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J Biol Chem, Vol. 274, Issue 49, 35023-35028, December 3, 1999
From the Lehrstuhl Biochemie I, Universität Regensburg,
Universitätsstrasse 31, D-93053 Regensburg, Germany
The extracellular matrix (ECM) of
Volvox is modified during development or in response to
external stimuli, like the sex-inducing pheromone. It has recently been
demonstrated that a number of genes triggered by the sex-inducing
pheromone are also inducible by wounding. By differential screening of
a cDNA library, a novel gene was identified that is transcribed in
response to the pheromone. Its gene product was characterized as an ECM
glycoprotein with a striking feature: it exhibits a hydroxyproline
content of 68% and therefore is an extreme member of the family of
hydroxyproline-rich glycoproteins (HRGPs). HRGPs are known as
constituents of higher plant ECMs and seem to function as structural
barriers in defense responses. The Volvox HRGP is also
found to be inducible by wounding. This indicates that the wound
response scenarios of higher plants and multicellular green algae may
be evolutionary related.
The evolution of a complex extracellular matrix
(ECM)1 from a simple cell
wall was one of the prerequisites to promote the transition from
unicellularity to multicellularity. The volvocine algae provide the
unique opportunity for exploring the pathways that led from a simple
cell wall to a complex ECM that stabilizes the shape of an organism and
mediates many developmental responses of cells to internal as well as
external stimuli. The volvocine algae range in complexity from
unicellular Chlamydomonas to multicellular organisms, with
differentiated cells and complete division of labor, in the genus
Volvox. The asexually growing organism of Volvox
carteri is composed of only two cell types: 2000-4000
biflagellate Chlamydomonas-like somatic cells are arranged
in a monolayer at the surface of a hollow sphere (1, 2) and 16 much
larger reproductive cells ("gonidia") lie just below the somatic
cell sheet. Volvox cells are surrounded and held together by
a glycoprotein-rich ECM (reviewed in Refs. 3 and 4). Cell walls and
ECMs of the volvocine algae are assembled entirely from glycoproteins (5) and a high content of hydroxyproline has been detected. Hydroxyproline-rich glycoproteins (HRGPs) represent a constituent of
higher plant ECMs, and much work has been done to analyze the structures of these proteins (6-11). However, there are few examples in the literature where multiple ECM proteins have been examined in
molecular detail from a single species or from closely related species.
This approach has been initiated with volvocine algae to allow a more
integrated approach to elucidate the structure, assembly, and function
of ECM proteins.
A remarkably rapid remodelling of the ECM is observed under the
influence of the sex-inducing pheromone (a glycoprotein) that triggers
initiation of the sexual life cycle of Volvox carteri (12-14). In particular, synthesis of some members of the pherophorin family of ECM proteins (15-17) is strongly induced by the pheromone. Pherophorins are ECM glycoproteins that contain a C-terminal domain with homology to the sex-inducing pheromone.
By differential screening of a cDNA library, additional genes were
recently identified that are transcribed under the control of the
sex-inducing pheromone (18). Unexpectedly, genes were found, in
addition to those encoding the pherophorins, that encode extracellular
chitinases and proteinases. In higher plants, similar protein families
are known to play an important role in defense against fungi. Indeed,
it could be demonstrated that the same set of genes triggered by the
sex-inducing pheromone is also inducible by wounding of
Volvox spheroids.
Pheromone-induced changes in the composition of the ECM have been
characterized in detail within the cellular zone of the ECM (12, 13,
15, 16, 19) and to a lesser extent within the deep zone (DZ) (17),
which contains all ECM components internal to the cellular zone (for
nomenclature see Ref. 3). The DZ appears as a relatively amorphous
component that fills the deepest regions of the spheroid and that may
constitute more than 90% of the total volume of the organism. In this
paper, we characterize a HRGP exhibiting an extreme composition that is
expressed in response to the sex-inducing pheromone and to wounding and
that is part of the DZ compartment of the ECM.
