair
within exons 1 (forward 5'-CCAAGATGAT(A/C)TGGGTAGC-3') and exon 5 (reverse 5'-TGTCAATGTGGATCCAGTC-3'). The forward primer had a single
degenerate nucleotide (internal parentheses (A/C)) to accommodate a
single base difference between the mouse and human/rat sequences. PCR
reactions were performed with a Pfu/Taq polymerase mixture (Roche Molecular Biochemicals Expand High Fidelity PCR system) for 5 min at 94 °C, then 40 cycles of 1 min at 94 °C,
1 min at 53 °C, and 2 min at 72 °C followed by a final 20 min
extension at 72 °C. Products were analyzed on 1.5% agarose gels,
and the major bands were excised and cloned into the pTOPO PCR blunt II
vector using a zero blunt TOPO PCR cloning kit (Invitrogen, Carlsbad,
CA) and then sequenced.
Human islet cDNA library screening using the mouse IGRP ORF probe
proved unsuccessful. However, once the genomic sequence and exon/intron
boundaries of human IGRP were established (Table I), each exon was
amplified using a series of PCR forward and reverse primers, each
incorporating 10 bp of the 5' sequence of the flanking exon and the
first 20 bp of the exon to be amplified. The products of these
reactions were gel-purified, mixed, and subjected to PCR using a
forward primer incorporating the start codon and Kozak sequence (33)
(5'-TCAAGATGGATTTCCACAGGA-3') and a compatible reverse primer located
just beyond the stop codon (5'-CAGAGCACTAACTCTAGGCACC-3'). The sequence
of the PCR product generated was confirmed and gave the expected
in vitro translation product. Human cadaveric islet RNA
became available at a later date and was used to amplify the coding
region of human IGRP cDNA with 40 cycles of RT-PCR with the above
forward primer and a reverse primer located further downstream in exon
5 (5'-GTGAAGTCGGATTAGAAGCC-3'). The PCR products were inserted into the
pTOPO blunt vector and then subcloned into pCDNA3.1 for expression
studies using EcoRI and XhoI sites common to both vectors.
Generation of Antisera and Immunoperoxidase Staining--
A
PstI fragment containing the majority of the mouse IGRP ORF
was inserted in-frame in the pUEX vector (34), generating a fusion
protein with Fusion Gene Plasmid Construction and Analysis--
The
construction of a mouse IGRP-chloramphenicol acetyltransferase (CAT)
fusion gene containing promoter sequence from
A human IGRP-CAT fusion gene was constructed in the pCAT(An) expression
vector such that the 5' and 3' end points were equivalent to those in
the mouse IGRP Protein Expression by in Vitro Translation and Cellular
Transfection--
In vitro transcription/translation assays
were performed using rabbit reticulocyte lysate with a TNT T7 Quick
translation kit (Promega) as previously described (37) using T7
polymerase transcripts from sequences cloned into the mammalian
expression vector pCDNA3.1 (Invitrogen). A number of mouse IGRP
constructs were analyzed in addition to the above-mentioned human IGRP
cDNA clones to evaluate the effects of 5'- and 3'-untranslated
region sequences on expression levels and the activity of two
alternative start codons in the sequence. These included: (i) the
full-length mouse IGRP cDNA (nt 1-1901) (21); (ii) a
PstI fragment (nt 110-1137) incorporating the second and
third AUG codons; (iii) a cloned PCR product (nt 220-1038) generated
using the primers 5'-TTGGAACCAAGATGATCTGG-3' (forward) and
5'-CAGAGCACTAACTCTAGGCACC-3') (reverse), which deleted the putative
start codon and second AUG codon but retained the third potential AUG
(nt 231) embedded in a Kozak sequence; (iv) a cloned PCR product (nt
59-1038) generated using the primers 5'-CAAGATGGATTTCCTTCATAGGAGT-3'
(forward) and 5'-CAGAGCACTAACTCTAGGCACC-3') (reverse), which contains
the entire ORF but with minimal flanking sequence.
The human IGRP protein was expressed by transient
transfection of COS 7 cells using the pCDNA3.1 vector full-length
construct (see above). A rat G6Pase catalytic subunit cDNA cloned
into the same vector was used as a positive control (21). Transfections were performed as previously described using a calcium phosphate precipitate containing 15 µg of a pCDNA 3.1 construct and 5 µg of pRSV Isolation of the Human and Rat IGRP Genes--
The human
IGRP gene was isolated from a human PAC library probed with
a mouse IGRP cDNA fragment (Fig. 1).
Two independent clones with similar restriction digestion patterns were
obtained from a screen of ~1010 bp of genomic sequence.
One of these clones, designated PAC 294, was selected for further
analysis and was subsequently found to contain the entire human IGRP
transcription unit (Fig. 1). A fragment of the rat IGRP gene
was generated by PCR using primers representing a region of the
promoter and exon 1 conserved in the mouse and human IGRP
genes. This was used to probe a rat BAC library to obtain a single
positive clone that was found to contain the entire rat IGRP
gene (Fig. 1).
Exon/Intron Structure of the Human and Rat IGRP Genes--
The
exon/intron structure of the human IGRP gene (Fig. 1; Table
I) was initially determined by comparing
the sequence of the human IGRP gene with that of the mouse
IGRP cDNA (21) and gene (28) and confirmed by subsequent sequence
analysis of human IGRP cDNAs generated by RT-PCR from human islet
RNA. The human and mouse IGRP genes are both composed of 5 exons, and the sizes of exons 2, 3, and 4 are identical (Fig. 1). The
exon/intron splice junctions are also well conserved in comparison with
the mouse gene and match the splice consensus sequence (38), with the exception of the boundary between the 3' end of intron C and the 5' end
of exon 4 (Table I). Both the human and mouse IGRP genes exhibit a change in the nucleotide at the 5' end of exon 4 from the
consensus G to an A (Table I). This change may explain the frequent
removal of exon 4 by differential splicing of the mouse (21) and human
IGRP mRNA (see below). The TATA motif identified in the
mouse IGRP gene promoter (28) is also conserved in the human
IGRP promoter (see below). Since this motif is critical for
determining the location of the transcription start site (39), we would
predict that the IGRP gene transcription start site is identical in the mouse and human genes. If correct, exon 1 in the human
gene will be 6 bp larger than in the mouse gene due to an insertion in
the 5'-untranslated leader sequence (Fig. 1). Thus, the length of
IGRP-coding sequence in exon 1 (218 bp) is identical between human and
mouse genes.
