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First published online October 6, 2006; 10.1105/tpc.106.041343

The Plant Cell 18:2554-2566 (2006)
© 2006 American Society of Plant Biologists

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A Posttranslationally Regulated Protease, VheA, Is Involved in the Liberation of Juveniles from Parental Spheroids in Volvox carteri[W]

Kazutake Fukada, Tan Inoue and Hideaki Shiraishi1

Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan

1 To whom correspondence should be addressed. E-mail siraisi{at}kuchem.kyoto-u.ac.jp; fax 81-75-753-3996.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
The lineage of volvocine algae includes unicellular Chlamydomonas and multicellular Volvox in addition to their colonial relatives intermediate in size and cell number. In an asexual life cycle, daughter cells of Chlamydomonas hatch from parental cell walls soon after cell division, while Volvox juveniles are released from parental spheroids after the completion of various developmental events required for the survival of multicellular juveniles. Thus, heterochronic change in the timing of hatching is considered to have played an important role in the evolution of multicellularity in volvocine algae. To study the hatching process in Volvox carteri, we purified a 125-kD Volvox hatching enzyme (VheA) from a culture medium with enzymatic activity to degrade the parental spheroids. The coding region of vheA contains a prodomain with a transmembrane segment, a subtilisin-like Ser protease domain, and a functionally unknown domain, although purified 125-kD VheA does not contain a prodomain. While 143-kD VheA with a prodomain is synthesized long before the hatching stage, 125-kD VheA is released into the culture medium during hatching due to cleavage processing at the site between the prodomain and the subtilisin-like Ser protease domain, indicating that posttranslational regulation is involved in the determination of the timing of hatching.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Green alga Volvox carteri, one of the simplest multicellular organisms, consists of only two cell types: immortal reproductive cells called gonidia and mortal somatic cells (Starr, 1969Go; Kirk and Harper, 1986Go; Kirk, 1998Go; Shimizu et al., 2001Go). In asexual adult spheroids, ~16 large nonmotile reproductive cells lie just beneath a monolayer of ~2000 small biflagellate somatic cells morphologically similar to the unicellular green alga Chlamydomonas reinhardtii. In the volvocine lineage, colonial relatives intermediate in size, cell number, and complexity between Chlamydomonas and Volvox are present. These colonial intermediates and Volvox are estimated to have diverged from a Chlamydomonas-like unicellular ancestor as recently as 50 ± 20 million years ago (Kirk and Harper, 1986Go; Rausch et al., 1989Go; Schmitt et al., 1992Go; Kirk, 1998Go). These volvocine algae provide a great opportunity to study the evolution of multicellularity (Kirk, 1998Go).

In the evolution of multicellularity in volvocine algae, delay in the timing of hatching is considered to have allowed embryos to perform various developmental events before maturation. In C. reinhardtii, hatching of daughter cells from their mother cell walls occurs within several hours after cell division (Harris, 1989Go). By contrast, in V. carteri, the hatching of juveniles occurs more than a day after the first cleavage of gonidia (Kirk, 1998Go). Various developmental events take place between the first cell division and hatching to configure the mature spheroid of V. carteri, including an increase in cell number, asymmetric cell division, morphogenesis of the blastomere, called inversion, which establishes juvenile configuration, germ-soma differentiation, the deposition of the extracellular matrix (ECM) for the expansion of spheroids, and juvenile flagellar development, which is essential for the survival of hatched juveniles (Coggin and Kochert, 1986Go; Tam and Kirk, 1991Go; Kirk, 1998Go; Sumper and Hallmann, 1998Go; Kirk et al., 1999Go; Miller and Kirk, 1999Go; Hallmann and Kirk, 2000Go; Nishii et al., 2003Go; Cheng et al., 2005Go; Kirk, 2005Go). Also, in colonial relatives of Volvox, a number of cell cleavages occur before hatching, increasing the cell numbers in individual organisms. Thus, heterochronic change in the timing of hatching is one prerequisite for the evolution of multicellularity in volvocine algae. Heterochronic mutants regarding the timing of hatching have been described for V. carteri (Huskey et al., 1979Go; Kirk, 1998Go). The timing of hatching is accelerated in premature-release mutants and retarded in delayed-release mutants, suggesting the existence of a genetic system that affects the timing of hatching.

Hatching in V. carteri is an event that degrades the ECM sheet of parental somatic cells to allow juveniles to be released. In a mature spheroid of V. carteri, ECM accounts for >99% of the total volume. Light and electron microscopy observations showed that ECM is a highly organized structure filling the space between neighboring cells and the internal portion of the spheroid (Kirk et al., 1986Go; Sumper and Hallmann, 1998Go). ECM is known to be mainly constituted of Hyp-rich glycoproteins, several of which have been unveiled in molecular detail (Sumper and Hallmann, 1998Go; Hallmann, 2003Go). Beneath the parental somatic ECM sheet by which gonidia and juvenile are protected, a series of embryonic developments, such as asymmetric cell division and inversion that do not occur in C. reinhardtii, take place until their liberation.

Approximately 36 h after the initiation of the cleavage of gonidia, V. carteri juveniles hatch from their parental spheroid by digesting the parental ECM (Jaenicke and Waffenschmidt, 1979Go; Kirk, 1998Go). Observation via light microscopy showed that portions of the parental somatic sheet, restricted just above each juvenile, collapse to create an opening (Kirk, 1998Go; Figure 1 ; see Supplemental Movie 1 online). Although hatching is one visible and conspicuous developmental process in the asexual life cycle of Volvox, few reports are available on this developmental stage. A 26-kD Ser protease was purified as a lytic enzyme from the culture medium after hatching, and the enzyme degraded the ECM of the parental somatic sheet to liberate juveniles in vitro (Jaenicke and Waffenschmidt, 1979Go, 1981Go); details of molecular properties, however, have not yet been reported. On the other hand, unicellular C. reinhardtii secretes a vegetative lytic enzyme (VLE) to digest the cell walls of mother cells, allowing daughter cells to be released after mitosis (Harris, 1989Go; Matsuda et al., 1995Go). Characterization of purified VLE showed that it is a Ser protease with a molecular mass of 130 kD on SDS-polyacrylamide gel. However, the amino acid sequence of VLE protein has also not yet been reported.