Culture Conditions--
The female V. carteri f.
nagariensis strains HK10 (wild type) and 153-48 (nitA Differential Screening of a cDNA Library--
Total RNA was
isolated from V. carteri spheroids (HK10) at various times
after the addition of the sex-inducing pheromone. RNA samples isolated
3, 6, and 12 h after the application of the pheromone were pooled,
and a cDNA library was prepared from the corresponding
poly(A)+ mRNA preparation (18) by using the Cloning of the dz-hrgp Gene--
The dz-hrgp cDNA
fragment obtained by differential screening of the cDNA library was
used as a probe to screen a V. carteri genomic library (19)
in PCR Amplification of dz-hrgp cDNA Fragments--
RNA from
sexually induced (2.5 h) V. carteri spheroids (HK10) 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), and cDNA fragments of dz-hrgp were amplified by PCR. Alternatively, cDNA
fragments were amplified by reverse transcription PCR as described
(25). RACE-PCR technique was performed as described (26).
DNA Sequencing--
Genomic and cDNA clones of
dz-hrgp were mapped with standard restriction enzymes, and
restriction fragments were subcloned. To create targeted breakpoints
for DNA sequencing, these subclones were digested unidirectionally with
exonuclease III (27). This was done from both sides of the subclones.
Products were transformed into ultracompetent Escherichia
coli cells (Epicurian Coli, Stratagene). The precise length of a
given insert was determined by gel electrophoresis. Sequencing from
both directions was done by cycle sequencing (28, 29) and isothermal
sequencing (30) using vector-based oligonucleotide primers that were
end labeled with [ Northern Blot Analysis--
Total RNA (10 µg) from V. carteri strain HK10 was separated on a 1.0% denaturing gel (23),
vacuum-blotted, UV-cross-linked onto Hybond-N membrane (Amersham
Pharmacia Biotech), and hybridized with a 0.6-kb
32P-labeled cDNA fragment of dz-hrgp. This
cDNA fragment came from the 3'-untranslated region of
dz-hrgp to prevent cross-reactions with other
polyproline-encoding genes.
Reverse Transcription-PCR--
Volvox spheroids were
incubated with or without the presence of the sex-inducing pheromone or
were wounded by forcing a concentrated Volvox suspension
through a 0.5-mm hypodermic needle. Reverse transcription-PCR from 20 Volvox spheroids was performed as described (25). The
antisense oligonucleotide primer 5'-GTGTTTCCACCAGTGCGA and the sense
oligonucleotide primer 5'-GAGCCATGTGGAAAGTCG were used for PCR
amplification of a 117-base pair dz-hrgp cDNA fragment. Products of PCR amplification were cloned and sequenced.
Construction of Chimeric Genes--
The fusion regions of the
chimeric dz-hrgp promoter-dz-hrgp gene or the
chimeric dz-hrgp-arylsulfatase gene were generated by the
recombinant PCR technique (33). The final construction was performed by
standard techniques (23).
Stable Transformation of Volvox--
Transformation of
Volvox was as described (34) but using a Biolistic
PDS-1000/He particle gun (Bio-Rad) (35) to bombard strain 153-48 (nitA Genomic PCR--
Genomic PCR was used to confirm stable
transformation of Volvox. 50 spheroids were selected under a
stereomicroscope and transferred into 10 µl of sterile lysis buffer
(0.1 M NaOH, 2.0 M NaCl, 0.5% SDS). After 5 min at 95 °C 200 µl of 50 mM Tris/HCl, pH 7.5, were added immediately. 2 µl of the resulting lysate was used for PCR (in
a total volume of 100 µl). PCR was performed by standard protocol. Products of PCR amplification were cloned into the SmaI site
of pUC18 and sequenced.
