J. Biol. Macromol., 7 (3), 35-48 (2007)

Regular Paper

Analysis of intron-exon positioning on glutamate decarboxylase
and its relation with evolution
Satoko Matsukawa and Hiroshi Ueno§
Laboratory of Applied Microbiology and Biochemistry, Graduate School of Humanities and Sciences,
Nara Women’s University, Nara 630-8506, JAPANReceived June 25, 2007; Accepted July 15, 2007

Glutamate decarboxylase (GAD) is an enzyme that catalyzes a production of GABA. The
positions of intron-exon boundaries are located on mammalian/fruit fly GAD genes and theirrelations with the evolution, function, and structure of the protein are considered. The intron-exonboundaries were assigned on the primary structures of GAD65 of human, rat, and mouse, GAD67 ofhuman, rat, mouse and chicken, and fruit fly GAD. GAD65 and 67 share an identical positions for
intron-insertion and similar amino acid sequences except highly heterogeneous exons 1-3 coding
region. When fruit fly GAD was compared with mammalian GADs, N-terminal region wasconstructed with only 20 amino acids for exon 1 in fruit fly GAD whereas 100 residues for exon 1-3 inmammalian GADs. The total number of introns in fruit fly GAD is significantly lower than that in
mammalian GADs as if fruit fly has lost its introns during the evolution. The presence of common
ancestral gene for fruit fly and mammalian GADs is assumed. The intron-exon boarders are
assigned on the predicted structures of the examined GADs. Most of the intron insertion positions
are not located directly on the defined secondary structural units or in the hydrophobic core. We
have examined the relationship between the intron-exon boundary and a module, a most compact
unit in protein structure proposed by Go [1], and have confirmed the boundary exhibits close
correlation with the module boundary.
Keywords: bioinformatics, evolution, exon, GAD, intron, module

Introduction
Glutamate decarboxylase (GAD, EC 4.1.1.15)
is an enzyme to synthesize γ-aminobutyric acid
(GABA), a major inhibitory neurotransmitter inhigher animals [2]. GAD is widely distributed
among living cells of various organisms frommammalians to single cell organisms [2, 3].
Recently, it has shown that mammals have two
distinct genes for GAD; for instance, human has
one on chromosome 2 and the other onchromosome 10, where each produces GAD67 andGAD65, respectively [4-6]. The two GADs
respond differently to their common cofactor________________________________________§To whom correspondences should be addressed.
E-mail: hueno@cc.nara-wu.ac.jp, Phone:+81-742-20-3493, Fax: +81-742-20-3448

pyridoxal 5’-phosphate (PLP) and differ insubcellular location [7, 8], which suggests that
GAD67 and GAD65 may have different
physiological roles. Therefore, the functionalroles of GAD isoforms have been investigated [3].
Obata and others have created knock-out mice
to demonstrate different functionality of GAD
isoforms. For example, GAD67-/- mice bornwith cleft palate are dead within a day after birth____________________________________
Abbreviations: 3D, three dimensional; DDC,
dihydroxyphenylalanine decarboxylase or
L-aromatic amino acid decarboxylase; GABA,
γ-amino butyric acid; GAD, glutamate
decarboxylase; PDB, Protein Data Bank; PLP,
pyridoxal 5’-phosphate

