Looking for the ancestry of the heavy-chain subunits of
heteromeric amino acid transporters rBAT and 4F2hc
within the GH13 a-amylase family
Marek Gabris
ˇ
ko
1
and S
ˇ
tefan Janec
ˇ
ek
1,2
1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
2 Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Trnava, Slovakia
Introduction
To fulfil its metabolic needs, a cell uses specialized
transport proteins to perform and control the uptake
and efflux of crucial compounds (e.g. sugars, amino
acids, nucleotides, inorganic ions and drugs) across
the plasma membrane. These proteins have been clas-
sified into the phylogenetically derived solute carrier
(SLC) families; current classification counts almost 50
SLC families [1,2]. The sequence similarity between
the heavy-chain subunits of heteromeric amino acid
transporters (hcHATs) and the a-glucosidases from
the a-amylase family [3] was first recognized more
than 15 years ago [4]. HATs are composed proteins
consisting of a light subunit (SLC7 members) and a
heavy subunit (known as rBAT or 4F2hc; SLC3
Keywords

hcHAT-like proteins, designated here as hcHAT1 and hcHAT2 groups,
were identified in basal Metazoa. Of the GH13 catalytic triad, only the cat-
alytic nucleophile (aspartic acid 199 of the oligo-1,6-glucosidase) could
have its counterpart in some 4F2hc proteins, whereas most rBATs contain
the correspondences for the entire GH13 catalytic triad. Moreover, the
4F2hc proteins lack not only domain B typical for GH13 enzymes, but also
a stretch of  40 amino acid residues succeeding the b4-strand of the cata-
lytic TIM barrel. rBATs have the entire domain B as well as longer loop 4.
The higher sequence–structural similarity between rBATs and GH13
enzymes was reflected in the evolutionary tree. At present it is necessary to
consider two different scenarios on how the chordate rBAT and 4F2hc
proteins might have evolved. The GH13-like protein from the cnidarian
Nematostella vectensis might nowadays represent a protein close to the
eventual ancestor of the hcHAT proteins within the GH13 family.
Abbreviations
ATG, amino acid transporter glycoprotein; CSR, conserved sequence regions; GH, glycoside hydrolase; HAT, heteromeric amino acid
transporter; hcHAT, heavy-chain subunits of heteromeric amino acid transporter; SLC, solute carrier.
FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS 7265
members), connected by a disulfide bridge [2].
Because of their significance in human pathology
(their defects lead to primary inherited aminoacidu-
rias, e.g. failed renal reabsorption of amino acids),
HATs have attracted much attention in medical stud-
ies (e.g. [2,5–7]). The light subunit is a nonglycosylat-
ed hydrophobic 12-helix transmembrane protein,
whereas the heavy subunit is a type II membrane
N-glycoprotein with an intracellular N-terminal end, a
single transmembrane region and a large extracellular
C-terminal domain [2]. It is the light subunit that
possesses the amino acid transportation activity,

overall sequence identity within the GH13 is extremely
low [13], it contains several groups of enzymes exhibit-
ing a higher degree of mutual sequence similarity so
that the family has recently been divided into subfami-
lies [18]. Of these, the best resemblance to hcHATs
was revealed for the members of the so-called oligo-
1,6-glucosidase subfamily [9,19,20]. This was recently
confirmed by solving the three-dimensional structure
of the C-terminal domain of 4F2hc [21], which most
resembles the oligo-1,6-glucosidase from Bacillus cereus
[22] and a-glucosidase from Geobacillus sp. HTA-462
[23].
In hcHATs, the regions of similarity cover the
sequence segments within the C-terminal extracellular
domain. The segments correspond, in fact, with some
of the a-amylase family CSRs, namely the b-strands
b2, b3, b4 and b8 of the (b ⁄ a)
8
barrel domain, and for
rBAT also with the short stretch near the C-terminus
of domain B [9,19]. From the sequence–structure point
of view, the basic difference between rBAT and 4F2hc
is that rBAT possesses the segment that corresponds
with domain B of GH13 enzymes, whereas 4F2hc does
not have it [9,19,24,25].
The main goal of the present study was to investi-
gate further the resemblance between hcHAT proteins
and the enzymes from the a-amylase family. We there-
fore carried out a bioinformatics study focused on a
detailed comparison of all available rBAT and 4F2hc

