Planta
Planta (1988) 173:373 384
9 Springer-Verlag 1988
The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins Jfirgen Voigt Institut ffir Allgemeine Botanik und Botanischer Garten, Universitfit Hamburg, Ohnhorststrage 18, D-2000 Hamburg 52, Federal Republic of Germany
Abstract. Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiC1 extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiC1 extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiC1 extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiC1 extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cellwall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with Abbreviations: BSA = bovine serum albumin ; IgG = immunoglobulin G; k D a - k i l o d a l t o n ; M r : r e l a t i v e molecular mass; P B S - phosphate-buffered saline; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris=2-amino2-(hydroxymethyl)-l,3-propanediol
cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii. Key words: Antibody specificity - Cell wall - Chlamydomonas (cell wall glycoproteins) - Glycoprotein (cell wall).
Introduction The cell wall of the unicellular green alga Chlamydomonas reinhardii does not contain cellulose or other polysaccharides, but is composed of several layers of hydroxyproline-rich glycoproteins whose overall composition resemble those of the cell-wall glycoproteins of higher plants (Chrispeels 1969; Roberts 1974; Catt et al. 1976; Stewart and Varner 1980; Smith 1981 ; Lamport and Catt 1981 ; Smith et al. 1984b; McNeil etal. 1984; Roberts et al. 1985). Cell walls isolated from the culture medium of vegetatively growing C. reinhardii cells can be separated into a soluble and an insoluble fraction by treatment with chaotropic agents like LiC1 or NaC104 (Hills 1973; Hills etal. 1973; Roberts 1974). The insoluble cell-wall component and the NaC104-soluble glycoproteins were found to be
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very similar with respect to amino-acid and sugar composition (Roberts 1974). The insoluble wall component, which can also be isolated from intact cells by extensive extractions with detergent-containing buffers (Voigt 1984), retains the overall saclike morphology of the wall, although it appears to be much thinner than the intact wall. Electron-microscopic examinations showed that this isolated cell-wall component is amorphous (Hills 1973; Hills et al. 1975) like the inner wall layer (W1) of intact cells (Roberts et al. 1972). Roberts and coworkers have therefore concluded that the inner wall layer is identical with the insoluble wall component (Cart et al. 1976, 1978; Roberts et al. 1985). The crystalline' outer wall layers are assumed to consist of salt-soluble cell-wall glycoproteins (Roberts 1974; Hills et al. 1975; Cart et al. 1976, 1978; Roberts et al. 1985). This assumption is, however, contradicted by the results of Goodenough and Heuser (t985) and my own data, which indicate that the insoluble cell-wall material is located in the central triplet of the wall. Pulse-labelling and pulse-chase experiments with [3t-I] proline and [asS] methionine demonstrated that the cell-wall glycoproteins, which can be solubilized by chaotropic salt solutions (Hills 1973; Hills etal. 1973; Roberts 1974; Voigt 1985a), are precursors of the insoluble wall layer (Voigt 1986). Extracts from isolated cell walls of C. reinhardii or C. eugametos contain high-molecular-weight glycoproteins (Davies and Lyall 1973; Roberts 1974; Catt et al. 1976; Musgrave et al. 1983; Smith et al. 1984a). Amino-acid analysis of these highmolecular-weight glycoproteins showed that they differ with respect to the proportion of hydroxyproline although the overall amino-acid compositions were found to be very similar (Goodenough et al. 1986). Recently, I have reported that the LiC1 extracts from intact wild-type cells contain some glycoproteins of considerably lower molecular weight which are missing in the LiC1 extracts from the cell-wall-deficient mutant CW-15 (Voigt 1985a). These polypeptides were found to be more strongly labelled with [3H] proline than with [35S] methionine (Voigt 1985a, 1986). Monk etal. (1983) have identified the same cell-wall-related polypeptides during their studies on the cell-surface polypeptides of C. reinhardii. Some of these cell-wall-related polypeptides have been purified to homogeneity and used to raise antibodies. These antibodies have been characterized with respect to their specificities against the polypeptide backbone and carbohydrate side
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
chains, and especially with respect to their crossreactivities with the different cell-wall glycoproteins of C. reinhardii. Material and methods Anti-rabbit immunoglobulin G (IgG) (biotinylated species-specific whole antibody from donkey), fluorescein-streptavidin and streptavidin-biotinylated horseradish peroxidase complex were purchased from Amersham-Buchler (Braunschweig, FRG). 3,3'-Diaminobenzidine was obtained from Sigma (Taufkirchen, FRG). All other chemicals (analytical grade) were purchased from Merck (Darmstadt, FRG) and Serva (Heidelberg, FRG).
Algal strains. The wild-type strain of Chlamydomonas reinhardii used in the present experiments was strain 137C (mating-type + ) obtained from Dr. R.P. Levine (Harvard University, Cambridge, MA, USA). The mutant strain "ls" (mating-type --) was isolated by Mergenhagen (1980), and the cell-wall-deficient mutant CW-15 (mating-type +), isolated by Davies and Plaskitt (1971), was obtained from Sammlung von Algenkulturen, G6ttingen, F R G (strain 83.8l). Media and growth conditions. Stock cultures were maintained on agar slants containing sodium acetate (2 g.1 -I) at 15~ and illuminated (4000 lx) using a 12 h light-12 h dark cycle. Cells were grown at 24 ~ C in high-salt medium (Sueoka 1960) with or without sodium acetate (2 g.l-~). The cultures were constantly bubbled with filtered air and constantly mixed by stirring bars. For asynchronous growth, the cells were continuously illuminated from the sides by a combination of white lamps (type 36W/25; Osram, Mfinchen, FRG) and "daylight" fluorescent lamps (Osram type 36W/11). The light flux was 10000 lx at the level of the cultures. Synchronous growth was achieved by the method of Surzycki (1971) using a 12 h light12 h dark illumination cycle. Cell concentrations were determined by duplicate hemocytometer counting. Cells were harvested at a cell density of 1. 1 0 6 - - 3 . 106 cells.ml 1.
Cellfractionation. The cultures were rapidly cooled to 0 ~ C and centrifuged at 6000 g for 10 rain. The culture medium was decanted immediately after centrifugation. The cells were twice washed and resuspended in high-salt medium to a final cell density of 1-108 cells-ml 1. Extraction of the polypeptide components of the LiCl-soluble wall layers was performed as recently described (Voigt 1985a) by addition of the same volume of ice-cold LiC1 (4 tool-1 1), incubation at 0 ~ C for 30 min and centrifugation at 10000-g for 20 rain. The supernatant was collected and used to isolate the salt-soluble cell-wall glyeoproreins. The insoluble cell-wall component was purified from the LiCl-extracted cells as recently described (Voigt 1984, 1985b). Gel exclusion chromatography. Gel exclusion chromatography of crude LiC1 extract from intact cells was performed on columns (2.5 cm i.d., 100 cm long) of Sepharose C1-4B, Sephacryl S-300 and Sephadex G-75, equilibrated with LiCI (1 m o l d - t ) 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-HC1 (20 mmol.1-1), pH 7.2. The crude LiC1 extract of intact cells was concentrated in dialysis bags with Aquacide II (Calbiochem, Marburg, FRG) prior to column chromatography. Treatment with Aquacide II had to be interrupted by dialysis against 0.5 mol-1 t LiC1 to concentrate the LiC1 extract efficiently. The columns were etuted with equilibration buffer. Fractions of 10 ml were collected and analyzed for absorbance at 280 nm, for protein concentration (according to Bradford 1976) and protein composition by sodium dodecyl sulfate
J. Voigt: Cell-wall glycoproteins of Chlamydomonas (SDS)-polyacrylamide gel electrophoresis (PAGE) (according to Laemmli 1970).
Sodium dodecyl su(fate-polyacrylamide gel electrophoresis. Analytical or preparative SDS-PAGE were performed on 14.12.0.:15 cm 3 slab gels according to Laemmli (1970). To obtain a sufficient separation of the glycoproteins, an urea-containing sample buffer had to be used as described by Voigt (1985a). The protein fractions were adjusted to 10% trichloroacetic acid (TCA) and incubated for 2 h at 4 ~ C. The precipitates were collected by centrifugation for :15 rain at 15000-g, twice washed with bidistilled water and redissolved overnight in a buffer consisting of urea (8 tool-1-1), SDS (2%, w/v), ethylenediamine tetraacetic acid (EDTA) (10 mmol.1-1), 2-mercaptoethanol (200 retool-l-i) and Tris-HC1 (20 retool.l-1, pH 7.5). After addition of sample buffer (1/4 volume), the protein mixtures were submitted to SDS-PAGE on slab gels according to Laemmli (1970). When the tracking dye, bromophenol blue, reached the front of the gel, electrophoresis was stopped. Analytical gel slabs were then stained with Coomassie Brillant Blue R-250. In the case of preparative gels, strips were cut from both sides of the gel slabs and stained with Coomassie Brillant Blue R-250. The main part of each gel slab was stored at 4 ~ C without acid treatment. Protein bands were cut from these untreated parts of the gel slabs according to the protein patterns of stained gel strips. The polyacrylamide pieces were homogenized in a buffer containing 0.1% (w/v) SDS and Tris-HC1 (20 mmol. 1-l, pH 8.0), and the homogenates filled into dialysis bags. The polypeptides were then eluted electrophoretically at 6 V.cm -1 overnight at room temperature using the electrophoresis buffer according to Laemmli (1970). The polypeptide solutions were concentrated against Aquacide II and stored at --20 ~ C. The eluted polypeptides were routinely checked for purity by analytical SDS-PAGE.