Figure 1
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  		Figure 1. Hatching of Juveniles from Parental Spheroid.

(A) to (C) Selected images taken during the hatching stage, showing a prehatched spheroid (A), liberation of a juvenile in progress ([B], arrow), and liberated juvenile leaving the parental spheroid (C). Complete animation is presented in Supplemental Movie 1 online in which images were taken every 8 s for 6.5 min and then compressed into a 19-s movie.

(D) A Coomassie blue–stained parental spheroid just after liberation of all juveniles, showing openings in the parental somatic sheet.

Bars = 0.1 mm.

 
Here, we focus on the molecular details of the Volvox hatching enzyme as a first step to elucidate the entire hatching process. We report the purification and characterization of a Volvox hatching enzyme (VheA) with a molecular mass of 125 kD from V. carteri. Sequence analysis revealed that vheA mRNA encodes a glycoprotein with homology to subtilisin Ser proteases and also with a single transmembrane segment at its NH2 terminus. The accumulation of vheA mRNA begins after inversion, and the accumulation level reaches maximal level during the hatching stage. Furthermore, while 125-kD VheA is released into a culture medium due to cleavage processing during hatching, a 143-kD form of VheA with prodomain is accumulated in juveniles before hatching.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
VheA Purification
During the hatching stage (Figure 1; see Supplemental Movie 1 online), an enzyme to degrade the ECM component of V. carteri is known to be secreted into the culture medium. The enzyme has been thought necessary for juveniles to hatch from their parental somatic sheet (Jaenicke and Waffenschmidt, 1979Go). However, to date, molecular characterization of the enzyme has not been performed. To investigate its molecular nature, first we purified it from a culture medium using ammonium sulfate precipitation, anion exchange chromatography on a DEAE Toyopearl 650M column (Figure 2A ) and a Source 15Q column (Figure 2B) and gel filtration on a Superdex 200HR 10/30 column (Figure 2C). While two major bands (125 and 61 kD) and a minor band (>200 kD) were detected in the active fraction after the Source 15Q column (Figure 2B), a single 125-kD protein was copurified with enzymatic activity when the fraction was further purified on Superdex 200HR 10/30 (Figure 2C). The final preparation was judged >98% pure by densitometric scanning of the stained SDS-polyacrylamide gel. We designated the enzyme VheA for Volvox hatching enzyme. The 125-kD VheA is only active to the parental somatic sheets, and the somatic sheet of juveniles liberated from the parental spheroid remains intact during the assay. Purified 125-kD VheA is most active to the ECM of the parental somatic sheet in the prehatching stage (35-h time point in the asexual life cycle shown in Figure 7B). When VheA preparation was applied to the spheroids in the 24-h stage (12 h before hatching), their susceptibility to the enzyme was lower than the spheroids in the prehatching stage (Table 1 ). The parental somatic sheets in this stage were degraded by VheA preparation regardless of heat-killed or intact spheroids, although the 26-kD protease described above was reported not to disintegrate living spheroids in vitro (Jaenicke and Waffenschmidt, 1979Go). Furthermore, VheA preparation did not work at all for 45 h spheroids, which are young adults after hatching (Table 1). In the natural hatching of V. carteri, only the portions of the parental somatic sheet just above the juveniles are degraded to make openings from which juveniles are liberated (Figure 1D). However, 125-kD VheA degraded the whole parental somatic sheet from the outside in the enzyme assay against prehatched spheroids, as is the case with the lytic enzyme reported by Jaenicke and Waffenschmidt (1979)Go. No preference to the roof of the juvenile compartment was observed even when serially diluted enzyme preparations were used in the assay.


Figure 2
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  		Figure 2. VheA Purification.

Pooled active fractions from DEAE Toyopearl 650M anion exchange chromatography (A) and Source 15Q anion exchange chromatography (B) and successive fractions from Superdex 200HR 10/30 gel filtration chromatography (C) were separated in a 7.5% SDS-polyacrylamide gel ([A] and [B]) or in a 4 to 20% gradient SDS-polyacrylamide gel (C) and then gels were silver stained. Numbers above the gel indicate fraction numbers, and numbers below represent ratio of released juveniles to total in the VheA assay.

 

Figure 7
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  		Figure 7. vheA mRNA Begins to Accumulate Long before Hatching Stage.

(A) Accumulation pattern of vheA mRNA detected by RNA gel blot hybridization using poly(A)+ RNA from various developmental stages. The probe used is shown in Figure 3. Probing for ribosomal protein S18 mRNA was performed as a loading control.

(B) Quantified signal intensities on blots were normalized using the signal of S18 mRNA as a loading control. Normalized values relative to that of the 36-h time point were plotted as a function of time during the asexual life cycle. Values are means ± SD of results from three independent experiments.

 

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  		Table 1. Degradation Activity against Spheroids at Various Developmental Stages

 
The effects of temperature and pH on the protease activity of the purified VheA were examined. VheA exhibited maximal enzymatic activity at 45°C and showed activity over a broad pH range between 7 and 11. Degrading activity for prehatch spheroids was inhibited in the presence of a metal ion chelator EDTA, suggesting that the enzymatic activity of VheA depended on divalent metal cations. To determine which divalent metal cations were required for enzymatic activity, the EDTA-inactivated protease was treated with various divalent metal ions (Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, or Zn2+). The enzymatic activity of VheA was restored by the addition of either calcium ion or, to a much lesser extent, magnesium ion.

