The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall*

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyprolinecontaining glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyprolinecontaining polypeptides of the insoluble wall fraction. Matrixassisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64-and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.
The extensin subgroup of the HRGP family includes proteins that are uniformly fibrous and devoted to meshwork formation, but several members of this particular subgroup, called chimeric extensins (8), contain both fibrous and globular domains, the globular portions presumably playing some additional role. These include some solanaceous lectins, in which the hydroxyproline-rich fibrous domains are assumed to anchor the protein to the wall and the globular domains are assumed to mediate sugar binding activity (1,2). Chimeric HRGPs are also expressed in reproductive tissues of higher plants (9 -13).
In the case of the volvocine green algae Chlamydomonas and Volvox, the vegetative and zygotic cell walls do not contain cellulose or other polysaccharides but consist exclusively of HRGPs (14,15). A multilayered architecture was observed for the walls of vegetative Chlamydomonas reinhardtii cells as revealed by electron microscopic studies (16 -19). In addition to an "inner wall" of covalently cross-linked HRGPs reminiscent of the higher plant meshworks (19), they posses an "outer wall" of HRGPs that co-assemble into crystalline arrays. The arrays can be solubilized by treatment with aqueous solutions of chaotropic salts, and they reassemble when the chaotropic salts are removed by dialysis (19 -24). Overall, the cell wall of vegetative C. reinhardtii cells is a multilayered extracellular matrix consisting of an insoluble, hydroxyproline-rich glycoprotein framework (17,25) and ϳ20 chaotrope-soluble, hydroxyproline-containing polypeptides (25). Formation of gametes and zygotes results in the activation of genes encoding additional hydroxyproline-containing cell wall polypeptides (26 -30). Three major constituents of this chaotrope-soluble cell wall fraction (GP1, GP2, and GP3) have been proved to be chimeric HRGPs (21). GP1 is a monomeric glycoprotein with a molecular mass of 272,359 Da and a carbohydrate content of 76% (31). Its polypeptide backbone (65,129 Da) (31) contains 32.3 mol % hydroxyproline (21), which corresponds to the hydroxyproline content of the insoluble cell wall fraction (32). The amino acid sequence of GP1 derived from the nucleotide sequence of the gp1 gene (31) consists of four domains. The (hydroxy)proline-rich sequences are located in domain 2, which contains 49 repeats of the PPSPX motif, and domain 3, which consists of PS repeats (31). Both domains form the 100-nm long fibrous structure of this molecule (21,31), whereas the globular head (21) can be attributed to the C-terminal domain (domain 4). MALDI-TOF analyses of native GP2 and chemically deglycosylated GP2 revealed molecular masses of 281,250 and 142,600 Da, respectively, indicating a carbohydrate content of 49% (33). A putative partial amino acid sequence derived from the nucleotide sequence of a cloned gene fragment was published by Adair and Apt (34). GP3 is a disulfide-linked heterodimer (21,33). MALDI-TOF analyses of GP3A and GP3B revealed molecular masses of 131,500 and 144,400 Da, respectively (33). After chemical deglycosylation, molecular masses of 70,550 and 102,290 Da were found for GP3A and GP3B (33), respectively, indicating that the carbohydrate content of GP3A is considerably higher than that of GP3B.
The insoluble wall fraction can be isolated from intact cells by successive extractions with different detergent-containing buffers (32). After chemical deglycosylation with hydrofluoride (HF)/pyridine, the "sac-like" morphology of the highly purified insoluble wall component is destroyed, and at least part of its polypeptide constituents become soluble in SDS-containing buffers (32). Isodityrosine has been detected in hydrolysates of Chlamydomonas cell walls (6), indicating that peroxidase-catalyzed cross-linking of cell wall glycoproteins via tyrosine side chains, which has been observed for the extensins of higher plants (35)(36)(37), also occurs in Chlamydomonas. Furthermore, it has been reported that a transglutaminase also catalyzes cross-linking of cell wall proteins in C. reinhardtii (7). However, both of these cross-links are not sensitive to HF treatment. Therefore, the observations that the sac-like morphology of the insoluble cell wall fraction is destroyed during chemical deglycosylation and that defined polypeptide constituents are released during this process indicate the existence of additional cross-links that are sensitive to HF treatment.
It has been reported recently that a 14-3-3 protein is a minor constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall (38). This particular 14-3-3 isoform was also shown to preferentially interact with the endoplasmic reticulum membranes of C. reinhardtii (39,40). Analyses of the amino acid composition of the insoluble fraction of the Chlamydomonas cell wall revealed a hydroxyproline content of Ͼ30% (32). Its HRGP constituents are, however, not yet characterized. Previous pulse-labeling and pulse-chase experiments and immunochemical studies indicated that the chaotrope-soluble cell wall fraction contains soluble precursors of the insoluble Chlamydomonas wall fraction (38,41,42). In this study, we provide evidence that the chaotrope-soluble HRGP GP2 is such a precursor of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

EXPERIMENTAL PROCEDURES
Strains and Growth Conditions-The C. reinhardtii Dangeard wild-type strain 137C (mating type ϩ) was obtained from Dr. R. P. Levine (Harvard University, Cambridge, MA). Cells were grown at 21°C and 20,000 lux in high salt medium supplemented with 0.2% (w/v) sodium acetate as described previously (43). Cell densities were determined by duplicate hemocytometer counting. Cells were harvested by centrifugation at 6000 ϫ g for 10 min at 4°C and washed twice with fresh culture medium.
