Báo cáo khoa học: Alternative splicing produces an H-protein with better substrate properties for the P-protein of glycine decarboxylase doc

Alternative splicing produces an H-protein with better
substrate properties for the P-protein of glycine
decarboxylase
Dirk Hasse
1
, Stefan Mikkat
2
, Martin Hagemann
1
and Hermann Bauwe
1
1 Department of Plant Physiology, University of Rostock, Germany
2 Core Facility Proteome Analysis, Medical Faculty, University of Rostock, Germany
Introduction
Several thousand plant genes are known to produce
multiple transcripts, but the precise function of most
of the alternatively encoded proteins is not known [1].
Alternative splicing has also been observed for H-pro-
tein, a component of the ubiquitous multi-enzyme sys-
tem glycine decarboxylase (GDC, EC 1.4.4.2) [2,3].
GDC is essential for photorespiratory and one-carbon
metabolism, and the vital function of this enzyme
complex is indicated by the fact that its inactivation is
fatal to plants [4] and animals [5].
The GDC H-protein (GLDH) has no catalytic activ-
ity itself but interacts via its lipoyl arm one after the
other with the three other GDC subunits, P-, T- and
L-protein. As a result of the GDC reaction cycle,
which requires the coenzymes NAD
+
and tetrahydro-

folate, methylene-tetrahydrofolate and NADH are
generated and CO
2
and NH
3
are released [6].
Plant H-proteins are often encoded by small multi-
gene families, and the individual H-proteins suppos-
edly fulfil various functions in plant metabolism [7–9].
Some plants, however, harbour only one H-protein
gene, and we have previously reported that C
4
species
of the genus Flaveria produce two H-proteins in an
organ-dependent manner by alternative 3¢ splice site
selection [2,10]. The alternatively encoded H-protein,
FtGLDH
AA
, harbours two additional alanine residues
very close to its N-terminus, and is by far the most
dominant H-protein type in leaf mitochondria of these
plants. Because of photorespiratory metabolism, leaf
mitochondria contain much more GDC than root
Keywords
alternative splicing; glycine decarboxylase;
H-protein; photorespiration
Correspondence
H. Bauwe, Department of Plant Physiology,
University of Rostock, Albert-Einstein-
Straße 3, D-18059 Rostock, Germany

Fax: +49 381 498 6112
Tel: +49 381 498 6110
E-mail:
Website: />pflanzenphysiologie
(Received 7 July 2009, revised 5 September
2009, accepted 25 September 2009)
doi:10.1111/j.1742-4658.2009.07406.x
Several thousand plant genes are known to produce multiple transcripts,
but the precise function of most of the alternatively encoded proteins is not
known. Alternative splicing has been reported for the H-protein subunit of
glycine decarboxylase in the genus Flaveria. H-protein has no catalytic
activity itself but is a substrate of the three enzymatically active subunits,
P-, T- and L-protein. In C
4
species of Flaveria, two H-proteins originate
from single genes in an organ-dependent manner. Here, we report on differ-
ences between the two alternative H-protein variants with respect to their
interaction with the glycine-decarboxylating subunit, P-protein. Steady-state
kinetic analyses of the alternative Flaveria H-proteins and artificially pro-
duced ‘alternative’ Arabidopsis H-proteins, using either pea mitochondrial
matrix extracts or recombinant cyanobacterial P-protein, consistently dem-
onstrate that the alternative insertion of two alanine residues at the N-ter-
minus of the H-protein elevates the activity of P-protein by 20% in vitro,
and could promote glycine decarboxylase activity in vivo.
Abbreviations
GDC, glycine decarboxylase; GLDH, GDC H-protein; GLDH
AA,
alternative H-protein; LplA, lipoate–protein ligase.
FEBS Journal 276 (2009) 6985–6991 ª 2009 The Authors Journal compilation ª 2009 FEBS 6985
mitochondria [11,12], but the relevance of alternative