36

Bioinformatic study on GAD

while GAD65-/- mice are survived with a slightly
increased tendency in having seizures [9].
GAD67 protein is a hydrophilic and soluble
polypeptide which is found mainly in the cellbodies of neurons and in the cytosol of other cells
particularly pancreatic β-cells. GAD65 is morehydrophobic and less soluble than GAD67 andreversibly anchored to the membrane of synaptic
vesicles of neurons and synaptic-like microvesicles
in pancreatic β-cells [7]. It has been suggested
that GAD67 is responsible for most of themetabolic GABA synthesis in the brain [8, 9].
Pancreatic GAD65 was identified as a target
antigen for autoantibody found in the blood
circulation of patients with insulin-dependent (typeI) diabetes [10, 11]. Accumulated evidences havesuggested that GAD isoforms play different roles
within a single cell. Moreover, it is probable toassume that the differences of functionality of theisoforms could be attributed to the differences intheir structures. However, for GADs from mostof the species, direct correlation of the exonboarders and the tertiary or secondary structure,
has not been established and needs to be elucidated.
Recently, an X-ray structure of GAD is reported
for E. coli GADB by Capitani et al. who performed
a homology modeling of GAD65 based onreported structures of pig DOPA decarboxylase(DDC) and E. coli GADB [PDB No. 1JS6 and
1PMM] [12, 13].
Eukaryotic genes encoding proteins are split
by introns, whereas prokaryotic genes are not split
by introns except for some cases such as plasmidof Agrobacterium rhizogenes [14]. The origin of
those spliceosomal introns and their functionalroles are still under investigation. It is still
controversial that whether or not 1) introns existed
in the ancestral genome of prokaryotes, eukaryotes,
or both, or 2) introns were inserted into eukaryotic
genomes in later stage during the evolution [15].
If it would be the former case, prokaryotes shouldhave lost the introns from their genomes andslimmed down the size of genomic sequences. If
the latter case is assumed, the introns could be
inserted randomly into the eukaryotic genes, where
the introns might have originated from theinsertion elements such as transposable elements.
Doolittle and Gilbert have suggested that theresulting introns would not contribute to thecreation or modification of the protein function [16,17].
A term “domain” is referred as a criticalstructural unit when the three-dimensional (3D)
structure of protein is discussed. In 1981, Go
introduced a concept of “module” which is a partof domain structure but more compact than domain[1]. A module is a peptide segment of about 15
residues long in an average and constructs acompact structural unit in the protein structure.
The term “module” is also referred to as the mostcompact functional unit of protein [1, 18, 19].
Recent studies have demonstrated that thelocation of introns in a gene exhibits close
correlation with module boundaries in a protein
structure, which has drawn an attention as theunderstanding of intron positions is crucial for
considering
theproteinstructure/function
relationships [1, 18, 20]. A protein evolutional
study allows the origin of intron to be traced backinto the ancestral gene; however, the roles of intron
as the mediator of exon shuffling have not been
fully realized. In this study, GAD genomes offruit fly, mouse, rat, chicken and human are
analyzed for the intron-exon borders as well as
their structural and functional relationships,
particularly linked to the evolution of GAD.

Materials and Methods
Multiple alignments - Primary amino acidsequences and intron-exon information on GADs
were obtained from the Ensemble genome browser(http://www.ensembl.org) for mammals and GTOP
(http://spock.genes. nig.ac.jp/ genome/gtop-j.html)
for fruit fly. The multiple alignments werecarried out using the CLUSTALW program(http://www.ebi.ac.uk/clustalw/). The accessnumbers in Ensemble for human, rat, mouse andchicken, and GTOP for fruit fly are as follows:
human GAD65: ENST00000259271, human
GAD67: ENST00000249471, rat GAD65:ENSRNOT0000000000824901, rat GAD67:ENSRNOT00000000008, mouse GAD65:
ENSMUST00000028123, mouse GAD67:
ENSMUST00000028507, chicken GAD67:
ENSGALT00000015628 , fruit fly: CG14994.
Structure analysis – Secondary structure
prediction was performed with the programS. Matsukawa and H. Ueno

37

Table 1 Comparison of amino acid sequences at the site where intron being allocated among mammalian andfruit fly GADs. The amino acid at the intron site is indicated in red (majority) or blue (minority) with its residue
number beneath.