The exact size of human IGRP exon 5 is unknown since a human
poly(A)+ cDNA was not isolated. However, the length of
the IGRP-coding sequence in exon 5 (512 bp) is identical in the human
and mouse genes, and the human genomic sequence could be aligned with
the mouse cDNA through to a conserved element preceding a consensus poly(A) addition site in mouse IGRP. The human IGRP genomic sequence up
to this point contained an additional 500 bp appearing as separate 400- and 100-bp inserts. On this basis, the expected human IGRP mRNA
would be larger than mouse IGRP mRNA, which is consistent with what
is seen on Northern blots (see below). The four introns in the
human IGRP gene, which were determined by direct sequencing, were similar in size to those of the mouse gene (Fig. 1).
The rat IGRP gene, by contrast, showed major differences
from the mouse and human genes. The exon/intron structure (Fig. 1; Table I), determined by comparison of the rat IGRP gene and
mouse IGRP cDNA (21) and gene sequences (28), showed that although exons 2 and 3 are identical in size, exon 4 is absent in the rat gene
(Fig. 1). With the exception of exon 4, the exon/intron splice junctions are otherwise conserved (Table I), and the equivalent of exon
5 was identifiable by sequence homology. Direct sequencing showed that
the intervening sequence between exons 3 and "5" in the rat
IGRP gene was 1830 bp in length; the equivalent in the mouse
and human was 3476 and 2951 bp, respectively. Alignment of the rat and
human gene sequences indicated that ~500 bp were deleted both
upstream and downstream of exon 4 (116 bp in both human and mouse).
Introns A and B were of a similar size to the corresponding human and
mouse introns (Fig. 1). The absence of exon 4 is consistent with other
observations (see below) that indicate that the rat gene is a
non-expressed pseudogene. The sizes for exons 1 and "5" in the rat
gene thus cannot be assigned (Fig. 1).
Chromosomal Mapping of the Human IGRP Gene--
Analysis of the
human genome high throughput sequence data base identified two BACs of
approximately 180 kbp, one of which contained the human IGRP
gene as a contiguous sequence. The two BACS contained the human Unigene
expressed sequence tag cluster markers H210260 and H101282, which
placed the gene on the distal end of chromosome 2 in the interval
D2S156 (microsatellite AFM211yd6) to D2S376 (microsatellite AFM319 × g1) (NCBI GeneMap 99 170.5-180.6 centimorgan) and close
to D2S399 at 174.8 centimorgan (microsatellite AFMa131wb9). Radiation
hybrid analysis placed the gene adjacent to the STS marker SHGC 13934 on chromosome 2 (LOD score 11.84 and 14.90 with exon 2 and exon 5 probes, respectively), which again lies in the D2S156 to D2S376
interval. The SHGC 13934 STS marker amplifies a 137-bp fragment of the
myosin heavy chain 1B gene, the sequence of which was found within the
same BAC as the human IGRP gene. Our previous mapping
studies with mouse IGRP gene (G6pc-rs) using two
interspecific back-cross DNA mapping panels located it on the proximal
portion of mouse chromosome 2 near the marker D2Mit11,
positioned at 39 centimorgan (28). The orthologous gene in humans would
be on chromosome 2q, consistent with our observations.
Sequence Analysis and Translation of Human IGRP
mRNA--
Attempts to clone a cDNA for human IGRP from a
series of human pancreatic islet cDNA libraries using a 1000-bp ORF
probe from the mouse IGRP cDNA were unsuccessful. cDNAs
encoding the ORF, however, could be generated either by PCR-based
ligation of the individual exons or by RT-PCR from cadaveric human
pancreatic islet total RNA. The synthetic and RT-PCR constructs were
identical in sequence and produced the same sized products upon
in vitro translation.
In vitro translation of a cRNA that incorporated the deduced
ORF (nt 43-1020 relative to the mouse cDNA sequence) (21)
generated a protein doublet of 37 kDa, a size consistent with the
predicted molecular mass (40,583 Da) (Fig.
2). In vitro translation of
the ORF of rat G6Pase catalytic subunit from the same vector gave a
somewhat smaller product (33 kDa versus 40,559 Da,
predicted) and raised the question as to whether the assigned human
IGRP start codon was correct. Exon 1 of human IGRP has an in-frame stop
codon at position 21, indicating that translation initiation upstream
was not possible. Downstream, however, there are two start codons that
are conserved in human IGRP and mouse IGRP, although not in the human
or mouse G6Pase catalytic subunits. The second of these has a strong
predicted Kozak sequence (33) that produced a protein of 28 kDa
(predicted size 33.8 kDa) from a 5'-truncated construct (Fig. 2). It
was concluded that the AUG codon at nt 43 is the preferred start site.
The size discrepancy between the predicted and observed molecular mass
of human IGRP probably relates to the hydrophobic nature of the
protein. The slower electrophoretic mobility of IGRP versus
the G6Pase catalytic subunit is conceivably related to its more acidic
nature (predicted pI 8.72 versus 9.22). The molecular sizes
of in vitro translated mouse IGRP and human IGRP were
indistinguishable and consistent with the native predicted molecular
mass (40,685 Da) and pI (8.62) of mouse IGRP and its size, determined
by Western blot analysis of islets (38 kDa) (data not shown).