splicing for cellular metabolism in these organs is not
known. An inspection of published structural data
[13,14] revealed that the N-terminus of the H-protein
is close to its lipoyl arm (Fig. S1). Hence, structural
alterations to this region could possibly provide GDC
variants that are fine-tuned for different metabolic
environments.
In this study, we examined whether the changes to
the N-terminus of Flaveria H-protein alter its reactivity
with P-protein, the actual glycine-decarboxylating sub-
unit of GDC. These experiments revealed that P-pro-
tein showed significantly higher activity with the
alternative H-protein than with the normal H-protein.
This was also found to occur with P-protein from
Pisum sativum (pea) leaf mitochondrial matrix extracts
as a source of native plant P-protein and with recom-
binant P-protein of the cyanobacterium Synechocystis
sp. strain PCC 6803 (referred to simply as Synecho-
cystis below). Further support came from experiments
with an engineered ‘alternative’ H-protein from Ara-
bidopsis thaliana,aC
3
plant. This artificially modified
H-protein also showed higher activity with P-protein
in comparison with the native H-protein. Hence, both
natural and artificial N-terminal insertion of two ala-
nyl residues result in higher P-protein activities in vitro,
and could increase GDC activity in vivo.
Results and Discussion
The two alternative H-proteins from the C

4
plant
Flaveria trinervia, FtGLDH and FtGLDH
AA
, were
overexpressed in Escherichia coli and obtained as
apparently pure recombinant proteins (Fig. 1). To be
functional, H-protein must be lipoylated at a specific
lysine residue [12,15]. While complete lipoylation can
be achieved by the addition of lipoic acid to the E. coli
growth medium in some expression systems [16], we
observed that lipoylation of H-protein was still incom-
plete in our system (Fig. S2). Therefore, all recombi-
nant H-proteins used in our study were treated with
E. coli lipoate–protein ligase A (LplA, EC 2.7.7.63)
[17]. Complete lipoylation was verified by native poly-
acrylamide electrophoresis in combination with use of
lipoate-specific antibodies, and further confirmed by
MALDI-TOF MS (Fig. 2).
Leaf mitochondria contain large amounts of all
GDC subunits and provide a convenient source of
native P-protein. Under in vitro assay conditions,
P-protein dissociates from the other GDC subunits
and its activity becomes sensitive to the addition of
extra H-protein [18,19]. Likewise, addition of 50 lm
recombinant FtGLDH to assays containing matrix
extracts prepared from purified pea leaf mitochondria
resulted in an approximately 50-fold stimulation of the
glycine–bicarbonate exchange rate [20] relative to the
rate measured in the absence of exogenous H-protein

(Table 1). Similar to previously reported data [21,22],
the glycine saturation kinetics were hyperbolic, with a
K
m
of 6 mm (Fig. S3).
We next examined the reactivity of the two F. triner-
via H-proteins in this system (Table 1). H-protein satu-
ration followed Michaelis–Menten kinetics for both
FtGLDH and FtGLDH
AA
. The K
m
values for H-pro-
tein were between 16 and 20 lm, and hence somewhat
larger than published values for H-proteins from other
sources [18,23]. This difference could be due, at least
in part, to the small amounts of pea H-protein intro-
duced into the assay with the mitochondrial extracts.
Notably, the V
max
values were significantly higher with
FtGLDH
AA
than they were with FtGLDH, indicating
that alternative splicing could produce a more efficient
H-protein. This possibility was examined in a more
precisely defined assay using recombinant cyanobacte-
rial P-protein.
As eukaryotic P-proteins cannot yet be produced by
recombinant DNA technology, we used the P-protein

from the cyanobacterium Synechocystis, which is
structurally very similar to eukaryotic P-proteins.
Moreover, recombinant Synechocystis P-protein is
enzymatically active and can be produced in large
enough quantities (lane 6 in Fig. 1). The enzymatic
activities obtained with recombinant plant H-proteins
as substrates were in a similar range to those measured
with Synechocystis H-protein, which served as an inter-
nal control (Table 1). This extends an earlier report
on the use of chicken H-protein as a substrate for
the P-protein from Arthrobacter globiformis [3,24]. In
Fig. 1. SDS–PAGE with recombinant H-proteins from Flave-
ria trinervia, Arabidopsis thaliana and Synechocystis, and recombi-
nant P-protein from Synechocystis. M, size markers (kDa); lane 1,
FtGLDH; lane 2, FtGLDH
AA
; lane 3, AtGLDH1; lane 4, AtGLDH1
AA
;
lane 5, Synechocystis H-protein; lane 6, Synechocystis P-protein.
Alternative splicing of glycine decarboxylase H-protein D. Hasse et al.
6986 FEBS Journal 276 (2009) 6985–6991 ª 2009 The Authors Journal compilation ª 2009 FEBS
comparison with Synechocystis H-protein, the plant
H-proteins showed lower affinities for the prokaryotic
P-protein; however, similar to our experiments with
mitochondrial matrix extracts, the P-protein activity
with saturating FtGLDH
AA
was about 20% higher
than that with saturating normal FtGLDH.