Extraction of Chaotrope-soluble Cell Wall Components-Saltsoluble cell wall components were extracted with aqueous LiCl (3 M) from intact cells as described previously (44). Cells were harvested by centrifugation at 6000 ϫ g for 10 min and resuspended in fresh culture medium to a final cell density of 2 ϫ 10 8 cells/ml. After the addition of the same volume of aqueous LiCl (6 M), the suspensions were incubated at 0°C for 3 h and subsequently centrifuged at 20,000 ϫ g for 30 min. The supernatants were stored at Ϫ20°C until used.
Isolation of the Insoluble Cell Wall Component-The insoluble cell wall component of C. reinhardtii was isolated from the LiCl-extracted intact wild-type cells by treatment with different detergent-containing buffers. The LiCl-extracted wild-type cells (corresponding to 2 g, fresh weight) were twice washed with distilled water and resuspended in 200 ml of phosphatebuffered saline (PBS) containing 2% (w/v) SDS and 200 mM 2-mercaptoethanol. After 30 min at room temperature, the suspension was centrifuged for 30 min at 20,000 ϫ g. The insoluble wall fraction was isolated from the supernatant by filtration using Stericup-VP filter units (250 ml) with 0.1-m Express (polyethersulfone) membranes (Millipore Corp., Bedford, MA). The filters were washed twice with 50 ml of 1% SDS in PBS, twice with 50 ml of PBS containing 1% (v/v) Triton X-100, and finally with 50 ml of distilled water. The filters were than cut into small pieces, vortexed in 5 ml of PBS, and shaken overnight to resuspend the insoluble cell wall material.
Purification of Chaotrope-soluble Cell Wall Polypeptides-The high molecular mass, chaotrope-soluble polypeptide fraction of the Chlamydomonas cell wall was isolated from the LiCl extracts of intact wild-type cells by hydrophobic chromatography using phenyl-Sepharose as a matrix and further purified by gel exclusion chromatography on a Sephacryl S-300 column. The fractions of the first peak of the Sephacryl S-300 column containing the high molecular mass cell wall glycoproteins were combined, desalted, and freeze-dried. The freeze-dried material was used as the starting material for chemical deglycosylation. The individual glycosylated or chemically deglycosylated cell wall polypeptides were isolated by preparative SDS-PAGE according to Laemmli (45) on gel slabs containing 7% (w/v) acrylamide.
Chemical Deglycosylation-Deglycosylation of the cell wall glycoproteins was performed by treatment with trimethylsilyltrifluoromethane sulfonate in anhydrous trifluoroacetic acid containing thioanisole. The reagent was prepared by mixing 2 g of trimethylsilyltrifluoromethane sulfonate with 6.5 ml of anhydrous trifluoroacetic acid and 0.5 ml of thioanisole, cooled to 0°C, and added to the freeze-dried cell wall glycoproteins (1 ml of reagent/10 mg of protein). After 5 h at 0°C, the reaction was quenched by the addition of 1 ml of distilled water and 8 ml of ice-cold acetone (100%). The precipitate was collected by centrifugation at 20,000 ϫ g for 30 min at 4°C. The supernatant was discarded, and the precipitate was washed twice with 80% (v/v) aqueous acetone and subsequently vacuum-evaporated until completely dry.
Determination of Protein-Quantitations of proteins were performed by the method of Bradford (46) using bovine serum albumin as a standard.
Digestion with Glu-C Protease-The 200-kDa component isolated from the chemically deglycosylated, chaotrope-soluble cell wall fraction (10 mg) was suspended in 10 ml of distilled water, denatured by boiling at 100°C for 30 min, and subsequently transferred to an ice bath. After the addition of 10 ml of 0.1 M ammonium bicarbonate buffer (pH 7.5) and 100 g of Glu-C protease, the reaction mixture was incubated in a water bath at 30°C. Another 100 g of Glu-C protease were added after 3 h. After 6 h, incubation was stopped by freezing. The reaction mixtures were freeze-dried and stored at Ϫ20°C until used.