JPRED (http://www.compbio.dundee.ac.uk/~www-jpred/). Relative solvent accessibility ofeach amino acid of pig DDC and E. coli GADB
was evaluated by the program DSSP
(http://swift.cmbi.ru.nl/gv/dssp/) based on their
X-ray structures.
X-ray structures of pig DDC and E. coli
GADB - The crystal structures of DDC from Sus
scrofa (Protein Data Bank (PDB) entry no. 1JS3)
and GADB from E. coli at low pH (active form,
PDB entry no. 1PMM) were obtained from PDB(http://www.rcsb.org/pdb/Welcome.do).
Structural alignments among E. coli GADB, pig
DDC and human GAD65 sequences were carriedout based upon Capitani’s study [12].
Evolutional tree - Evolutional analysis foramino acid sequences of mammalian and fruit fly
GADs was performed by the method ofneighbor-joining using CLASTALW. The
evolutional tree was generated by the software
Tree View (http://taxonomy.zoology.gla.ac.uk/rod/
treeview.html).

Results
Multiple alignments - The coding regions ofhuman, rat and mouse GAD65 genomes arecomposed with 16 exons (Fig. 1a). Exon 1
contains the 5’ untranslated region of GAD65mRNA, and exon 16 contains the protein’s
carboxyl terminal region and a small portion ofuntranslated region. Similarly, the coding regionof human GAD67 genomes consists of 16 exons.
Rat, mouse, and chicken GAD67 genomes contain17 exons, where an additional exon, exon 0,
together with a part of exon 1 contains the 5’
untranslated region of GAD67 mRNA (Table 1).
Exon 16 of GAD67 covers the entire 3’
untranslated region of GAD67 mRNA. The
genomes of both GAD isoforms from mammalian
species that we have examined exhibit highidentity in the intron insertion positions. The
comparison of the primary structures GAD65 frommammalian species showed over 90% identity (Fig.

38

Bioinformatic study on GAD

Fig. 1 Comparison of the amino acid sequences at the sites of intron intervention in the coding
region of mammalian GADs and fruit fly GAD cDNAs. (a) mammalian GAD65, (b)mammalian GAD67, (c) human GAD67 and GAD65, (d) human GAD67, GAD65 and fruit fly
GAD, respectively. Numbers on the right are residue positions. The intron insertion sites
are shown in gray highlight. The numbers of intron insertion sites are shown above withcircle or square for mammalian or fruit fly, respectively. Deduced protein sequences arealigned by using the program CLUSTALW.
Amino acid identity is indicated by asterisk
for the match, colon for strong similarity and dot for weak similarity. Absence of thecompatible residues at the position is indicated by blank space.

S. Matsukawa and H. Ueno

Fig. 1b. (continued)

39

40

Bioinformatic study on GAD

Fig. 1c. (continued)1a). Similar results were obtained for GAD67(Fig. 1b). On the other hand, when the primarystructures were compared between GAD67 andGAD65 from the same species, the sequencehomology declined to 65% [21]. Despite of thesequence diversities among mammalian species,
the positions of the exon-intron boundaries in all
mammalian GADs were virtually identical (Table
1 and Fig. 1d). It is of interest to note that mostof the heterogeneity was found within N-terminal
100 residues. The N-terminal regions of humanas well as the other mammalian GADs have beencoded by exons 1-3 (Fig. 1c). When details ofexons 1-3 were compared, the level ofheterogeneity appeared to be different for each of
the exons. While there are about 30% residues
conserved between GAD65 and GAD67 on exon 1
and exon 2, the frequency of the conservation
becomes extremely low in case of exon3.
Fruit fly GAD genomes include seven exons,of which exon 0 and a part of exon 1 contain the 5’
untranslated region of fruit fly GAD mRNA (Table

S. Matsukawa and H. Ueno

41

Fig. 1d. (continued)1). The exons 2-7 of fruit fly GAD and exons
4-16 of mammalian GADs show high amino acidsequence homology. Also fruit fly andmammalian GADs share almost identicalexon-intron boundaries within this region (Fig. 1d).
The number of exons in fruit fly GAD issignificantly smaller than that of mammalians.

