The VSFASSQQ motif confers calcium sensitivity to the intracellular apyrase LALP70.

Abstract

Apyrases are divalent ion dependent tri- and dinucleotide phosphatases with different substrate specificity. The intracellular lysosomal apyrase LALP70 is also expressed as a splice variant (LALP70v) lacking a VSFASSQQ motif in the center of the molecule (aminoacids 287-294). However, the functional significance of this motif is unknown. In this report we used a thin layer chromatography approach to study separately the UTPase and UDPase activity of the two LALP-enzymes.
We show, that LALP70 and LALP70v cleaved UTP to UDP in a calcium independent manner. In contrast, the cleavage of UDP to UMP was strongly calcium dependent for LALP70, but calcium independent for LALP70v.
The VSFASSQQ motif not only influences the substrate specificity of LALP70, but it confers calcium sensitivity to LALP70 during the UDP cleavage. Whether this is due to direct binding of calcium to this motif or to a conformational change of the enzyme, remains to be elucidated.

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BioMed Central
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BMC Biochemistry
Open Access
Research article
The VSFASSQQ motif confers calcium sensitivity to the
intracellular apyrase LALP70
Annette Biederbick1, Ralf Rösser1, Jörg Storre2 and Hans-Peter Elsässer*1
Address: 1Institute for Cytobiology and Cytopathology, University of Marburg, Germany and 2Institute for Molecular Biology and Tumor Research,
University of Marburg, Germany
Email: Annette Biederbick - biederbi@mailer.uni-marburg.de; Ralf Rösser - roesser@med.uni-marburg.de; Jörg Storre - storre@odin.imt.uni-
marburg.de; Hans-Peter Elsässer* - elsaesse@mailer.uni-marburg.de
* Corresponding author
Abstract
Background: Apyrases are divalent ion dependent tri- and dinucleotide phosphatases with
different substrate specificity. The intracellular lysosomal apyrase LALP70 is also expressed as a
splice variant (LALP70v) lacking a VSFASSQQ motif in the center of the molecule (aminoacids 287–
294). However, the functional significance of this motif is unknown. In this report we used a thin
layer chromatography approach to study separately the UTPase and UDPase activity of the two
LALP-enzymes.
Results: We show, that LALP70 and LALP70v cleaved UTP to UDP in a calcium independent
manner. In contrast, the cleavage of UDP to UMP was strongly calcium dependent for LALP70, but
calcium independent for LALP70v.
Conclusions: The VSFASSQQ motif not only influences the substrate specificity of LALP70, but
it confers calcium sensitivity to LALP70 during the UDP cleavage. Whether this is due to direct
binding of calcium to this motif or to a conformational change of the enzyme, remains to be
elucidated.
Background
Apyrases, which are mostly ectoenzymes, are a protein
family of enzymes capable of cleaving nucleotide tri- and
diphosphates in a calcium or magnesium dependent
manner, not being altered by P-type, F-type or V-type
NTPase inhibitors [1]. We have previously identified the
lysosomal apyrase of 70 kDa (LALP70, Acc. No.
NP_004892; NTPDase4α, [2]) and its splice variant
LALP70v (NTPDase4β, [2]), which are one of the first
intracellular apyrases being described [3,4]. The difference
between LALP70 and LALP70v is the 8 amino acid motif
VSFASSQQ missing in the splice variant LALP70v [5]. This
motif is encoded at the end of exon 7 and is located after
the fourth apyrase conserved region of LALP 70 [3,5]. The
VSFASSQQ motif is also present in the mouse homologue
of LALP70 (Acc. No. NP_080450), which shows 93%
identity with the human sequence. No other apyrase
deposited in the common databases contains this motif.
In the original description LALP70v was located in the
Golgi apparatus [4], while LALP70 was identified as a lys-
osomal membrane protein [3], implicating that the
VSFASSQQ motif might be involved in the sorting of the
LALP protein. However, analysis of the membrane topol-
ogy of LALP70 indicated, that the spliced motif is on the
site of the organelle lumen and not on the cytoplasmic
site, making at least classical sorting mechanisms unlikely
[6]. Furthermore, both the Golgi localisation of LALP70v
Published: 16 June 2004
BMC Biochemistry 2004, 5:8
Received: 31 March 2004
Accepted: 16 June 2004
This article is available from: http://www.biomedcentral.com/1471-2091/5/8
© 2004 Biederbick et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.