Cloning of Full-Length cDNA
An amino acid sequence of 31 amino acid residues from the NH2 terminus of purified 125-kD VheA was determined by Edman degradation (VheA-N, Table 2 ). The 125-kD band on an SDS-polyacrylamide gel was excised and subjected to in-gel tryptic digestion followed by HPLC separation to determine the internal amino acid sequence. Using de novo tandem mass spectrometry (MS/MS) sequencing, amino acid sequences of three peptides were determined: VheA-C1, VheA-C2, and VheA-C3 (Table 2).


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  		Table 2. Determined NH2-Terminal and Internal Sequence

 
Two degenerate primers were designed based on amino acid sequences derived from the VheA-N (LSRAYWFNKI) and VheA-C1 (QPTSNFNVFR) fragments. RT-PCR with these degenerated primers resulted in the production of a 1.3-kb cDNA fragment (Figure 3 ). Nucleotide sequencing of the 1.3-kb fragment showed that this fragment contained the coding region for all the determined amino acid sequences. The 5'- and 3'-rapid amplification of cDNA ends (RACE) reactions resulted in the isolation of a full-length vheA cDNA of 6753 bp, including a 5'-untranslated region (UTR) of 190 bp, a coding region of 3381 bp, and a 3'-UTR of 3166 bp (Figure 4 ). vheA cDNA contained a putative polyadenylation signal (UGUAA) characteristic of V. carteri (Schmitt et al., 1992Go; Kirk, 1998Go) at 10 bp upstream from the poly(A) tail (Figure 4). In the deduced amino acid sequences of the coding region of vheA (Figure 4), the determined internal sequences of VheA-C1, VheA-C2, and VheA-C3 and the determined NH2-terminal sequence of VheA-N were found in positions 625 to 635, 535 to 550, 337 to 354, and 180 to 210, respectively. Also, we found that the vheA gene had several tandem repeat sequences in its 3'-UTR (GTAT repeat at nucleotide positions 3842 to 3881 and 4241 to 4381, CTGTGCGGCACACG repeat at 5458 to 5497, TCC repeat at 5503 to 5539, GAG repeat at 5666 to 5680, and TGTC repeat at 5830 to 5853) (Figure 4).


Figure 3
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  		Figure 3. Strategies for Cloning vheA cDNA.

Arrowheads mark locations and directions of primers. The coding region and UTRs are indicated by a closed box and lines, respectively. Thick lines indicate probes used for RNA gel blot (Northern) and DNA gel blot (Southern) analyses.

 

Figure 4
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  		Figure 4. Nucleotide Sequence and Deduced Amino Acid Sequence of vheA cDNA.

A cleavage processing site and stop codon are shown by an arrowhead and an asterisk, respectively. Putative N-glycosylation sites and tandem repeat sequences in 3'-UTR are indicated by open and shaded boxes, respectively. The putative polyadenylation signal UGUAA is underlined.

 
VheA Is a Subtilisin-Like Ser Protease with a Transmembrane Segment
The NH2-terminal amino acid sequence (VheA-N, Table 2) of purified 125-kD VheA was found from Asp-180 to Met-210 in the deduced amino acid sequence (Figure 4). This result suggested that Met-1 to Ser-179 was first synthesized as a prodomain. No typical NH2-terminal signal sequence was found in the deduced amino acid sequence (Bendtsen et al., 2004Go); however, a Kyte-Doolittle hydropathy plot (Figure 5A ) (Kyte and Doolittle, 1982Go) and a topological algorithm based on a hidden markov model (Krogh et al., 2001Go) suggested that a single obvious hydrophobic region (amino acid residues 41 to 63), probably a transmembrane segment, was present near the NH2-terminal end of the prodomain and that residues 1 to 40 were located on the cytoplasmic side of the membrane (Figure 5E). Therefore, the VheA precursor is classified as a type II integral membrane protein and is thought to be synthesized as a membrane-bound protein translocated to the cell surface.


Figure 5
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  		Figure 5. VheA Is a Type II Membrane Protein and Subtilisin-Like Ser Protease.

(A) Hydropathy plot of deduced amino acid sequence of VheA. The algorithm of Kyte and Doolittle (1982)Go was used with averaging over a window of nine residues. The hydrophobic region is shown by a bar.

(B) Purified 125-kD VheA was separated by SDS-PAGE and analyzed by the modified PAS method. The 125-kD VheA was detected as a glycoprotein under UV illumination (left) and then stained by Coomassie blue (right).

(C) Comparisons of the deduced amino acid sequence of the catalytic portion of VheA with Tg SUB1, thermitase, subtilisin Carlsberg, and hypothetical protein in C. reinhardtii genome scaffold_1. Protein sequences were aligned with a T-COFFEE algorithm (Notredame et al., 2000Go). Identically conserved amino acid residues, highly conserved amino acid residue, and weakly conserved amino acid residues are indicated by asterisks, colons, and periods, respectively. The catalytic triad of His, Asp, and Ser is shaded.

(D) Amino acid sequence alignment of calcium binding sites of thermitase with VheA using the Blosum62 algorithm (Henikoff and Henikoff, 1992Go). Strong and medium-strength calcium binding sites of thermitase are shown by filled and shaded boxes, respectively.

(E) Domain structure of VheA. Relative position of transmembrane segment, prodomain, subtilisin-like Ser protease domain, functionally unknown domain, and putative N-glycosylation sites (represented by Y) are indicated.

