From the Lehrstuhl Biochemie I, Universität Regensburg, D-93053 Regensburg, Germany
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ABSTRACT |
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The volvocine algae provide the unique
opportunity for exploring development of an extracellular matrix.
Volvox is the most advanced member of this family and
represents the simplest multicellular organism, with differentiated
cells, a complete division of labor, and a complex extracellular
matrix, which serves structural and enzymatic functions. In
Volvox carteri a glycosylated extracellular phosphatase was
identified, which is partially released from the extracellular matrix
into the growth medium. The phosphatase is synthesized in response to
inorganic phosphate starvation and is strictly
calcium-dependent. The metalloenzyme has been purified to
homogeneity and characterized. Its gene and cDNA have been cloned.
Comparisons of genomic and cDNA sequences revealed an extremely
intron-rich gene (32 introns). With an apparent molecular mass of 160 kDa the Volvox extracellular phosphatase is the largest phosphatase cloned, with no sequence similarity to any other
phosphatase. This enzyme exhibits a modular composition. There are two
large domains and a small one. The large domains are highly homologous to each other and therefore most likely originated from gene
duplication and fusion. At least one EF-hand motif for calcium binding
was identified in this extracellular protein. Volvox
extracellular phosphatase is the first calcium-dependent
extracellular phosphatase to be cloned.
One of the prerequisites to promote the transition from
unicellularity to multicellularity during evolution was the development of a complex extracellular matrix
(ECM)1 from a simple cell
wall. The ECM of a multicellular organism is a complex organelle that
serves structural and enzymatic functions (reviewed in Ref. 1). It
mediates many developmental responses including regulation of growth
and differentiation, wound repair, and pathogen defense. The ECM also
plays a key role when adaptations to environmental changes are required.
The evolutionary transition from unicellularity to multicellularity can
be analyzed among the volvocaceae, a family of photoautotrophic green
algae. The complexity within this family progresses gradually from
unicellular Chlamydomonas via Gonium and
Pandorina to Eudorina, forming colonies with an
increasing cooperation of individual cells. The most advanced member of
the volvocaceae, Volvox, has developed a multicellular form
of organization with a complete division of labor between two cell
types, somatic and reproductive. In Volvox carteri, about
2000 cells are somatic and only 16 cells are reproductive (2, 3).
The ECM of Volvox exhibits anatomically distinct structures;
based on light and electron microscopic observations the
Volvox ECM was divided into four main zones and several
subzones (reviewed in Ref. 4). The Volvox ECM lacks
cellulose, pectins, or lignin but mainly consists of glycoproteins, and
the structures of several ECM glycoproteins are known in molecular
detail (reviewed in Ref. 5).
In the cell walls of higher plants several enzymes such as peroxidases,
invertases, mannosidases, glucanases, arabinosidases, galactosidases,
polygalacturonase, pectin methylesterases, malate dehydrogenase,
proteases, and phosphatases have been identified (reviewed in Ref. 1).
However, research on these individual proteins has occurred in
different plant species and all sorts of plant organs and cell types.
Since higher plant ECMs are extremely complex amalgams, relatively
little is known about the precise functions and intermolecular
interactions. In these complex systems, it is also difficult to
investigate in vivo enzymatic reactions to environmental
changes. Therefore, a simple model organism as Volvox might
be advantageous for ECM studies. So far, the only ECM glycoproteins of
Volvox with proven enzymatic activity are a
lysozyme/chitinase (6) and an arylsulfatase (7).
I am interested in adaptations within the Volvox ECM in
response to environmental stimuli. Since phosphorus is an important nutrient and often limiting in many ecosystems, the role of the ECM
during phosphorus deprivation was investigated. In this report an
atypical calcium-dependent extracellular phosphatase was
identified in Volvox, which is synthesized in response to
phosphate deprivation. The enzyme has been isolated and characterized.
Likewise, the cloning and characterization of its gene is described.
Culture Conditions--
The female V. carteri f.
nagariensis strains "HK10" (wild type),
"dissociator," and "153-48" were obtained from R. C. Starr (Culture Collection of Algae, University of Texas, Austin, TX) or from
D. L. Kirk (Washington University, St. Louis, MO). Synchronous cultures were grown in Volvox medium (8) at 28 °C in an
8-h dark/16-h light (10,000 lux) cycle (9). Strain 153-48 was grown in
the presence of 1 mM NH4Cl.
Standard Volvox medium contains 0.16 mM
glycerophosphate. Due to an orthophosphate (Pi)
contamination in the glycerophosphate (Sigma) used, standard
Volvox medium also contains ~25 µM
orthophosphate. In (glycero)phosphate-free Volvox medium
also no orthophosphate was detectable.
Assay of Activity--
Routinely, phosphatase activity was
measured using p-nitrophenyl phosphate (pNPP) as a
substrate. The assay contained 40 mM pNPP, 200 mM Tris-HCl, pH 9.5, and enzyme in a final volume of 1 ml.
When the metalloenzyme nature of the phosphatase was discovered, the
assay was supplemented with 5 mM CaCl2. After
incubation at 37 °C for 15 min, the reaction was stopped by adding
100 µl of 4 M NaOH. Enzyme activity was quantified by
measuring the absorbance at 405 nm.