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and the lysosomal localisation of LALP70 were proposed
on evidences from transfection experiments using either
myc-tagged or the GFP-tagged proteins, respectively. Thus,
the intracellular localisation of the two endogenous splice
variants is still uncertain and it is open whether the
reported differences are based on technical reasons.
Apyrases are capable of cleaving NTPs and/or NDPs with
different substrate specificities [1]. Using a variety of com-
mon NTPs and NDPs, LALP70 was found to be a UTPase
or a TTPase [5], whereas LALP70v had a rather broad sub-
strate specificity with preferences for CTP or UDP [4,5].
This point to a function of the VSFASSQQ motif concern-
ing the preferred substrate of the enzyme. A further char-
acteristic feature of apyrases is their dependence on
calcium or magnesium ions [1]. Here we show, by dissect-
ing the NTPase activity from the NDPase activity, that
LALP70 has a calcium sensitive NDPase activity which is
not present in LALP70v. Our data show, that the
VSFASSQQ motif confers calcium sensitivity to LALP70 as
a NDPase.
Results and Discussion
To investigate separately the cleavage of NTP to NDP and
of NDP to NMP, UTP was chosen as a substrate, since both
enzymes, LALP70 and LALP70v, revealed a similar sub-
strate specificity for this nucleotide [5]. We used tritiated
UTP, which together with its cleavage products 3H-UDP
and 3H-UMP, could be detected by a quantitative thin
layer chromatographic approach (Fig. 1A,1B). When
crude membrane fractions from mock transfected cells
were used in this assay, less than 5% of the tritiated UTP
initially present was cleaved during a 30 minute incuba-
tion (Fig. 1C), when 5 mM calcium and 500 µM magne-
sium were present. Moreover, when only 10 µM calcium
were used in the presence of 500 µM magnesium, no
cleavage of tritiated UTP could be detected (data not
shown). This indicates that only a small amount of endog-
enous activity was present in the sample. Note that all
experiments shown were done in triplicate. The standard
deviation are not given graphically to keep the figures
clearly arranged. The numerical mean values and standard
deviations are given in table 1.
When membrane fractions were used from cells trans-
fected with LALP70, tritiated UTP decreased rapidly and
exponentially. This decrease was not dependent on the
used calcium concentration (Fig. 2A). At lower calcium
concentrations (1 – 100 µM) UDP increased rapidly until
a steady state concentration. In contrast, at calcium con-
centrations higher than 100 µM the UDPase activity was
stimulated in a dose dependent manner (Fig. 2B). Accord-
ingly, we observed a more rapid increase of UMP at these
higher calcium concentrations (Fig. 2C). Thus, LALP70
has a UDPase activity which is calcium sensitive, while the
UTPase activity is independent on the calcium concentra-
tion. In contrast, when membrane fractions were used
from cells transfected with LALP70v (Fig. 3A,3B,3C), the
UDPase enzyme activity was almost identical for 10 µM or
5 mM calcium, respectively (Fig. 3B), indicating that the
loss of the VSFASSQQ motif abrogates the calcium sensi-
tivity of the UDPase activity. Since in all experiments mag-
nesium ions were present in a concentration of 500 µM,
the dependencies observed must be specific for calcium
rather than for divalent cations in general.
It is not known how the VSFASSQQ motif influences the
calcium binding abilities of LALP70. Three general types
of calcium binding sides (class I to III) have been
described, depending on the positioning of the ligands in
the amino acid chain [12,13]. The best documented cal-
cium binding protein domain is the EF-hand motif [14-
16]. This motif provides, in a 12 amino acid stretch, 6 to
7 oxygen ligands to complex one Ca2+ ion [12,17]. How-
ever, using the PROSITE software, no EF-hand motif could
be detected in the predicted LALP70 protein sequence.
Other calcium binding motifs use amino acid side chains
which are more or less scattered over the protein back-
bone (class II and III type) and which can hardly detected
by a sequence analysis in silico. Using CLUSTALW, align-
ments of the LALP70 amino acid sequence with sequences
of different calcium binding proteins from the three
classes mentioned did not result in significant homolo-
gies (data not shown). Recently, the calcium coordination
of a soluble form of the ecto-nucleoside triphosphate
diphosphorylase I (CD39), a typical membrane anchored
apyrase [18,19], was investigated by electron paramag-
netic resonance (EPR) spectroscopy [20]. Chen and Gui-
dotti provided evidence that calcium is complexed by
protein ligands as well as by oxygen atoms of the substrate
molecule ATP or ADP, respectively. However, under our
experimental conditions an excess of magnesium was
present, which usually constitute strong complexes with
NTPs or NDPs [12]. At least for lower calcium concentra-
tions it is unlikely that magnesium is substituted by cal-
cium according to the model proposed by Chen and
Guidotti [20]. Although this model might describe cor-
rectly the involvement of divalent cations in the cleavage
mechanism of NTPs and NDPs, our results indicate a fur-
ther binding of calcium to LALP70. However, whether the
VSFASSQQ motif contributes directly to the binding of
calcium, or if it changes the structure of the protein, so
that other potential calcium ligands are properly posi-
tioned for calcium binding, remains open.
The absence of the VSFASSQQ motif abrogates the cal-
cium susceptibility of the UDPase activity (Fig. 2B and
3B). NTP/NDP binding in apyrases has been contributed
to the apyrase conserved regions (ACRs) 1 and 4, which
are homologous to the β- and γ-phosphate binding