Preparation of Anti-DZ-HRGP Antiserum--
The peptide
PRRSPVVALVETC (amino acids 26-37 of DZ-HRGP) with an artificial
cysteine at the C-terminal end was synthesized by using Fmoc
(9-fluorenylmethyloxycarbonyl) amino acid derivatives. The peptide was
purified on a reversed phase C18 HPLC column (Nucleosil 100-7, 7 µm; Macherey-Nagel, Düren, Germany). The predicted
molecular mass and the sequence of the peptide were confirmed by
electrospray mass spectrometry and by Edman degradation. The synthetic
peptide was covalently linked to a maleimide-activated carrier protein (keyhole limpet hemocyanin) via the SH group of the artificial cysteine
and used to raise polyclonal antibodies in rabbit. Antibodies were
purified by protein G-Sepharose column chromatography (Amersham Pharmacia Biotech). Further purification of anti DZ-HRGP antiserum was
on an affinity column (Sulfolink Coupling Gel, Pierce) with covalently
linked DZ-HRGP peptide. The column was produced and handled as
described (36). The antibodies were tested by Western blot analysis,
using the synthetic peptide (this time covalently linked to bovine
serum albumin) as an antigen.
Localization of DZ-HRGP--
Whole Volvox spheroids
were separated into defined fractions to allow localization of DZ-HRGP.
A DZ extract was prepared as described below. The remaining material
(intact cells and cell-bound ECM) was extracted with 2 M
NaCl (2 h) and then disrupted ultrasonically (Sonifier B15, Branson,
Danbury, CT). Soluble and insoluble components were separated by
ultracentrifugation (100,000 × g, 30 min). All fractions were lyophilized, deglycosylated by anhydrous HF (37), and
analyzed by a Western blot using the polyclonal DZ-HRGP antibody.
Purification of DZ-HRGP from Volvox--
Sexually induced
Volvox spheroids from three 20 l cultures were
harvested at the stage of embryogenesis by filtration on a 100-µm
mesh nylon screen. The spheroids were broken up by forcing them through
a 0.5-mm hypodermic needle. The disrupted spheroids were centrifuged at
25,000 × g for 30 min. The supernatant (DZ extract)
was brought to 50 mM Tris/HCl, pH 9.0, 10 mM
NaCl and applied to a QAE-Sephadex A-25 anion exchange column (Amersham Pharmacia Biotech) equilibrated with the same buffer. Elution was
performed with 250, 500 and 800 mM NaCl. Fractions
containing DZ-HRGP antigen were dialyzed against 4 mM
NH4HCO3 and lyophilized. The dried material was
deglycosylated by anhydrous HF (37), applied to SDS-PAGE, and blotted
onto polyvinylidene difluoride membrane. The membrane was stained with
Coomassie Blue R-250 (Serva, Heidelberg, Germany) in 50% methanol,
destained with 50% methanol/10% acetic acid, and washed with water.
The band corresponding to the DZ-HRGP antigen was cut out and sequenced
using an automated gas-phase peptide sequencer (Applied Biosystems,
Foster City, CA).
Radioactive labeling of DZ-HRGP with
[14C]Bicarbonate--
Pulse labeling with
[14C]bicarbonate was performed in vivo as
described (38).
Amino Acid Analysis and Mass Spectrometry--
Amino acid
analysis was performed as described by Cohen and Strydom (39).
Molecular masses of fractions of interest were determined by
electrospray mass spectrometry using a SSQ 7000 mass spectrometer (Finnigan).
Differential Screening--
Total RNA was isolated from V. carteri spheroids (female HK10) harvested at various times after
the addition of the sex-inducing pheromone. RNA samples isolated 3, 6, and 12 h after the application of the pheromone were pooled, and a
cDNA library ( DNA Sequencing and Deduced Amino Acid Sequence--
To extend the
cDNA sequence information obtained from the originally isolated
0.6-kb cDNA, the RACE-PCR (26) was used to obtain the missing 5'
stretches. But the RACE-PCR yielded only a 0.25-kb fragment because a
stretch of unusually high (G)C-content caused premature termination of
reverse transcription. To circumvent this problem, the
dz-hrgp cDNA fragment was used to clone the corresponding genomic DNA. Sequencing of this unusual stretch of
genomic DNA also produced particular problems and was only possible by
the application of special techniques like creation of targeted
breakpoints for DNA sequencing by exonuclease III digestion of
subclones (for details, see "Experimental Procedures"). The
strategy applied to collect the complete nucleotide sequence of
dz-hrgp cDNA is shown in Fig.