Purification of Phosphatase--
Volvox spheroids
(HK10) from three 20-liter cultures were collected on a 100-µm mesh
nylon screen. After extensive washing with Volvox medium
lacking phosphate, the algae were incubated in phosphate-free medium
under standard conditions for further 24 h. The Volvox
algae were collected as described and disrupted ultrasonically
(Sonifier B15; Branson, Danbury, CT), and the lysate was cleared by
centrifugation (24,000 × g, 20 min). The crude enzyme
solution was fractionated and precipitated with ammonium sulfate
(30-80% saturation; centrifugation at 24,000 × g, 20 min). The precipitated protein was dissolved, desalted by dialysis (20 mM Tris-HCl, pH 8.0) for 15 h, and passed through a
Sep-Pak Plus C18 reversed phase cartridge (Waters, Milford,
MA). The flow-through was loaded on a QAE-Sephadex A-25 anion exchange
column (Pharmacia, Uppsala, Sweden), and the column was developed by
applying a NaCl gradient (up to 1 M) in 20 mM
Tris-HCl, pH 8.8, at 1 ml/min. Fractions containing enzyme activity
were concentrated by ammonium sulfate precipitation as described. The
dissolved protein was further fractionated by molecular-sieve
chromatography on a HiLoad 16/60 Superdex 200 preparative grade fast
protein liquid chromatography column (Pharmacia, Uppsala, Sweden) in 20 mM Tris-HCl, pH 8.8, 200 mM NaCl at a flow rate
of 1 ml/min. Fractions containing the enzyme solution were concentrated
using a Centriplus-30 concentrator (Amicon, Beverly, MA). After a short
ultracentrifugation step (60,000 × g, 10 min)
phosphatase was still in solution, but after a long run (90,000 × g, 18 h) the soluble phosphatase was sedimented. Both
centrifugation steps were repeated twice. Finally the enzyme pellet was
dissolved in 20 mM Tris-HCl, pH 8.8.
Proteolytic Digestion and Separation of Peptides--
50 µg of
phosphatase were applied to a SDS-polyacrylamide gel electrophoresis
(6%). The gel was stained with 0.25% Coomassie Blue, 45% methanol,
19% acetic acid, and destained with 30% methanol, 7% acetic acid.
The gel slice containing the extracellular phosphatase was excised, cut
into small pieces, and crushed. The gel pieces were treated as follows:
2 times for 1 h 30% methanol, 7% acetic acid; 3 times for 1 h 50% methanol, 10% acetic acid; 2 times for 1 h 90% ethanol.
Then the gel pieces were dried in vacuo, subsequently soaked
in 4% trypsin, 0.2 M NH4HCO3 and
incubated at 37 °C for 14 h. The suspension was centrifuged,
and the supernatant was kept. After three subsequent washing steps (2 times 0.2 M NH4HCO3; 1 time 0.2 M NH4HCO3, 50% acetonitrile), the
supernatant and the wash eluates were pooled, filtered (0.22-µm
Millex-GV4 filter; Millipore Waters, Bedford, MA), brought to 0.1%
trifluoroacetic acid, and dried by lyophilization. The peptides were
dissolved in 6 M guanidine HCl, 0.1%
CF3CO2H and fractionated by reversed phase HPLC
(SMART system; Pharmacia, Uppsala, Sweden) on a µRPC C2/C18 (3 µm) column (Pharmacia, Uppsala,
Sweden). Peptides were eluted by a 30-min linear gradient from 5 to
40% acetonitrile in 0.1% CF3CO2H with a flow
rate of 200 µl/min. Peptides were rechromatographed on a µRPC
C18 (3 µm) column (Pharmacia, Uppsala, Sweden) under the
same conditions. Peptides were sequenced by Edman degradation using an
automated gas phase peptide sequencer (Applied Biosystems, Foster City, CA).
Generation of a cDNA Probe by PCR--
RNA was extracted
from phosphate-starved (5 h) Volvox as described (10).
RT-PCR was as described previously (11, 12) using the antisense
primer 5'-TCRTANGTRAAYTG (amino acid positions 13-17 of peptide 6),
the sense primer 5'-GARGAYATHGGNGG (amino acid positions 3-7 of
peptide 6), and an Expand High Fidelity polymerase mix (Boehringer
Mannheim, Mannheim, Germany). Forty cycles of PCR amplification
(94 °C, 10 s; 40 °C, 15 s; 72 °C, 5 s) were
performed on a GeneAmp PCR System 2400 (Perkin-Elmer). The resulting
44-bp DNA fragment was ligated into the SmaI site of pUC18
vector and sequenced.
Cloning of the Gene--
The V. carteri genomic
library (13) in PCR Amplification of cDNA Fragments--
RNA from
phosphate-starved (5 h) Volvox was used to construct a
cDNA library covalently linked to magnetic beads according to the
instructions of the manufacturer of the beads (Deutsche Dynal, Hamburg,
Germany). The cDNA fragments of phosphatase were amplified by PCR,
using this library. Rapid amplification of cDNA ends (RACE)-PCR
technique was performed as described (17). PCR fragments were ligated
into the SmaI site of pUC18 or pBluescript II SK vector
(Stratagene, La Jolla, CA). Synthetic oligonucleotides were used to
sequence the phosphatase cDNA fragments in both directions.