SDS-PAGE and Protein Gel Blot
Analyses-Macromolecules present in the LiCl extracts were precipitated by the addition of trichloroacetic acid (10% (w/v) final concentration) in the cold, and the precipitates were collected by centrifugation. The pellets were washed twice with distilled water and redissolved in a small volume of urea/SDS buffer (44). After the addition of sample buffer (45) containing bromphenol blue as a tracking dye, the polypeptides were separated by SDS-PAGE according to Laemmli (45) on gel slabs (83 ϫ 65 ϫ 0.15 mm) containing 7.5 or 12.5% (w/v) acrylamide. After electrophoresis, the gels were either stained with Coomassie Brilliant Blue G-250 or blotted electrophoretically onto polyvinylidene difluoride membranes (Porablot, Macherey-Nagel, Düren, Germany) as described by Towbin et al. (47). After pretreatment of the blots with 3% (w/v) bovine serum albumin in PBS for 3 h at 37°C or overnight at 7°C, the protein gel blots were probed with polyclonal antibodies raised in rabbits against different cell wall polypeptides diluted 1:300 with PBS containing 1% (w/v) bovine serum albumin. After 1 h at 37°C, the blots were washed four times with PBS for 30 min at room temperature, followed by treatment for 1 h at 37°C with alkaline phosphatase-coupled goat anti-rabbit IgG antibodies (Amersham Biosciences, Braunschweig, Germany) diluted 1:800 with PBS containing 1% (w/v) bovine serum albumin. After incubation, the blots were washed at least twice with PBS and subsequently twice with Tris-buffered saline for 30 min at room temperature. Finally, the indirectly bound alkaline phosphatase was detected by its enzyme activity using 5-bromo-4-chloro-3-indolyl phosphate as a substrate in the presence of nitro blue tetrazolium chloride (48).
Epitope Analysis-A total of 407 overlapping pentadecapeptides representing the whole amino acid sequence of the Chlamydomonas GP2 protein derived from the open reading frame of the GP2 cDNA (GenBank TM /EBI accession number BN001086) were generated by spot synthesis using a cellulose paper sheet as a solid support (49). These overlapping pentadecapeptides were used to determine the epitope specificities of the different antibodies as described previously (38,49). After incubation with the antibody and extensive washing, bound rabbit IgG were measured by incubation with alkaline phosphatase-coupled goat anti-rabbit IgG antibody and subsequent detection of the indirectly bound alkaline phosphatase via its enzyme activity using 5-bromo-4-chloro-3-indolyl phosphate as a substrate in the presence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. After documentation of the results, the membrane was stripped as described previously (49).
Determination of N-terminal Amino Acid Sequences-The untreated and chemically deglycosylated cell wall polypeptides and their fragments obtained by cleavage with Glu-C protease were purified by SDS-PAGE according to Laemmli (45) and blotted electrophoretically onto polyvinylidene difluoride membranes as described by Towbin et al. (47). After staining with Coomassie Brilliant Blue G-250, the protein bands were cut from the membrane and completely destained with methanol. N-terminal amino acid sequences were determined by automated Edman degradation using a Model 470A gas-phase sequencer (Applied Biosystems, Foster City, CA).
Mass Spectrometry-Proteolysis with trypsin of the purified insoluble cell wall fraction was performed essentially as described for in-gel digestion of protein bands cut from SDSpolyacrylamide gels (50). The cell wall material was digested for 3 h with porcine trypsin (sequencing grade, modified; Promega Corp., Madison, WI) at a concentration of 67 ng/l in 25 mM ammonium bicarbonate (pH 8.1) at 37°C. Prior to peptide mass mapping and sequencing of tryptic fragments by tandem mass spectrometry, peptide mixtures were extracted from the insoluble cell wall material by 1% formic acid, followed by two changes of 50% acetonitrile. The combined extracts were vacuum-dried until only 1-2 l were left and desalted using ZipTip pipette tips (Millipore Corp.) according to the manufacturer's instructions. MALDI-reflector-TOF analysis of the matrix ␣-cyano-4-hydroxycinnamic acid-nitrocellulose prepared on the target using the fast evaporation method (51) was performed on a Bruker Daltoniks REFLEX IV equipped with an N 2 337-nm laser and gridless pulsed ion extraction. Sequence verifications of some fragments were performed by nanoelectrospray tandem mass spectrometry on a Q-Tof I mass spectrometer (Micromass, Manchester, UK) equipped with a nanoflow electrospray ionization source. Gold-coated glass capillary nanoflow needles were obtained from Protana (medium NanoES spray capillaries). Data base searches (NCBInr nonredundant protein data base) were done using the MASCOT software from Matrix Science (52).
Isolation of DNA and RNA-RNA, high molecular mass Chlamydomonas DNA, and plasmid DNAs were isolated according to Maniatis et al. (53).
PCR-Primers for the rapid amplification of cDNA ends (54) were derived from the nucleotide sequences of scaffold 3 of the C. reinhardtii genome sequence (genome.jgi-psf.org/Chlre3/ Chlre3.home.html) starting at positions 1890535-1890594 and The Chlamydomonas Cell Wall Precursor GP2 OCTOBER 19, 2007 • VOLUME 282 • NUMBER 42 from GP2-encoding expressed sequence tags (ESTs). Reverse transcription-PCR was performed with Pwo DNA polymerase (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Genomic PCR was performed with high molecular mass Chlamydomonas DNA as a template and Pwo DNA polymerase as recommended by the supplier. Rapid amplification of gene ends was performed as described by Liu and Baird (55).