Preparation of antibodies. Antibodies against the different polypeptides were raised in rabbits. Aliquots of the polypeptide solutions corresponding to 20-50 tag protein were diluted with 1 nrl phosphate-buffered saline (PBS) and mixed with 1 ml Freund's complete adjuvans (Calbiochem). Injections were performed every two weeks intraperitoneally and subcutaneously at multiple sites over a period of three to four months. Serum samples were taken 6 d after each booster injection starting two months after the primary injection and immunization controlled by Ouchterlony double-diffusion tests in 1% agarose gels prepared in PBS, pH 7.2 (Ouchterlony 1948). When the double-diffusion assay was positive, the animals were bled and the immune sera fractionated by differential (Ntt4)2SO 4 precipitation and by passing the redissolved and dialysed protein through a colunm of Whatman DE-52 equilibrated with :10 retool-1-1 sodium phosphate, pH 6.8 (Levy and Sober 1960). The unbound material was concentrated by (NH4)2 SOs precipitation; the redissolved precipitate was extensively dialysed against 0.9% (w/v) NaCI containing 10 retool. 1 1 sodium phosphate, pH 7.2, and stored at - 2 0 ~ C. Preparation of cell-wall-derived glycopeptides. Cell-wall glycopeptides were prepared by successive digestion with pronase E and proteinase K of polypeptide mixtures extracted from intact ceils of C. reinhardii with aqueous LiCI (Voigt :1985a). The LiCI extracts of intact wild-type cells were dialysed overnight at 4 ~ C against :100 vol. of a buffer containing NaC1 (150 m m o l . l - 1), E D T A (10 mmol-1-1) and Tris-HC1 (20mmol'1-1, pH7.5). The suspension was concentrated against Aquacide II and then dialysed against the same buffer. The polypeptides were then incubated with :1 rag. m l - i pronase
375 E (Sigma, Dreieich, FRG) for 30 rain at 55 ~ C, followed by digestion with 2 mg.ml -~ proteinase K (Merck) at 37~ for 6 h. The reaction mixtures were then adjusted to 10% (w/v) trichloroacetic acid. After 2 h at 0 ~ C, the precipitated protein components were removed by centrifugation at 20000.g for 30 min. The supernatant was passed through a column (100 cm long, 2.5 cm i.d.) of Biogel P :10 (Bio Rad, Mfinchen, FRG) equilibrated with acetic acid (0.1 mol.l-1). The columns were eluted with the same solvent. Fractions of 5 ml were collected and the pH values measured. Aliquots were hydrolysed with 80% (v/v) H2SO~ for 10 rain at 100~ and the hydrotysates analyzed for sugars by the phenol-sulfuric acid method (Dubois et al. 1956). Neutral fractions containing glycopeptides were combined and freeze-dried.
Chemical modification of cell-wall glycoproteins. Partially purified cell-wall glycoproteins which were obtained by gel exclusion chromatography of LiC1 extracts from intact C. reinhardii wild-type cells were used as starting material for the preparation of chemically modified cell-wall glycoproteins. (i) Periodate oxidation. A solution of partially purified cell-wall glycoproteins (protein concentration 0.2mg.m1-1) containing LiCI (1 mol.1-1) and Tris-borate (10 retool-1-1, pH 8.3) was mixed with the same volume of a solution of sodium metaperiodate (40 mmol-1-1) and incubated for 2 h at 0 ~ C in the dark. The reaction mixture was then diluted with Tris-borate (10 mmol. 1 1, pH 8.3), extensively dialysed against the same buffer and concentrated against Aquacide II. (ii) MiM alkaline hydrolysis: A solution of partially purified cell-wall glycoproteins (protein concentration 0.2 m g m l - i ) was adjusted to 2 tool-1-1 KOH and incubated at room temperature for 2 h. The solution was neutralized by addition of HC1 and extensively dialysed against a buffer containing 0.1% (w/v) SDS and Tris-borate (10 mmol.1-1, pH 8.3). (iii) Deglycosylation: 2 mg freeze-dried cell-wall glycoproteins were chemically deglycosylated by the method of Edge et at. (1981) using trifluoromethane-sulfonic acid and anisol. After 4 h, the reaction was stopped and reagents and sugars removed by method A of Edge et al. (1981). The proteins were dialysed against 2 mmol-1-1 pyridine acetate (pH 5.5), lyophilised and redissolved in Tris-borate (10 retool. t - i , pH 8.3). Immunoblot analysis. Analytical SDS-PAGE was performed as described above. The gels were then placed adjacent to a sheet of nitrocellulose (BA 85, Schleicher & Schfill, Dassel, FRG) and electrophoretically blotted for 18 h at 15 V, 300 mA (Towbin et al. 1979). The nitrocellulose sheet was then dried, washed with PBS, blocked with 3% (w/v) bovine serum albumin (BSA) in PBS and successively incubated for 16 h at 4 ~ C with rabbit immune IgG (diluted l:100 with 1% BSA-PBS), biotinylated donkey anti-rabbit-IgG serum (diluted 1:200 with 1% BSAPBS) and for I h at room temperature with preformed streptavidin-horseradish-peroxidase complex (diluted 1:200 with 1% BSA-PBS). After each treatment, the filter blots were washed four times with 1% (v/v) NP40 in PBS for 30 rain at room temperature. Colour was developed by the method of Hawkes et al. (1982) using 3,3'-diaminobenzidine in the presence of COC12. Dot-immunobinding assays were performed on nitrocellulose filters containing 10 ~tg of untreated or chemically modified cell-wall polypeptides per spot. The filters were processed as described for the Western-blot analysis.
Immunofluorescence studies. Chlamydomonas reinhardii cells were fixed with 4% (w/v) formaldehyde in PBS for 4 h at 4 ~ C and washed four times with PBS. Formaldehyde-fixed cells or purified insoluble wall material were incubated with 3% (w/v)
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
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BSA in PBS overnight at 4 ~ C and then treated 16-20 h with either preimmune serum or antibodies (50 ~tg rabbit IgG per ml J% BSA-PBS). After washing with 1% NP40-PBS (four times), the samples were successively incubated for 4 h at room temperature with biotinylated donkey anti-rabbit IgG serum (diluted 1 : 200 with 1% BSA-PBS) and fluorescein isothiocyanate (FITC)-conjugated streptavidine (diluted 1:100 with J% BSA-PBS). After each treatment, the samples were washed four times with 1% (v/v) NP40 in PBS. Samples were examined using a Zeiss epifluorescence optics with filter set 10 for FITC (Zeiss, Oberkochen, FRG).