Construction of the Chimeric Stable Transformation of Volvox--
All transformation
experiments were performed with the Volvox nitrate reductase
gene as a selectable marker on a separate plasmid (21). Plasmid DNAs
for transformation of Volvox were purified using anion
exchange columns (Qiagen, Hilden, Germany). Gold microprojectiles (1 µm in diameter; Bio-Rad) were coated with the required targeting
plasmids, and stable transformation of Volvox strain 153-48 was performed as described (21), but using a Biolistic PDS-1000/helium
particle gun (Bio-Rad). Strain 153-48 carries a stable loss-of-function
mutation within the nitrate reductase gene (nitA) (22). Out
of 30 NitA-positive transformants, two transgenic Volvox
clones were recovered which expressed the phoX gene constitutively.
Preparation of Anti-phosphatase Antiserum--
The peptide
AIYFPNIPPAVTEEEKC was synthesized on a SP4000 peptide synthesizer
(Labortec, Bubendorf, Switzerland) by using Fmoc
(N-(9-fluorenyl)methyloxycarbonyl) amino acid derivatives. The peptide corresponds to the N terminus of the mature phosphatase with an artificial cysteine at the C-terminal end. The predicted molecular mass and the sequence of the peptide were confirmed by
electrospray mass spectrometry using a Single-Stage Quadrupole 7000 mass spectrometer (Finnigan, San Jose, CA) and by Edman degradation using an automated gas phase peptide sequencer (Applied Biosystems, Foster City, CA). The peptide was covalently linked to a
maleimide-activated carrier protein (keyhole limpet hemocyanin; Pierce)
via the SH group of the artificial cysteine. A New Zealand rabbit was
immunized (intradermally) with 100 µg of phosphatase peptide coupled
to 100 µg of carrier protein in 250 µl of Freund's complete
adjuvant (Sigma). The rabbit was boostered three times at the 20th day (subcutaneously), 30th day (intradermally), and 40th day
(subcutaneously) from the first immunization on with 100 µg of
phosphatase peptide coupled to 100 µg of carrier protein in 250 µl
of Freund's incomplete adjuvant (Sigma). At the 61st day 20 ml of
blood were withdrawn from the rabbit's ear vein. After blood clotting
had taken place, the blood was centrifuged for 10 min at 2,000 × g and the supernatant was kept. 0.02% sodium
ethylmercurithiosalicylate were added to the supernatant and stored at
4 °C.
Western Blot Analysis--
About 4000 Volvox
spheroids of wild-type or transformed algae were harvested by
filtration on a 100-µm mesh nylon screen and disrupted
ultrasonically. The lysate was cleared by centrifugation (20,000 × g, 3 min) and used for Western blot analysis. Deep zone extracts were as described previously (23). Samples were separated on a
5% standard SDS-polyacrylamide gel, electroblotted to a polyvinylidene fluoride membrane (0.45 µm; Millipore Waters, Bedford, MA), and probed using the polyclonal rabbit anti-phosphatase peptide antibody or
a mouse anti-FLAG monoclonal antibody M2 (Eastman Kodak Co.). Signals
were visualized using the enhanced chemiluminescence Western blotting
analysis system (Pharmacia, Uppsala, Sweden).
Assay of Substrate Specificity--
Different possible
substrates (p-nitrophenyl phosphate, glycerophosphate,
phosphoenolpyruvate, ribose 5-phosphate, phosphorylcholine, O-phospho-L-tyrosine, AMP, cAMP, NADPH, NADH,
1,2-diacyl-glycero-3-phosphate, L-
Radioactive labeling of ECM extracts, SSG 185, or pherophorin-S with
[33P]phosphate was performed in vivo as
described before (23). The different ECM glycoproteins were assayed for
remaining [33P]phosphate by comparing the incorporated
radioactivity prior to and after incubation with the phosphatase on a
SDS-polyacrylamide gel followed by fluorography.
Identification of an Inducible Extracellular Phosphatase in
Volvox--
Phosphorus is an important and often limiting nutrient
(25). Analyzing the kinetics of the Pi decrease in the
medium during growth of Volvox, I observed contrary to my
expectations an increasing orthophosphate concentration. When
Volvox colonies were grown for 6 days (or 3 generations) in
standard Volvox medium, the orthophosphate concentration
increased from ~25 to ~100 µM, whereas the number of
colonies rose by 2 orders of magnitude (up to six colonies/ml). Glycerophosphate supplied with the growth medium is the only possible source for orthophosphate production, indicating the action of a potent
(extracellular) phosphatase. Thus, the growth medium and different
extracts from Volvox spheroids were assayed for this
activity using pNPP as a chromogenic substrate. A high level of
phosphatase activity was identified in phosphate-deprived
Volvox, independent of the developmental stage used. This
activity correlates with the appearance of a ~160-kDa protein band on
SDS-polyacrylamide gels (Fig.