DNA Sequencing-Sequencing of PCR fragments cloned into the pCAPs vector (Roche Applied Science, Mannheim, Germany) and direct sequencing of PCR products were performed with a BigDye terminator cycle sequencing kit (Applied Biosystems) and a Model 377 automated DNA sequencer (Applied Biosystems).

Characterization of the Insoluble Cell Wall Fraction of C.
reinhardtii-The insoluble cell wall fraction of C. reinhardtii isolated from intact cells by successive treatments with different detergent-containing buffers revealed a sac-like morphology as revealed by phase-contrast microscopy (Fig. 1A). This morphology was completely destroyed by chemical deglycosylation with HF/pyridine accompanied by solubilization of distinct polypeptides (Fig. 1B, lane 1). The pattern of polypeptides released from the insoluble cell wall fraction by treatment with HF/pyridine was highly reproducible. Two polypeptides with apparent molecular masses of 64 and 45 kDa were the most prominent polypeptide constituents (Fig. 1B, lane 1). When these deglycosylation products were subjected to protein gel blot analyses, all of the solubilized polypeptides (Fig. 1B, lane 1) were recognized by the antisera against the chemically deglycosylated, insoluble cell wall fraction (referred to as anti-dICW) and its purified 64-and 45-kDa constituents (referred to as anti-dICW-64kDa and anti-dICW-45kDa, respectively) (lanes 3, 5, and 7). None of these polypeptides released from the insoluble wall fraction (Fig. 1A) by treatment with HF/pyridine (Fig.   1B, lane 1) reacted with any of the various preimmune sera (lanes 2, 4, and 6).
As reported previously, the constituents of the insoluble glycoprotein framework of the C. reinhardtii cell wall are crosslinked via isodityrosine formation (6) and as a consequence of transglutaminase activity (7). Both cross-links are resistant to treatment with HF. Therefore, the reproducible release of a set of defined polypeptides from the insoluble cell wall fraction by treatment with HF/pyridine indicates the presence of additional cross-links that are sensitive to HF. Prominent polypeptides with apparent molecular masses of Ͼ300, 150 -220, 110 -125, 64, and 45 kDa were released from the insoluble cell wall fraction under these conditions as revealed by both Coomassie Brilliant Blue G-250 staining of the SDS-polyacrylamide gels (Fig. 1B, lane 1) and protein blot analyses using the anti-dICW, anti-dICW-64kDa, and anti-dICW-45kDa antibodies (lanes 3, 5, and 7, respectively. The 64-and 45-kDa components released from the insoluble cell wall fraction by chemical deglycosylation are not individual polypeptides as reported previously (38). Edman degradation of these particular constituents of the insoluble wall fraction revealed that their N-terminal amino acid sequences are rather heterogeneous (38). Mixtures of several amino acid residues are cleaved off during each round of Edman degradation (38). This holds true also for other constituents of the insoluble cell wall fraction released during chemical deglycosylation (data not shown). These findings are at least partly due to the fact that the polypeptide constituents of the insoluble wall fraction are cross-linked not only via the HF-sensitive bonds but also via HF-resistant isodityrosine formation and transglutaminase-dependent reactions (6,7). Therefore, we decided to look for putative precursors of the insoluble glycoprotein framework of the insoluble cell wall fraction.
Periplasmic Precursors of the Insoluble Cell Wall Fraction-Because of its sac-like morphology (Fig. 1A), the insoluble glycoprotein framework of the cell wall limits the size of the Chlamydomonas cell, which increases considerably during the cell enlargement period before the onset of the cell division phase, resulting in the formation of up to 16 zoospores surrounded by the wall of the mother cell (43). A prerequisite for this considerable enlargement of the insoluble glycoprotein framework of the Chlamydomonas cell wall is the availability of its soluble precursors in the periplasmic compartment of the Chlamydomonas cell. For this reason, we analyzed the LiCl extracts of intact wild-type cells for the presence of putative precursors of the insoluble cell wall fraction (Fig. 2). Protein blot analyses using polyclonal antibodies raised against the unfractionated deglycosylation products of the insoluble wall fraction revealed that these particular antibodies reacted with the high molecular mass, chaotrope-soluble polypeptides with apparent molecular masses of Ͼ140 kDa present in the LiCl extracts of intact wild-type cells (Fig. 2, lanes 2 and 3). Furthermore, these high molecular mass, chaotrope-soluble polypeptides were also recognized by antibodies raised against the 64and 45-kDa polypeptides purified from the deglycosylation products of the insoluble wall fraction (Fig. 2, lanes 5, 6, 8, and  9). The latter antibodies did not reveal comparably strong signals with smaller constituents of the LiCl extracts of intact wild- type cells (Fig. 2, lanes 5, 6, 8, and 9). Therefore, we concluded that the high molecular mass polypeptides present in the LiCl extracts of intact cells with apparent molecular masses of Ͼ140 kDa were precursors of the 64-and 45-kDa deglycosylation products of the insoluble cell wall fraction.