 
A homology search of the GenBank protein database for the deduced amino acid sequence revealed that residues 180 to 537 of VheA identified 180 hits with E-values <1E–5. All of these proteins are subtilisins and subtilisin-like Ser proteases from Eukaryota, Bacteria, and Archaea, including the three most similar annotated proteins: Tg SUB1, a subtilisin-like Ser protease from Toxoplasma gondii (29% identity) (Miller et al., 2001Go), thermitase, a subtilisin-type Ser protease from Thermoactinomyces vulgaris (28% identity) (Teplyakov et al., 1990Go), and subtilisin Carlsberg, one of the most characterized enzymes in the subtilisin family from Bacillus licheniformis (25% identity) (Jacobs et al., 1985Go). Homology with Ser proteases suggests that VheA allows juveniles to be released by digesting the protein(s) in the ECM of parental spheroids. As shown in Figure 5C, sequence alignment of these proteases indicated that VheA contains a catalytic triad consisting of three amino acid residues (His-303, Asp-226, and Ser-502) conserved in these Ser proteases (Siezen and Leunissen, 1997Go). By contrast, the COOH terminal of VheA had no homology to any proteins in the current GenBank protein database. This COOH-terminal functionally unknown domain (amino acid residues 538 to 1127) accounts for 52% of the deduced amino acid sequence of VheA (Figure 5E).

Calcium ions are known to bind to three calcium binding sites of thermitase to increase protein stability (Frommel and Hohne, 1981Go; Teplyakov et al., 1990Go; Gros et al., 1991Go). Amino acid alignment of VheA and thermitase showed that strong and medium-strength calcium binding sites of thermitase were well conserved in VheA (Figure 5D). Of the six amino acid residues that constitute strong calcium binding sites in thermitase, four amino acid residues were conserved in VheA. In addition, all amino acid residues in the medium-strength calcium binding site were conserved as well (Figure 5D).

A TBLASTN search of the sequenced genome of C. reinhardtii v3.0 at the Joint Genome Institute (JGI) (http://genome.jgi-psf.org/cgi-bin/runAlignment?db=Chlre3&advanced=1) with an E-value cutoff of 1E–10 identified a hypothetical gene in scaffold_1 with significant similarity to VheA. The retrieved sequence was analyzed using the gene-finding algorithm Genscan to predict cDNA sequences (Burge and Karlin, 1997Go). The protein sequence predicted from the cDNA sequence was homologous to VheA throughout the three domains and showed 50.0% identity and 64.8% similarity to VheA as a whole. The conserved catalytic triad of the Ser protease domain in the identified C. reinhardtii gene is shown in Figure 5C.

The vheA gene contained 24 putative N-glycosylation sites (Asn-X-Ser/Thr, where X is not Pro) in both the subtilisin-like Ser protease domain and the functionally unknown domain, but the prodomain, including the transmembrane segment, contained no putative N-glycosylation site (Figures 4 and 5E). To determine whether VheA was a glycoprotein, we used the modified periodic acid–Schiff (PAS) method (Zacharius et al., 1969Go), which was designed to selectively stain glycoproteins using a fluorescent probe. Purified 125-kD VheA was efficiently detected under UV irradiation (Figure 5B), indicating that VheA is a glycoprotein.

vheA Is a Single-Copy Gene
Genomic DNA gel blot analysis was performed to determine the copy number of the vheA genes in the V. carteri genome. As shown in Figure 6 , the observed hybridization patterns showed that digestion of genomic DNA with HindIII, PstI, or BglII gave single bands. When the blot was rehybridized with another probe corresponding to a nucleotide position at 1 to 842 of the vheA cDNA, digestion of genomic DNA with HindIII, PstI, or BglII also resulted in single bands. The lack of additional hybridizing fragments in these digestions indicates that vheA is encoded by a single-copy gene. To further confirm this result, we used BLASTN to compare the vheA cDNA sequence to the ~454,000 V. carteri genomic sequence reads in the JGI database (http://genome.jgi-psf.org/cgi-bin/runAlignment?db=Chlre3&advanced=1) with an E-value cutoff of 1E–5 and identified 13 records with E-values of 0.0 to 5E–25. All identified records have >95% identity with vheA cDNA. These records would correspond to vheA genomic sequence, since most nonidentical nucleotides are located near the end of the shotgun sequence reads; therefore, these nonidentities seem to be caused by errors in the shotgun sequencing. Thus, the fact that other vheA-related sequences were not detected in the V. carteri genomic sequence reads strongly supports the notion that vheA is a single-copy gene.


Figure 6
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  		Figure 6. vheA Is a Single-Copy Gene.

Genomic DNA of V. carteri was digested with HindIII (H), PstI (P), or BglII (B) and hybridized with the vheA probe shown in Figure 3.

 
vheA mRNA Maximally Accumulates around Hatching Stage
Poly(A)+ RNAs isolated from spheroids at various developmental stages of the asexual life cycle were subjected to RNA gel blot analysis to investigate the accumulation level of vheA mRNA during the life cycle. vheA RNA levels in the RNA gel blot were quantified and normalized to levels of ribosomal protein S18 mRNA, which is highly expressed throughout its developmental cycle regardless of the cell type and has been used as an internal control in past studies (Kirk et al., 1999Go; Shimizu et al., 2002Go). As shown in Figure 7A, a single mRNA species of ~7.0 kb in length was detected in accordance with the length of the obtained full-length cDNA (6.75 kb). The accumulation level of vheA mRNA was very low at the inversion stage (10 h), gradually rising during cytodifferentiation and expansion and then reaching maximal level during hatching (35 h). Thereafter, it declined to a lower level during maturation and embryogenesis of the gonidia in the daughter spheroids (Figure 7B).