Results
Isolation of cell-wall glycoproteins from LiCl extraets of intact wild-type cells. T o o b t a i n the saltsoluble cell-wall g l y c o p r o t e i n s , LiC1 e x t r a c t s f r o m i n t a c t w i l d - t y p e cells were f r a c t i o n a t e d b y gel ex-
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Fig. 1 A, B. Fractionation of LiC1 extracts from intact cells of C. reinhardii by gel exclusion chromatography on a Sephadex G-75 column equilibrated with 2 mol. 1-1 LiC1 buffered with 20 mmol-1 1 Tris-HC1, pH 7.2. A Elution profile. Eluate fractions were analyzed for absorbance at 280 nm ( ) and protein concentration (. . . . . ). B Sodium dodecyl sulfate-PAGE of column fractions. Aliquots (200 gl) from every second column fraction were analyzed by SDS-PAGE on slab gels containing 17% acrylamide. The gels were stained with Coomassie Brillant Blue. a, b-marker proteins (1(~20 ~tg each) bovine serum albumin (Mr 68000), 3-phosphoglycerate kinase (Mr 47000), aldolase ( M r 40000), glyceraldehyde-3-phosphate dehydrogenase (Mr 36000), hemoglobin (Mr 15 600)
c l u s i o n c h r o m a t o g r a p h y . T h e best results were achieved with Sephadex G-75 columns equilibrated a n d eluted w i t h a b u f f e r c o n t a i n i n g 2 m o l " 1- t LiC1 (Fig. 1 A). E l u a t e f r a c t i o n s were a n a l y s e d f o r p r o tein c o m p o s i t i o n b y S D S - P A G E (Fig. 1 B). T h e h i g h - m o l e c u l a r - w e i g h t cell-wall g l y c o p r o t e i n s were eluted in the v o i d v o l u m e t o g e t h e r w i t h s o m e smaller p o l y p e p t i d e s [relative m o l e c u l a r m a s s ( M r ) = 6 6 , 35, 26 a n d 12 k i l o d a l t o n s (kDa)]. C h r o m a t o g r a p h y o n S e p h a r o s e C1-4B c o l u m n s yielded a similar e l u t i o n p a t t e r n , the h i g h - m o l e c u l a r - w e i g h t cell-wall g l y c o p r o t e i n s a n d the a s s o c i a t e d smaller p o l y p e p t i d e s being eluted in a f r a c t i o n with a n M r o f 300000. W h e n LiC1 extracts f r o m s t a t i o n a r y cells w e r e f r a c t i o n a t e d b y gel e x c l u s i o n c h r o m a t o g r a p h y , a n a d d i t i o n a l p o l y p e p t i d e o f 38 k D a w a s f o u n d t o be a s s o c i a t e d w i t h the h i g h - m o l e c u l a r -
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 2. Analytical SDS-PAGE of the polypeptides used to raise antibodies. The polypeptides were isolated from LiC1 extracts of intact C. reinhardii cells by gel exclusion chromatography (Fig. 1) and subsequent preparative SDS-PAGE. a = 150-kDa glycoprotein; b = 38-kDa glycoprotein; e = 35-kDa glycoprotein ; d = 26-kDa component; e = 12-kDa component; f = mixture of marker proteins (5-15 gg each) myosin (Ms 205000), fl-galactosidase (Mr 116000), phospborylase b (Mr 97000), bovine serum albumin (Mr 68000), ovalbumin (Mr 45000), carbonic anhydrase (Mr 29 000)
weight cell-wall glycoproteins. One of the high-molecular-weight glycoproteins and some of the smaller polypeptides were further purified by preparative SDS-PAGE. An analytical SDS-PAGE of these purified polypeptides, which were used to raise antibodies, is shown in Fig. 2. Characterisation of antibodies. Antibodies against the five isolated polypeptides were raised in rabbits and preliminarily analyzed by double-diffusion assays with crude LiC1 extracts of intact wild-type cells (Fig. 3). Independent of the arrangement of the different antibodies in the peripheral wells, one fused precipitation line was observed for all the antibody preparations (wells I-5) but not for the preimmune serum (well 6). In the case of the antibodies raised against the 12-kDa and the 26-kDa component, respectively, an additional precipitation line was found which did not fuse with the precipitation line formed by the other antibodies, indicating that these two antibodies are not monospecific. To ascertain that the different antibodies really react specifically with the cell surface of C. reinhar-
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Fig. 3. Double diffusion of antibodies raised against different isolated polypeptides with crude LiC1 extract from C. reinhardii wild-type cells. Centre well: unfractionated LiC1 extract; outer wells: l = a n t i - 1 2 k D a IgG, 2 = a n t i - 2 6 k D a IgG, 3=anti35 kDa IgG, 4=anti-38 kDa IgG, 5-anti-150 kDa IgG, 6 = preimmune IgG
dii, formaldehyde-fixed wild-type cells were incubated with preimmune IgG or with the different antibody preparations. The bound rabbit IgG molecules were detected by immunofluorescence after successive incubations with biotinylated speciesspecific anti-rabbit IgG antibodies from donkey and fluorescein-conjugated streptavidine. As shown in Fig. 4, cell-surface-bound rabbit IgG molecules could be detected by immunofluorescence when the cells were incubated with the antibody against the 150-kDa cell-wall glycoprotein (C, D). Cells incubated with preimmune serum, on the other hand, did not bind rabbit IgG (Fig. 4 A, B), indicating that the binding of the antibodies to the cell surface of C. reinhardii is the result of specific antigen-antibody interactions. Binding of rabbit IgGs was also detected, when the cells were incubated with the other antibody preparations. No immunofluorescence was observed when the purified insoluble wall component was analyzed (data not shown). Furthermore, the cell surface of the cell-wall-deficient mutant CW-I 5 did not react with any of the antibodies (data not shown).
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J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 4A-D. Indirect
immunofluorescence staining of formaldehyde-fixed C. reinhardii cells using preimmune IgG (A, B) or anti-150 kDa IgG (C, D). A, C: fluorescein optics to visualize bound rabbit IgGs; B, D: phase contrast. Bar = 10 pm
Double-diffusion assays performed with purified polypeptides demonstrated that all the antibodies showed cross-reactivity with the different antigens and were found to be also reactive against some other polypeptides present in the LiC1 extracts from intact wild-type cells (Fig. 5). However, differences were observed with respect to the relative intensities of the precipitation lines when constant amounts of the antibodies were used (Fig. 5). This finding indicates that in spite of the crossreactivities the different antibodies do not have exactly the same specificities. Immunoblot analyses likewise showed that the LiC1 extracts from intact wild-type cells contained several polypeptide coinponents which cross-reacted with the different antibodies (Figs. 6, 8). The pattern of antigens present in the LiC1 extracts was found to be dependent on growth conditions and cell-cycle stage (Fig. 6, 8). The predominant part of the polypeptides present in the LiC1 extracts did not react with the antibodies (Fig. 6). The LiC1 extracts from intact cells of the cell-wall-deficient mutant CW-15 contained extremely low levels of cross-reacting polypeptides. Large quantities of LiCl-extracted macromolecules from this mutant had to be submitted to Westernblot analysis in order to detect these antigens (Fig. 6, slots d and i). Only two cross-reacting polypeptides of Mr 32 k D a and 27 kDa were found
in LiC1 extracts from CW-15 cells (Fig. 6, slot d). These findings indicate that the antibodies have a specificity for a limited number of polypeptides present in the LiC1 extracts from intact wild-type cells and that these cross-reacting polypeptides are located in the cell wall and- or the periplasmic space.
Binding of antibodies to chemically modified cellwalIpolypeptides. Dot-immunobinding assays with chemically modified cell-wall polypeptides were performed to investigate the specificities of the different antibodies (Fig. 7). The cell-wall material used for these studies was isolated by gel exclusion chromatography of LiC1 extracts from intact wildtype cells (Fig. 1), The polypeptide mixture eluted in the void volume of a Sephadex G-75 column was desalted and spotted onto nitrocellulose filters either without pretreatment (row a) or after treatment with sodium metaperiodate (row b), treatment with 2 M K O H (row c) or chemical deglycosylation (row d). After incubation with 3% BSAPBS, the nitrocellulose filters were analyzed with the different antibodies. Treatment of the cell-wall polypeptides with sodium metaperiodate did not appreciably reduce the binding of the different antibodies (row b). Preincubation of the cell-wall polypeptides with 2 mol.1 1 K O H reduced the bind-
J. Voigt: Cell-wail glycoproteins of Chlamydomonas
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Fig. 5. Double-diffusion assays of antibodies raised against different putative cell-wall polypeptides of C. reinhardii with several polypeptide components isolated from LiC1 extracts of intact wild-type cells. The polypeptides were isolated from the LiCI extracts by gel exclusion chromatography (Fig. I) and subsequent preparative SDS-PAGE. Atiquots (40 ~1) of the isolated polypeptides (protein concentration 0.1 0.3 rag. ml-1) were placed into the central wells. a = 280-kDa glycoprotein; b = 150-kDa glycoprotein; c - 97-kDa polypeptide; d= 66-kDa polypeptide; e = 56-kDa polypeptide; f = 43-kDa polypeptide; g = 38-kDa glycoprotein; h = 35kDa glycoprotein; i=26-kDa component. The antibody loads (40 gl; protein concentration 5 mg.ml-1) in the peripheral wells were: 1 =preimmune IgG; 2=anti-12 kDa IgG; 3=anti-26 kDa IgG; 4=anti-35 kDa IgG: 5=anti-38 kDa IgG; 6=anti-150 kDa IgG
ing capacity for the a n t i b o d y against the 1 5 0 - k D a g l y c o p r o t e i n a n d d e s t r o y e d the antigenic sites for the a n t i b o d y against the 2 6 k D a c o m p o n e n t , whereas the binding o f the o t h e r antibodies was essentially not affected (row c). Only the antibodies raised against the 150 k D a glycoprotein a n d the 3 5 - k D a c o m p o n e n t were able to recognize the chemically deglycosylated cell-wall p o l y p e p t i d e s (row d). T h e binding capacity for b o t h antibodies,
however, was strongly reduced by chemical deglycosylation o f the cell-wall polypeptides.