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domains of the actin/hsp70/hexokinase superfamily [21-
23]. In accordance to this observation mutations in this
regions led to changes in the substrate specificity of the
apyrases tested [24,25]. However, deletions or mutations
of the sequence outside the ACRs 1 and 4 were found to
influence the substrate specificity, even if the mutated part
of the protein belongs to a membrane spanning region
[26-28]. This suggests, that substrate binding and cleavage
of NTPs/NDPs are related to the tertiary structure of the
enzyme in a highly complex manner. The functional inter-
relationship between calcium binding, substrate binding
Detection of mono- and diphosphate nucleotides generated by LALP70 or LALP70v apyrase activity
Figure 1
Detection of mono- and diphosphate nucleotides generated by LALP70 or LALP70v apyrase activity. Crude membrane prepa-
rations containing expressed LALP70 were incubated with 3H-UTP/UTP in the presence of 5 mM Ca2+ and 500 µM Mg2+. After
indicated time points, samples were separated by thin layer chromatography and tritiated nucleotides were detected with a
thin layer radioactivity scanner (A). Peak 1 contains 3H-UTP, peak 2 3H-UDP and peak 3 3H-UMP, and the integrated amount
of counts per peak represents the amount of product generated by the enzyme in a certain time intervall. The peak positions
were calibrated using unlabeled nucleotides, which were separated onto fluorescence covered HPTLC plates and detected by
UV-light (λ = 260 nm; (B)). When a crude membrane preparation from cells transfected with the expression vector only were
used in the assay described in (A), less than 5% of the 3H-UTP/UTP was cleaved during a 30 minute incubation (C).