Partial Amino Acid Sequences of a 190-kDa Deglycosylation Product of the Chaotrope-Soluble Cell Wall Fraction-The high molecular mass, chaotrope-soluble cell wall glycoproteins were isolated from the LiCl extracts of intact wild-type cells by phenyl-Sepharose chromatography and further purified by gel exclusion chromatography as described previously (42). When the obtained mixture of chaotrope-soluble cell wall glycoproteins (Fig. 3, lane 1) was subjected to chemical deglycosylation by treatment with trimethylsilyl trifluoromethanesulfonate (38,42), four prominent deglycosylated polypeptides with apparent molecular masses of 190, 130, 100, and 65 kDa were obtained (lane 2). Almost the same patterns were observed by protein blot analyses using polyclonal antibodies raised against the chemically deglycosylated, high molecular mass constituents of the chaotrope-soluble cell wall fraction (referred to as anti-dCSCW) (data not shown) or against the deglycosylation products of the insoluble cell wall fraction (anti-dICW, anti-dICW-65kDa, and anti-dICW-45kDa) (data not shown). Attempts to determine the N-terminal amino acid sequences of these chaotrope-soluble cell wall polypeptides by Edman degradation were successful for the GP3 subunits and their 100and 65-kDa deglycosylation products (Table 1). In the case of all the other cell wall polypeptides, this experimental approach was unsuccessful even after chemical deglycosylation, with the exception of the 190-kDa deglycosylation product, in which an N-terminal Ser residue was detected (Table 1). However, subsequent rounds of Edman degradation did not reveal additional sequence data for this 190-kDa deglycosylation product. How-  Fig. 1). Lane 1, gel containing 20 g of protein stained with Coomassie Brilliant Blue G-250; lanes 2 and 3, protein gel blots containing 20 and 10 g of protein, respectively, probed with the anti-dICW antibody; lanes 5 and 6, protein gel blots containing 20 and 10 g of protein, respectively, probed with the anti-dICW-64kDa antibody; lanes 8 and 9, protein gel blots containing 20 and 10 g of protein, respectively, probed with the anti-dICW-45kDa antibody; lanes 4, 7, and 10, prestained protein molecular mass markers.

FIGURE 3. SDS-PAGE analyses of purified, high molecular mass, LiClsoluble cell wall glycoproteins and their deglycosylation products.
Untreated (lane 1) and chemically deglycosylated (lane 2) cell wall glycoproteins (30 g of protein) were separated by SDS-PAGE on slab gels containing 12.5% (w/v) acrylamide under reducing conditions. After electrophoresis, the gels were stained for protein with Coomassie Brilliant Blue. The chaotrope-soluble cell wall glycoproteins GP2, GP3A, and GP3B are indicated, as are molecular mass markers.

TABLE 1 N-terminal amino acid sequences of Glu-C peptide fragments of the 190-kDa deglycosylation product of the chaotrope-soluble cell wall fraction
dGP, deglycosylated GP. ever, Edman degradation of some polypeptide fragments obtained by partial degradation with Glu-C protease of the 190-kDa deglycosylation product revealed N-terminal amino acid sequences of 13-20 residues. The longest amino acid sequence (20 residues) was determined for the 91-kDa fragment ( Table  1). The same N-terminal amino acid sequences were observed for the 74-and 53-kDa fragments (Table 1). Additional amino acid sequences of the 200-kDa deglycosylation product (Fig. 3, lane 2) were obtained by Edman degradation of its 82-, 66-, and 39-kDa fragments ( Table 1). The same N-terminal amino acid sequences were obtained for the 82-and 66-kDa fragments ( Table 1). Nucleotide Sequences of the GP2 cDNA and the Corresponding gp2 Gene and the Derived Amino Acid Sequence-The genome of C. reinhardtii has been sequenced almost completely, and an EST library is also available (56,57). Therefore, we analyzed the amino acid sequences shown in Table 1 against scaffold 3 of the Chlamydomonas genome sequence (genome. jgi-psf.org/Chlre3/Chlre3.home.html) using the tBLASTn program. The only hit was obtained for the N-terminal sequence of the 91-kDa fragment obtained by degradation with Glu-C protease of the 190-kDa deglycosylation product of the chaotropesoluble cell wall fraction ( Table 1). The nucleotide sequence corresponding to this particular amino acid sequence (TNNAFYPAYTMLGVKMFFDG) was detected in scaffold 3 at positions 1890535-1890594. By the same approach, EST sequences BP094641.1, BP094998.1, and AY596305.1 were shown to encode this particular amino acid sequence. The cDNA corresponding to AY596305.1 was already annotated to encode the 1146 C-terminal amino acid residues of GP2. Comparing the nucleotide sequence of AY596305.1 with scaffold 3 of the C. reinhardtii genome sequence, we found that the corresponding area of the gp2 gene is not interrupted by intron sequences. This finding was corroborated by the results of comparative reverse transcription-PCR and genomic PCR experiments (data not shown). Nucleotide sequences located upstream of this particular sequence were amplified by the 5Ј-rapid amplification of cDNA ends (54) and 5Ј-rapid amplification of gene ends (55) techniques. Although additional sequence data were obtained by this approach (GenBank TM / EBI accession numbers BN001086 and AM419024), the 5Ј-end of the corresponding mRNA and the encoded N-terminal end of GP2 are still unknown. As shown in Fig. 4B, the additional 5Ј-coding nucleotide sequence of the gp2 gene is interrupted by an intron and preceded by another intron sequence.