In order to determine whether this finding can be
generalized beyond Flaveria H-proteins, we engineered
a splice variant of the H-protein AtGLDH1 from the
C
3
plant A. thaliana (AtGLDH1
AA
). This artificial
‘alternative’ H-protein differs from the naturally occur-
ring Arabidopsis H-protein only by the insertion of
two additional alanyl residues near the N-terminus.
Kinetic analysis of the these two H-proteins fully con-
firmed our findings with the two Flaveria H-proteins,
i.e. the AtGLDH1
AA
-saturated specific reaction rate
was significantly higher than that with the normal
AtGLDH1 (Table 1). This corroborated our view that
the alternatively spliced Flaveria H-protein is a better
substrate for P-protein, and indicated that this
improvement is exclusively caused by a slight modifica-
tion to its N-terminus.
It should be noted that our method for producing
recombinant H-proteins leaves a small extension of
four amino acid residues at the N-terminus of each
recombinant protein after removal of the His tag (Fig.
S4). The kinetic differences between the normal and
alternative H-proteins, either from Flaveria or from
Arabidopsis, were very consistent. Nonetheless, to fully
exclude the possibility that this artificial extension

could bias our results, all four plant H-proteins were
re-cloned into the tagless expression vector pET-3a.
The biochemical parameters obtained with these
tagless H-proteins were almost identical to those for
the respective thrombin-cleaved H-proteins (Table 1).
This confirmed that the higher V
max
values measured
Fig. 2. Correct structure of H-proteins was confirmed by MALDI-
TOF MS, with differences between theoretical and measured
molecular masses always below 1 Da. The signal at m ⁄ z 14 230
corresponds to the singly charged ion of lipoylated mature Flave-
ria trinervia H-protein GLDH (mass shift of 188 Da relative to unli-
poylated H-protein). H-protein FtGLDH
AA
shows the expected mass
increase of 142 Da, which is due to the insertion of two extra alanyl
residues. Lipoylated mature Arabidopsis thaliana H-protein GLDH1
also shows the expected signal at m ⁄ z 14 417 Da. The Arabidopsis
H-protein variant AtGLDH1
AA
was produced by mutagenesis, and
the two extra alanyl residues result in the calculated mass increase.
Small satellite peaks and shoulders are artefacts that originated
during sample preparation or the desorption ⁄ ionization process.
Table 1. Kinetic parameters for Pisum sativum and Synechocystis
P-protein with H-protein from Flaveria trinervia, Arabidopsis thaliana
or Synechocystis as substrate. H-proteins carrying an N-terminal
extension of four amino acids, which remain after thrombin cleav-
age of the His tag, are labelled ‘+4’. Values are means ± SE from

three (mitochondrial matrix P-protein) or four (purified Synechocys-
tis P-protein) independent experiments.
K
m (H-protein)
(lM)
V
max
(nmolÆCO
2
Æmg
)1
Æmin
)1
)
Mitochondrial P-protein
FtGLDH +4 20.4 ± 2.1 9.4 ± 0.1
FtGLDH
AA
+4 15.9 ± 1.6 11.2 ± 0.4
AtGLDH1 + 4 23.5 ± 3.1 9.8 ± 0.5
AtGLDH1
AA
+4 26.7 ± 3.0 12.2 ± 0.6
No exogenous protein – 0.2 ± 0.03
Synechocystis P-protein
SyGLDH +4 1.6 ± 0.1 5.5 ± 0.12
FtGLDH +4 106 ± 17 3.4 ± 0.11
FtGLDH
AA
+4 93 ± 15 4.1 ± 0.07