Specificities o f antibodies saturated with cell-wallderived glycopeptides. Nitrocellulose filters containing m a c r o m o l e c u l e s , which were extracted by a q u e o u s LiC1 f r o m C. reinhardii wild-type cells a n d s e p a r a t e d by S D S - P A G E , were p r o b e d with different antibodies p r e i n c u b a t e d in the presence or ab-
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J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 6. Immunoblot analysis of LiCl-extracted polypeptides from C. reinhardii wild-type strain 137C and the cell-wall-deficient mutant CW-15 using anti-150-kDa antibodies. Macromolecules extracted by treatment with aqueous LiC1 from intact C. reinhardii wild-type or CW-15 cells were separated by SDS-PAGE on slab gels containing 15 % acrylamide and electrophoretically transferred to nitrocellulose filters. One part of each gel (lanes f - k ) was stained with Indian ink, the other part (lanes a-e) was successively treated with anti-150-kDa rabbit IgG, biotinylated anti-rabbit IgG (donkey), and peroxidase-conjugated streptavidine. Finally, the blots were stained by incubation with enzyme substrate solution containing diaminobenzidine and H202. Lanes a, f." extract from 2.10 v synchronized wild-type cells harvested at the end of the growth period; lanes b, g: extract from 2.107 synchronized wild-type cells harvested during cell division; lanes c, h: extract from 2.10 v wild-type cells asynchronously grown under mixotrophic conditions; lanes d, i: extract from 5-107 CW-15 cells asynchronously grown under mixotrophic conditions; lanes e, j : extract from 1.107 wild-type cells asynchronously grown under autotrophic conditions; lane k: mixture of marker proteins (1-3 I~g each) phosphorylase (Mr 97000), bovine serum albumin (Mr 68 000), ovalbumin (Mr 46000), carbonic anhydrase (Mr 29000)
Fig. 7. Dot-immunobinding assays on chemically modified cell-wall polypeptides isolated from LiCl-extracts from intact wild-type C. reinhardii cells by gel exclusion chromatography. Each row was dotted with 3 gl of partially purified cell-wall potypeptides (protein concentration 50 gg.ml 1) which were either untreated (a), incubated for 2 h with sodium metaperiodate (b), treated with 2 mold-1 KOH for 2 h (c) or chemically degtycosylated with trifluoromethane-suifonic acid/anisol (d), Prior to use, the differentially pretreated polypeptides were extensively dialysed against 10 mmo1.1-x Tris-borate, pH 8.3, and adjusted to a protein concentration of 50 gg-ml ~. Each dot in a column was assayed with one of the different antibodies as indicated in the figure; pre = preimmune IgG
s e n c e o f c e l l - w a l l - d e r i v e d g l y c o p e p t i d e s ( F i g . 8). The same components were found to be labelled by the polyclonal antibodies raised against the 150 k D a , t h e 35 k D a a n d t h e 26 k D a c o m p o n e n t s ( F i g . 8). T h e s a m e l a b e l l i n g p a t t e r n w a s a l s o o b s e r v e d w h e n t h e a n t i b o d i e s a g a i n s t t h e 38 k D a a n d the 12kDa components were used (data not shown). Preincubation of the different antibodies
with cell-wall-derived glycopeptides reduced the a m o u n t s o f I g G m o l e c u l e s r e a c t i n g w i t h t h e LiC1extracted macromolecules. Staining of the bands was observed after a longer incubation period than in t h e c a s e o f u n t r e a t e d a n t i b o d i e s . L a b e l l i n g o f the various macromolecules by the antibodies a g a i n s t t h e 150 k D a a n d t h e 35 k D a c o m p o n e n t s w a s m u c h less a f f e c t e d b y t h e p r e i n c u b a t i o n w i t h
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 8. Effect of preincubation with cell-wall-derived glycopeptides on the reactivity of different antibodies with polypeptides present in the LiC1 extract of C. reinhardii wild-type cells. Macromolecules extracted by aqueous LiC1 from synchronously growing wild-type cells, which were harvested in the middle of the ceil-enlargement period, were separated by SDS-PAGE on slab gels containing 10% acrylamide and electrophoretically transferred to nitrocellulose paper. The strips were treated with different antibodies which were preincubated in the presence (+) or absence ( - ) of cell-wall-derived glycopeptides. Bound rabbit IgGs were detected as described in the legend of Fig. 6
cell-wall-derived glycopeptides than labelling by the a n t i b o d y against the 26 k D a c o m p o n e n t (Fig. 8). Labelling o f the LiCl-extracted polypeptides with the antibodies against the 38 k D a and the 12 k D a c o m p o n e n t s was suppressed a h n o s t completely when these antibodies were saturated with cell-wall-derived glycopeptides (data not shown). Therefore, the antibodies raised against the 38 kDa, the 26 k D a and the 12 k D a c o m p o nents, seem to be p r e d o m i n a n t l y reactive against the c a r b o h y d r a t e side chains, whereas the polyclonal antibodies against the 150 k D a and the 35 k D a c o m p o n e n t s are also reactive against the polypeptide backbone. F u r t h e r m o r e , these results indicate that all the immunologically related polypeptides present in the LiC1 extracts contain b o t h similar c a r b o h y d r a t e side chains a n d similar structures within the non-glycosylated stretches o f the polypeptide chains, because the labelling pattern by the antibodies against the 150 k D a and the 35 k D a c o m p o n e n t s was not changed by the prein-
381
Fig. 9. Immunoblot analysis of polypeptides present in purified cell walls from growing tobacco pollen tubes with antibodies to the 150-kDa cell-wall glycoprotein from C. reinhardii. Polypeptides extracted from two cell-wall preparation (lanes a, c and b, d) of growing tobacco pollen tubes were separated by SDS-PAGE on slab gels containing 15% (w/v) acrylamide and subsequently transferred electrophoretically to nitrocellulose filters. One part of each filter blot was stained with Indian ink (lanes a, b), the other part was probed using anti-150-kDa IgGs (lanes c, cO. Lane e. marker proteins (1 3 g each) phosphorylase (M~ 97000), bovine serum albumin (Mr 68000), ovalburain (Mr 46000), carbonic anhydrase (Mr 29000)
cubation o f the antibodies with the cell-wall-derived glycopeptides. Cross-reactivity o f antibodies against cell-wall glycoproteins f r o m C. reinhardii with a potypeptide present in the cell wall o f growing tobacco pollen tubes. Isolated cell walls f r o m growing tobacco pollen tubes were extracted with urea-SDS buffer and the extracted macromolecules submitted to Western-blot analysis using the a n t i b o d y against the 150 k D a cell-wall glycoprotein o f C. reinhardii (Fig. 9). Only one o f the different polypeptides (Fig. 9, slots a, b) was f o u n d to be reactive against the a n t i b o d y (Fig. 9, slots c, d). This cross-reacting polypeptide was estimated to have an Mr o f 75 000. Discussion Antibodies were raised against five putative cellwall glycoproteins which were isolated f r o m LiC1 extracts o f intact wild-type cells o f C. reinhardii.
382
All these antibodies specifically reacted with the cell surface of wild-type cells. They showed crossreactivity with the different antigens and were also found to be reactive against some other polypeptides present in the LiC1 extracts from intact wildtype cells, as shown by double-diffusion assays and immunoblot analyses. The corresponding antigens were largely missing in LiC1 extracts from the cellwall-deficient mutant CW-15. These findings indicate that the immunologically related polypeptides present in the LiC1 extracts from intact wild-type cells are intrinsic constituents of the cell wall of C. reinhardii. Investigating the cell-surface polypeptides of C. reinhardii by in-vivo vectoral labelling by glucose-oxidase-coupled lactoperoxidase-dependent 125I-iodination, M o n k et al. (1983) have also identified those polypeptides which are associated with the cell wall. Seventeen cell-wall-associated polypeptides with Mr values between 26000 and 280000 have been identified by these authors. Essentially the same components were found in cell walls shed into the culture medium by the C. reinhardii mutant imp-I (Matsuda et al. 1985) and in cell walls mechanically isolated from gametes of C. reinhardii (Imam et al. 1985). All these polypeptides were also found in the LiC1 extracts from intact wild-type cells (Voigt 1985a, 1986). The main components of the cell-wall-associated macromolecules described by M o n k et al. (1983), Matsuda et al. (1985) and Imam et al. (1985) were found to be more strongly labelled with [3H] proline than with [3s S] methionine (Voigt 1985 a, 1986) and were therefore assumed to be structural polypeptides of the wall. The pattern of these polypeptides which are more strongly labelled with [3H] proline than with [35S] methionine agrees very well with the pattern of immunologically related polypeptides present in the LiC1 extracts from C. reinhardii wild-type cells (this paper). All the obtained polyclonal antibodies predominantly reacted with the carbohydrate side chains of the analyzed glycoproteins. Therefore, the results of the present investigation primarily indicate that the glycosylation patterns of the immunologically related polypeptides are very similar. Only two of the five antibody preparations were found to be reactive against chemically deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides, they still recognized the same polypeptides as did the untreated antibodies. Reactivity of the other antibody preparations, on the contrary, was largely suppressed by preincubation with cell wall-derived glycopeptides. These findings indicate that the an-