The deduced human IGRP ORF encoded a 355-amino acid protein of a
generally hydrophobic character (Fig. 3).
Three consensus sites for NH2-linked glycosylation were
present (amino acids 50, 92, and 287), and the protein had a
COOH-terminal consensus sequence (KKXX) characteristic of an
ER-resident transmembrane protein. The hydrophobic amino acids were
arranged in nine major stretches, eight of which were predicted to be
able to span a phospholipid bilayer as an
The human IGRP ORF sequence could be aligned with 2 gaps with residues
7-359 of the human G6Pase catalytic subunit (359 aa) (Fig. 3). The
sequence was 50.4% identical (75.2% similarity) and homologous over
the full length of the molecules including the putative transmembrane
domains. The conservation of these hydrophobic segments and the charged
residues within them suggests that they may have a function other than
simple membrane spanning. Of the three potential sites of
N-glycosylation, only the site located in the putative
second lumenal domain (amino acid 92) was conserved. Both the human
IGRP and G6Pase catalytic subunit molecules contain the COOH-terminal
ER membrane protein retention motif. The human IGRP ORF was identical
in length to the previously cloned mouse IGRP (21) and was closely
homologous (84.8% identity; 92.9% similarity). The homology is
similar to that between the human and mouse G6Pase catalytic subunits
and argues for conservation of function of IGRP. The G6Pase catalytic
subunit shares an extended sequence motif that is found in bacterial
vanadate-sensitive haloperoxidases and mammalian phosphatidic acid
phosphatases (41, 42) (Fig. 3). This motif, which incorporates the
active site of these enzymes, is conserved in human IGRP
(Lys72 ... Arg-Pro80 ...
Pro-Ser-Gly-His115 ... Ser-Arg168 ...
His174) with the same alignment. Amino acids within the
G6Pase catalytic subunit sequence whose mutation results in loss of
function in glycogen storage disease type 1a (Fig. 3) were generally
conserved with the exception of Gly33 (Ala),
Ser120 (Ala), and Lys209 (Leu); the amino acid
present in the G6Pase catalytic subunit is shown in parentheses. Human
and mouse IGRP were identical in sequence at all these positions.
No cDNA for rat IGRP could be isolated by screening either rat
insulinoma or rat islet cDNA libraries with a mouse IGRP probe nor
generated by RT-PCR of rat islet total RNA using rat-specific primers
deduced from the genomic sequence. This contrasted with the relative
ease with which the mouse IGRP cDNA was obtained from these sources
(21). The alignment of the coding regions of the rat gene and those of
mouse and human IGRP furthermore suggested that even if such a cDNA
existed, it would not encode a protein of similar size and sequence to
mouse or human IGRP. Thus, although regions of the rat IGRP
gene corresponding to exons 1, 2, 3, and 5 of mouse IGRP were highly
homologous at the nucleotide level (89.9% identity), there were 3 catastrophic changes within the deduced reading frame. First, a
deletion of 2 nucleotides within exon 1 (mouse nt 73 and 89) changes
the rat coding sequence beyond amino acid 10 even though the reading
frame remains open to the end of the exon. Second, exon 4 (116 bp) is
absent, and splicing of exons 3 and 5, if it occurred, would alter the
reading frame of exon 5. Third, an additional base is present in exon 5 (mouse nt 917), altering the reading frame and producing a premature stop codon. The change in sequence of exon 1 would be circumvented if
the alternate start site at Met57 were used; however, this
would only generate a 11.8-kDa protein because of the exon 4 deletion.
Tissue Distribution and Expression of Human IGRP
mRNA--
Northern blot analyses of human tissue
poly(A)+ mRNA with a human IGRP ORF probe showed the
presence of a single ~3100-bp hybridizing species in pancreas (Fig.
4A). Testis produced a weaker
signal (10% of the pancreas signal) from hybridizing species of
~2400 and 1000 bp (Fig. 4B), whereas 14 other major human
tissues were negative (<3% of the pancreas signal). The same probe
used under low stringency conditions (42 °C, 0.2× SSC) showed a
strong signal from human pancreatic islet total RNA, an even stronger
signal with the equivalent loading of mouse islet RNA, but no signal from rat islets or the rat insulinoma cell lines, RIN (Fig.
4C) and INS-1 (data not shown).
IGRP mRNA expression was further investigated by RT-PCR using
highly conserved primer pairs within exons 1 and 5. As in the case of
Northern blotting, strong signals were detected from mouse and human
islet RNA preparations, but none were detected from rat islets (Fig.
4D). A series of RT-PCR products that were ~100, 200, and
350 bp shorter than the expected 595-bp target were also obtained using
RNA from human islets and human Islet-specific Pancreatic Expression of Mouse IGRP--
Although
Northern blotting indicated a pancreas-specific pattern of IGRP
mRNA expression (Fig. 4A), this analysis did not determine whether IGRP was expressed in pancreatic endocrine or exocrine cells. To address this issue antibodies were raised to recombinant mouse IGRP, and immunoperoxidase staining of mouse pancreas
was performed. The result shows that the antigen was localized to islet
cells with no reactivity evident in the acinar tissue or ductal
elements (Fig. 5). Very few IGRP-negative
cells were observed within the islet, suggesting that alpha and beta cells were certainly immunoreactive and that possibly all four endocrine cell types expressed the protein (Fig. 5).