2a. The sequence was submitted to the GenBankTM/EBI Data Bank with accession number
AJ242540. The dz-hrgp gene contains a single intron within
its coding region. The deduced amino acid sequence for the DZ-HRGP is
shown in Fig. 2b. This amino acid sequence exhibits striking
features. The open reading frame encodes a polypeptide 409 amino acid
residues in length, including a typical signal sequence.
(Hydroxy-)proline constitutes 68% of the amino acid residues of the
mature polypeptide. Several stretches of up to 14 (hydroxy)- proline
residues and numerous repeats of Ser-(Pro)3 as well as
(Ser/Arg)-(Pro)4 elements are special features of this gene
product. These data (and the data presented below) indicate a close
relation of this novel Volvox protein to a well known
protein family, namely, to the HRGPs of higher plants (6-11). The
reason for giving DZ-HRGP its name was this relationship and its
localization within the DZ of the Volvox ECM (see
below).
Because of the extended proline stretches of DZ-HRGP there are only
short amino acid sequences near both the N and C termini of the
polypeptide, which could serve as a suitable antigen in DZ-HRGP
antibody production to prevent cross-reactions with other proline-rich
ECM glycoproteins. Therefore, a sequence derived from the N-terminal
end of DZ-HRGP was used to synthesize the peptide PRRSPVVALVETC. An
artificial cysteine at the C terminus of this peptide simplified
coupling to a carrier protein. Peptide specific polyclonal antibodies
were raised in rabbit.
Identification and Homologous Overexpression of DZ-HRGP--
The
deduced DZ-HRGP amino acid sequence includes a typical signal peptide
indicating an extracellular localization of DZ-HRGP. The
peptide-specific antibody was used to search for DZ-HRGP in different
extracts prepared from sexually induced Volvox spheroids. However, neither complete lysates nor ECM fractions produced any signal
in immunodetection experiments. The putative extracellular localization
of DZ-HRGP suggests extensive glycosylation of hydroxyproline residues,
and this in turn could prevent immunodetection by our peptide-specific
antibody. Therefore, the components of Volvox extracts were
deglycosylated by treatment with anhydrous HF. Indeed, after
deglycosylation, positive signals at ~150 kDa could be obtained in
extracts from complete Volvox spheroids as well as in an ECM extract representing the DZ of Volvox ECM. The material of
the DZ is selectively released by mild mechanical stress as may be exerted by forcing Volvox spheroids through a hypodermic needle.
Because Western blots yielded only a weak DZ-HRGP signal,
overexpression of DZ-HRGP in Volvox was thought to get
sufficient amounts of DZ-HRGP for structural studies. Random
integration by illegitimate recombination events is the preferred mode
of DNA integration into the Volvox genome, and transformants
often integrate multiple copies of the plasmids used for transformation (34). Therefore, transgenic Volvox were generated that
express additional copies of the dz-hrgp gene under the
control of its own promoter. Stable transformants were produced as
described previously (25, 34, 35, 40). The transgenic Volvox
strain did not show any visible change in phenotype, but the expression rate of DZ-HRGP was clearly higher than in wild-type algae. Again, the
polyclonal DZ-HRGP antibody leads to a positive immunosignal at ~150
kDa after deglycosylation of extracts from sexually induced transformants. ~50% of DZ-HRGP is liberated just by forcing the Volvox spheroids through a hypodermic needle (DZ extract);
all of the remaining DZ-HRGP is extracted in the presence of 2 M NaCl.
A molecular mass of 39.6 kDa is calculated for the mature polypeptide
chain of DZ-HRGP (Fig. 2b), but this is much less than the
apparent molecular mass of deglycosylated DZ-HRGP (~150 kDa) shown on
Western blot gels (Fig. 3b).
The extreme (hydroxy-)proline content of DZ-HRGP explains the
difference between the observed and calculated molecular masses,
because stretches of poly-(hydroxy-)proline have a reduced ability to
bind SDS (41).