This is because exon 3 in the fruit fly is extended
so that it covers exons 5–10 of the mammalians(Table 1, Fig. 1d). In other words, fruit fly lacks
introns corresponding to introns 5–10 ofmammalians. Similarly, exon 6 of the fruit fly
GAD, matching to the exons 13–15 of the42

Bioinformatic study on GAD

Fig. 2 Predicted secondary structures assigned on the aligned mammalian and fruit fly GAD
amino acid sequences. Secondary structures predicted by JPRED: α-helix is indicated by H,
β-structure by E and random structure by -. Numbers on the right are residue positions. The
intron sites are shown in gray highlight. The numbers of intron insertion sites are shown above
with circle or square for mammalian or fruit fly, respectively.

S. Matsukawa and H. Ueno

43

Fig. 2. (continued)
mammalian GADs, does not have corresponding
introns 13 and 14 in the mammalian GADs (Fig.
1d). The lengths of exons tend to be longer infruit fly than those in mammals: The results are
consistent with Go’s description [22]. Exons 1-3
of mammalian GADs and exon 1 of fruit fly GAD
were not homologous; however, a short segment at
the end of exon 1 of fruit fly is conserved in both
fruit fly and mammalian GADs (Fig. 1d).
Position of introns and its relationship withthe secondary structure – The predicted secondary
structure of the examined GADs are summarized
in Fig. 2. In mammalian GADs, the position of
introns in the secondary structures on the aminoacid sequences and the intron-exon boundaries arehighly conserved. The predicted secondary
structure of fruit fly GAD was similar to exon 4
and thereafter of mammalian GADs. When thesecondary structure assigned to N-terminal low
homology region was examined, a segment
covering entire exon 2 takes α-helical structure(Fig. 2). The secondary structure predicted forthe remaining region exhibited close resemblance
between the two isoforms. Therefore, theheterogeneous nature at the N-terminal region isconsidered.

The locations of introns are closely examinedas they locate at the sites where no significant
secondary structures being defined. Only two offifteen mammalian GAD boundaries, #13 and #15,
are located in the middle of their predicted long
α-helical structure at the C-terminus region. In
fruit fly, only intron #6 is equivalent to the two
introns of mammals, locating at the middle ofα-helix, and other five intron sites are outside of
defined secondary structures.
Solvent accessibility of GAD and thepositions of intron-exon boundaries – The solvent
accessibility is one of good indicators that
represent the spatial characteristics of theindividual amino acid in the protein [22]. A
three-dimensional model for human GAD65 wasconstructed by Capitani et al. based upon aminoacid homology between human GAD65 and pig
DDC and/or E. coli GADB, where X-ray structuresof pig DDC and E. coli GADB were used as
templates [12]. We have employed a similar
approach as reported by Capitani et al. [12]. At
first, an image of human GAD65 structure wassuperimposed onto pig DDC and E. coli GADB .
Then, the spatial positioning of the intron-exonboundaries of GAD65 was assigned based on the

44

Bioinformatic study on GAD

Fig. 3 Solvent accessibility profiles of (a) pig DDC and (b) E. coli GADB.
Horizontal axis is amino acid residue numbers. Vertical axis is a relative solvent
accessibility derived from crystal structure as calculated by DSSP program.
Diamond marks show the amino acid compatible to the intron insertion sites of human
GAD65.
solvent accessibility plots obtained from pig DDC
13, and 15, are buried in the hydrophobic coreand E. coli GADB as shown in Figs. 3a and 3b,
regions. Remaining three intron sites, #7, 9, andrespectively. The solvent accessibility plots for
14 do not belong to either areas. In Fig. 3b, out
pig DDC and E. coli GADB were obtained byof ten available intron sites, seven intron sites,
using DSSP program (Web site:#4-7, 11, 13 and 15, are positioned inside and the
http://swift.cmbi.ru.nl/gv/dssp/, Figs. 3a, 3b). In
rest three introns, #8, 12, and 14 are found on theFigs. 3a and 3b, the sites corresponding to thesurface of the GAD molecule.
intron-insertion positions on human GAD65 wereindicated. As a result, out of twelve available
intron sites in Fig. 3a, three introns, #8, 11, and 12,
Discussion
are on the surface and six intron sites, #4, 5, 6, 10,
Glutamate decarboxylase (GAD) areS. Matsukawa and H. Ueno