Enzymic Activity of Human IGRP--
Enzyme activity studies were
performed by transiently transfecting COS 7 cells with various
pCDNA 3.1 constructs; G6P hydrolytic activity was then assessed in
a micosomal fraction prepared from lysed cells. A construct encoding
the rat G6Pase catalytic subunit served as a positive control, and the
efficiency of transfection was evaluated by co-transfection of a Rous
sarcoma virus- Transcriptional Activity of the Proximal Mouse, Rat, and Human IGRP
Gene Promoters--
We have previously shown that the proximal mouse
IGRP promoter region, located between
The observation that several putative cis-acting elements
are conserved in the rat IGRP promoter (Fig. 6) was surprising given that the rat IGRP gene is not expressed (Figs. 4,
C and D). In contrast, the TATA box motif
identified in the mouse IGRP promoter is not conserved in the rat
promoter (Fig. 6). Wobbe and Struhl (43) have shown that the sequence
TGTA found in the rat promoter directs a greater than 20-fold lower
level of in vitro transcription than the TATA motif. To
determine whether this and other sequence variations affect the
relative activity of the mouse, rat, and human IGRP promoters, we
constructed fusion genes in which these promoters were ligated to the
CAT reporter gene. Basal IGRP-CAT fusion gene expression was then
assayed after transient transfection of the HIT cell line. Fig.
7A shows that the human IGRP
promoter sequence located between
A concern with all the data obtained on the rat IGRP gene
and its transcription or translation was the fact that a single BAC
clone served as the source of sequence data and fusion gene constructs.
It was conceivable that a pseudogene was selected in the original
screen and/or that sequence artifacts were introduced during the
construction of the BAC library. These concerns were addressed by
sequence analysis of critical regions of the rat IGRP gene
amplified by PCR using primer sets based on conserved rat/mouse
sequences with genomic DNA isolated from adult rat spleen and liver as
the template. These regions included the promoter and the TGTA motif,
the deletions and insertion affecting the reading frame in exons 1 and
5 and the entire intervening sequence between exons 3 and 5. In every
case the BAC sequence was confirmed. The rat BAC sequencing and
PCR-based gene sequencing were performed in Nashville, TN and Denver,
CO, respectively using independent primer sets to avoid any risk of contamination.
Two remarkable features of the IGRP molecule are the focus of the
current investigation. The first is its structural similarity to the
G6Pase catalytic subunit, a molecule that plays a central role in
glucose homeostasis and the pathophysiology of diabetes mellitus. The
second is its restricted expression to pancreatic islets. A comparative
analysis of the human, mouse, and rat gene structures was undertaken to
define the conserved primary sequence of the protein, which could
impact on the expression of IGRP catalytic activity and conserved
promoter sequences that could be important in tissue-specific and
physiological transcriptional regulation.
Multiple shared sequences were identified in the promoters of the
mouse, rat, and human IGRP genes (Fig. 6) that, along with our previous in situ footprinting studies (36), will
facilitate the identification of cis-acting elements, which
are important for basal and islet-specific IGRP gene
expression. Of specific interest for future mutagenesis are two
putative E-box elements (Fig. 6). Such an element is one of the major
sites that contributes to basal insulin gene expression mediated by a
heterodimeric complex of the basic helix-loop-helix proteins
BETA2/NeuroD and a ubiquitous factor, either E2A or HEB (44, 45).
Surprisingly, both the putative IGRP E-box motifs are conserved in the
rat promoter despite the fact that the TATA box in the rat IGRP
promoter is mutated (Fig. 6) and the gene is not expressed (Fig.
4D). An E-box motif in the human transcobalamin II promoter
has been shown to mediate bidirectional transcription in the absence of
a TATA box motif (46). However, in the case of the rat IGRP promoter,
the two E-box motifs are not sufficient to confer high promoter
activity in the absence of the TATA box (Fig. 7B). The
proximal region of the mouse IGRP promoter that contains the two
putative E-box motifs by itself confers very low basal fusion gene
expression (36); thus, even if these elements are found to contribute
to basal IGRP gene expression, their activity will depend on
additional distal elements.
Several protein binding sites have been identified in the mouse IGRP
promoter by in situ footprinting (36) (Fig. 6) for which no
candidate trans-acting factor can be identified by computer analysis using the MatInspector software (47). These conceivably represent binding sites for novel islet-enriched
trans-acting factors, a hypothesis that gains further
credence from the observation that these regions are highly conserved
in the rat and human IGRP promoters (Fig. 6). The search for such
elements and their associated binding proteins is of particular
interest given the emerging realization that such factors include
potential diabetogenes (48, 49), many of which also play a critical
role in pancreatic ontogeny (50-52).
Since the human IGRP gene, its mRNA, and the encoded
protein showed great similarity to the corresponding mouse IGRP
molecules this suggests conservation of biological function. The
full-length human IGRP protein, like the mouse protein, possessed
characteristic structural features of an ER-localized transmembrane
protein with hydrolytic properties (41, 42). Yet as previously
observed, no catalytic activity was obtained with either G6P or a
generic phosphatase substrate (Table II). Nevertheless, the present
documentation of the human IGRP protein sequence will assist in future
structure/function studies using site-directed mutagenesis and chimeric
IGRP/G6Pase catalytic subunit proteins. Immunohistochemical analyses
with antibodies directed toward recombinant mouse IGRP confirm that the
IGRP protein is expressed endogenously in mouse islets (Fig. 5).
Moreover, Western blotting analyses with antibodies directed toward
Myc-tagged mouse IGRP show that it can be expressed in COS 7 cells by
transient transfection.3 Thus our inability to
detect enzymatic activity cannot be related to failure of protein expression.