of comparable positions would be < 100.
Figure 1 illustrates the evolutionary relationships
between the studied hcHAT proteins and GH13
enzymes from the a-amylase family. The tree was
Origin of rBAT and 4F2hc within the GH13 a-amylase family M. Gabris
ˇ
ko and S
ˇ
. Janec
ˇ
ek
7266 FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS
M. Gabris
ˇ
ko and S
ˇ
. Janec
ˇ
ek Origin of rBAT and 4F2hc within the GH13 a-amylase family
FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS 7267
calculated using the neighbour-joining method [26].
Other approaches, such as maximum likelihood [27],
maximum parsimony [28], minimum evolution [29] and
upgma [30] were also used, but they delivered basically
similar topologies (not shown).
The two main groups of hcHATs (Fig. 1), i.e. those
of rBAT and 4F2hc, form their own clusters within
which taxonomy is respected: (a) for the rBATs from
human via representatives of mammals, birds, lizard,
frogs and fishes to Urochordata (sea squirts) and

other by the GH13 enzymes.
All remaining sequences (except those from nema-
todes) that were not possible to classify as true rBAT
and true 4F2hc proteins were first designated as
hcHAT-like proteins. Then, based on an approximate
alignment, which served to construct a preliminary evo-
lutionary tree, these hcHAT-like proteins were divided
into hcHAT1 and hcHAT2 groups (Table 1). It is
worth mentioning that most of them are hypothetical
proteins that in some cases were retrieved from recent
complete genome sequencing projects containing raw
sequence data still without appropriate annotation.
Most hcHAT1 proteins cover the insects and, in a
wider sense, the Arthropoda (daphnia), which are com-
pleted by Cephalochordata and Echinodermata (both
Deuterostomia) and one representative from Cnidaria
(Nematostella). The group of hcHAT2 proteins also
consists of Arthropoda, i.e. insects accompanied by
Daphnia and Ixodes, and two representatives of schisto-
somes. Interestingly, although present in the subgenus
Drosophila, hcHAT2 proteins seem to be lacking in the
melanogaster group (subgenus Sophophora).
With regard to hcHAT1 from Aeges aegypti [31] and
Drosophila melanogaster [32], these two proteins have
already been experimentally confirmed as heavy-chain
subunits (CD98hc, i.e. 4F2hc) in the amino acid trans-
porter system analogous to that known in mammals
[2,21]. A similar observation was reported for the
SPRM1hc from Schistosoma mansoni [33], which in the
present study is classified in the hcHAT2 group

presence of full GH13 domain B in hcHAT1 and the
absence of its parts in hcHAT2 indicate the eventual
intermediary or primordial character of both hcHAT1
and hcHAT2 with regard to the appearance of typical
rBAT and typical 4F2hc proteins in animals. This
seems to be obvious, according to our present knowl-
edge, from Urochordata (Fig. 1).
The second sequence feature clearly visible from the
alignment is whether the individual catalytic residues,
or even the entire catalytic triad of the GH13 a-amy-
lase family, could be found in the hcHAT representa-
tives. Fort et al. [21] reported that the human 4F2hc
does not exhibit any a-glucosidase activity. This is con-
sistent with almost a complete lack of the catalytic
triad in all 4F2hc proteins (Fig. 2). It is worth men-
tioning that, especially in higher animals (mammals
and also in frogs and fishes), an aspartate (aspartic
acid 248 in human 4F2hc; aspartic acid 380 in Fig. 1
as both the N-terminal and transmembrane segments
are involved) could be a relic of the GH13 b4-strand
catalytic nucleophile [3,11–13], although shifted one
position to the C-terminus (Fig. 2). On the other hand,
most rBAT representatives contain all three catalytic
residues (Fig. 2) with the exception of those from
birds, lizards and frogs (lacking both essential aspar-
tates at the b4- and b7-strands) and also from some
fishes (lacking the b4-strand aspartate). This may mean
that the eventuality of a-glucosidase activity of true
rBATs cannot be unambiguously eliminated.
The selected CSRs (Fig. 2) characteristic of the

glucosidase and neopullulanase subfamilies.
Origin of rBAT and 4F2hc within the GH13 a-amylase family M. Gabris
ˇ
ko and S
ˇ
. Janec
ˇ
ek
7270 FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS
tional sequence features conserved mutually between
the hcHAT and hcHAT-like proteins and GH13
enzymes, as well as within the individual groups of
hcHAT representatives, i.e. rBAT, 4F2hc, hcHAT1,
hcHAT2 and ATG groups (Table 1). Overall, and
interestingly, the residues that have not yet been
revealed to be essential for the GH13 enzymes seem to
be well conserved, e.g. (a) a stretch of three hydro-
phobic aliphatic residues (207_LII in human rBAT)
preceding the important aspartate (aspartic acid 98 in
oligo-1,6-glucosidase) in region I covering the
b3-strand; (b) a segment of up to five residues
(307_GVDGF in human rBAT) preceding the func-
tional arginine (arginine 197 in oligo-1,6-glucosidase)
in region II of the b4-strand; and (c) more or less the
entire region VII, i.e. the b8-strand. The fact that
rBATs exhibit more sequence similarities with the
GH13 enzymes than the 4F2hc proteins is also clearly
and easily visible in selected CSRs (Fig. 2). It concerns
mainly: (a) tryptophan (tryptophan 161 in human
rBAT) in region VI (b2-strand); (b) histidine (histidine