distributed among the wide varieties of organisms
including microorganisms, insects, plants, andanimals [3]. Despite of the diverse distribution,
GAD shows high amino acid sequence
resemblances among species [3]. Althoughhomology studies on GAD have been extensively
carried out [23], little is understood how
intron-exon shuffling has occurred in mammals
and the relationship between the localization of
intron-exon boarders and their positions in thesecondary and tertiary structures. In this paper,
we have assigned intron-exon boarders into theamino acid sequences of mammalian and fruit fly
GADs and have tried to address the molecular
evolution and predicted structure on mammalian
GAD65.
Our comparative study on amino acidsequence homology between GAD65 and GAD67
has revealed a significant homology between two
isoforms. Intron positions are almost identicalbetween the isoforms. Since these facts cansuggest that there was a common ancestral genefor mammalian GADs [21], we have looked into
the evolutional process of GAD by examining thesequence homology and the intron positioning.
The heterogeneity between GAD65 and 67
lies mostly upon their N-terminal regions, where
Bu and Tobin have noted the first 100 amino acid

Table 2. Comparison of alignment score
between each exon. The score was yieldedby alignment of each exon of human GAD65
and 67 using ClustalW program.

45

residues as a low homology region [21]. WhenFig. 4 Phylogenetic tree for mammalian
GADs and fruit fly GAD. The presented
tree was generated by the program of Tree
View
(http://taxonomy.zoology.gla.ac.uk/rod/treev
iew.html) based on the calculation by themethod of neighbor-joining.
the exon boarders are marked upon the amino acidsequences, an interesting correlation between the
low homology region and the exon boarder has
become apparent; the low homology region is
totally included within the exons 1-3. (Table 2, Fig.
1c). The remaining amino acid sequence thatcovers exons 4-16 is conserved between theisoforms (Fig. 1c). Table 2 summarizes thesequence alignment score for each exon
component where exons 1-3 exhibits significantly
low score. When the low homology region is
examined further, the first 50 amino acids and thesecond 50 amino acids show significantly different
homology patterns. Within the first 50 aminoacid residues, the conserved amino acids are about
30%, as indicated * in Fig. 1c and this region
originates from exons 1-2. On the other hand,within the second 50 amino acid residues thatoriginated from exon 3, very little residues areconserved. Since the differences between
mammalian GAD isoforms resides on the segment
encoded by exons 1-3, this uneven distribution
could lead us to explain how GAD65 and 67 would
be evolved from the common mammalian ancestralgene.
The heterogeneity in exon 3 sequences for
GAD65 and 67 provokes suspicion that this exon