Although the data obtained on human IGRP were consistent with the
hypothesis that IGRP plays an important tissue-specific function in the
adult pancreatic islet, the results with the rat IGRP gene
paradoxically indicate that IGRP is a non-functional or perhaps a
vestigial gene product. We found no evidence for the existence of a
second, functional IGRP gene in the rat, and mRNA
expression could not be detected in rat islets or rat islet-derived cell lines using either probes that hybridize with the highly homologous mouse sequence or by RT-PCR analyses with exact primers (Fig. 4). The rat IGRP gene nucleotide sequence was more
closely related to the mouse than the human sequence, as expected from the taxonomic divergence of these species, and clearly it was the rat
equivalent of the IGRP gene. A possible explanation of this
paradox is that selection has occurred in the rat IGRP gene for a series of mutational events that have ensured its inactivation. The deletion of exon 4 in the rat IGRP gene removes
sequences that would be essential for hydrolytic activity (53),
although it could be argued that, like the common
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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cell-derived
cell lines that encodes an islet-specific G6Pase catalytic
subunit-related protein (IGRP) (21). IGRP is a putative ER membrane
protein that is similar in size (38 kDa), topology, and sequence
(~50% identity at the amino acid level) to the G6Pase catalytic
subunit (21). The function of IGRP, however, is uncertain since its overexpression in fibroblast or endocrine cell lines does not increase
rates of G6P hydrolysis in tissue homogenates (21). The mouse
IGRP gene has a chromosomal locus that is distinct from the
G6Pase catalytic subunit but it has a similar exon/intron structure,
suggesting that the genes arose from an ancient gene duplication/transposition event (28). To date, IGRP gene
expression has only been detected in pancreatic endocrine cells (21), a feature that is reflected in the islet-specific activity of the IGRP
promoter as assessed using fusion gene constructs. Thus, the IGRP
promoter is inactive in human HepG2 hepatoma cells but is ~150-fold
more active than the G6Pase catalytic subunit promoter in hamster
insulinoma tumor (HIT) cells (28).
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-32P]dATP (>3000 Ci
mmol
1) and [
-32P]ATP (>6000
Ci mmol
1) were obtained from Amersham
Pharmacia Biotech, and [3H]acetic acid, sodium salt (>10
Ci mmol
1), was obtained from ICN. All
individually specified reagents were of analytical grade and purchased
from Sigma.
-32P]dATP using the
Stratagene Prime-It II random primer labeling kit according to the
manufacturer's instructions (final specific radioactivity, 0.2-1.2
Ci.nmol
1). DNA sequencing was performed using
the U. S. Biochemical Corp. Sequenase kit or by automated sequencing
using an ABI 377 DNA analyzer. All DNA sequences are numbered relative
to the experimentally determined mouse IGRP gene
transcription start site, designated +1 (28).
80 °C to Kodak
X-Omat AR film. Specific hybridization signals were identified as
positive replicates from the filter map, and the corresponding PAC
clone was then used for large scale isolation of plasmid by cesium
chloride gradient centrifugation (29). Two independent clones
designated PAC 294 (~90 kbp) and PAC 299 (~110 kbp) were isolated.
They were digested with a panel of restriction enzymes, and Southern
blot analysis was performed with fragments corresponding to the 5' or
3' end of the mouse IGRP gene (28). These were, respectively, a SalI-PvuII fragment of a mouse
IGRP cDNA clone (pSV.SPORT 1B1) containing exons 1, 2, and part of
exon 3 (21) and an XbaI-XbaI fragment of the
mouse IGRP gene, isolated from the pGEM-BAC 4.5 plasmid
(28), containing exons 4 and 5 and the intervening intron sequence. The
hybridization analysis indicated that both PACs contained the entire
human IGRP gene, which was then isolated within two
overlapping genomic sub-clones, a ~9-kbp XhoI-XhoI fragment containing the promoter and
exons 1-4 and an ~6-kbp EcoRI-EcoRI fragment
containing exons 3-5 and 3'-flanking sequence. The fragments were
sub-cloned from PAC294 into pGEM7 (Promega, Madison, WI) and sequenced
on both strands over their entire length. The identification of the
exon/intron boundaries (Table I) and the sizes of the four introns in
the IGRP gene (Fig. 1) were initially determined by
comparison with the mouse IGRP gene sequence (28) and
subsequently from the human cDNA sequence (see below).
TC3, Min6, RIN, INS1, HIT, and
TC1 as well as COS 7 cells were
passaged as subconfluent cultures (8.5-cm dishes) in Dulbecco's
modified Eagle's medium supplemented with 100 units/ml penicillin and
100 µg/ml streptomycin. INS-1 cultures contained in addition 50 µM mercaptoethanol. All cell cultures were also
supplemented with 10% (v/v) fetal bovine serum except HIT cell
cultures, which were supplemented with 2.5% (v/v) fetal bovine serum
and 15% (v/v) horse serum.
-galactosidase, which was purified by isolation of
inclusion bodies and preparative SDS-gel electrophoresis. Antibodies were raised in New Zealand white rabbits by immunization in complete Freund's adjuvant followed by boosting in incomplete Freund's adjuvant at six weekly intervals. Balbc mouse pancreas was
perfusion-fixed with 4%(w/v) paraformaldehyde and subjected to
standard paraffin embedding and sectioning before immunoperoxidase
staining using the primary antiserum diluted 1:100 in PBS (2 h at room
temperature) and a secondary donkey anti-rabbit antibody conjugated to
horseradish peroxidase (Jackson Laboratories).