AtGLDH1 + 4 71 ± 4.5 4.8 ± 0.15
AtGLDH1
AA
+4 55 ± 5.4 6.0 ± 0.26
FtGLDH 140 ± 9.6 3.5 ± 0.20
FtGLDH
AA
125 ± 10.4 4.2 ± 0.23
AtGLDH1 51 ± 4.8 4.9 ± 0.16
AtGLDH1
AA
60 ± 2.2 5.8 ± 0.07
D. Hasse et al. Alternative splicing of glycine decarboxylase H-protein
FEBS Journal 276 (2009) 6985–6991 ª 2009 The Authors Journal compilation ª 2009 FEBS 6987
with the alternative H-proteins, both the naturally
occuring FtGLDH
AA
and the artificial AtGLDH1
AA
,
are indeed exclusively due to the insertion of two
additional alanyl residues into the N-terminus of the
H-protein.
In conclusion, the substrate properties of the alter-
natively encoded Flaveria H-protein FtGLDH
AA
in the
P-protein reaction of GDC differ significantly from
those of the normal Flaveria H-protein. This was
found in two independent assay systems, using either

mitochondrial matrix extracts or recombinant cyano-
bacterial P-protein. It was further substantiated by
analysis of an engineered ‘alternative’ Arabidopsis
H-protein. Hence, several lines of independent experi-
mental evidence consistently demonstrated that
alternative 3¢ splice site selection results in a more
efficient H-protein.
As fas as the physiological relevance of this effect is
concerned, several other important variables are still
unknown and require further research. From a
methodical point of view, the GDC concentration is
extremely high in leaf mitochondria but very much
lower in mitochondria from heterotrophic tissue.
Although root mitochondria have not yet been exam-
ined for this feature, H-protein is present at levels of
approximately 1 mm in the matrix of pea leaf mito-
chondria, which is 7-fold higher than the concentration
of P-protein [12] and 40-fold higher then the K
m
values
determined in vitro. This could indicate that H-protein
is possibly saturating in the leaves of C
3
plants, but
unfortunately in vitro assays are very difficult to per-
form at such high protein concentrations. Our assay
conditions may be closer to the situation in C
4
leaf
mitochondria, which contain distinctly less GDC, and

even closer to that of root mitochondria.
Furthermore, it is not known whether alternative
splicing of H-protein pre-mRNA is a particular feature
of Flaveria C
4
species or also occurs in other C
4
plants. The genomes of maize (Zea mays, http://www.
maizesequence.org) and Sorghum bicolor [25] do not
indicate the presence of alternative H-proteins in these
monocot C
4
plants, and sequence data for other dicot-
yledonous C
4
plants are not yet available. Our data
show that the alternative H-protein enhances P-protein
activity by about 20% in vitro. Although this is not a
very large effect, it could have significance for both C
3
and C
4
plants in vivo. For C
3
plants, this may be
assumed from effects on photosynthesis observed after
antisense suppression of P-protein in potato, which
indicated co-limitation of leaf metabolism by GDC
activity [26]. In C
4

plants, the photorespiratory carbon
flux is low, but nevertheless essential [27]. C
4
plants
hence require less GDC than C
3
plants. However, the
photorespiratory flux can considerably increase under
conditions of low water supply and high temperature
[28]. Under such conditions, which occur intermittently
or may even prevail in many habitats typical for C
4
plants, the use of alternative H-protein could counter-
act limitations to photosynthetic ⁄ photorespiratory
metabolism. Possible enhancement effects on T- and
L-protein remain to be examined, but could add to a
larger overall activity enhancement.
Finally, alternative splicing of H-protein pre-mRNA
is regulated organ-dependently in C
4
Flaveria species
[2]. It appears that C
4
plants very often harbor only
one H-protein gene (Zea mays,C
4
Flaveria) or two
H-protein genes (Sorghum bicolor), whereas up to four
H-protein genes occur in C
3