46

Bioinformatic study on GAD

could be inserted after the ancestral gene wasevolved. However, the prediction of thesecondary structure as shown in Fig. 2 strongly
suggests that exons 1-3 have existed in the
mammalian ancestral gene prior to the evolution.
Therefore, it is reasonable to assume that theheterogeneity should be attributed to an
accumulation of point mutations. It is still inquestion when and how the point mutations having
been accumulated on exons 1-3. Nevertheless,the idea of comparing homology of the primarystructures and exon positions would be a useful
tool to study molecular evolution.
Fruit fly GAD and mammalian GADs exhibit
high homology in their primary structures andgood agreements on intron-insertion positions. It
is strongly suggested that fruit fly GAD and the
mammalian GAD isoforms share a common origin
as shown in the evolutionary tree (Fig. 4) [21, 23].
However, there are several points that makes fruit
fly GAD different from the mammalian isoforms.
First, nine introns are missing in the fruit fly GAD:The number of introns in mammalian GADs is 15
whereas only 6 for fruit fly (Fig. 1). This could
be explained by either fruit fly had lost nine introns
or mammalians had gained them. It would bereasonable to assume that an ancestral gene alreadyhad introns prior to the evolution, so thatmammalian GADs inherited those introns but fruit
fly lost some of them during the evolution [18, 22].
Second point is that a large deletion at the
N-terminal region of fruit fly GAD is observed andthose deleted segments are corresponding to theentire exon 2 and most of exon 3 of mammalian
GADs (Fig. 1d). Since there are few conserved
residues near the first exon-intron boarder of fruit
fly GAD, it could be assumed that the large
deletion occurred after the evolution or extra
sequences were gained in mammalian GADs after
the evolution. By any means, the heterogeneity
observed at the N-terminal regions of fruit fly andmammalian GADs couldn’t be explained bymutations occurred at amino acid level. It is
probable to assume that any divergence introducedin GADs could have taken place at DNA level,such as exon shuffling.
As discussed above, it is apparent that exons
1-3 of mammalian GADs are highly heterogeneous,
probably due to their susceptibility to mutation.

Exons 1-3 encode for the N-terminal 100 aminoacid residues; the region is believed to act as thedetermining role for GAD protein localization in
the cell. It has been shown that GAD67 localizes
in the cytosol and GAD65 associates withvesicular membrane. GAD65 has three Cys andone Thr residues at the N-terminal region which
are susceptible to palmitoylation andphosphorylation, respectively [24-26]. The entire
N-terminal 100 residues may not be essential forthe catalytic activity since the recombinant feline
GAD67, that expressed with its N-terminal 80
residues truncated, exhibited almost full enzymatic
activity [27]. Furthermore, other decarboxylases
belong to the same vitamin B6 family, such as
DOPA decarboxylase, and histidine decarboxylase,
do not have the N-terminal segments constructedwith exons 1-3 of GADs [3]. It is reasonable tothink that the role(s) of the N-terminal region of
mammalian GADs would be the determining
factor for cellular localization. The signal can bepalmitoylation, phosphorylation, pI, hydrophobic,
and/or hydrophilic nature built in the N-terminal
region.
In addition to the unique primary structure at
the N-terminal region of mammalian GADs, there
are other characteristic differences between GAD
isoforms. GAD67 seems to be tightly bind to
pyridoxal 5’-phosphate (PLP), a cofactor of GAD,
to form holo enzyme whereas GAD65 shows
weaker binding to PLP in forming half-apo
enzyme [4] [7, 8]. It could be assumed that thedifferences in N-terminal sequences would cause
the altering effects on an affinity toward PLP on
the mammalian GAD isoforms. Answer to thispoint may have to wait for the results of X-ray
crystallographic analysis.
Go and her colleagues have reported that
intron-exon boarders tend to appear right at thesecondary structure and/or at the hydrophobic coreregion [22]. In our present analysis on GADs
(Fig. 1b), two out of sixteen boundaries, #13 and#15, of mammals and one out of six, #6, of fruit fly
were found at the middle of long helical structure.
Most of the boundaries we have analyzed seem tobe localized outside of the defined secondary
structure. However, boundaries examined for the
structures of pig DDC (Fig. 3a) and E. coli GADB
(Fig.3 b) are found in the hydrophobic core, andS. Matsukawa and H. Ueno

somewhat buried. This is consistent with Go’s
report [22], in which she has proposed a term
“module”, a minimum element of protein structure.
The position of module in the protein structure isoften correlated close to the intron-insertion
positions [1, 18, 20].
Therefore, structural
information on module can be provided by locating
the intron-exon boundaries. Our current study
has indicated that the intron-exon boundaries
appear at the hydrophobic core regions.

89

ACKNOWLEDGEMENTS
We wish to thank Ms. Yumiko Ueno for help in
preparing the English manuscript, Ms. Hiromi
Okigami for helpful discussions, the members of
the laboratory for their kindness.
10
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Communicated by Ohnishi Masatake