1A). The 160-kDa protein band
is not detectable in Volvox grown in the presence of 1 mM orthophosphate. Phosphate-starved wild-type algae
secrete about 50% of the activity into the growth medium, and about
5% are in the deep zone of the Volvox ECM (for nomenclature
see Ref. 4). The remaining ~45% of activity are bound within the
cellular zone of the ECM. In a mutant Volvox strain called
"dissociator" (26), which is unable to produce a structurally
intact ECM and consequently dissociates into single cells shortly after
the end of embryogenesis, about 90% of phosphatase activity and most
of the ~160-kDa protein are secreted into the growth medium.
These results suggested the existence of an extracellular 160-kDa
phosphatase in Volvox, which is inducible by phosphate deprivation.
Purification of the Enzyme--
In my experiments, purification of
phosphatase from large volumes of growth medium caused problems.
Therefore, the phosphatase bound within the cellular zone of the ECM
served as the starting material. Volvox spheroids were
cultivated in phosphate-free medium for 24 h and then harvested.
The Volvox algae were disrupted ultrasonically, and the
lysate was cleared by centrifugation. The crude extract was
fractionated and precipitated with ammonium sulfate (30-80% saturation), desalted by dialysis, and passed through a reversed phase
C18 cartridge that did not adsorb the enzyme. The
flow-through was loaded on an anion exchange column (QAE-Sephadex), and
the column was developed with a NaCl gradient. Fractions containing enzyme activity were concentrated and then further fractionated by
molecular sieve chromatography (Superdex 200). Due to self-aggregation, the soluble extracellular phosphatase could be sedimented by
ultracentrifugation. This final centrifugation step was repeated twice,
leading to a homogenous phosphatase preparation (Fig.
1B).
Protein Chemical Studies--
Automated Edman degradation of the
purified Volvox phosphatase showed that its N-terminal amino
acid sequence is AIYFPNI. In order to get additional sequence
information from internal peptides, Volvox phosphatase,
localized in a SDS-polyacrylamide gel by Coomassie stain, was digested
by incubating the corresponding gel slice with trypsin. The resulting
peptide mixture was eluted from the polyacrylamide gel and
chromatographed by reversed phase C2/C18 HPLC.
All peaks were rechromatographed by C18 HPLC. Samples of
well separated peaks were subjected to amino acid sequence analysis on
an automated gas-phase sequencer. The amino acid sequence data obtained
are summarized in Table I. Peptide 10 includes a N-glycosylation motif, and amino acid 10 of this
peptide gave no signal in automated Edman degradation, which indicates
N-glycosylation at this site.
Amplification of a cDNA Fragment by PCR--
The amino acid
sequence of the tryptic peptide 6 (Table I) was used to synthesize
degenerated oligonucleotide primers. The sense primer 5'-GARGAYATHGGNGG
was designed from amino acid positions 3-7. The antisense primer
5'-TCRTANGTRAAYTG was designed from amino acid positions 13-17 of the
same peptide. The latter primer was used to reverse transcribe total
RNA of phosphate-starved V. carteri. The resulting cDNA
was amplified by PCR using both the sense and antisense primers. Forty
cycles of amplification yielded a cDNA fragment of 44 bp in length,
which was cloned and sequenced. The deduced amino acid sequence matched
the sequence of peptide 6, with glycine at position 8.
Genomic and cDNA Clones--
A genomic library of V. carteri constructed in the replacement vector
The complete 5.1-kb cDNA-sequence of the Volvox
phosphatase was obtained by RT-PCR and RACE-PCR (17) technology. The
3'-RACE-PCRs revealed two different ends of the extracellular
phosphatase mRNA, one 45 bp longer than the other. The applied
strategy to collect the complete nucleotide sequence of the phosphatase
cDNA is summarized schematically in Fig. 2B. The
sequence was submitted to the GenBankTM EBI data bank with
accession number AJ012458.
The exon-intron organization of the phoX gene was deduced by
comparison of genomic and cDNA sequences. Fig. 2A
summarizes the relative locations of the 33 exons and 32 introns within
~15 kb of phosphatase sequence. The smallest exon encodes for only 11 amino acids. 73% of the genomic DNA sequence between start and stop
codon is derived from introns.
The Phosphatase Polypeptide--
The deduced amino acid sequence
of the Volvox phosphatase is shown in Fig.
3. The PSORT protein localization program
(27) predicts a cleavable hydrophobic leader sequence (amino acids 1-23) which is in accordance with the sequenced N terminus of the
mature phosphatase. A total of eight possible
N-glycosylation sites (NXT and NXS)
were found. As mentioned above, glycosylation of one of those sites has
been confirmed by protein chemistry (peptide 10, Table I). Based on the
cDNA data the molecular mass of the mature phosphatase protein
should be 144 kDa, but SDS-gel analysis revealed a somewhat higher
molecular mass (~160 kDa). The difference may result from
post-translational modification, e.g. glycosylation.