N-terminal amino acid sequence
The derived amino acid sequence of GP2 (Fig. 4B) includes all of the amino acid sequences obtained by Edman degradation of the peptide fragments obtained by Glu-C protease treatment of the 190-kDa deglycosylation product of the chaotrope-soluble cell wall fraction of C. reinhardtii (Fig. 4B and Table 1). As expected, all of these partial sequences are preceded by Glu residues. The known part of the GP2 protein contains four (hydroxy)proline-rich domains (domains II (residues 288 -339), IV (residues 576 -623), VI (residues 772-819), and VIII (residues 949 -1054)) (Fig. 4, A and B), which contain 13, 11, 8, and 20 XP n motifs (n Ͼ 1), respectively (including the SP n motifs). Additional XP n motifs also occur in domains I (residues 1-287) and III (residues 340 -575) (Fig. 4A).
Amino Acid Sequences Related to GP2 Occur in the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall-Fourteen fragments putatively derived from GP2 were identified By MALDI-TOF analysis of the tryptic peptides released from the insoluble cell wall fraction (Table 2). However, these GP2related tryptic peptides represent only ϳ15% of the entire polypeptide backbone of this particular chaotrope-soluble cell wall glycoprotein. Evidence that almost the whole GP2 polypeptide occurs in the insoluble glycoprotein framework of the Chlamydomonas cell wall was obtained by an immunological approach. To this end, 407 overlapping pentadecapeptides, together representing almost the entire polypeptide backbone of GP2, were synthesized on a solid support (Fig. 5, A and B) and comparatively analyzed for binding of polyclonal antibodies raised against the chemically deglycosylated, chaotrope-soluble and -insoluble cell wall fractions (Fig. 5C, upper panels). The polyclonal antibody raised against the total deglycosylation products of the insoluble cell wall fraction (anti-dICW) revealed more or less strong signals with 297 of the 407 GP2derived pentadecapeptides, whereas only 165 of these peptides reacted with the antibody against the deglycosylated, chaotrope-soluble cell wall fraction (anti-dCSCW) (Fig. 5C). In both cases, the epitopes recognized by these antibodies were distributed over the entire GP2 backbone (Fig. 5, A-C). Most of the peptides covering the proline-rich domains of the GP2 backbone (domains II, IV, VI, and VIII) (Fig. 5A) did not react with either antibody (Fig. 5, B and C).
As shown in Fig. 1B, polypeptides with apparent molecular masses of 64 and 45 kDa were the most prominent in the mixture of polypeptides released from the insoluble cell wall frac-  Table 1) are shown in boldface underlined letters. The polyadenylation sequence is shown in boldface italic letters. This sequence is available as GenBank TM /EBI accession number AM419024. The Chlamydomonas Cell Wall Precursor GP2 OCTOBER 19, 2007 • VOLUME 282 • NUMBER 42 ) and representing the whole known amino acid sequence of the mature GP2 polypeptide were synthesized by spot synthesis using cellulose paper as a solid support (49). C, after treatment with blocking solution, the cellulose-bound scan peptides were incubated with the different antibodies. After extensive washing, bound IgGs were detected by incubation with alkaline phosphatase-coupled anti-rabbit IgG antibodies and subsequent visualization of the indirectly bound alkaline phosphatase as described under "Experimental Procedures." tion by chemical deglycosylation. These particular constituents of the insoluble wall fraction are not individual polypeptides, as revealed by the results of Edman degradation, but mixtures of polypeptides of the same size (38). Polyclonal antibodies raised against these polypeptides cross-reacted with GP2 as shown by protein blot analyses (Fig. 2, lanes 5, 6, 8, and 9). Therefore, we investigated the epitope specificities of the anti-dICW-64kDa and anti-dICW-45kDa antibodies using the 407 GP2-derived scan peptides (Fig. 5B). It is astonishing that both antibodies reacted with more GP2-derived pentadecapeptides (Fig. 5C, lower panels) than the polyclonal antibodies raised against the total deglycosylation products of the insoluble wall fraction and the deglycosylated, chaotrope-soluble cell wall fraction (anti-dICW and anti-dCSCW, respectively) (lower panels). The patterns of GP2-derived pentadecapeptides recognized by the anti-dICW-64kDa and anti-dICW-45kDa antibodies differed both from each other and from that recognized by the anti-dICW antibody (Fig. 5C), and the recognized peptide sequences are distributed over the entire GP2 backbone (Fig. 5, A-C).