Expression of vheA Is Restricted to Juvenile Cells
To examine the cell-type specificity of the expression of vheA mRNA, we performed semiquantitative RT-PCR on the total RNA isolated from juveniles, parental somatic cells, juvenile somatic cells, or juvenile gonidia (Figure 8C ). As shown in Figure 8A, vheA transcript was detected in juveniles but not in parental somatic cells. Thus, vheA is expressed in juveniles but not in parental somatic cells. When gonidia and somatic cells of just-hatched juveniles were separated, vheA transcript was detected in both the gonidia and the somatic cells of the juvenile (Figure 8B).


Figure 8
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  		Figure 8. Expression of vheA Is Specific to Juveniles.

(A) Semiquantitative RT-PCR analysis of vheA and S18 expression in parental somatic cells (P) and juveniles (J). Numbers above gels indicate PCR cycles. Minus-RT controls showed no genomic DNA contamination in original RNA samples.

(B) Semiquantitative RT-PCR analysis of vheA and S18 expression in juvenile somatic cells (S) and juvenile gonidia (G).

(C) Schematic representation of cell types in a prehatched spheroid.

 
VheA Is Synthesized with Transmembrane Domain before Hatching and Then Released Outside by Cleavage Processing during Hatching
As mentioned above, VheA deduced from the cDNA sequence contained a single transmembrane segment at the NH2 terminus (Figure 4), while VheA purified from culture medium did not. To examine whether vheA mRNA was initially translated into a precursor VheA with the transmembrane domain, and then 125-kD VheA was released by cleavage at the site between the prodomain and the subtilisin-like Ser protease domain, protein gel blot analysis was performed using cell lysates extracted before hatching and the culture medium collected after hatching. Although the anti-VheA antibody detected a single band of 125-kD VheA in the culture medium collected after hatching, VheA extracted from spheroids before hatching had a molecular mass of 143 kD on the blot (Figure 9A ). This 18-kD difference indicates that the vheA gene is primarily synthesized as a precursor with prodomain containing a single transmembrane segment and then VheA is released into the culture medium after cleavage processing.


Figure 9
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  		Figure 9. 143-kD VheA with Transmembrane Segment Is Synthesized Long before Hatching Stage and Then 125-kD VheA Is Released into Culture Medium Due to Cleavage Processing.

(A) Immunoblot analysis of cellular extracts before hatching and culture medium after hatching with anti-VheA antibody.

(B) Immunoblot analysis of cellular extracts at various developmental stages. Numbers above blot indicate time points when samples were prepared. Cellular extracts before hatching (0 to 35 h) were prepared from intact spheroids, although those after hatching (36 to 42 h) were prepared from a mixture of hatched juveniles and parental somatic cells. As an internal control, actin was detected (bottom panel).

(C) Immunoblot analysis of culture medium samples collected at various developmental stages.

 
To further analyze the accumulation and release patterns of VheA during asexual development, cell extracts and culture mediums were collected at various stages. Although 143-kD VheA was detected throughout the life cycle, its accumulation levels were slightly lower and higher around inversion and hatching stages, respectively (Figure 9B). As shown in Figure 9C, the release of 125-kD protein outside the spheroids started at 36 h, and the accumulation level reached a plateau at 38 h. Note here that while juveniles had not hatched yet at 35 h, all had already hatched at 36 h in the developmental cycle under our culture conditions. Thus, the timing of the release of 125-kD VheA exactly corresponds to hatching.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
Enzymatic Activity of 125-kD VheA for the ECM of the Parental Somatic Sheet
From the culture medium after hatching, we purified a 125-kD protein with degrading activity for the ECM of the parental somatic sheet in vitro. The enzyme is more active to the ECM of the parental somatic sheet in the prehatching stage than in the 24-h stage (Table 1). In addition, it cannot work at all for the young hatched adult spheroids in the 45-h stage. Several possibilities may explain these results. One differential susceptibility to VheA is controlled by an inhibitor or activator for VheA (Jaenicke and Waffenschmidt, 1981Go; Kirk, 1998Go). In the case of control by inhibitor, immature spheroids in the young adult and postinversion stages are protected by an unidentified inhibitor against VheA that is degraded toward the hatching stage. Alternatively, an unidentified activator for VheA is synthesized around the hatching stage. Another possibility is that the substrate(s) of VheA is synthesized toward the hatching stage, becoming an integral component of the ECM of parental spheroids. It is known that ECM is not static during the asexual life cycle of V. carteri and that young spheroids expand by depositing ECM components during the expansion stage (Sumper and Hallmann, 1998Go). Therefore, it is possible that a substrate(s) for VheA is synthesized and incorporated into the ECM of a parental somatic sheet during the expansion stage as an integral component(s) of ECM. Thus, when the substrate(s) in the ECM is degraded by VheA during the hatching stage, other ECM components are also dissociated from ECM, resulting in the breakdown of ECM.

VheA exhibits calcium-dependent activity over a broad range of pH between 7 and 11, with an optimum temperature of 45°C. Although the enzymatic property resembles the VLE of C. reinhardtii (Matsuda et al., 1995Go), the temperature optimum of VheA is higher than VLE, whose temperature optimum is 35°C. As described above, VheA is a subtilisin-like Ser protease, and Ser proteases, including subtilisins, are known to be active at highly alkaline pH and higher optimal temperatures than general enzymes (Rao et al., 1998Go). These properties are consistent with our results.