There is an internal sequence similarity within the phosphatase
sequence. Amino acids 30-596 show 46% identity to amino acids 610-1185, indicating a modular composition of phosphatase; there are
two quite similar large domains, I and II, each about 570 amino acids
in length, and a small C-terminal domain III, which is about 180 amino
acids in length (Fig. 3).
Comparison of the Volvox phosphatase sequence with other
phosphatases or all the sequences of the Swiss-Prot protein sequence data base (BLASTP search; Ref. 28) did not reveal any significant sequence similarities.
Immunological Detection--
Rabbit polyclonal antibodies were
raised against the synthetic N-terminal phosphatase peptide
AIYFPNIPPAVTEEEKC. The artificial cysteine at the C terminus of this
peptide was used for covalent attachment to a carrier protein (keyhole
limpet hemocyanin). The peptide antibody specifically recognizes the
160-kDa phosphatase band in lysates of phosphate-deprived
Volvox (Fig. 4A).
The quantitative distribution of the immunopositive material in
different extracts from phosphate-starved Volvox (growth
medium, whole cell lysate, deep zone extract) was just as found for the
phosphatase activity described above (data not shown). Identical
amounts of phosphatase activity from these extracts or from the
purified enzyme were loaded on a SDS-polyacrylamide gel and analyzed by
Western blotting (Fig. 4B). There is no significant
difference in intensity between the immunopositive band derived from
the purified phosphatase and the immunopositive bands derived from
different extracts, indicating that the purified extracellular
phosphatase is the predominant or sole phosphatase enzyme in these
extracts.
Constitutive Overexpression of Phosphatase in
Volvox--
Transgenic Volvox were generated which express
the phoX gene with a C-terminal epitope tag (FLAG tag). To
allow an enzymatic differentiation between endogenous and recombinant
phosphatase, the recombinant phosphatase was constitutively expressed
using the strong
Phosphatase release from the ECM into the growth medium exhibited a
striking behavior; the higher the density of the Volvox spheroids the more phosphatase was released (the cell membranes remained undamaged). Up to 80% total phosphatase activity could be
found in the growth medium if Volvox colonies were incubated in close contact to each other. The concentrated growth medium originating from the same number of transgenics incubated at different cell densities is shown in a Western blot analysis in Fig.
4D. Later I recognized that this
density-dependent effect is not only a property of
transgenics grown in phosphate-sufficient medium but also of wild-type
algae grown in phosphate-deficient medium (data not shown). Possibly,
the Volvox ECM is partially degraded by the action of
enzymes, if the colonies are in close contact and more and more
phosphatase can be released.
The Metalloenzyme Nature--
To determine whether divalent
cations were required for the activity of the extracellular phosphatase
of Volvox, the purified enzyme was treated with the metal
ion chelators EDTA or EGTA. Concentrations as low as 5 µM
EDTA or EGTA resulted in complete inactivation of the enzyme. To
investigate whether the phosphatase activity could be recovered after
inactivation, the enzyme was treated with 2 mM EGTA,
followed by the addition of a variety of metal ion species at 10 mM, namely Cu2+, Mg2+,
Ca2+, Fe2+, Mn2+, Zn2+,
and Co2+. Only calcium permitted 70% reactivation of the
EGTA-inactivated enzyme (Fig.
5A). Reconstitution of
activity by calcium was investigated in detail by adding varying
concentrations of CaCl2 to the EGTA-inactivated enzyme and
measuring phosphatase activity (Fig. 5B). All of the metals
used were in the form of chloride salts and chloride ions had no
detectable effect on phosphatase activity at the concentrations used
(data not shown). Accordingly, the extracellular phosphatase of
Volvox is a calcium-dependent metalloenzyme. The
amino acid sequence of extracellular phosphatase reveals at least one
typical "EF hand" motif (Fig. 5C). This motif is
localized at amino acid positions 210-239 (Fig. 3) within domain I of
phosphatase.
Substrate Specificity and Inhibitors--
The extracellular
phosphatase of Volvox has a broad substrate specificity
(Table II). The best substrates are
p-nitrophenyl phosphate, glycerophosphate, and
phosphoenolpyruvate, but all the other phosphomonoesters tested were
also accepted. Since the phosphomonoester within the amino acid
O-phospho-L-tyrosine is a substrate, the
extracellular phosphatase exhibits a protein phosphatase activity as
well. Contrary to these broad phosphomonoesterase activities, the
phosphatase is unable to hydrolyze the phosphodiester in cAMP or the
phosphoanhydride bond in NADH. Therefore, Volvox extracellular phosphatase is a phosphomonoesterase.
Several phosphatases can effectively hydrolyze the phosphomonoester
bond of isolated phosphoproteins (29-31). The ECM of Volvox owns several phosphorus-containing glycoproteins (32-34). Their quality as a substrate for the Volvox phosphatase was
investigated. The 33P-labeled ECM glycoproteins
pherophorin-S (23), SSG 185 (13), or whole 33P-labeled ECM
extracts were tested for 33P liberation by the phosphatase
(Table II). No 33P was liberated with any of these
substrates, indicating the absence of phosphomonoesters in the ECM
structure of Volvox. Since a phosphodiester bridging two
arabinose residues via their 5-positions was isolated from hydrolysates
of SSG 185 (35) and pherophorin-S (23), this type of phosphodiester
might be the predominant or sole phosphorus compound in the ECM of
Volvox.