tifiable, even for the enzymatically active GH13 mem-
bers [13]. Therefore, one of the goals was to align
correctly the b5-strands of the hcHAT sequences,
which was especially problematic for the 4F2hc pro-
teins completely lacking the catalytic glutamate
(Fig. 2). In this regard, the putative GH13-like
sequence from the cnidarian Nematostella vectensis
containing the b5-strand segment 273_RLLIGE
(Fig. 2) should be of special importance from an evo-
lutionary point of view, as it contains the features of
both the GH13 enzymes (i.e. the glutamic acid residue
in a corresponding position) and typical 4F2hc
proteins (i.e. arginine or lysine followed by the stretch
of three aliphatic hydrophobic residues, e.g.
405_RLLIAG in human 4F2hc; Fig. 2). This segment
preceding the catalytic b5-strand glutamate is also con-
served in most insect a-glucosidases, supporting the
possibility that the ancestry of the hcHAT proteins
within the GH13 a-amylase enzyme family could be
rooted in basal metazoans, currently represented by
Nematostella vectensis.
A comparison of the three-dimensional structures of
representatives of hcHATs (human 4F2hc, 417 residues
[21] and a model of the human rBAT with 535 resi-
dues) and GH13 enzymes (Geobacillus sp. HTA-46
a-glucosidase; 531 residues [23]) confirmed the
expected higher similarity between rBAT proteins and
GH13 enzymes (root-mean-square deviation 1.62 A
˚
between the C

Selection pressure
With regard to close sequence similarity between the
GH13 enzymes and the hcHAT proteins (especially
rBATs), it is interesting to compare the selection pres-
M. Gabris
ˇ
ko and S
ˇ
. Janec
ˇ
ek Origin of rBAT and 4F2hc within the GH13 a-amylase family
FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS 7271
Origin of rBAT and 4F2hc within the GH13 a-amylase family M. Gabris
ˇ
ko and S
ˇ
. Janec
ˇ
ek
7272 FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS
sure acting on corresponding stretches of amino acid
sequences. For this purpose, the selecton tool [38]
was chosen. Figure 3 illustrates the similarities and dif-
ferences in selection pressure acting on the three stud-
ied protein groups – mammalian 4F2hc proteins,
vertebrate rBATs and insect a-glucosidases. In agree-
ment with the higher degree of sequence similarity
between rBAT proteins and GH13 enzymes, the selec-
tion pressure was also found to be more similar for
these two groups than that observed for 4F2hc and

a-amylase family. In fact, two different evolutionary
scenarios could be taken into account: (a) in one single
event in basal Metazoa and a subsequent split into
rBAT and 4F2hc (probably via hcHAT1 group) in
chordates; and (b) in two independent branching
events, i.e. 4F2hc in the basal Metazoa via HAT-like
proteins and rBAT directly from enzymes in deuterost-
omes. It is worth mentioning here that both scenarios
reflect the ancestry of both rBATs and 4F2hc proteins
anchored within the GH13 a-amylase family. The dif-
ference is only in the way leading from the GH13
enzymes either to rBAT and 4F2hc together or to
rBAT and 4F2hc separately. At present it is not possi-
ble to draw the evolutionary picture unambiguously.
The first evolutionary scenario, basically consistent
with the one proposed originally [9], means that in
basal Metazoa an ancestor of both the present-day
4F2hc and rBAT proteins was separated from the
GH13 enzymes. The ancestor acquired the N-terminal
and transmembrane segments and, eventually (in most
taxa), duplicated and evolved to give in chordates: (a)
rBATs that have kept most of the GH13 sequence–
structural features, including domain B as well as cata-
lytic residues (often the entire catalytic triad); and (b)
4F2hc that has consecutively lost almost all of the
GH13 characteristic sequence–structural features,
including domain B as well as functional residues
(mainly the catalytic triad). The weak points of this
scenario are: (a) the striking similarity between rBATs
and GH13 enzymes; (b) the higher similarity between