The molecular mass of VheA, 125-kD, is not coincident with hatching-lysin, 26-kD, which was purified as a hatching enzyme of V. carteri (Jaenicke and Waffenschmidt, 1979Go). Although the discrepancy is difficult to explain since the amino acid sequence of 26-kD protein has not been reported, one possibility is that the 26-kD protein is a degradation product of VheA. It appears that this possibility is very low since 125-kD VheA is quite stable after release into the culture medium and no degradation product is detected on the SDS-polyacrylamide gel. Another possibility is that the preparation of 26-kD protein contained a faint amount of 125-kD VheA undetected by the staining method. In fact, a low concentration of purified VheA undetectable even by silver staining exhibited degrading activity for ECM of a parental somatic sheet (data not shown). Also, note that the molecular mass of C. reinhardtii hatching enzyme VLE has been reported to be 130 kD (Matsuda et al., 1995Go). Although a direct comparison between VLE and VheA is not possible since the amino acid sequence of VLE has not been reported yet, the Chlamydomonas hatching enzyme, which plays a homologous role with the Volvox hatching enzyme, has close molecular mass. This may support the notion that the hatching enzyme of V. carteri is not a 26-kD but a 125-kD protein.

Domain Structure of VheA
Homology searching with deduced amino acid sequence indicates that 125-kD VheA is comprised of a subtilisin-like Ser protease domain and a functionally unknown domain (Figure 5E). Subtilisin is one of the largest families of Ser proteases. Amino acid alignment analysis suggests that VheA has an active triad comprised of Ser, His, and Asp (Figure 5C), which are conserved in Ser proteases. It is known that calcium ions bind to thermitase of T. vulgaris and subtilisins to stabilize their structure against thermal denaturation and proteolytic degradation (Voordouw et al., 1976Go; Frommel and Hohne, 1981Go; Gros et al., 1991Go). Amino acid alignment to thermitase shows that VheA has two putative calcium binding sites corresponding to thermitase (Figure 5D) but no calcium binding sites corresponding to subtilisins, suggesting that calcium ions are likely to stabilize VheA structure in the same way as thermitase.

The functionally unknown region is a large domain accounting for 52% of the translated VheA (Figure 5E). Nevertheless, the domain does not show homology to any other proteins in databases. Further analysis is required to determine whether this domain plays any role in the hatching process and enzymatic activity.

A search of the sequenced genome of C. reinhardtii at JGI identified a hypothetical protein with significant similarity to VheA. The predicted amino acid sequence of the identified protein has similarity with VheA throughout the whole amino acid sequence, and overall identity and similarity are 50.0 and 64.8%, respectively. Since no other genes with E-values <1E–10 are found in the genomic sequence of C. reinhardtii, it is likely that the identified gene in C. reinhardtii is an ortholog of the vheA gene. Thus, it seems that V. carteri uses an enzyme that was present in a unicellular ancestor(s) for the hatching process of multicellular juveniles.

Cleavage Processing of VheA
Change in molecular mass from the 143-kD VheA extracted from spheroids to the 125-kD VheA collected from culture medium (Figure 9A) suggests that membrane-bound VheA is processed and released into the culture medium as 125 kD. The 143-kD VheA extracted from spheroids is almost consistent with the expected molecular mass of 145 kD calculated from the apparent molecular mass of the purified VheA (125 kD) on the SDS-polyacrylamide gel and the theoretical molecular mass of the prodomain (20 kD).

The NH2-terminal amino acid sequence of the processing site of VheA (QTSS/DVGL, Figure 4) resembles TgSUB1 (NTSS/KGSN) (Miller et al., 2001Go). The cleavage processing site of VheA (46 residues upstream of the catalytic Asp) corresponds to an appropriate location for autocatalytic cleavage sites of subtilisins (approximate range is between 30 and 50 residues upstream of the catalytic Asp) (Siezen and Leunissen, 1997Go). As described above, VheA appears to be released into the culture medium following processing. This mode of synthesis resembles Bacillus amyloliquefaciens subtilisin (Power et al., 1986Go), which is synthesized as a membrane-bound precursor and released outside the cell after autocatalytic cleavage.

Many, though not all, prosequences of secreted proteases, including Ser, Cys, aspartyl, and metalloproteases, act as intramolecular chaperones and are essential for folding into the active enzyme (Silen and Agard, 1989Go; Smith and Gottesman, 1989Go; Zhu et al., 1989Go; van den Hazel et al., 1993Go; Marie-Claire et al., 1999Go). Upon completion of folding into the active form, autoproteolytic cleavage of the covalent linkage between the prosequence domain and the mature enzyme occurs to remove their prosequences. By contrast, prosequences in human matrix metalloproteinases are known to maintain them in an inactive state instead of acting as intramolecular chaperones (Van Wart and Birkedal-Hansen, 1990Go; Morgunova et al., 1999Go). The prodomain of VheA does not show any homology to the prosequences of the described proteases, including subtilisin. Since the amino acid sequence of prosequences among homologous proteases is less conserved than protease domains (Shinde and Inouye, 2000Go), it is difficult to predict the role of the prosequence from the amino acid sequence in the function of VheA. Further studies are needed to determine the role of the cleavage processing of VheA in the function of VheA.

Expression Profile of VheA
RNA gel blot analysis shows that the accumulation level of vheA starts to increase at the end of the inversion stage, continues increasing for almost a day, and then reaches maximum at the hatching stage; after the liberation of juveniles, it declines (Figure 7B). As shown in Figure 9B, 143-kD VheA with prodomain accumulates throughout the life cycle. However, protein gel blot analysis of the collected culture medium shows that 125-kD VheA starts to be released into the culture medium at 36 h and is detected from 36 to 42 h on the blot (Figure 9C). Considering the observation that no juvenile hatched until 35 h and liberation was completed within 1 h after 35 h, the release time of 125-kD VheA corresponds exactly to the liberation time of juveniles from parental spheroids. As shown in Figure 8, vheA is expressed only in the somatic cells and the gonidia of juveniles but not in parental somatic cells. Thus, it seems that concentration of active VheA becomes high on the juvenile surface during the natural hatching, resulting in the degradation of the roof of the juvenile compartment that allows juveniles to be released from parental spheroids.