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and substrate cleavage has to be further elucidated by
direct biochemical and structural approaches using the
purified LALP70 and LALP70v proteins.
Conclusions
The VSFASSQQ motif, which is lacking in the LALP70
splice variant LALP70v, plays an important function for
enzymatic properties of this apyrase. This was shown by a
new invented thin layer chromatographic approach which
allows the simultaneous quantification of the UTPase and
UDPase activity. Both apyrases were not calcium sensitive
in their UTPase function. However, UDP cleavage was cal-
cium sensitive in a concentration dependent manner in
LALP70, but not in LALP70v. We hypothesize that the
VSFASSQQ motif is a new calcium sensitive regulatory
domain for the UDPase activity in LALP70. However,
whether the calcium sensitivity depends on a direct cal-
cium binding to the VSFASSQQ motif or is due to confor-
mational changes of the protein induced through the
VSFASSQQ motif remains to be elucidated.
Methods
Materials
All nucleotide phosphates and the 25-kDa polyethylene-
imine were from Sigma-Aldrich (Taufkirchen, Germany),
and the 3H-UTP (1 µCi/µl; 25 Ci/mmol) were from
Hartmann (Braunschweig, Germany). HPTLC plates Silica
gel 60 (10 × 10 cm) with concentrating zone and fluores-
cence covered HPTLC plates Silica gel 60 F254 (10 × 10 cm)
were from Merck (Darmstadt, Germany).
Expression vectors
Both the human full-length LALP70 cDNA and the
LALP70v cDNA missing the 24 bp between nucleotides
1028–1051 were generated and cloned into the mamma-
lian expression vector pCL-Neo (Promega) as described
elsewhere [5].
Cell culture and transfection
COS-7 cells were cultivated in Dulbecco's modified
Eagle's medium supplemented with 2 g/liter Hepes, 10%
fetal calf serum, 50 µg/ml gentamycin, and incubated at
37°C in a humidified chamber equilibrated with 5% CO2.
COS-7 cells were transfected with the mammalian pCL-
Neo expression vector alone or with this vector containing
either the cDNA for LALP70 or for LALP70v, respectively,
using a 25-kDA polyethyleneimine (Sigma-Aldrich) as
transfection reagent [7]. Transfection procedure was per-
formed as described elsewhere [3]. Transfected cells were
harvested for nucleotide phosphatase assays 40 h after
transfection.
Table 1: Mean values and standard deviation in %cpm of 3H-UTP hydrolysis experiments, obtained with membrane fractions from COS
cells transfected with LALP70 or LALP70v cDNA, respectively.
05 101520 2530
mean ±
S.E.M.
73 ± 7,5
91 ± 1,7
27 ± 7,5
9 ± 1,7
0
0
62 ± 12
67 ± 2,0
73 ± 8,0
62 ± 3,5
72 ± 0,7
38 ± 10
33 ± 2,0
27 ± 8,5
35 ± 2,1
27 ± 0,7
0,3 ± 0,7
0,5 ± 0
0
0
1,8 ± 0,4
mean ±
S.E.M.
67 ± 5,5
80 ± 4,0
31 ± 4,9
18 ± 5,0
3 ± 1,2
2,3 ± 1,1
40 ± 9,0
41 ± 10
44 ± 4,0
43 ± 5,0
40 ± 7,0
58 ± 9,0
58 ± 10
53 ± 4,0
51 ± 5,0
44 ± 7,7
2 ± 0,3
2 ± 0,6
3 ± 0,6
7 ± 1,8
16 ± 1,0
mean ±
S.E.M.
58 ± 4,9
70 ± 7,0
36 ± 4,2
25 ± 7,0
6,3 ± 1,5
5 ± 2,5
27 ± 1,7
31 ± 2,1
31 ± 8,0
23 ± 6,1
42 ± 8,0
68 ± 2,0
64 ± 0,5
65 ± 7,0
59 ± 2,5
27 ± 8,5
5 ± 2,0
6 ± 2,0
5 ± 1,0
18 ± 3,5
31 ± 0,5
mean ±
S.E.M.
48 ± 8,3
57 ± 4,1
42 ± 6,6
36 ± 7,5
10 ± 1,7
7 ± 3,5
21 ± 0,6
27 ± 2,0
26 ± 8,0
22 ± 1,4
28 ± 3,5
72 ± 3,0
65 ± 2,0
65 ± 8,0
54 ± 2,1
25 ± 8,5
7 ± 1,5
8 ± 3,0
8 ± 1,0
24 ± 3,9
48 ± 4,9
mean ±
S.E.M.
40 ± 2,0
51 ± 5,0
45 ± 2,6
35 ± 5,5
16 ± 4,3
14 ± 4,5
19 ± 4,5
26 ± 2,0
24 ± 5,0
17 ± 0,6
27 ± 2,1
71 ± 2,5
65 ± 5,4
65 ± 0,5
49 ± 4,9
18 ± 6,4
10 ± 1,7
10 ± 4,0
12 ± 4,0
34 ± 4,9
56 ± 3,9
mean ±
S.E.M.
32 ± 5,8
46 ± 5,7
46 ± 4,7
37 ± 6,9
22 ± 4,3
17 ± 4,9
19 ± 6,0
22 ± 0,5
24 ± 4,0
20 ± 2,8
22 ± 2,5
70 ± 6,5
67 ± 3,0
61 ± 1,0
38 ± 3,9
15 ± 4,5
11 ± 1,1
12 ± 3,7
14 ± 5,0
42 ± 1,0
63 ± 2,1
mean ±
S.E.M.
32 ± 2,0
36 ± 4,3
40 ± 6,5
42 ± 8,5
28 ± 5,8
21 ± 8,4
16 ± 5,0
19 ± 1,0
23 ± 5,0
19 ± 3,1
23 ± 3,5
69 ± 5,0
66 ± 4,5
61 ± 0,5
19 ± 2,8
14 ± 7,0
14 ± 1,0
16 ± 5,6
17 ± 6,0
51 ± 5,6
63 ± 3,5
Lalp70vUTP 10 µM Ca2+
5 mM Ca2+
10 µM Ca2+
5 mM Ca2+
10 µM Ca2+
5 mM Ca2+
1 µM Ca2+
10 µM Ca2+
100 µMCa2+
1 mM Ca2+
5 mM Ca2+
1 µM Ca2+
10 µM Ca2+
100 µMCa2+
1 mM Ca2+
5 mM Ca2+
1 µM Ca2+
10 µM Ca2+
100 µMCa2+
1 mM Ca2+
5 mM Ca2+
UDP
UMP
Lalp70UTP
UDP
UMP

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Ca2+ dependent generation of mono- and diphosphate nucleotides by LALP70 phosphatase activity
Figure 2
Ca2+ dependent generation of mono- and diphosphate nucleotides by LALP70 phosphatase activity. Crude membrane prepara-
tions containing expressed LALP70 were incubated with 3H-UTP/UTP, 500 µM Mg2+ and various concentration of Ca2+. Apy-
rase activity products were separated by thin layer chromatography and 3H-labeled nucleotides were detected by a thin layer
radioactivity scanner. At timepoints indicated cpm of each peak were counted and are given as percentage of all counted cpm.
Mean values from three separate measurements are given. While dephosphorylation of 3H-UTP by LALP70 is independent of
the Ca2+concentration (A), dephosphorylation of 3H-UDP (B) and thereby generation of 3H-UMP (C) depends on the Ca2+con-
centration.