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
tibodies against the 35kDa and the 150 kDa components also recognize non-glycosylated stretches of the polypeptide backbone, which are present in all the immunologically related cell-wall polypeptides. Therefore, the question arises whether all the immunologically related cell-wall glycoproteins are derived from a single polypeptide chain by differential post-translational modification or whether they contain different polypeptide chains which are encoded by a gene family. Goodenough etal. (1986) have isolated and characterized the high-molecular-weight cell-wall glycoproteins of Chlamydomonas reinhardii. The amino-acid compositions of these wall constituents were found to be rather similar. However, considerable differences were observed with respect to the proportions of histidine and hydroxyproline, indicating that the different cell-wall polypeptides of C. reinhardii are also encoded by different members of a gene family. This interpretation is supported by the observation that the pattern of immunologically related cell-wall glycoproteins is dependent on cell cycle and growth conditions (Figs. 6, 8). A relatively large number of different, but immunologically related salt-soluble cell-wall glycoproteins have been found in C. reinhardii (this paper). Analyses by SDS-PAGE of the polypeptides released by chemical glycosylation from the insoluble cell-wall component of C. reinhardii yielded a polypeptide pattern of very similar complexity (Vogeler et al. 1986). In higher plants, the number of salt-soluble cell-wall glycoproteins ( = extension precursors) is considerably lower (Smith 1981; Smith et al. 1984b, 1986). The extensins of carrot and tomato contain polypeptides whose amino-acid sequences show some similarities (Epstein and Lamport 1984; Smith et al. 1984b, 1986; Chen and Varner 1985 a, b). The molecular weights of the polypeptide chains, however, were found to be rather different. The cell wall of C. reinhardii contains both a glycoprotein whose molecular weight (35000) corresponds to that of the carrot extensin precursor (Smith 1981; Chen and Varner 1985a) and polypeptides whose molecular weights are comparable to those of the tomato extensin precursors (Smith et al. 1984b, 1986). Therefore, the question arises whether or not the cell-wall polypeptides of C. reinhardii are related to the extensins of higher plants. Recently, it has been reported that the prolyl hydroxylase of C. reinhardii which is required for the posttranslational processing of the cell-wall polypeptides has the same substrate specificity as the corresponding enzyme of higher plants (Tanaka
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
eta1. 1981; Erickson etal. 1984; Blankenstein et al. 1986). This enzyme only hydroxylates prolyl residues which are localized in oligo prolyl clusters. This finding is a first indication that the cell-wall glycoproteins of C. reinhardii contain amino-acid sequences which are similar to the extensins of higher plants. It is, therefore, an important observation that the cell wall of growing tobacco pollen tubes contains a single polypeptide ( M r 7 5 0 0 0 ) which cross-reacts with the antibodies raised against one of the cell-wall glycoproteins of C. reinhardii (Fig. 9). Efforts are presently being made to clarify the question of whether or not this polypeptide is a precursor of tobacco extensin. Furthermore, it has to be clarified whether the cross-reactivity with the antibodies against C. reinhardii cell-wall glycoproteins depends on a similar glycosylation pattern or whether this cross-reactivity is caused by homologous amino-acid sequences. I wish to thank Mrs. Petra Mfinzner for her excellent technical assistance and Mr. D. Janke for his generous gift of purified cell walls from growing tobacco pollen tubes. I am also indepted to Hans-Peter Vogeler for chemical deglycosylation of cell-wall glycoproteins and Dr. M. Meyberg and R. Kappler for their help during the immunofluorescence studies. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
References Blankenstein, P., Lang, W.C., Robinson, D.G. (1986) Isolation and characterization of prolyl hydroxylase from Chlamydomonas reinhardii. Planta 169, 238-244 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Catt, J.W., Hills, G.J., Roberts, K. (1976) A structural glycoprotein, containing hydroxyproline, isolated from the cell wall of Chlamydomonas reinhardii. Planta 131, 165-171 Cart, J.W., Hills, G.J., Roberts, K. (1978) Cell wall glycoproteins from Chlamydomonas reinhardii, and their self-assembly. Planta 138, 91-98 Chen, J., Varner, J.E. (1985a) Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein. Proc. Natl. Acad. Sci. USA 82, 4399-4403 Chen, J., Varner, J.E. (1985b) An extracellular matrix protein in plants: characterization of a genomic clone for carrot extensin. EMBO J. 4, 2145-2151 Chrispeels, M.J. (1969) Synthesis and secretion of hydroxyproline containing macromolecules in carrot. I. Kinetic analysis. Plant Physiol. 44, 1187-1193 Davies, D.R., Lyall, V. (1973) The assembly of a highly ordered component of the cell wall: The role of heritable factors and of physical structure. Mol. Gen. Genet. 124, 21 34 Davies, D.R., Plaskitt, A. (1971) Genetical and structural analysis of cell-wall formation in Chlamydomonas reinhardii. Genet. Res. 17, 33 43 Dubois, M.K., Gilles, K.A., Hamilton, J.K., Reber, P.A., Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356 Edge, A.S.B., Faltynek, C.R., Hof, L., Reichert, L.E., Weber,
383 P. (1981) Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal. Biochem. 118, 131-137 Epstein, L., Lamport, D.T.A. (1984) An intramolecular linkage involving isodityrosine in extensin. Phytochemistry 23, 1241 1246 Erickson, S.S., Fukudal, M., Varner, J.E. (1984) Specificity of prolyl hydroxylase from carrot root discs. (Abstr.) Plant Physiol. 75, Suppl., 28 Goodenough, U.W., Gebhart, B., Mecham, R.P., Heuser, J.E. (1986) Crystals of the Chlamydomonas reinhardii cell wall: polymerization, depolymerization and purification of glycoprotein monomers. J. Cell Biol. 103, 405-417 Goodenough, U., Heuser, J.E. (1985) The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quickfreeze, deep-etch technique. J. Cell Biol. 101, 1550-1568 Hawkes, R., Nidag, E., Gordon, J. (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142-147 Hills, G.J. (1973) Cell wall assembly in vitro from Chlamydomonas reinhardii. Planta 115, 12-23 Hills, G.J., Gurney-Smith, M., Roberts, K. (1973) Structure, composition, and morphogenesis of the cell wall of Chlamydomonas reinhardii. II. Electron microscopy and optical diffraction analysis. J. Ultrastruct. Res. 43, 179 192 Hills, G.J., Phillips, J.M., Gay, M.R., Roberts, K. (1975) Selfassembly of a plant cell wall in vitro. J. Mol. Biol. 96, 431-441 Imam, S.H., Buchanan, M.J., Shin, H.-C., Snell, W.J. (1985) The Chlamydomonas cell wall : Characterization of the wall framework. J. Cell Biol. 101, 1599 1607 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685 Lamport, D.T.A., Catt, J.W. (1981) Glycoproteins and enzymes of the cell wall. In: Encyclopedia of plant physiology, N.S., vol. 13B: Plant carbohydrates II, pp. 133-165, Tanner, W., Loewus, F.A., eds. Springer, Berlin Heidelberg New York Levy, H.B., Sober, H.A. (1960) A simple chromatographic method for preparation of gamma globulin. Proc. Soc. Exp. Biol. Med. 103, 250-252 Matsuda, Y., Saito, T., Yamaguchi, T., Kawase, H. (1985) Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardii is a metalloprotease and digests the sodium perchlorate insoluble component of cell walt. J. Biol. Chem. 260, 6373-6377 McNeil, M., Darvill, A.G., Fry, S.C., Albersheim, P. (1984) Structure and function of the primary cell walls of plant. Annu. Rev. Biochem. 33, 625-663 Mergenhagen, D. (1980) Die Kinetik der Zoosporenfreisetzung bei einem Mutantenstamm yon Chlamydomonas reinhardii. Mitt. Staatsinst. Allg. Bot. Hamburg 17, 18-26 Monk, B.C., Adair, W.S, Cohen, R.A., Goodenough, U.W. (1983) Topography of Chlamydomonas: Fine structure and polypeptide components of the gametic flagellar membrane surface and the cell wall. Planta 158, 517 533 Musgrave, A., de Witdt, P., Broekman, R., van den Ende, H. (1983) The cell wall of Chlamydomonas eugametos. Immunological aspects. Planta 158, 82-89 Ouchterlony, O. (1948) In vitro method for testing the toxinproducing capacity of diphtheria bacteria. Acta Pathol. Microbiol. Scand. 25, 186-191 Roberts, K. (1974) Crystalline glycoprotein walls of algae: their structure, composition, and assembly. Phil. Trans. R. Soc. London, Ser. B 268, 129-146 Roberts, K., Grief, C., Hills, G.J., Shaw, P.J. (1985) Cell wall glycoproteins: structure and function. J. Cell Sci. Suppl. 2, 105-127
384 Roberts, K., Gurney-Smith, M., Hills, G.J. (1972) Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. I. Ultrastructural and preliminary chemical analysis. J. Ultrastruct. Res. 40, 599-613 Smith, M.A. (]981) Characterization of carrot cell wall protein. I. The effect of ~,c(-dipyridyl on cell wall protein biosynthesis and secretion in incubated carrot discs. Plant Physiol. 68, 956-963 Smith, E., Roberts, K., Hutchings, A., Galfre, G. (1984a) Monoclonal antibodies to the major structural glycoprotein of the Chlamydornonas cell wall. Planta 161,330-338 Smith, J.J., Muldoon, E.P., Lamport, D.T.A. (1984b) Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures. Phytochemistry 23, 1233-1239 Smith, J.J., Muldoon, E.P., Willard, J.J., Lamport, D.T.A. (1986) Tomato extensin precursors P1 and P2 are highly periodic structures. Phytochemistry 25, 1021 1030 Stewart, D.A., Varner, J.E. (1980) Purification and characterization of a salt extractable hydroxyproline-rich glycoprotein from aerated carrot discs. Plant Physiol. 66, 787 792 Sueoka, N. (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardii. Proc. Natl. Acad. Sci. USA 46, 83-91 Surzycki, S. (1971) Synchronously grown cultures of Chlamydomonas reinhardii. Methods Enzymol. 23, 67-73 Tanaka, M., Sato, K., Uchido, T. (1981) Plant prolyl hydroxy-