Purification of DZ-HRGP--
To prove the identity of the
immunoreactive material and the DZ-HRGP, the components of the DZ
extract of the ECM were fractionated by ion exchange chromatography
(QAE-Sephadex). Because proteins from the DZ are hardly stained with
standard procedures on SDS-PAGE gels, the Volvox spheroids
were grown in the presence of [14C]bicarbonate prior to
preparation of the DZ extract, and the SDS-PAGE gels were analyzed by
fluorography. As demonstrated by analytical SDS-PAGE, the QAE-Sephadex
chromatography separated two main protein species of the deep zone
extract completely from each other. These protein species exhibit
apparent molecular masses of ~300 and ~240 kDa and elute at 500 and
at 800 mM NaCl, respectively (Fig. 3a). The
material of both fractions was then deglycosylated by treatment with
anhydrous HF and fractionated by 6% SDS-PAGE. After blotting, an
immunoreactive polypeptide with an apparent molecular mass of ~150
kDa could be detected in the 500 mM NaCl fraction (Fig.
3b). To confirm that it is indeed the ~300-kDa protein
(glycosylated) that produces the ~150-kDa immunosignal (deglycosylated), the ~300-kDa protein was further purified by excision from a SDS-polyacrylamide gel. After elution the ~300-kDa protein was deglycosylated and subjected to SDS-PAGE. Again, the ~150-kDa immunosignal was detectable in a Western blot (data not shown). The immunoreactive polypeptide was subjected to automated Edman
degradation, resulting in the N-terminal sequence
Ala-Hyp-Ala-Arg-Lys-Hyp-Hyp-Hyp-Arg-Arg-Ser-Hyp, matching the
N-terminal sequence deduced for mature DZ-HRGP. Remarkably, even the
very first proline residue of the polypeptide turned out to be
posttranslationally modified to hydroxyproline. Amino acid analyses of
DZ-HRGP resulted in molar ratios of the predominant amino acids
hydroxyproline, arginine, and serine of 10:1:1.4. On the basis of the
cDNA sequence the ratios of (hydroxy)-proline, arginine, and
serine were calculated as 10.2:1:1.6. All the other amino acids within
DZ-HRGP could not be quantified exactly in amino acid analyses because
of the small amounts detected. As shown in Fig.
4, all of the prolines of DZ-HRGP are
found to be modified to hydroxyproline.
Carbohydrate Composition of DZ-HRGP--
The carbohydrate
composition of DZ-HRGP was determined by radio gas chromatography.
DZ-HRGP purified from Volvox spheroids grown in the presence
of [14C]bicarbonate was hydrolyzed, and the resulting
monosaccharides were analyzed as the alditol acetates. DZ-HRGP contains
the neutral sugars arabinose and galactose in a 2:1 ratio (Fig.
5).
The Promoter of dz-hrgp Gene Mediates
Pheromone-dependent Transcription--
To examine the
properties of the dz-hrgp promoter, the 5'-nontranslated
region (~3 kb) of dz-hrgp was placed in front of a reporter gene, the arylsulfatase gene from Volvox (25, 42) (Fig. 6a). In wild-type
Volvox, arylsulfatase is only expressed under sulfur
starvation; no activity is detectable in organisms grown in
sulfate-containing medium (42).
After transformation of Volvox with the chimeric
dz-hrgp/arylsulfatase gene, the reverse transcription-PCR
technique was used to verify the existence of hybrid mRNA in
transformants (see "Experimental Procedures"). Volvox
transformants containing the arylsulfatase gene under the control of
the dz-hrgp promoter were incubated with or without the
sex-inducing pheromone in the presence of the chromogenic enzyme
substrate 4-nitrocatechol sulfate. Arylsulfatase activity was
determined photometrically by measuring the absorbance of the liberated
4-nitrocatechol. After treatment with the sex-inducing pheromone, only
transformants exhibited enzyme activity (Fig. 6b). Thus, the
promoter fragment from the dz-hrgp gene is sufficient to
mediate transcription in response to the sex-inducing pheromone.