Only (hydroxy)proline-rich domains II, IV, VI, and VIII (Fig. 5,  A and B) were not recognized by the anti-dICW-64kDa and anti-dICW-45kDa antibodies (Fig. 5C, lower panels). Because the antigens used for the generation of the anti-dICW-64kDa and anti-dICW-45kDa antibodies are considerably smaller than GP2, these data further corroborate our previous findings that the 64-and 45-kDa deglycosylation products of the insol-uble cell wall fraction are not individual polypeptides but mixtures of several polypeptides of the same size (38).

DISCUSSION
As shown in this study, MALDI-TOF analyses of peptide fragments released by trypsin treatment from the purified insoluble cell wall fraction of C. reinhardtii revealed the presence of 14 peptides that could be attributed to specific non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2 (Table 2). However, these 14 putatively GP2-derived peptides cover only 15% of this particular cell wall polypeptide. This is obviously due to the fact that GP2 is highly glycosylated (33). Furthermore, the experimental proof of its presence in the insoluble glycoprotein framework of the Chlamydomonas cell wall is hindered by cross-linking via intermolecularisodityrosineformation(6),transglutaminasedependent reactions (7), or carbohydrate side chains (32). Therefore, we used an immunological approach to circumvent this problem. To this end, 407 overlapping pentadecapeptides that together cover the whole known amino acid sequence of GP2 were synthesized on a solid support (Figs. 4B and 5B). These chemically synthesized pentadecapeptides were used for epitope mapping of polyclonal antibodies raised against the deglycosylation products of the purified insoluble cell wall fraction (Fig. 5C). The polyclonal antibody raised against the total deglycosylation products of the insoluble cell wall fraction (anti-dICW) revealed signals with 297 of the 407 GP2-derived pentadecapeptides (Fig. 5C). The epitopes detected by this particular antibody (Fig. 5C) are distributed over the whole GP2 backbone (Figs. 4B and 5B). A considerable proportion (77 peptides) that did not react with this antibody (peptides 99 -114, 190 -208, 259 -271, and 326 -354) (Fig. 5C) are derived from the (hydroxy)proline-rich domains of GP2 (Fig. 5, A and B). Proline residues located in proline-rich motifs are usually substrates for the proline hydroxylases (58 -60). After transformation to hydroxyproline, these residues are further modified by addition of oligoarabinosyl side chains (8,17,(61)(62)(63). For these reasons, it is not astonishing that the proline-rich domains of the GP2 precursor are not recognized by polyclonal antibodies raised against the corresponding deglycosylation products. Therefore, our findings that 297 of the 407 GP2-derived pentadecapeptides are recognized by the polyclonal antibody raised against the total deglycosylation product of the insoluble cell wall fraction and that the epitopes recognized by this particular antibody are distributed over the whole GP2 backbone clearly show that the whole GP2 molecule is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall.
As reported previously, the constituents of the insoluble glycoprotein framework of the C. reinhardtii cell wall are cross-linked through the formation of isodityrosine bridges (6) and as a consequence of transglutaminase activity (7). Both cross-linking types are resistant to treatment with HF/pyridine. Nevertheless, a reproducible set of polypeptides was released from the purified insoluble cell wall fraction during chemical deglycosylation with HF/pyridine (  The Chlamydomonas Cell Wall Precursor GP2 OCTOBER 19, 2007 • VOLUME 282 • NUMBER 42 of additional cross-links that are sensitive to HF. It has been reported recently that C. reinhardtii contains a prolyl 4-hydroxylase that is essential for proper cell wall assembly (64). Because hydroxyproline residues are glycosylated (8,17,(61)(62)(63), these findings indicate that some of the hydroxyproline-bound carbohydrate side chains are involved in the cross-linking of the constituents of the insoluble cell wall fraction. Cross-links via carbohydrate side chains have been described previously for the extracellular matrix of Volvox carteri (65). On the other hand, neither the 190-kDa deglycosylation product of GP2 nor other deglycosylation products of the chaotrope-soluble cell wall fraction (Fig. 3, lane 2) could be identified in the mixture of polypeptides released from the insoluble cell wall fraction by treatment with HF/pyridine (Fig. 1B), with the apparent exception of the 65-kDa deglycosylation product of GP3A (33). The molecular mass of deglycosylated GP3A corresponds roughly to that of the prominent 64-kDa component found in the polypeptides released from the insoluble wall fraction by treatment with HF/pyridine (Fig. 1B). However, Edman degradation of the 65-kDa deglycosylation product of GP3A revealed a defined N-terminal amino acid sequence (Table 1), in contrast to the 64-kDa constituent of the chemically deglycosylated, insoluble cell wall fraction (38). As reported previously, the 64-and 45-kDa components released from the insoluble glycoprotein framework of the C. reinhardtii cell wall are not individual polypeptides as shown by N-terminal sequencing; mixtures of several amino acids are cleaved off during each round of Edman degradation (38). This holds true also for other constituents of the insoluble wall fraction released by chemical deglycosylation (data not shown). These findings are essentially due to the fact that that the constituents of the insoluble wall fraction are cross-linked not only via the HF-sensitive bonds but also via HF-resistant isodityrosine bridges and cross-links formed by the action of transglutaminase (6,7).