Protein gel blot analysis (Figure 9B) shows that 143-kD VheA is synthesized long before hatching. Early expression of hatching enzyme genes has also been reported for medaka (Yasumasu et al., 1992bGo), sea urchin (Lepage and Gache, 1990Go), masu salmon (Araki et al., 1996Go), and crayfish (Geier and Zwilling, 1998Go), whose developing embryos degrade their extraembryonic matrix to allow the embryo to be hatched. In medaka and sea urchin, hatching enzymes are synthesized long before hatching and are stored in secretory granules in the hatching gland cells of the medaka embryo (Yasumasu et al., 1992aGo) and probably in the vesicles of sea urchin embryo cells (Lepage et al., 1992Go) until secretion. Although a direct comparison between hatching from eggs in metazoa and from the parental body in protozoa is not feasible, it is interesting that the hatching enzymes in both systems are accumulated far before the actual hatching event. Future analysis, including subcellular localization of membrane-bound VheA, would be informative about the regulation of hatching in Volvox until the appropriate stage.

Finally, since hatching is one important aspect of multicellularity in volvocine algae, and the controlling mechanism of the timing of the hatching event is inferred to exist from heterochronic mutants, further investigation about hatching in volvocine algae will deepen our understanding of the evolution of multicellularity.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
Strain and Cultivation Conditions
Volvox carteri forma nagariensis (female strain HK10) was obtained from D.L. Kirk (Washington University). Synchronous cultures were maintained in modified standard Volvox medium (SVM) at 30°C under a 16-h-light/8-h-dark (10,000 lux) cycle (Kirk and Kirk, 1983Go; Aono et al., 2005Go).

Assay for VheA Activity
Enzymatic activity was assayed by a modification of the method described by Jaenicke and Waffenschmidt (1979)Go. Prehatched parental spheroids were killed by heating at 55°C for 10 min unless otherwise described. Spheroids were added to the VheA preparation dialyzed against VheA buffer (1.678 mM piperazine, 0.5 mM calcium nitrate, and 0.162 mM magnesium sulfate, pH 9.5) and then incubated at 30°C. Incubation time was for 1 h unless otherwise described. Counting the liberated juveniles and total juveniles gave the ratio (%) of the released juveniles as an index of the enzymatic activity of VheA.

VheA Purification and Glycoprotein Detection
Ten hours before hatching, synchronized spheroids (15 liters) were settled and transferred to a flask containing 1.5 liters of SVM using glass pipettes with wide openings. Three hours after hatching, the culture medium was filtrated through a 30-µm-mesh nylon membrane followed by centrifugation (18,500g, 15 min). The proteins precipitated with a 40 to 70% saturation of ammonium sulfate (shifting molarity method) were dissolved in the VheA buffer and then dialyzed against 3 liters of VheA buffer in dialysis membrane (Spectra/Por MWCO 50,000; Spectrum Laboratories) (Coligan et al., 1995Go). The sample was loaded onto a DEAE Toyopearl 650M column (1.5 x 46 cm; Tosoh). Bound proteins were eluted with VheA buffer containing 100 mM NaCl. The active fraction was pooled and dialyzed against the VheA buffer. After applying the dialysate onto a Source 15Q column (Amersham Biosciences), bound proteins were eluted with the VheA buffer containing 50 mM NaCl. The active fraction was loaded onto a Superdex 200HR 10/30 column (Amersham Biosciences). Preequilibration and elution of columns (Source 15Q and Superdex 200HR 10/30) were performed at a flow rate of 1.0 and 0.5 mL/min, respectively, using AKTA explorer 10S (Amersham Biosciences). All purification steps were performed at 4°C.

Glycosylated protein in 7.5% SDS-polyacrylamide gel was detected using the GlycoProfile III fluorescent glycoprotein detection kit (Sigma-Aldrich) according to the manufacturer's instructions.

Temperature and pH Optimum and Effect of Divalent Cations
Temperature optimum was determined using a QuantiCleave protease assay kit (Pierce) and a VheA buffer as an assay buffer. The pH optimum was determined using heat-killed spheroids as substrates. The buffers used in this experiment were 1.678 mM Bis-Tris, pH 6 to 7, 1.678 mM Tris-HCl, pH 8, or 1.678 mM piperazine, pH 9 to 12, each containing 0.5 mM calcium nitrate and 0.162 mM magnesium sulfate. Purified sample with a Source 15Q was dialyzed against each assay buffer and then used in the assay. In the assay of metal ions, aliquots of the sample pretreated with 0.4 mM EDTA were incubated for 4 h with 0.5 mM metal ion (Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, or Zn2+).

Amino Acid Sequencing
N-terminal protein sequence analysis by automated Edman degradation was done by APRO Life Science of Japan. Internal sequence analysis by MS/MS was performed by ProPhoenix of Japan.

RNA Isolation and cDNA Cloning
Total RNA was isolated as described by Kirk and Kirk (1985)Go. Poly(A)+ RNA was purified using Oligotex-dT30 Super beads (Takara). Reverse transcription was performed using Moloney murine leukemia virus reverse transcriptase (RNaseH–) (ReverTra Ace; Toyobo). PCR amplification with degenerate primers (HE-N2-U, 5'-YTIWSIMGIGCITAYTGGTTYAAYAARAT-3', and HE-C2-R, 5'-CGRAAIACRTTRAARTTISWIGTIGGYTG-3') was performed with conditions consisting of 94°C for 2 min, followed by 45 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 3 min. The 5'- and 3'-RACE was performed using the 5'-RACE system Version 2.0 (Invitrogen) and FirstChoice RLM-RACE (Ambion) according to the manufacturers' instructions. The following oligonucleotides were used for RACE: 5'RACE (5'-TCAATTGCTGCTGCGGATTCGAC-3'), 3'RACE4 (5'-CGAACCGTCGTAAAACGAAC-3'), and 3'RACE12 (5'-GCTGGTAGCCCTGTTCTGAC-3').