Transcription of dz-hrgp Gene Is Cell Type-specific--
In the
Northern blotting experiments described above whole Volvox
spheroids were used for RNA preparation. To investigate whether
transcription of the dz-hrgp gene is cell type-specific, both cell types of (sexually induced) Volvox spheroids,
somatic and reproductive, were separated from each other by size
fractionation. RNA from both cell types was extracted and reverse
transcribed, and a dz-hrgp cDNA fragment was amplified
by PCR. Transcription of the dz-hrgp gene was detectable
mainly in somatic cells, as shown in Fig.
7a.
Wounding Induces Transcription of dz-hrgp Gene--
As
demonstrated recently (18), all genes known so far to be under the
control of the sex-inducing pheromone are also triggered by wounding.
This observation led us to investigate whether transcription of
dz-hrgp gene is also responsive to wounding.
Volvox spheroids were slit by mild mechanical stress simply
by forcing them through a hypodermic needle. 2.5 h later total RNA
from 20 spheroids was extracted and reverse transcribed, and a
dz-hrgp cDNA fragment amplified by PCR. As shown in Fig.
7b, transcription of dz-hrgp gene is not
detectable in asexually growing Volvox colonies. In contrast, the sex-inducing pheromone as well as wounding induces transcription of this gene (Fig. 7b). Wounding even appears
to stimulate a higher rate of transcription as compared with the induction by the sex-inducing pheromone.
This paper describes the structure and properties of a novel
component of the Volvox ECM with the striking feature of
being composed of 68% hydroxyproline. Some of these hydroxyprolines are arranged in Ser-(Pro)3 and Ser-(Pro)4
elements that are typical of higher plant extensins (9). Bradley
et al. (43) noted that tissue wounding in higher plants
selectively stimulates the expression of tyrosine-rich extensins. In
this respect, the algal DZ-HRPG is different because it contains only a
single tyrosine residue. Similarly, it does not perfectly fit into the
general class of higher plant extensins because the predominant basic amino acid lysine is replaced by arginine in the algal polypeptide. To
our knowledge, there is no other protein described with a
(hydroxy)-proline content as high as 68%. For example, the higher
plant extensins with the highest (hydroxy)-proline contents are
those of Vigna unguiculata (44), Nicotiana
tabacum (45), and Gossypium barbadense (46), exhibiting
53, 44, and 40%, respectively. In the animal kingdom the mini-collagen
of Hydra attenuata (47) exhibits 44%.
A typical feature of algal ECM proteins characterized so far is a
strictly modular composition, where hydroxyproline-rich (HR) sequences
seem to serve as rod-shaped spacers separating modules that are
completely devoid of hydroxyproline residues. It has been suggested
that these hydroxyproline-rich stretches define a HR module family
combining more specialized modules to yield chimeric and
multifunctional ECM proteins (4). In this sense, DZ-HRGP represents the
first volvocacean ECM glycoprotein being only composed of one (or
more?) HR module(s). Electronmicroscopic studies have shown that main
parts of the volvocacean ECM consist of a network of fibrous structures
(48, 49). Most probably, the rod-shaped HR modules mainly have a
structural function and serve as building blocks to create these
defined framework of the ECM. Where analyzed in more detail, these
modules were found to be targets for extensive posttranslational
modifications. Among the modifications identified in Volvox
are O-glycosylations with oligoarabinosides, attachment of
saccharides containing phosphodiester bridges between arabinose
residues, and in a single case, the additional attachment of a highly
sulfated arabinomannan (19). As analyzed in more detail for the ECM
protein SSG 185, the HR module is also involved in covalent
cross-linking of the monomeric units (19). If DZ-HRPG serves a similar
function, this rod-shaped molecule could be involved in the creation of
the fibrous networks observed in the deep zone compartment (3). The
fact that overexpression of DZ-HRPG did not create an aberrant ECM
morphology could indicate the participation of a second ECM molecule in
the cross-linking reaction. In this case, only the concomitant
overexpression of both ECM partners interacting in a stoichiometric
relation would be expected to cause a visible phenotype.
The natural resistance of higher plants to diseases involves an array
of inducible defense responses, including synthesis of extracellular
hydrolytic enzymes such as proteases and chitinases and the
accumulation of HRGPs within the ECM. The latter glycoproteins are
hypothesized to function in defense as structural barriers (50) or as
specific microbial agglutinins (51, 52) against pathogen attack.