J. Voigt: Cell-wall glycoproteins of Chlamydomonas lase recognizes poly(1-proline)II helix. J. Biol. Chem. 256, 2397 2400 Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354 Vogeler, H.-P., Voigt, J., Wachholz, I., Manshard, E., K6nig, W.A., Mix, M. (1986) Changes of the cell wall structure during the vegetative cell cycle of Chlamydomonas reinhardii. In: Cell walls '86, pp. 40-43, Vian, B., Reis, D., Goldberg, R., eds. Groupe Parois, Paris Voigt, J. (1984) A study of cross-links between the glycoprotein subunits within the insoluble inner cell wall layer of Chlamydomonas reinhardii. Mitt. Staatsinst. Allg. Bot. Hamburg 19, 83-98 Voigt, J. (1985 a) Extraction by lithium chloride of hydroxyproline-rich glycoproteins from intact cells of Chtamydomonas reinhardii. Ptanta 164, 379-389 Voigt, J. (1985b) Macromolecules released into the culture medium during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardii. Biochem. J. 266, 259-268 Voigt, J. (1986) Biosynthesis and turnover of cell wall glycoproteins during the vegetative cell cycle of Chlamydomonas reinhardii. Z. Naturforsch. 41c, 885-896 Received 29 May; accepted 20 August 1987
Planta (1988) 173:373 384
9 Springer-Verlag 1988
The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins Jfirgen Voigt Institut ffir Allgemeine Botanik und Botanischer Garten, Universitfit Hamburg, Ohnhorststrage 18, D-2000 Hamburg 52, Federal Republic of Germany
Abstract. Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiC1 extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiC1 extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiC1 extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiC1 extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cellwall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with Abbreviations: BSA = bovine serum albumin ; IgG = immunoglobulin G; k D a - k i l o d a l t o n ; M r : r e l a t i v e molecular mass; P B S - phosphate-buffered saline; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris=2-amino2-(hydroxymethyl)-l,3-propanediol
cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii. Key words: Antibody specificity - Cell wall - Chlamydomonas (cell wall glycoproteins) - Glycoprotein (cell wall).
Introduction The cell wall of the unicellular green alga Chlamydomonas reinhardii does not contain cellulose or other polysaccharides, but is composed of several layers of hydroxyproline-rich glycoproteins whose overall composition resemble those of the cell-wall glycoproteins of higher plants (Chrispeels 1969; Roberts 1974; Catt et al. 1976; Stewart and Varner 1980; Smith 1981 ; Lamport and Catt 1981 ; Smith et al. 1984b; McNeil etal. 1984; Roberts et al. 1985). Cell walls isolated from the culture medium of vegetatively growing C. reinhardii cells can be separated into a soluble and an insoluble fraction by treatment with chaotropic agents like LiC1 or NaC104 (Hills 1973; Hills etal. 1973; Roberts 1974). The insoluble cell-wall component and the NaC104-soluble glycoproteins were found to be
374
very similar with respect to amino-acid and sugar composition (Roberts 1974). The insoluble wall component, which can also be isolated from intact cells by extensive extractions with detergent-containing buffers (Voigt 1984), retains the overall saclike morphology of the wall, although it appears to be much thinner than the intact wall. Electron-microscopic examinations showed that this isolated cell-wall component is amorphous (Hills 1973; Hills et al. 1975) like the inner wall layer (W1) of intact cells (Roberts et al. 1972). Roberts and coworkers have therefore concluded that the inner wall layer is identical with the insoluble wall component (Cart et al. 1976, 1978; Roberts et al. 1985). The crystalline' outer wall layers are assumed to consist of salt-soluble cell-wall glycoproteins (Roberts 1974; Hills et al. 1975; Cart et al. 1976, 1978; Roberts et al. 1985). This assumption is, however, contradicted by the results of Goodenough and Heuser (t985) and my own data, which indicate that the insoluble cell-wall material is located in the central triplet of the wall. Pulse-labelling and pulse-chase experiments with [3t-I] proline and [asS] methionine demonstrated that the cell-wall glycoproteins, which can be solubilized by chaotropic salt solutions (Hills 1973; Hills etal. 1973; Roberts 1974; Voigt 1985a), are precursors of the insoluble wall layer (Voigt 1986). Extracts from isolated cell walls of C. reinhardii or C. eugametos contain high-molecular-weight glycoproteins (Davies and Lyall 1973; Roberts 1974; Catt et al. 1976; Musgrave et al. 1983; Smith et al. 1984a). Amino-acid analysis of these highmolecular-weight glycoproteins showed that they differ with respect to the proportion of hydroxyproline although the overall amino-acid compositions were found to be very similar (Goodenough et al. 1986). Recently, I have reported that the LiC1 extracts from intact wild-type cells contain some glycoproteins of considerably lower molecular weight which are missing in the LiC1 extracts from the cell-wall-deficient mutant CW-15 (Voigt 1985a). These polypeptides were found to be more strongly labelled with [3H] proline than with [35S] methionine (Voigt 1985a, 1986). Monk etal. (1983) have identified the same cell-wall-related polypeptides during their studies on the cell-surface polypeptides of C. reinhardii. Some of these cell-wall-related polypeptides have been purified to homogeneity and used to raise antibodies. These antibodies have been characterized with respect to their specificities against the polypeptide backbone and carbohydrate side
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
chains, and especially with respect to their crossreactivities with the different cell-wall glycoproteins of C. reinhardii. Material and methods Anti-rabbit immunoglobulin G (IgG) (biotinylated species-specific whole antibody from donkey), fluorescein-streptavidin and streptavidin-biotinylated horseradish peroxidase complex were purchased from Amersham-Buchler (Braunschweig, FRG). 3,3'-Diaminobenzidine was obtained from Sigma (Taufkirchen, FRG). All other chemicals (analytical grade) were purchased from Merck (Darmstadt, FRG) and Serva (Heidelberg, FRG).
Algal strains. The wild-type strain of Chlamydomonas reinhardii used in the present experiments was strain 137C (mating-type + ) obtained from Dr. R.P. Levine (Harvard University, Cambridge, MA, USA). The mutant strain "ls" (mating-type --) was isolated by Mergenhagen (1980), and the cell-wall-deficient mutant CW-15 (mating-type +), isolated by Davies and Plaskitt (1971), was obtained from Sammlung von Algenkulturen, G6ttingen, F R G (strain 83.8l). Media and growth conditions. Stock cultures were maintained on agar slants containing sodium acetate (2 g.1 -I) at 15~ and illuminated (4000 lx) using a 12 h light-12 h dark cycle. Cells were grown at 24 ~ C in high-salt medium (Sueoka 1960) with or without sodium acetate (2 g.l-~). The cultures were constantly bubbled with filtered air and constantly mixed by stirring bars. For asynchronous growth, the cells were continuously illuminated from the sides by a combination of white lamps (type 36W/25; Osram, Mfinchen, FRG) and "daylight" fluorescent lamps (Osram type 36W/11). The light flux was 10000 lx at the level of the cultures. Synchronous growth was achieved by the method of Surzycki (1971) using a 12 h light12 h dark illumination cycle. Cell concentrations were determined by duplicate hemocytometer counting. Cells were harvested at a cell density of 1. 1 0 6 - - 3 . 106 cells.ml 1.
Cellfractionation. The cultures were rapidly cooled to 0 ~ C and centrifuged at 6000 g for 10 rain. The culture medium was decanted immediately after centrifugation. The cells were twice washed and resuspended in high-salt medium to a final cell density of 1-108 cells-ml 1. Extraction of the polypeptide components of the LiCl-soluble wall layers was performed as recently described (Voigt 1985a) by addition of the same volume of ice-cold LiC1 (4 tool-1 1), incubation at 0 ~ C for 30 min and centrifugation at 10000-g for 20 rain. The supernatant was collected and used to isolate the salt-soluble cell-wall glyeoproreins. The insoluble cell-wall component was purified from the LiCl-extracted cells as recently described (Voigt 1984, 1985b). Gel exclusion chromatography. Gel exclusion chromatography of crude LiC1 extract from intact cells was performed on columns (2.5 cm i.d., 100 cm long) of Sepharose C1-4B, Sephacryl S-300 and Sephadex G-75, equilibrated with LiCI (1 m o l d - t ) 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-HC1 (20 mmol.1-1), pH 7.2. The crude LiC1 extract of intact cells was concentrated in dialysis bags with Aquacide II (Calbiochem, Marburg, FRG) prior to column chromatography. Treatment with Aquacide II had to be interrupted by dialysis against 0.5 mol-1 t LiC1 to concentrate the LiC1 extract efficiently. The columns were etuted with equilibration buffer. Fractions of 10 ml were collected and analyzed for absorbance at 280 nm, for protein concentration (according to Bradford 1976) and protein composition by sodium dodecyl sulfate
J. Voigt: Cell-wall glycoproteins of Chlamydomonas (SDS)-polyacrylamide gel electrophoresis (PAGE) (according to Laemmli 1970).