Separation of Cell Types, Their RNA Isolation, and Semiquantitative RT-PCR
Parental somatic sheets and juveniles were individually collected just after hatching using a Pasteur pipette under a microscope. Somatic cells and gonidia of juveniles were separated using a 5-µm-mesh nylon screen as described by Hallmann et al. (2001)Go. By microscopic inspection, it was confirmed that these cell preparations were not cross-contaminated by other cell types. RNA was purified from each cell type as described (Hallmann and Sumper, 1994Go), followed by treatment with DNase I (Cloned DNase I; Takara). The total RNA was reverse-transcribed using ReverTra Ace (Toyobo) in 20 µL of reaction buffer according to the manufacturer's instructions. One microliter of cDNA was used as a template for semiquantitative RT-PCR in 50 µL of reaction buffer (Aono et al., 2005Go). Separate reaction tubes were used for the PCR reactions of different cycle numbers. PCR cycles for vheA were 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. PCR cycles for S18 were 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Following the PCR reactions, 5-µL aliquots were analyzed on agarose gels followed by staining with ethidium bromide. The following oligonucleotides were used: HE-fraC-for (5'-ATGGTGACCACGCCTAACGTG-3'), HE-fraC-rev (5'-TCCGAACATAGAAGTCCACGAAATTG-3'), S18f (5'-ATGGGCTCTCTGGTCCACGGC-3'), and S18r (5'-CGGATCTTCTTCAGGCGCTCC-3').

DNA Gel Blot Hybridization
V. carteri genomic DNA was purified as described by Mages et al. (1988)Go. Digests (3 µg) with restriction enzymes separated in 0.8% agarose gel electrophoresis were transferred onto a Hybond-N membrane (Amersham Biosciences) (Sambrook et al., 1989Go). The membrane was probed with a 685-bp fragment corresponding to nucleotide positions 2376 to 3060 of vheA cDNA. The probe was labeled with alkaline phosphatase (AlkPhos Direct; Amersham Biosciences), and hybridization was performed according to the manufacturer's instructions. The signals were detected using CDP-Star chemiluminescent reagent (Amersham Biosciences).

RNA Gel Blot Hybridization
The poly(A)+ RNA (3 µg) was electrophoresed in a 1% agarose gel containing formaldehyde. RNA was transferred to a Hybond-N+ membrane (Amersham Biosciences) (Sambrook et al., 1989Go). The probe corresponding to nucleotide positions 1 to 842 of vheA cDNA was used for hybridization. Labeling and detection were performed as described above.

Antibodies and Protein Gel Blotting
Polyclonal antibody was raised against two synthetic peptides corresponding to amino acid residues 180 to 193 and 602 to 615 of VheA (Operon Biotechnologies). The antibody was affinity purified using peptides immobilized on solid support.

For protein gel blotting, spheroids were harvested by filtration on a 30-µm-mesh nylon screen and then washed with SVM. The spheroid preparations before hatching (0 to 35 h) contained only intact spheroids, while those after hatching (36 to 42 h) contained hatched juveniles and parental somatic cells. Proteins in culture medium were concentrated using microcon YM-50 (Millipore). An equal volume of 2x SDS sample buffer (4% SDS, 12% 2-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, and 100 mM Tris-HCl, pH 6.8) was added to the collected samples and then boiled for 3 min. After centrifugation at 15,000g for 2 min, supernatant was collected and filtrated through a 0.45-µm filter. Extracts (0.8-µg protein) and proteins from culture medium concentrated from 6 mL of the medium were separated on a 7.5% SDS-polyacrylamide gel. After being transferred to a Hybond-P membrane (Amersham Biosciences), nonspecific binding sites on the membranes were blocked with TBS containing 0.1% Tween 20 and 5% nonfat dry milk (TBSTM) and then incubated with rabbit anti-actin antibody (Sigma-Aldrich) or rabbit anti-VheA polyclonal antibody (1:20,000 dilution in TBSTM) (Harlow and Lane, 1988Go). Immunoreactivity was visualized with alkaline phosphatase–conjugated anti-rabbit IgG secondary antibody (1:20,000 dilution in TBSTM; Promega) and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium.

Accession Numbers
Sequence data of vheA cDNA can be found in the GenBank/EMBL data libraries under accession number AB242141. Other sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers AY043483 for TgSUB1 and X03341 for subtilisin Carlsberg and in the Protein Data Bank data library under identification number 1THM for thermitase.

Supplemental Data
The following material is available in the online version of this article.

Supplemental Movie 1. Time-Lapse Series of Images during Hatching Stage.


   Acknowledgments
 
We thank David L. Kirk (Washington University, St. Louis, MO) for giving us the Volvox strain and Naoki Aono (National Institute for Basic Biology, Okazaki, Japan) for helpful advice and comments. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Takeda Science Foundation.


   Footnotes
 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hideaki Shiraishi (siraisi{at}kuchem.kyoto-u.ac.jp).

[W] Online version contains Web-only data. Back

www.plantcell.org/cgi/doi/10.1105/tpc.106.041343

Received January 22, 2006; Revision received August 2, 2006. accepted September 18, 2006.


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