Some substances, besides EGTA and EDTA, were identified that affect
phosphatase activity negatively: phosphate, sulfate, and vanadate are
inhibitors of the phosphatase from Volvox. In the presence
of 20 mM phosphate, sulfate, or vanadate activity decreases to 3, 27, or 15%, respectively. Vanadate is known to be a
protein-phosphotyrosine phosphatase inhibitor (36). Different amino
acids at concentrations up to 20 mM, such as
L-homoarginine, L-cysteine,
L-phenylalanine, that inhibit other phosphatases
(37, 38) had no significant effect on the activity of the
Volvox phosphatase (Table II).
Additional Enzymatic Properties--
The Volvox
extracellular phosphatase exhibits a broad temperature optimum at
25-45 °C and is rather heat-sensitive; after 10 min at 56 or
65 °C only 35 or 1% of the initial activity remains.
Phosphatases are routinely categorized by the pH required for optimal
activity. The extracellular phosphatase of Volvox showed maximum activity at pH 9.5, with only 5% of the maximum activity at pH
7.0. Therefore, the Volvox phosphatase was classified as an
alkaline phosphatase (EC 3.1.3.1).
Extracellular phosphatase from Volvox is insensitive
toward non-ionic detergents. With 10% Triton X-100 no decrease of
activity was detectable. Contrary to non-ionic detergents, the ionic
detergent SDS (0.3%) led to complete inactivation/denaturation.
In nearly all lakes, phosphorus is the limiting element that
determines the total biomass of algae (25, 39-43). Most of the soluble
phosphate in a lake has been washed out of the sediments. But in many
soils most of this soluble phosphate is esterified to organic
compounds, making it impossible for many organisms to absorb it
directly (44). Since in lakes overgrown with algae the orthophosphate
concentration falls into the submicromolar range (40), the possession
of an extracellular phosphatase with a broad substrate specificity is a
great advantage for algae like Volvox. Besides the synthesis
of an extracellular phosphatase, Volvox shows an additional
adaptation to phosphorus deprivation. When phosphorus becomes limiting
in a lake, Volvox makes use of its capacity for phototaxis
and chemotaxis; Volvox accumulates near the surface during
the day and swims downward at night to zones near the ground where
phosphate or phosphate ester concentrations are clearly higher
(45-48). Due to the coordinated power of 4000-8000 beating flagellas
a multicellular Volvox alga reaches a speed of 5 m/h and
comes as deep as 30-40 m below the surface, in contrast to the much
slower unicellular algae (48).
It was speculated that Volvox has made another adaptation to
phosphorus deprivation (49)2:
the ECM of Volvox owns many phosphorus-containing
glycoproteins (32-34), and these glycoproteins could be an
extracellular phosphorus store. However, no 33P was
liberated from whole 33P-labeled ECM extracts by the
extracellular phosphatase. A phosphodiester bridging two arabinose
residues was isolated from several ECM glycoproteins of
Volvox (23, 35). Thus, all phosphate within the ECM of
Volvox could be in the form of phosphodiesters, which have
structural or functional tasks, but represent no phosphorus store.
Unlike most phosphatases, the Volvox enzyme requires
Ca2+ ions for its activity. The calcium dependence,
together with the high pH optimum of the enzyme, might be an adaptation
to activity in hard water, where the pH and calcium concentrations
(CaCO3) are high. A high concentration of calcium results
in a very low concentration of soluble orthophosphate, making an
extracellular phosphatase advantageous.
Only two other extracellular phosphatases are known that are also
calcium-dependent, one from Haloarcula
marismortui and one from Chlamydomonas reinhardtii. The
extracellular phosphatase from the halophilic archaebacterium H. marismortui (50) has the same molecular weight and a comparable pH
optimum as the Volvox enzyme, but this phosphatase requires
4 M salt for activity. The Chlamydomonas
extracellular phosphatase (51) is somewhat larger (190 kDa) than the
Volvox enzyme but has the same pH optimum and a comparable
substrate specificity. Unfortunately, both these enzymes have not yet
been cloned, excluding a comparison at the molecular level. Cloning of
the Chlamydomonas phosphatase would be of particular
interest for evolutionary studies.
Apart from the requirement for calcium the size of the
Volvox phosphatase is striking. Subunit molecular masses of
most alkaline phosphatases range from 47 to 87 kDa (52, 53). Beside the Volvox enzyme (160 kDa) there are only the two large
extracellular phosphatases of Chlamydomonas (190 kDa) and
Haloarcula (160 kDa) mentioned above and another of the
unicellular cyanobacterium Synechococcus (145 kDa) (54, 55)
with a high molecular weight. The Synechococcus phosphatase
has been cloned (55), but there is no similarity to the
Volvox enzyme, and the Synechococcus enzyme has
no modular structure.