Surprisingly, the simple multicellular green alga Volvox
responds to wounding in much the same way as observed in higher plants.
As was demonstrated recently (18), Volvox responds to
wounding with the synthesis of a chitinase as well as a protease that
is combined with chitin-binding modules. With the additional
demonstration of a typical HRGP that is produced in response to
wounding, it now appears that much of the response scenario found in
higher plants already exists in multicellular green algae. Even more
surprising is the fact that these algal pathways are also triggered by
the sex-inducing pheromone. However, Kirk and Kirk (53) were able to
demonstrate that synthesis of the sex-inducing pheromone can be
triggered in somatic cells by a short heat shock applied to asexually
growing organisms. This response induces the production of dormant
zygotes that survive unfavorable conditions like drought. Although
wounding is unable to induce pheromone production, similar biochemical
responses are observed after wounding and pheromone application (18). As induction of sexuality and subsequent production of zygotes obviously is part of the strategy of the organism to escape from environmental stress, it appears to make sense that apparently completely different stimuli (wounding and pheromone treatment) cause
up-regulation of the same set of genes. Further studies on this algal
system should confirm or deny an evolutionary relation of both the
pheromone and the wound response systems. In addition, the possible
relation of this algal system and the wound healing reactions in higher
plants deserves further investigation.
We thank Dr. R. Deutzmann and E. Hochmuth for
mass spectrometry and for protein sequencing.
*
This work was supported by Grant SFB 521 from the Deutsche
Forschungsgemeinschaft.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) AJ242540.
§
To whom correspondence should be addressed. Tel.:
49-941-943-2835; Fax: 49-941-943-2936; E-mail:
armin.hallmann@vkl.uni-regensburg.de.
The abbreviations used are:
ECM, extracellular
matrix;
HRGP, hydroxyproline-rich glycoprotein;
DZ, deep zone;
PCR, polymerase chain reaction;
RACE, rapid amplification of cDNA
ends;
kb, kilobase(s);
HPLC, high pressure liquid chromatography;
PAGE, polyacrylamide gel electrophoresis;
HF, hydrogen fluoride;
HR, hydroxyproline-rich.
Response to the Sexual Pheromone and Wounding in the Green
Alga Volvox: Induction of an Extracellular Glycoprotein
Consisting Almost Exclusively of Hydroxyproline*
,§,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) 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 (20) at 28 °C in a
8 h dark/16 h light (10000 lux) cycle (21). Strain 153-48 was
grown in the presence of 1 mM NH4Cl. The
sex-inducing pheromone was used as described (22).
ZAP
cDNA synthesis kit (Stratagene, La Jolla, CA). Replica filters were
probed with 32P-labeled cDNA prepared from
polyadenylated RNA extracted from sexually induced or asexually growing
V. carteri spheroids. Hybridization was performed according
to standard procedures (23). The cDNA fragment of
dz-hrgp yielded only a 32P signal from sexually
induced spheroids, not from asexually growing organisms.
EMBL 3 (24). Cloning of the dz-hrgp gene followed
standard techniques (23).
-32P]ATP. Sequencing of GC-rich
stretches was improved by the addition of Me2SO (31) and by
using nucleotide analogues (32).
) with DNA-coated microprojectiles.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP) was constructed from the corresponding
poly(A)+ mRNA preparation. Repeated differential
screenings of the cDNA library with cDNA derived from asexual
versus sexually induced organisms resulted in the detection
of novel clones in addition to the already known members of the
pherophorin family (15-17) and to the chitinase and the cysteine
protease described recently (18). One of these novel clones was named
dz-hrgp for reasons explained below. The kinetics of
dz-hrgp mRNA accumulation in response to the
sex-inducing pheromone was analyzed by Northern blotting. As shown in
Fig. 1, hybridizing RNA started to
accumulate only about 30 min after pheromone treatment and reached its
maximum 2 h later. No significant signals were observed in
asexually growing organisms. The mRNA detected by the
dz-hrgp cDNA fragment is ~2.0 kb in length.