plants [7,9]. Interestingly,
transcript analyses revealed that at least one member
of these gene families is preferentially expressed in
photosynthesizing tissues [7,8]. This is why some
researchers differentiate between two classes of H-pro-
teins: class I H-proteins, which are preferentially
expressed in tissue with high photorespiration, and
class II H-proteins, which are more strongly involved
in one-carbon metabolism of non-photorespiratory tis-
sues [9]. Similar to this, alternative H-proteins could
possibly allow fine adjustment of GDC to the parti-
cular metabolic requirements in various organs of C
4
plants.
Experimental procedures
Overexpression constructs
Overexpression constructs for Synechocystis P-protein
(SwissProt P74416) and H-protein (Swissprot P73560) have
been described previously [29]. Coding regions of the
mature F. trinervia H-proteins [2] FtGLDH and
FtGLDH
AA
(SwissProt P46485) were ligated into the
expression vectors pET-28a (tagged) and pET-3a (tagless)
(Merck, Darmstadt, Germany) via the NdeI and BamHI
restriction sites.
For the A. thaliana H-proteins AtGLDH1 (At2g35370,
SwissProt P25855) and AtGLDH1
AA
, cDNA was prepared

from 2.5 lg of purified leaf RNA (NucleospinÔ RNA plant
kit, Macherey-Nagel, Du
¨
ren, Germany) using the Revert-
AidÔ H Minus cDNA synthesis kit (MBI Fermentas, St
Leon-Rot, Germany). The coding region of the mature
H-protein AtGLDH1 was PCR-amplified using the gene-
specific primers (with underlined restriction sites) 5¢-
CATAT
GTCCACAGTTTTGGA-3¢ (sense, for native AtGLDH1),
5¢-
CATATGTCCACAGCTGCAGTTTTGGA-3¢ (sense, for
the artificial AtGLDH1
AA
) and 5¢-GGATTCCCTAGTGA
GCAGCATCT-3¢ (antisense), and the ElongaseÔ enzyme
mix (Invitrogen, Karlsruhe, Germany). The PCR product
Alternative splicing of glycine decarboxylase H-protein D. Hasse et al.
6988 FEBS Journal 276 (2009) 6985–6991 ª 2009 The Authors Journal compilation ª 2009 FEBS
was ligated via pGEM-T (Promega, Mannheim, Germany)
into pET-28a, and recloned into pET-3a as described above
for F. trinervia H-protein overexpression constructs.
The E. coli lplA gene (SwissProt P32099) [17] was ampli-
fied by PCR (sense primer, 5¢-
CATATGTCCACATTAC
GCCTGCT-3¢; antisense primer, 5¢-
AAGCTTCTACCTTA
CAGCCCCCG-3¢). The sense and antisense primers were
extended by NdeI and HindIII sites (underlined), respec-
tively. After initial cloning of the PCR amplificates into

pGEM-T, coding sequences were excised and ligated into
corresponding cloning sites of pET-28a.
Recombinant proteins
Synechocystis P-protein was produced essentially as
described previously [29]. Major modifications to this
earlier procedure comprised addition of 200 lm pyridoxal-
phosphate and 15 mm 2-mercaptoethanol to all buffers.
E. coli strain BL21 (DE3) cells overexpressing H-pro-
teins were induced using 1 mm isopropyl-b-d-thiogalacto-
pyranoside for 16 h at room temperatures in 2YT medium
supplemented with 0.4 mm lipoic acid, harvested, and soni-
cated in 20 mm Bis ⁄ Tris (pH 6). Cleared lysates were then
loaded onto a Q-SepharoseÔ column (GE Healthcare,
Munich, Germany), and eluted using a linear buffered
0–0.5 m NaCl gradient. Fractions containing H-protein
were pooled, concentrated, and further purified on a Seph-
acrylÔ S-100 column (GE Healthcare) equilibrated in the
same buffer.
E. coli LplA was overexpressed and purified by metal
affinity chromatography similar to Synechocystis P-protein
but with 10% glycerol added to all buffers. The enzyme was
stored at )20 °C in a buffer containing 10 mm Tris ⁄ 10 mm
Mops (pH 7.5), 0.5 mm MgSO
4
, 50% glycerol and 5 mm
2-mercaptoethanol. Prior to use, it was activated for at least
30 min by the addition of 200 mm 2-mercaptoethanol.
For complete lipoylation, recombinant H-proteins (at
0.6 mgÆmL
)1