306 to +3 in the
pCAT(An) expression vector (35) has been previously described (28). A
rat IGRP-CAT fusion gene was constructed in the pCAT(An) expression
vector as follows. The rat IGRP gene promoter was isolated
as a HindIII-PstI fragment and subcloned into
HindIII-PstI-digested pSP72 (Promega). The
promoter fragment was then isolated from the pSP72 plasmid as a
HindIII-BamHI fragment and ligated into HindIII-BglII-digested pCAT(An). The resulting
plasmid contains rat IGRP promoter sequence from
900 to +3, relative
to the position of the mouse transcription start site. The
PstI site used in this cloning is conserved in the mouse
IGRP gene, and the same strategy was used in the
construction of the previously described full-length mouse IGRP-CAT
fusion gene (28). Therefore, the same 3' polylinker sequence between
position +3 and the CAT reporter gene is present in the mouse and rat
fusion gene constructs. A truncated rat IGRP-CAT fusion gene was then
generated by restriction enzyme digestion of the
900 IGRP-CAT
construct with HindIII and NheI followed by
Klenow treatment of the noncompatible ends and blunt-end ligation. The
resulting plasmid has a calculated 5' end point of
321. The TGTA
sequence in the rat IGRP promoter was mutated to a TATA box by
site-directed mutagenesis within the context of the
321 to +3
promoter fragment using PCR and the following oligonucleotide as the 3'
primer:
5'-AACTGCAGGGCTCAGAGTTCGGTTGTCTTTATAGGGTCCCCTTGTGATG-3'. A PstI site used for cloning purposes and the mutated base
are underlined. The 5' PCR primer
(5'-CGGGATCCAAGCTCTAGCCAAGC-3'), with the
BamHI cloning site underlined, was designed to conserve the
junction between the IGRP promoter and pCAT(An) vector to be the same
as that in the wild-type rat
321 IGRP-CAT fusion gene
construct; the HindIII-NheI junction is shown in
italics. The PCR fragment was digested with BamHI and
PstI and subcloned into
BamHI-PstI-digested pSP72 for sequencing. The
promoter fragment was then re-isolated from the pSP72 plasmid as a
BamHI-PstI fragment and ligated into
BamHI-PstI-digested
321 rat IGRP-CAT. This
BamHI site is located immediately 5' of the
HindIII cloning site in the pCAT(An) vector (35).
306 IGRP-CAT and rat
321 IGRP-CAT constructs (Fig. 7).
This was achieved using PCR in conjunction with the following 5'
(5'-CCCAAGCTTCACCAAACATAGAAATTGC-3') and 3'
(5'-AACTGCAGTGCTCTGATTCCCACCG-3') primers.
HindIII and PstI sites used for cloning purposes
are underlined. A single base pair change (italics) at position
1 in
the human promoter was introduced into the 3' primer to restore the
PstI site such that the subsequent sub-cloning of the PCR fragment generated a fusion gene construct with the same 3' polylinker sequence between position +3 and the CAT reporter gene as found in the
mouse and rat fusion gene constructs. Thus, the PCR fragment was
digested with HindIII and PstI and subcloned into
HindIII-PstI-digested pSP72. The promoter
fragment was then isolated from the pSP72 plasmid as a
HindIII-BamHI fragment and ligated into
HindIII-BglII-digested pCAT(An). The resulting
plasmid contains human IGRP promoter sequence from
324 to +3,
relative to the position of the mouse transcription start site.
Promoter fragments generated by PCR were completely sequenced to ensure
the absence of polymerase errors, whereas promoter fragments generated
by restriction enzyme digestion were only sequenced to confirm the 5'
end points. All plasmid constructs were purified by centrifugation
through cesium chloride gradients (29). For fusion gene analyses, HIT
cells were grown and co-transfected as previously described using a
calcium phosphate precipitate containing 15 µg of a CAT construct and
2.5 µg of a Rous sarcoma virus-
galactosidase fusion gene
construct (36). After transfection,
-galactosidase and CAT activity
were assayed as described (36). To correct for variations in
transfection efficiency, the results are expressed as a ratio of
CAT:
-galactosidase activity. In addition, three independent
preparations of each plasmid construct were analyzed in quadruplicate
in separate experiments.
-galactosidase followed by culture for a further 48-72 h in
Dulbecco's modified Eagle's medium with serum (36). Cells were
harvested using a non-enzymic procedure (Life Technologies, Inc. cell
dissociation buffer), rinsed twice in 0.3 M sucrose, 10 mmol l
1 MES-K+, 2 mmol l
1 EGTA, 1 mmol
l
1 MgSO4 (pH 6.5), and then sonicated for 20 s in 1 ml of the same media. The sonicate was centrifuged at 800 × g for 6 min to remove unbroken cells and debris, and a
particulate fraction was prepared by further centrifugation of the
supernatant at 214,000 × g for 30 min in a Beckman
TLN-55 rotor. The pellet was resuspended in 300-500 µl of
homogenization media (~0.3-1 mg/ml protein) and assayed for G6P
hydrolytic activity (21). The supernatant was assayed for
-galactosidase activity using the spectrophoto- metric assay
previously described (21).
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Fig. 1.
Structure of the mouse, rat, and human
IGRP genes. The IGRP gene exon and
intron sizes were determined by a combination of direct DNA sequencing
and PCR as described under "Experimental Procedures."
Comparison of the exon/intron boundaries of the mouse, rat, and human
IGRP genes
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Fig. 2.
In vitro translation of human
(h) and mouse (m) IGRP. T7
polymerase-derived transcripts from cloned pCDNA3.1 constructs were
translated in vitro in the presence of
[35S]methionine using rabbit reticulocyte lysate and
analyzed by SDS-PAGE. Mouse IGRP constructs included the original
cDNA (mIGRP cDNA; nt 1-1901), a PstI
fragment containing the second and third alternative start codons
(mIGRP Met 2&3; nt 110-1137), a PCR construct containing
the third start codon only (mIGRP Met 3; nt 220-1038), a
PCR construct containing the ORF but with minimal flanking sequence
(mIGRP Met1; nt 59-1038), and the exon 4 form of IGRP
(mIGRP exon 4).
-helix (TMAP (40)).
Each stretch, however, contained a charged amino acid(s) (aa): the
sequence aa 25-47, Asp34 and Arg36; aa
57-77, Asp65 and Lys72; aa 116-138,
His133; aa 148-173, Arg168; aa 179-193,
Glu191, aa 210-230, Arg227; aa 255-280,
Arg261 and Glu276; aa 288-307,
Arg293; and residues 319-343, Lys327. The
NH2 terminus of the protein did not bear a consensus signal sequence for import into the ER, but it is conceivable that the putative transmembrane segments could function in this regard.