Sodium dodecyl su(fate-polyacrylamide gel electrophoresis. Analytical or preparative SDS-PAGE were performed on 14.12.0.:15 cm 3 slab gels according to Laemmli (1970). To obtain a sufficient separation of the glycoproteins, an urea-containing sample buffer had to be used as described by Voigt (1985a). The protein fractions were adjusted to 10% trichloroacetic acid (TCA) and incubated for 2 h at 4 ~ C. The precipitates were collected by centrifugation for :15 rain at 15000-g, twice washed with bidistilled water and redissolved overnight in a buffer consisting of urea (8 tool-1-1), SDS (2%, w/v), ethylenediamine tetraacetic acid (EDTA) (10 mmol.1-1), 2-mercaptoethanol (200 retool-l-i) and Tris-HC1 (20 retool.l-1, pH 7.5). After addition of sample buffer (1/4 volume), the protein mixtures were submitted to SDS-PAGE on slab gels according to Laemmli (1970). When the tracking dye, bromophenol blue, reached the front of the gel, electrophoresis was stopped. Analytical gel slabs were then stained with Coomassie Brillant Blue R-250. In the case of preparative gels, strips were cut from both sides of the gel slabs and stained with Coomassie Brillant Blue R-250. The main part of each gel slab was stored at 4 ~ C without acid treatment. Protein bands were cut from these untreated parts of the gel slabs according to the protein patterns of stained gel strips. The polyacrylamide pieces were homogenized in a buffer containing 0.1% (w/v) SDS and Tris-HC1 (20 mmol. 1-l, pH 8.0), and the homogenates filled into dialysis bags. The polypeptides were then eluted electrophoretically at 6 V.cm -1 overnight at room temperature using the electrophoresis buffer according to Laemmli (1970). The polypeptide solutions were concentrated against Aquacide II and stored at --20 ~ C. The eluted polypeptides were routinely checked for purity by analytical SDS-PAGE.
Preparation of antibodies. Antibodies against the different polypeptides were raised in rabbits. Aliquots of the polypeptide solutions corresponding to 20-50 tag protein were diluted with 1 nrl phosphate-buffered saline (PBS) and mixed with 1 ml Freund's complete adjuvans (Calbiochem). Injections were performed every two weeks intraperitoneally and subcutaneously at multiple sites over a period of three to four months. Serum samples were taken 6 d after each booster injection starting two months after the primary injection and immunization controlled by Ouchterlony double-diffusion tests in 1% agarose gels prepared in PBS, pH 7.2 (Ouchterlony 1948). When the double-diffusion assay was positive, the animals were bled and the immune sera fractionated by differential (Ntt4)2SO 4 precipitation and by passing the redissolved and dialysed protein through a colunm of Whatman DE-52 equilibrated with :10 retool-1-1 sodium phosphate, pH 6.8 (Levy and Sober 1960). The unbound material was concentrated by (NH4)2 SOs precipitation; the redissolved precipitate was extensively dialysed against 0.9% (w/v) NaCI containing 10 retool. 1 1 sodium phosphate, pH 7.2, and stored at - 2 0 ~ C. Preparation of cell-wall-derived glycopeptides. Cell-wall glycopeptides were prepared by successive digestion with pronase E and proteinase K of polypeptide mixtures extracted from intact ceils of C. reinhardii with aqueous LiCI (Voigt :1985a). The LiCI extracts of intact wild-type cells were dialysed overnight at 4 ~ C against :100 vol. of a buffer containing NaC1 (150 m m o l . l - 1), E D T A (10 mmol-1-1) and Tris-HC1 (20mmol'1-1, pH7.5). The suspension was concentrated against Aquacide II and then dialysed against the same buffer. The polypeptides were then incubated with :1 rag. m l - i pronase
375 E (Sigma, Dreieich, FRG) for 30 rain at 55 ~ C, followed by digestion with 2 mg.ml -~ proteinase K (Merck) at 37~ for 6 h. The reaction mixtures were then adjusted to 10% (w/v) trichloroacetic acid. After 2 h at 0 ~ C, the precipitated protein components were removed by centrifugation at 20000.g for 30 min. The supernatant was passed through a column (100 cm long, 2.5 cm i.d.) of Biogel P :10 (Bio Rad, Mfinchen, FRG) equilibrated with acetic acid (0.1 mol.l-1). The columns were eluted with the same solvent. Fractions of 5 ml were collected and the pH values measured. Aliquots were hydrolysed with 80% (v/v) H2SO~ for 10 rain at 100~ and the hydrotysates analyzed for sugars by the phenol-sulfuric acid method (Dubois et al. 1956). Neutral fractions containing glycopeptides were combined and freeze-dried.
Chemical modification of cell-wall glycoproteins. Partially purified cell-wall glycoproteins which were obtained by gel exclusion chromatography of LiC1 extracts from intact C. reinhardii wild-type cells were used as starting material for the preparation of chemically modified cell-wall glycoproteins. (i) Periodate oxidation. A solution of partially purified cell-wall glycoproteins (protein concentration 0.2mg.m1-1) containing LiCI (1 mol.1-1) and Tris-borate (10 retool-1-1, pH 8.3) was mixed with the same volume of a solution of sodium metaperiodate (40 mmol-1-1) and incubated for 2 h at 0 ~ C in the dark. The reaction mixture was then diluted with Tris-borate (10 mmol. 1 1, pH 8.3), extensively dialysed against the same buffer and concentrated against Aquacide II. (ii) MiM alkaline hydrolysis: A solution of partially purified cell-wall glycoproteins (protein concentration 0.2 m g m l - i ) was adjusted to 2 tool-1-1 KOH and incubated at room temperature for 2 h. The solution was neutralized by addition of HC1 and extensively dialysed against a buffer containing 0.1% (w/v) SDS and Tris-borate (10 mmol.1-1, pH 8.3). (iii) Deglycosylation: 2 mg freeze-dried cell-wall glycoproteins were chemically deglycosylated by the method of Edge et at. (1981) using trifluoromethane-sulfonic acid and anisol. After 4 h, the reaction was stopped and reagents and sugars removed by method A of Edge et al. (1981). The proteins were dialysed against 2 mmol-1-1 pyridine acetate (pH 5.5), lyophilised and redissolved in Tris-borate (10 retool. t - i , pH 8.3). Immunoblot analysis. Analytical SDS-PAGE was performed as described above. The gels were then placed adjacent to a sheet of nitrocellulose (BA 85, Schleicher & Schfill, Dassel, FRG) and electrophoretically blotted for 18 h at 15 V, 300 mA (Towbin et al. 1979). The nitrocellulose sheet was then dried, washed with PBS, blocked with 3% (w/v) bovine serum albumin (BSA) in PBS and successively incubated for 16 h at 4 ~ C with rabbit immune IgG (diluted l:100 with 1% BSA-PBS), biotinylated donkey anti-rabbit-IgG serum (diluted 1:200 with 1% BSAPBS) and for I h at room temperature with preformed streptavidin-horseradish-peroxidase complex (diluted 1:200 with 1% BSA-PBS). After each treatment, the filter blots were washed four times with 1% (v/v) NP40 in PBS for 30 rain at room temperature. Colour was developed by the method of Hawkes et al. (1982) using 3,3'-diaminobenzidine in the presence of COC12. Dot-immunobinding assays were performed on nitrocellulose filters containing 10 ~tg of untreated or chemically modified cell-wall polypeptides per spot. The filters were processed as described for the Western-blot analysis.
Immunofluorescence studies. Chlamydomonas reinhardii cells were fixed with 4% (w/v) formaldehyde in PBS for 4 h at 4 ~ C and washed four times with PBS. Formaldehyde-fixed cells or purified insoluble wall material were incubated with 3% (w/v)
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
376
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BSA in PBS overnight at 4 ~ C and then treated 16-20 h with either preimmune serum or antibodies (50 ~tg rabbit IgG per ml J% BSA-PBS). After washing with 1% NP40-PBS (four times), the samples were successively incubated for 4 h at room temperature with biotinylated donkey anti-rabbit IgG serum (diluted 1 : 200 with 1% BSA-PBS) and fluorescein isothiocyanate (FITC)-conjugated streptavidine (diluted 1:100 with J% BSA-PBS). After each treatment, the samples were washed four times with 1% (v/v) NP40 in PBS. Samples were examined using a Zeiss epifluorescence optics with filter set 10 for FITC (Zeiss, Oberkochen, FRG).