, corresponding to approximately 50 lm) were
incubated at 30 °C for 3 h in 10 mm Tris ⁄ 10 mm Mops
(pH 7.5), 5 mm MgSO
4
,5mm ATP, 5 lm E. coli LplA and
200 lm lipoic acid (which is saturating for LplA). LplA
was removed by chromatography through Q-SepharoseÔ
and SephacrylÔ S-100 columns as described above. Purified
H-proteins were used immediately for kinetic assays, or
flash frozen in liquid nitrogen after addition of 20% v ⁄ v
glycerol and stored at )80 °C.
MALDI-TOF MS
The purity of recombinant proteins was assessed by SDS–
PAGE [30], and the molecular identity of all H-proteins
including completeness of lipoylation was further verified
by MALDI-TOF MS. Sample aliquots were desalted using
ZipTip pipette tips containing C
18
reverse-phase medium
(Millipore Corp., Bedford, MA, USA) and applied to a
polished steel MALDI target by mixing with an equal
volume of saturated a-cyano-4-hydroxycinnamic acid in
35% acetonitrile ⁄ 0.1% trifluoroacetic acid. Data were
acquired in linear mode using a Reflex III mass spectro-
meter (Bruker Daltonics, Bremen, Germany). For exact
determination of the mean mass, the protein sample was
mixed with an aliquot of protein standard I (Bruker
Daltonics) to allow internal calibration, resulting in a mean
mass error of < ±1 Da at the investigated mass range.
Matrix extracts from pea leaf mitochondria

Mitochondria were prepared from young garden pea plants
and purified as described previously [31] on self-generating
density gradients. Matrix extracts were prepared by lysis in
four freeze–thaw cycles, followed by centrifugation at
44 000 g for 30 min to remove mitochondrial membranes.
The supernatant was concentrated using VivaspinÔ col-
umns with a 10 kDa exclusion limit (Sartorius, Go
¨
ttingen,
Germany), and adjusted to a protein concentration of
3.5 mgÆmL
)1
. All steps were performed at 4 °C.
Determination of P-protein activity
The recombinant P- and H-proteins were equilibrated in
20 mm sodium phosphate (pH 7.5) using pre-packed PD-10
columns (SephadexÔ G-25 M; Pharmacia, Freiburg,
Germany). After determination of protein concentration
[32], P-protein activity was assayed using the glycine–bicar-
bonate exchange reaction [20]. Assays with Synechocystis
P-protein contained 100 mm sodium phosphate (pH 6.0),
0.1 mm pyridoxalphosphate, 2 mm dithiothreitol, 20 mm
glycine, 30 mm NaH
14
CO
3
(2.5 lCi), 2.5 lg P-protein
(0.04 lm P-protein dimer) and 0–250 lm H-protein in a
total volume of 300 lL [29]. Assays with mitochondrial
matrix extracts contained 50 mm sodium phosphate (pH

6.0), 0.1 mm pyridoxalphosphate, 2 mm dithiothreitol,
30 mm glycine, 30 mm NaH
14
CO
3
(2.5 lCi), 9 lg matrix
protein and 0–100 lm H-protein in a total volume of
150 lL. H-protein concentrations varied as indicated in the
figures. Reactions were run at 30 °C in the linear response
range for 20 min (varying glycine) or 30 min (varying
H-protein). Rates obtained without substrate were sub-
tracted, and kinetic parameters were calculated by nonlin-
ear regression analysis using the software package
graphpad prism (GraphPad Software, San Diego, CA,
USA). All kinetic data are means ± SD from three or four
analyses over the full substrate range.
Acknowledgements
Financial support to D.H. by the Landesgraduier-
tenfo
¨
rderungsprogramm Mecklenburg-Vorpommern is
gratefully acknowledged.
D. Hasse et al. Alternative splicing of glycine decarboxylase H-protein
FEBS Journal 276 (2009) 6985–6991 ª 2009 The Authors Journal compilation ª 2009 FEBS 6989
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Supporting information
The following supplementary material is available:
Fig. S1. Three-dimensional structure of pea H-protein
showing the N-terminus and the position of the lipoyl
arm.
Fig. S2. Overexpression and effects on H-protein of
E. coli lipoate–protein ligase A.
Fig. S3. Glycine saturation kinetics of pea mitochon-
drial matrix P-protein.

Fig. S4. N-terminal sequences of thrombin-cleaved
recombinant H-proteins and mass spectrometric verifi-
cation of the recombinant Arabidopsis H-proteins.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
D. Hasse et al. Alternative splicing of glycine decarboxylase H-protein
FEBS Journal 276 (2009) 6985–6991 ª 2009 The Authors Journal compilation ª 2009 FEBS 6991