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Fig. 3.
Alignment of the deduced peptide sequences of
human and mouse IGRP and the corresponding G6Pase catalytic subunit
sequences. The predicted amino acid sequence of the human IGRP
protein was aligned using CLUSTAL with mouse IGRP, human G6Pase
catalytic subunit (accession number U01120), and mouse G6Pase catalytic
subunit (accession number U00445). Putative transmembrane segments are
shaded, and conserved charged residues within them are
designated +/ . Consensus sites for NH2-linked
glycosylation are also shown (#) below the sequence block. Residues
defined as being of key catalytic importance in haloperoxidases and
related phosphatases are boxed, as is the COOH-terminal
endoplasmic reticulum retention signal. Point mutations in the human
liver enzyme that give rise to type 1a glycogen storage disease are
indicated by the depiction of the mutant residue above the sequence
block. Black dots (
) indicate amino acids that are identical between
the human and mouse IGRP and the human and mouse G6Pase catalytic
subunit.
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Fig. 4.
Analysis of mouse, rat, and human IGRP
mRNA expression by Northern blotting and RT-PCR. Panels
A and B, Northern blotting analysis was performed on
2-µg samples of poly(A) + mRNA
(CLONTECH MTN and MTN1 blots) hybridized with a
human IGRP cDNA spanning the ORF. Blots were washed in 2× SSC at
42 °C and visualized by phosphorimaging (72 h exposure). The imaging
sensitivity range is 3-fold higher for the blot shown in panel
B relative to that in panel A. PBMC,
peripheral blood mononuclear cell. Panel C, Northern
blotting analysis was performed on 5-µg samples of total RNA from the
indicated tissues hybridized with a human IGRP cDNA spanning the
ORF. Blots were washed in 2× SSC at 42 °C and visualized by
phosphorimaging (72-h exposure). Panel D, samples (300 ng)
of total RNA were reverse-transcribed using random nonamers, then
amplified by PCR for 30 cycles using conserved primers within IGRP
exons 1 and 5. A full-length mouse IGRP clone (0.1 ng) was used as a
reference template. Products included the expected 595-bp product and a
series of shorter amplicons. Human islets were obtained from cadaveric
donors (numbers 144, 1174, and 1232) and cultured for 24-72 h before
analysis. Mouse and rat islets were freshly prepared. RNA isolated from
two human cell lines (NesY2 (human nesidioblastoma-derived
insulin-secreting cell line) A and B) derived from a single
nesidioblastoma patient was also analyzed.
cell-derived cell lines. Cloning
and sequencing of these products from human sample 144 and 1174 (Fig.
4D) identified them as alternatively spliced variants, the
most prominent of which is an exon 4 deletion equivalent to that
previously documented in mouse IGRP (21, 28). Other variants included
deletions of exon 2, exons 3 plus 4, and exons 2, 3, and 4 together.
Splicing occurred accurately at the donor/splice junctions shown in
Table I. Only one of the alternatively spliced products (
exons 3 and
4) maintained the reading frame of the full-length molecule and could
potentially generate a 32.2-kDa protein. The relative proportions of
alternately spliced forms varied from individual to individual and
between oligo(dT) versus randomly primed transcripts. This
variation did not appear to correlate with the age, sex, and weight of
the donor nor the warm ischemia time before islet isolation or duration of tissue culture before RNA extraction (Fig. 4D). Without
reference to fresh material from healthy individuals, the significance
of these alternatively spliced products is unclear.
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Fig. 5.
Immunoperoxidase staining of mouse pancreas
with antibodies raised to recombinant IGRP. Immunoperoxidase
staining was performed as described under "Experimental
Procedures." IGRP was localized by immunoperoxidase labeling
(brown) and the sections were counterstained with Gill's
hematoxylin.
galactosidase fusion gene construct. Transfection
with the G6Pase catalytic subunit construct resulted in an ~25-fold
increase in G6P hydrolysis over basal activity (Table
II). In contrast, transfection with a
construct encoding human IGRP produced no detectable change in G6P
hydrolytic activity, as previously observed for mouse IGRP (Table II
and Ref. 21). Transfection of COS 7 cells with constructs encoding
truncated forms of mouse IGRP in which the putative start codon was
deleted but which contained either the second or third putative start
site also failed to increase basal G6P hydrolytic activity (Table II).
The rates of hydrolysis of the generic phosphatase substrate,
p-nitrophenol phosphate were not altered in human IGRP- or
mouse IGRP-transfected COS 7 cells, although they were good substrates
for the G6Pase catalytic subunit (data not shown and Ref. 21).
G6P hydrolytic activity in transiently transfected COS7 cells
-galactosidase (5 µg). Enzyme activities were determined in cell
homogenates after 48 h. Mouse IGRP constructs (see Fig. 2 legend
for details) included the designated full-length construct (mouse
IGRP), a PstI fragment containing the second and third
alternative start codons (Met 2+3), and a PCR construct containing the
third start codon only (Met 3). Each tabulated result represents the
mean ± S.E. of duplicate determinations from four separate
experiments.
306 and +3, is sufficient to
confer maximal IGRP-CAT fusion gene expression in HIT cells (28). The level of basal mouse IGRP-CAT fusion gene expression in both HIT and
TC-3 cells decreases gradually upon deletion of the IGRP promoter
sequence between
306 and
66, indicating that multiple cis-acting elements contribute to maximal fusion gene
expression (36). An alignment of the equivalent human and rat IGRP
promoter regions revealed multiple regions of conserved sequence (Fig. 6). We previously determined the location
of several transcription factor binding sites in the mouse IGRP
promoter using the ligation-mediated PCR in situ
footprinting technique; these binding sites correlated with regions of
the IGRP promoter, identified as being important for basal IGRP-CAT
fusion gene expression (36). Fig. 6 shows that many of the residues in
the mouse IGRP promoter that are contacted by transcription factors in
TC-3 cells in situ are also conserved in the human and
rat promoters. In addition, a hepatocyte nuclear factor-3
binding site identified in the mouse IGRP promoter, which binds a
hepatocyte nuclear factor-3 in vitro (36), is also conserved
in the rat and human promoters (Fig. 6).