The size of the Volvox extracellular phosphatase could play
an important role within the ECM. Small soluble proteins leave the ECM
mesh-work if they are not cross-linked or differently bound, but ECM
enzymes need some mobility for activity (56). Due to its size
sufficient amounts of the Volvox phosphatase might be kept
back within the ECM mesh-work. In this context, the intramolecular domain duplication found in domains I and II of the Volvox
phosphatase could be understood as a way to raise the molecular weight
during evolution. Simultaneously this duplication could have given the opportunity to obtain two active centers within one polypeptide. During
evolution these domains could have developed a different substrate
specificity to reach the broad substrate spectrum of today's
phosphatase. The positions and sequences of the 32 introns within the
phosphatase gene indicate that the intramolecular domain duplication of
domains I and II is not a recent event; only three introns are at the
same positions in domains I and II (intron 1 in domain I and intron 16 in domain II, 3 in I, and 18 in II, 9 in I, and 23 in II), and the
intron sequences are completely different. Interestingly, there are
only few genes having more introns than this phosphatase gene,
e.g. the human or mouse genes for the major fibrillar
collagens contain 51-54 introns within ~30 kb (57).
There is another explanation for the intramolecular domain duplication.
Some phosphatases like the alkaline phosphatase of Escherichia
coli exist as dimers of identical subunits (52). Instead of using
two identical molecules to create a functional enzyme, two (nearly)
identical domains fused within a single polypeptide could fulfill the
same purpose.
This extracellular phosphatase can be used as a tool for genetic
experiments in Volvox. The phosphatase structural gene with its C-terminal epitope tag could serve as a reporter gene that results
in an enzymatically and immunologically detectable gene product.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
EMBL 3 (14) was used to clone the phosphatase gene,
named phoX. The screening and cloning procedures followed
standard techniques (15). DNA sequencing was performed by the chain
termination method (16) using T7 DNA polymerase (Pharmacia, Uppsala,
Sweden). Synthetic oligonucleotides were used throughout to sequence
the phoX gene.
-Tubulin Phosphatase Gene with
Epitope Tag--
To achieve high and constitutive phosphatase
production, the phosphatase phoX gene was placed under the
control of the Volvox
-tubulin promoter (18). For
construction of the chimeric gene, genomic clones of Volvox
-tubulin (nucleotides 40-501 of tubulin sequence as deposited in
the GenBankTM data base, accession number L24547) and
phosphatase were used. An additional EcoRV site was
generated by PCR directly upstream of the start codons both of the
phoX gene and the Volvox
-tubulin gene as
described (19), to facilitate ligation of the parent DNAs. To
introduce a short marker peptide (DYKDDDDK, FLAG epitope tag) at the C
terminus of the phosphatase, the gene splicing by overlap extension, or
recombinant PCR technique, was performed as described (20). The first
PCR was performed on a genomic phosphatase plasmid with a 19-mer 5'
sense primer (5'-CTGTTAACCTCTTGCTGAT) and a 44-mer 3' antisense primer
(5'-CTACTTGTCATCGTCGTCCTTGTAGTCATCCCAGGACGATACGC) with only half of the primer matching; the other half carries the
sequence coding for the epitope tag (tag sequence in bold and stop
codon in italics). The second PCR was performed on the same template
with a 42-mer 5' sense primer
(5'-GACTACAAGGACGACGATGACAAGTAGATGCGCGCACGTGAC, in which the underlined nucleotides are complementary to the
corresponding region of the antisense primer of the first PCR), and a
20-mer 3' antisense primer (5'-AGTGATGCAATGATCTTACA). The third PCR was carried out with both of these PCR products and only the flanking primers. PCRs were performed as described (11). All fragments were
joined together by standard techniques (15). The complete construct was
confirmed by restriction analysis and sequencing.
-phosphatidylcholine)
were assayed by the Pi release during incubation in the
presence of the phosphatase (15 min at 37 °C). For that purpose 100 µl of enzyme/substrate solution containing liberated phosphate were
mixed with 1 ml of a solution containing 3 mM ammonium
molybdate, 80 mM ascorbic acid, and 0.7 M
sulfuric acid (24), and the solution was incubated for 20 min at
37 °C. Negative controls were performed without the enzyme. The
absorbance at 820 nm was measured, and the liberated phosphate was
determined diagrammatically by comparison with a standard curve of
phosphate. Pi concentrations of the growth medium were
determined in the same way.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Detection and purification of extracellular
phosphatase. A, wild-type Volvox were grown
in (ortho)phosphate-sufficient (+) or -deficient ( ) medium for
24 h. The (concentrated) growth medium was applied to an
SDS-polyacrylamide gel (5%) and stained with silver. B,
purified phosphatase was loaded on a SDS-polyacrylamide gel (5%) and
stained with Coomassie Blue.
Amino acid sequences of tryptic peptides derived from extracellular
phosphatase of Volvox
EMBL 3 was
screened using the 44-bp cDNA fragment as a probe. Eighty-five
positive clones were identified out of ~60,000 phages screened. The
~16-kb insert of one of these phage clones was subcloned and
sequenced (
phoX-1 in Fig.