. Janec
ˇ
ek Origin of rBAT and 4F2hc within the GH13 a-amylase family
FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS 7273
only one possible place for rooting the tree that is on
the branch leading to the enzymes originating from
non-Metazoa (the eventual outgroup).
It is worth mentioning, however, that if the evolu-
tionary tree of all proteins studied here is based on the
alignment of CSRs (Fig. 2), the ATG proteins from
nematodes [34] and all hcHAT-like proteins designated
here as hcHAT1 group (Table 1), i.e. hcHAT-like pro-
teins covering the basal Metazoa and Arthropoda,
cluster together with both the rBAT proteins and
GH13 enzymes, leaving the 4F2hc proteins with the
hcHAT2 group at a different branch (tree not shown).
It should be pointed out that despite the fact the
GH13 CSRs could be considered to be something like
sequence fingerprints of the GH13 a-amylase family
members [13], the tree based on the CSRs is supported
by low bootstrap values. It is thus not possible to say
which one, hcHAT1 or hcHAT2, is orthologous to
rBAT or 4F2hc, if any. Although both hcHAT1
(insect) and hcHAT2 (schistosoma) representatives
have already been shown to function rather as 4F2hc
than as rBAT [31–33], their rBAT-like role has not as
yet been investigated. However, as seen in Fig. 1 (the
tree based on the complete alignment), the hcHAT2
group (Arthropoda) cluster with both ATG1 and
ATG2 (Nematoda), indicating that the hcHAT2 and

cated by small yellow boxes. The individual structural parts of the proteins, i.e. the N-terminal and the transmembrane segments, domain A
(TIM barrel), domain B and domain C, are indicated by green, yellow, blue and grey shadowing, respectively.
Origin of rBAT and 4F2hc within the GH13 a-amylase family M. Gabris
ˇ
ko and S
ˇ
. Janec
ˇ
ek
7274 FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS
in fruit flies (Diptera) was revealed [42,43]. Moreover,
the events of horizontal gene transfer have also been
discussed, as animal-like and plant-like a-amylase
genes were found in actinomycetes and other bacteria
[44,45]. However, the main goal of the present work
was to shed more light on the early history of extant
rBAT and 4F2hc proteins. Many sequences of hypo-
thetical hcHAT-like proteins, covering the basal meta-
zoans and arthropods that are very probably
homologues to heavy-chain partners of the light-chain
subunits of the HAT system of chordates, have been
identified and analysed, which should enable the exper-
imentalists in the field to direct their research more
appropriately. The significance of this study could be
in the field of: (a) hcHAT protein research (expanding
the taxonomy spectrum and eventually focusing on
more simple model organisms, e.g. Nematostella vect-
ensis); (b) the a-amylase GH13 family enzymes (identi-
fying the sequence features often omitted in analyses
of GH13 enzymes, but also clearly conserved in rBAT

like proteins hcHAT1 (22) and hcHAT2 (14).
Eight key enzyme specificities from the oligo-1,6-glu-
cosidase subfamily (the GH13 subfamilies 4, 16–18,
29–31 and also until now some unassigned GH13
enzymes) [18,20] were selected (Table 1) and retrieved
from GenBank [47]; the proteins with known three-
dimensional structure being preferred. In addition, the
three representatives of the neopullulanase subfamily
(GH13_20) [20] were added together with the three
neopullulanase-like (intermediary character; GH13_36)
enzymes. Because a-glucosidases are produced by a
wide spectrum of diverse taxonomic groups, these
GH13 enzymes from bacteria (6), fungi (2) and insects
(16) were also involved.
The set of studied proteins was completed by four
hypothetical proteins (marked as GH13-like in
Table 1), which could not be distinguished as hcHAT
proteins or GH13 enzymes. Despite possessing the cat-
alytic triad and domain B (typical for GH13 enzymes),
they may also possess the transmembrane region (typi-
cal for hcHAT proteins) because at present their
sequences are apparently (sea urchin) and potentially
(Nematostella) incomplete. Concerning the sequences
from Trichoplax, they contain the N-terminal part pre-
ceding the TIM barrel, but it is sequentially dissimilar
to that found in hcHATs.
All sequence alignments were performed using the
program clustalx2 [52] and then manually tuned with
regard to CSRs, domain borders and tertiary struc-
tures known from the literature [3,9,12–14,20–22]. Sev-

ek Origin of rBAT and 4F2hc within the GH13 a-amylase family
FEBS Journal 276 (2009) 7265–7278 ª 2009 The Authors Journal compilation ª 2009 FEBS 7275
In order to compare selection pressure acting on
hcHAT proteins and a-amylase family enzymes, three
different groups – mammalian 4F2hc, vertebrate rBAT
and insect a-glucosidases (Table 1) – were analysed
using the selecton tool [38] at />The ratio between nonsynonymous (Ka) and synony-
mous (Ks) substitutions was calculated for every codon
using the default M8 codon-substitution model [58] on
aligned nucleotide sequences. In the analysis, the
default neighbour-joining tree made by the selecton
itself was used, the selection pressure not being
mapped on a three-dimensional structure, and the
precision level was set as the highest.
Acknowledgement
This work was supported in part by VEGA grant no.
2 ⁄ 0114 ⁄ 08 from the Slovak Grant Agency for Science.
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