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Fig. 6.
Alignment of the mouse, rat, and human
IGRP gene promoter sequences. The human, mouse,
and rat IGRP promoter sequences were aligned using the IntelliGenetics,
Inc. IFIND program and labeled relative to the experimentally
determined transcription start site of mouse IGRP (28) designated as
+1. Increases ( ) or decreases (
) in dimethyl sulfate methylation
of the mouse IGRP promoter comparing in situ
versus in vitro methylated
TC-3 cell DNA were
determined by ligation-mediated PCR (36). The TATA box, two E-box
motifs, and a hepatocyte nuclear factor-3 binding site, which
are conserved between the mouse and human promoters, are
boxed.
324 and +3 confers a slightly
higher level of basal fusion gene expression than the equivalent mouse promoter sequence located between
306 and +3. By contrast, neither the equivalent rat IGRP promoter sequence located between
321 and +3
(Fig. 7B) nor a longer fragment of the promoter containing sequence located between
900 and +3 (Fig. 7A) confers
appreciable fusion gene expression. However, mutating the rat TGTA
motif back to the consensus TATA motif markedly enhances basal rat
IGRP-CAT fusion gene expression (Fig. 7B). Nevertheless, the
actual level of rat promoter activity remains ~3-fold lower than that
of the mouse (Fig. 7B), suggesting that changes in elements
other than the TATA box contribute to the low activity of the rat
promoter.
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Fig. 7.
Basal activities of the mouse, rat, and human
IGRP gene promoters. HIT cells were transiently
co-transfected, as described under "Experimental Procedures," with
mouse, rat, or human IGRP promoter-CAT fusion gene constructs (15 µg)
containing the promoter sequence shown together with a reference vector
encoding -galactosidase (
gal; 2.5 µg). The rat
constructs had either the wild-type (TGTA) or back-mutated (TATA) box
motif. After transfection, cells were cultured for 18-20 h in
serum-free medium, and CAT and
-galactosidase activity was
determined (36). The mean ratio of CAT:
-galactosidase activity
±S.E. is presented from three transfection experiments, each using
independent preparations of each CAT plasmid.
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exon 4 splice
variant of mouse (21) and human IGRP (Fig. 4D), such a molecule has a
regulatory function. However, the reading frame mutations in the 5' and
3' ends of the ORF of rat IGRP and the change in the TATA box indicate
that, at some point in evolution, expression of IGRP in the rat islet
was turned off because the protein was unnecessary, redundant, or deleterious.
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ACKNOWLEDGEMENTS |
---|
We thank Roland Stein and Eva Henderson for providing the HIT cell line, Rebecca Taub for the rat G6Pase construct, Kevin Docherty for NesY2 (human nesidioblastoma-derived insulin-secreting cell line) RNA, and Donna Curtis for assistance with the data base searching. Human islets were obtained from Miami, FL, Rochester, MN and Boston, MA through the Juvenile Diabetes Foundation International islet consortium.
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FOOTNOTES |
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* This work was supported by a grant from Juvenile Diabetes Foundation International (JDFI) and Vanderbilt Diabetes Core Laboratory Grant P60 DK20593 (to R. O'B.), American Diabetes Association Grant 9901-116 and Barbara Davis Center Diabetes and Endocrinology Research Center Grant P30 DK57516 (to J. C. H.), and by a grant from the Nord-Pas de Calais Region (to F. L.).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) AF283835 (human IGRP gene excluding the promoter), AF283575 (human IGRP promoter), AF321459-AF321463 (human IGRP exons 1-5, respectively), AF323433 (rat IGRP promoter and exon 1), AF323434-AF323436 (rat IGRP exons 2, 3, and 5, respectively), NM021331 (mouse IGRP cDNA), AF118761 (mouse IGRP promoter), and AF 118762-AF118766 (mouse IGRP exons 1-5, respectively).
§ Supported by Vanderbilt Viruses, Nucleic Acids, and Cancer Training Program 5T32 CA09385-17).
Recipient of Vanderbilt Molecular Endocrinology Training
Program Award 5 T 32 DK07563-12).
To whom correspondence may be addressed: Dept. of Molecular
Physiology and Biophysics, 761 MRB II, Vanderbilt University Medical School, Nashville, TN 37232-0615. Tel.: 615-936-1503; Fax:
615-322-7236; E-mail: richard.obrien@mcmail.vanderbilt.edu (R. O'B.)
or Tel.: 303-315-8197; Fax: 303-315-4892; E-mail:
john.hutton@uchsc.edu (J. C. H.).
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101549200
2 Children's Hospital Oakland Research Institute BACPAC Resources Home Page.
3 B. Bergman and J. C. Hutton, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: IGRP, islet-specific G6Pase catalytic subunit-related protein; G6Pase, glucose-6-phosphatase; ER, endoplasmic reticulum; G6P, glucose 6-phosphate; ORF, open reading frame; CAT, chloramphenicol acetyltransferase; RIN and INS-1, rat insulinoma-derived cell lines; contig, group of overlapping clones; HIT, hamster insulinoma tumor; bp, base pair(s); kbp, kilobase pair(s); PCR, polymerase chain reaction; RT, reverse transcription; nt, nucleotides; MES, 4-morpholineethanesulfonic acid; aa, amino acid(s).
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