View larger version (53K):
[in a new window]
Fig. 1.
Northern blot analysis of dz-hrgp
mRNA. The accumulation of dz-hrgp mRNA in
vegetative or sexually induced organisms (by treatment with
10 12 M sex-inducing pheromone at 0 h)
was determined. Equal amounts of total RNA isolated after different
periods of incubation from vegetative or sexually induced
Volvox organisms were hybridized with a 0.6-kb cDNA
fragment of dz-hrgp (see cDNA clone in Fig.
2).
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Fig. 2.
Strategy applied to collect the complete
nucleotide sequence of dz-hrgp cDNA and the
deduced amino acid sequence of DZ-HRGP. a, completion
of cDNA was achieved by 5'-RACE-PCR, by reverse transcription-PCR,
and by sequence analysis of a genomic clone. The position of an intron
is indicated by an arrowhead. The sequence was submitted to
the GenBankTM/EBI Data Bank with accession number AJ242540.
b, deduced amino acid sequence of DZ-HRGP. All prolines are
shown in white letters on a black background. An
arrow marks the signal peptidase cleavage site.
View larger version (28K):
[in a new window]
Fig. 3.
Purification of DZ-HRGP. Volvox
algae were labeled with 14C in vivo.
Fractions of the following steps were subjected to 6% SDS-PAGE: crude
DZ extract, DZ extract applied to a QAE-Sephadex column and eluted at
250, 500, or 800 mM NaCl. a, analysis by
fluorography; x-ray film after a 15-h exposure. b,
analysis by Western blot using anti DZ-HRGP antibodies for detection.
The antigen-antibody complex was detected using
alkaline-phosphatase-conjugated secondary antibodies. The material
analyzed in b was deglycosylated by anhydrous HF.
View larger version (18K):
[in a new window]
Fig. 4.
Identification of hydroxyproline in acid
hydrolysates of DZ-HRGP. a, reversed phase HPLC
analysis of amino acids, which were modified by phenylisothiocyanate to
facilitate detection. The positions of hydroxyproline (hyp)
and proline (pro) are indicated. b,
characterization of the hydroxyproline peak from a by
electrospray mass spectrometry. The predicted mass for the
phenylisothiocyanate derivative of hydroxyproline is 267.2 Da.
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[in a new window]
Fig. 5.
Sugar analysis of DZ-HRGP by radio gas
chromatography. 14C-Labeled alditol acetates derived
from DZ-HRGP (radioactivity) after acid hydrolysis are
shown. The scan on top (mass) records the mass
signal of internal standards.
View larger version (18K):
[in a new window]
Fig. 6.
The promoter region of the dz-hrgp
gene mediates transcription control by the sex-inducing
pheromone. a, structure of the chimeric gene containing
the dz-hrgp promoter region and the arylsulfatase reporter
gene (genomic clone). b, photometric arylsulfatase activity
assay (25) from transformants. Algae were disrupted by ultrasonic
treatment, and the lysates were assayed using 4-nitrocatechol sulfate
as a chromogenic substrate (optical density at 515 nm produced in 30 min by 50 algae/ml). Extracts from asexually grown ( 
) or sexually
induced (+) Volvox algae are shown. Transformants
(T1 and T2) are compared with the
nitA strain 153-48 (N) used as DNA recipient and to wild-type
HK10 algae (WT).
View larger version (61K):
[in a new window]
Fig. 7.
Cellular localization and induction of
dz-hrgp mRNA. dz-hrgp mRNA was detected
by reverse transcription and subsequent PCR amplification. A 117-base
pair cDNA fragment of dz-hrgp was expected if the intron
within this fragment was spliced correctly. a, somatic and
reproductive cells of sexually induced Volvox spheroids were
investigated separately to identify the cellular localization of
dz-hrgp mRNA. b, vegetatively grown
Volvox spheroids, spheroids after incubation with the
sex-inducing pheromone (for 2.5 h), or vegetatively grown
spheroids 2.5 h after wounding were investigated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
These authors contributed equally to this work and are considered
first authors.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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