2A). It was noticed that the
~16-kb insert did not contain the 3'-terminal end of the phosphatase
gene. Therefore, a genomic probe, derived from the outermost 3'-end of
the sequenced phage insert, was used for an additional screening of the
EMBL 3 library. This produced ~90 positive clones, and 5 were
characterized. One of these phage clones contained the missing
3'-terminal end of the phosphatase gene and, therefore, was subcloned
and sequenced (
phoX-2 in Fig. 2A). The
partially overlapping inserts of the phages
phoX-1 and -2 allowed the determination of the complete sequence of the V. carteri phosphatase gene, named phoX, including the 5'-
and 3'-untranslated regions (Fig. 2A).
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Fig. 2.
Genomic and cDNA clones encoding
phosphatase. A, physical map of two overlapping
extracellular phosphatase genomic clones ( phoX-1 and
phoX-2) and the intron-exon structure of the complete
extracellular phosphatase phoX gene below. SalI
sites linking insert and vector DNA are in bold.
B, BamHI; E, EcoRI;
H, HindIII; K, KpnI;
N, NotI; S, SalI;
X, XbaI. B, PCR strategy applied to
collect the complete extracellular phosphatase cDNA. Positions of
32 introns are given by arrowheads. B,
BamHI; EI, EcoRI; EV,
EcoRV; H, HindIII; K,
KpnI.
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Fig. 3.
Deduced amino acid sequence of Volvox
extracellular phosphatase. Identical amino acid sequences of
domains I and II are highlighted (black or gray
background). The cleavage site of the hydrophobic leader sequence
is marked by an arrowhead.
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Fig. 4.
Western blot analysis of wild-type or
recombinant extracellular phosphatase after SDS-polyacrylamide gel
electrophoresis (5%). A, wild-type Volvox were
grown in phosphate-sufficient (+) or -deficient ( ) medium for 24 h and then disintegrated by ultrasonic treatment. The crude cell
lysates were analyzed by a Western blot using anti-extracellular
phosphatase antibodies for detection. B, identical amounts
of phosphatase activity (measured at pH 9.5) from purified phosphatase
enzyme (pure) and from different extracts of phosphate-starved
wild-type Volvox, namely concentrated growth medium
(1), an ultrasonic lysate of whole cells (2), and
a deep zone extract (3) were analyzed by a Western blot
using anti-extracellular phosphatase antibodies for detection.
C, wild-type algae and transformants expressing the
recombinant FLAG-tagged phosphatase were grown in phosphate-sufficient
medium, and lysates of whole cells were analyzed by a Western blot
using anti-FLAG antibodies. D, the same number of algae
constitutively expressing the tagged phosphatase were incubated in
different volumes (density: 100 colonies/ml or 700 colonies/ml) in
phosphate-sufficient medium for 12 h. Then the media of both
samples were concentrated and analyzed by a Western blot using
anti-FLAG antibodies.
-tubulin promoter of Volvox (for details
see "Experimental Procedures"). Transgenic Volvox algae
were produced as described previously (21), and the presence of the
chimeric mRNA in transformants was confirmed by RT-PCR (data not
shown). The total phosphatase activity in transgenics was about
50-80-fold higher than in the parent strain (153-48) when both were
grown in phosphate-sufficient medium. These results confirm the
identity of the phoX gene as the phosphatase gene. In
Western blot analysis a monoclonal antibody raised against the FLAG
epitope tag stains a 160-kDa band in lysates of the transgenics (Fig.
4C).
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Fig. 5.
The metalloenzyme nature of Volvox
extracellular phosphatase. A, effect of different
metal ion species on the activity of Volvox extracellular
phosphatase after inactivation of the enzyme by the addition of 2 mM EGTA. pNPP hydrolysis by the phosphatase was assayed at
pH 9.5 in the presence of 10 mM of the actual metal ion
species. B, following inactivation of the phosphatase by the
addition of 2 mM EGTA, varying concentrations of
CaCl2 were added, and the extracellular phosphatase was
assayed for pNPP hydrolysis at pH 9.5. C, consensus sequence
for calcium-binding EF hands (58) and the sequence found in
Volvox phosphatase (standardized and precise sequence). The
symbol n denotes a nonpolar side chain, and o
denotes an oxygen-containing side chain (D, N, E, Q, S, and
T).
Substrate specificity and inhibitors of Volvox extracellular
phosphatase
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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I am indebted to Dr. M. Sumper for critical reading of the manuscript. I thank Dr. R. Deutzmann and E. Hochmuth for sequencing peptides. I also thank N. Eichner and C. Friederich for their expert technical assistance.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 521).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) AJ012458.
To whom correspondence should be addressed: Lehrstuhl Biochemie I,
Universität Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany. Tel.: 49 941 943 2835; Fax: 49 941 943 2936; E-mail: armin.hallmann{at}vkl.uni-regensburg.de.
The abbreviations used are: ECM, extracellular matrix; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; pNPP, p-nitrophenyl phosphate; RACE, rapid amplification of cDNA ends; RT, reverse transcription; kb, kilobase pairs; bp, base pairs.
2 A. Hallmann, unpublished data.
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