The polyclonal antibodies raised against the purified 64-kDa (anti-dICW-64kDa) and 45-kDa (anti-dICW-45kDa) constituents of the chemically deglycosylated, insoluble cell wall fraction (Fig. 1B) cross-reacted with several high molecular mass polypeptides (Ͼ140 kDa) present in the LiCl extracts of intact cells, including GP2 (Fig. 2, lanes 5, 6, 8, and 9). As shown in Fig.  5C (lower panels), both antibodies reacted with more GP2-derived pentadecapeptides than the polyclonal antibodies raised against the total deglycosylation products of the insoluble wall fraction (anti-dICW) and against the deglycosylated, chaotrope-soluble cell wall fraction (anti-dCSCW) (Fig. 5C, upper  panels). Only (hydroxy)proline-rich domains II, IV, VI, and VIII (Fig. 5, A and B) were not recognized by the anti-dICW-64kDa and anti-dICW-45kDa antibodies (Fig. 5C, lower panels). Because the antigens used for the generation of the anti-dICW-64kDa and anti-dICW-45kDa antibodies are considerably smaller than GP2, these data further corroborate our previous findings that the 64-and 45-kDa deglycosylation products of the insoluble cell wall fraction are not individual polypeptides but mixtures of several polypeptides of the same size (38). Interestingly, the 45-kDa component purified from the deglycosylation products of the insoluble wall fraction was found to be FIGURE 6. Sequence alignment of C. reinhardtii GP2 and a V. carteri GP2 homolog. The GP2 amino acid sequence was analyzed with regard to the recently released V. carteri genomic sequence (genome.jgi-psf.org/ Volca1/Volca1.home.html) using the tBLASTn program. The only hit obtained was detected in scaffold 1 at positions 2007075-2010858. By the same approach, the EST sequences CBHO2940, CBHO15012, CBHO15996, CBHO17719, CBGZ16240, and CBGZ20812 were shown to encode this particular amino acid sequence. These EST sequences were used to identify overlapping additional ESTs. Sequences from the BLAST search were translated with Baylor College of Medicine six-frame utilities (searchlauncher.bcm.tmc.edu/seq-util/seq-util.html), aligned using ClustalW, and refined manually. Conserved sequences are boxed. resistant to in vitro proteolysis with several endoproteinases, whereas the 64-kDa component was degraded to a 45-kDa fragment under the same conditions (32). Overall, these data indicate that, after cross-linking of GP2 and other precursors to the insoluble glycoprotein framework, the size of their polypeptide backbones must be reduced by proteolytic processes to reveal the 64-and 45-kDa fragments. Partial proteolysis of the insoluble cell wall fraction is a prerequisite for cell enlargement (41,42,66) and is accompanied by release of macromolecules into the culture medium (41,66). The size of the polypeptide fragments retained in the insoluble cell wall layer is apparently determined not only by the distribution of putative cleavage sites but also by the carbohydrate chains and the associated chaotrope-soluble cell wall glycoproteins.
During the last year, the genomic sequences of two additional green algae, Ostreococcus tauri (genome.jgi-psf.org/Ostta4/ Ostta4.home.html) and V. carteri (genome.jgi-psf.org/Volca1/ Volca1.home.html), became available. No homolog to the C. reinhardtii gp2 gene was detected in either the genomic or EST sequences of O. tauri, a wall-less unicellular green alga belonging to the Prasinophyceae occurring in the upper water columns of the oceans. By the same approach, a gene encoding a GP2 homolog was detected, however, in the genome of V. carteri and the corresponding EST sequences (Fig. 6). The presence of the corresponding ESTs show that this GP2 homolog is expressed. The derived amino acid sequence of the V. carteri GP2 homolog revealed ϳ50% sequence identity to Chlamydomonas GP2 and contains the same number of (hydroxy)proline-rich sequences at conserved positions (Fig. 6). Furthermore, most of the Cys residues and the surrounding amino acid sequences are strongly conserved (Fig.  6). As reported previously (23), the V. carteri GP2 homolog is able to co-crystallize with the chaotrope-soluble cell wall glycoproteins of C. reinhardtii. Therefore, the question arises at to whether or not the V. carteri GP2 homolog is also a constituent of the insoluble fraction of the extracellular matrix of V. carteri.