Results
Isolation of cell-wall glycoproteins from LiCl extraets of intact wild-type cells. T o o b t a i n the saltsoluble cell-wall g l y c o p r o t e i n s , LiC1 e x t r a c t s f r o m i n t a c t w i l d - t y p e cells were f r a c t i o n a t e d b y gel ex-
90
Fig. 1 A, B. Fractionation of LiC1 extracts from intact cells of C. reinhardii by gel exclusion chromatography on a Sephadex G-75 column equilibrated with 2 mol. 1-1 LiC1 buffered with 20 mmol-1 1 Tris-HC1, pH 7.2. A Elution profile. Eluate fractions were analyzed for absorbance at 280 nm ( ) and protein concentration (. . . . . ). B Sodium dodecyl sulfate-PAGE of column fractions. Aliquots (200 gl) from every second column fraction were analyzed by SDS-PAGE on slab gels containing 17% acrylamide. The gels were stained with Coomassie Brillant Blue. a, b-marker proteins (1(~20 ~tg each) bovine serum albumin (Mr 68000), 3-phosphoglycerate kinase (Mr 47000), aldolase ( M r 40000), glyceraldehyde-3-phosphate dehydrogenase (Mr 36000), hemoglobin (Mr 15 600)
c l u s i o n c h r o m a t o g r a p h y . T h e best results were achieved with Sephadex G-75 columns equilibrated a n d eluted w i t h a b u f f e r c o n t a i n i n g 2 m o l " 1- t LiC1 (Fig. 1 A). E l u a t e f r a c t i o n s were a n a l y s e d f o r p r o tein c o m p o s i t i o n b y S D S - P A G E (Fig. 1 B). T h e h i g h - m o l e c u l a r - w e i g h t cell-wall g l y c o p r o t e i n s were eluted in the v o i d v o l u m e t o g e t h e r w i t h s o m e smaller p o l y p e p t i d e s [relative m o l e c u l a r m a s s ( M r ) = 6 6 , 35, 26 a n d 12 k i l o d a l t o n s (kDa)]. C h r o m a t o g r a p h y o n S e p h a r o s e C1-4B c o l u m n s yielded a similar e l u t i o n p a t t e r n , the h i g h - m o l e c u l a r - w e i g h t cell-wall g l y c o p r o t e i n s a n d the a s s o c i a t e d smaller p o l y p e p t i d e s being eluted in a f r a c t i o n with a n M r o f 300000. W h e n LiC1 extracts f r o m s t a t i o n a r y cells w e r e f r a c t i o n a t e d b y gel e x c l u s i o n c h r o m a t o g r a p h y , a n a d d i t i o n a l p o l y p e p t i d e o f 38 k D a w a s f o u n d t o be a s s o c i a t e d w i t h the h i g h - m o l e c u l a r -
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 2. Analytical SDS-PAGE of the polypeptides used to raise antibodies. The polypeptides were isolated from LiC1 extracts of intact C. reinhardii cells by gel exclusion chromatography (Fig. 1) and subsequent preparative SDS-PAGE. a = 150-kDa glycoprotein; b = 38-kDa glycoprotein; e = 35-kDa glycoprotein ; d = 26-kDa component; e = 12-kDa component; f = mixture of marker proteins (5-15 gg each) myosin (Ms 205000), fl-galactosidase (Mr 116000), phospborylase b (Mr 97000), bovine serum albumin (Mr 68000), ovalbumin (Mr 45000), carbonic anhydrase (Mr 29 000)
weight cell-wall glycoproteins. One of the high-molecular-weight glycoproteins and some of the smaller polypeptides were further purified by preparative SDS-PAGE. An analytical SDS-PAGE of these purified polypeptides, which were used to raise antibodies, is shown in Fig. 2. Characterisation of antibodies. Antibodies against the five isolated polypeptides were raised in rabbits and preliminarily analyzed by double-diffusion assays with crude LiC1 extracts of intact wild-type cells (Fig. 3). Independent of the arrangement of the different antibodies in the peripheral wells, one fused precipitation line was observed for all the antibody preparations (wells I-5) but not for the preimmune serum (well 6). In the case of the antibodies raised against the 12-kDa and the 26-kDa component, respectively, an additional precipitation line was found which did not fuse with the precipitation line formed by the other antibodies, indicating that these two antibodies are not monospecific. To ascertain that the different antibodies really react specifically with the cell surface of C. reinhar-
377
Fig. 3. Double diffusion of antibodies raised against different isolated polypeptides with crude LiC1 extract from C. reinhardii wild-type cells. Centre well: unfractionated LiC1 extract; outer wells: l = a n t i - 1 2 k D a IgG, 2 = a n t i - 2 6 k D a IgG, 3=anti35 kDa IgG, 4=anti-38 kDa IgG, 5-anti-150 kDa IgG, 6 = preimmune IgG
dii, formaldehyde-fixed wild-type cells were incubated with preimmune IgG or with the different antibody preparations. The bound rabbit IgG molecules were detected by immunofluorescence after successive incubations with biotinylated speciesspecific anti-rabbit IgG antibodies from donkey and fluorescein-conjugated streptavidine. As shown in Fig. 4, cell-surface-bound rabbit IgG molecules could be detected by immunofluorescence when the cells were incubated with the antibody against the 150-kDa cell-wall glycoprotein (C, D). Cells incubated with preimmune serum, on the other hand, did not bind rabbit IgG (Fig. 4 A, B), indicating that the binding of the antibodies to the cell surface of C. reinhardii is the result of specific antigen-antibody interactions. Binding of rabbit IgGs was also detected, when the cells were incubated with the other antibody preparations. No immunofluorescence was observed when the purified insoluble wall component was analyzed (data not shown). Furthermore, the cell surface of the cell-wall-deficient mutant CW-I 5 did not react with any of the antibodies (data not shown).
378
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 4A-D. Indirect
immunofluorescence staining of formaldehyde-fixed C. reinhardii cells using preimmune IgG (A, B) or anti-150 kDa IgG (C, D). A, C: fluorescein optics to visualize bound rabbit IgGs; B, D: phase contrast. Bar = 10 pm
Double-diffusion assays performed with purified polypeptides demonstrated that all the antibodies showed cross-reactivity with the different antigens and were found to be also reactive against some other polypeptides present in the LiC1 extracts from intact wild-type cells (Fig. 5). However, differences were observed with respect to the relative intensities of the precipitation lines when constant amounts of the antibodies were used (Fig. 5). This finding indicates that in spite of the crossreactivities the different antibodies do not have exactly the same specificities. Immunoblot analyses likewise showed that the LiC1 extracts from intact wild-type cells contained several polypeptide coinponents which cross-reacted with the different antibodies (Figs. 6, 8). The pattern of antigens present in the LiC1 extracts was found to be dependent on growth conditions and cell-cycle stage (Fig. 6, 8). The predominant part of the polypeptides present in the LiC1 extracts did not react with the antibodies (Fig. 6). The LiC1 extracts from intact cells of the cell-wall-deficient mutant CW-15 contained extremely low levels of cross-reacting polypeptides. Large quantities of LiCl-extracted macromolecules from this mutant had to be submitted to Westernblot analysis in order to detect these antigens (Fig. 6, slots d and i). Only two cross-reacting polypeptides of Mr 32 k D a and 27 kDa were found
in LiC1 extracts from CW-15 cells (Fig. 6, slot d). These findings indicate that the antibodies have a specificity for a limited number of polypeptides present in the LiC1 extracts from intact wild-type cells and that these cross-reacting polypeptides are located in the cell wall and- or the periplasmic space.
Binding of antibodies to chemically modified cellwalIpolypeptides. Dot-immunobinding assays with chemically modified cell-wall polypeptides were performed to investigate the specificities of the different antibodies (Fig. 7). The cell-wall material used for these studies was isolated by gel exclusion chromatography of LiC1 extracts from intact wildtype cells (Fig. 1), The polypeptide mixture eluted in the void volume of a Sephadex G-75 column was desalted and spotted onto nitrocellulose filters either without pretreatment (row a) or after treatment with sodium metaperiodate (row b), treatment with 2 M K O H (row c) or chemical deglycosylation (row d). After incubation with 3% BSAPBS, the nitrocellulose filters were analyzed with the different antibodies. Treatment of the cell-wall polypeptides with sodium metaperiodate did not appreciably reduce the binding of the different antibodies (row b). Preincubation of the cell-wall polypeptides with 2 mol.1 1 K O H reduced the bind-
J. Voigt: Cell-wail glycoproteins of Chlamydomonas
379
Fig. 5. Double-diffusion assays of antibodies raised against different putative cell-wall polypeptides of C. reinhardii with several polypeptide components isolated from LiC1 extracts of intact wild-type cells. The polypeptides were isolated from the LiCI extracts by gel exclusion chromatography (Fig. I) and subsequent preparative SDS-PAGE. Atiquots (40 ~1) of the isolated polypeptides (protein concentration 0.1 0.3 rag. ml-1) were placed into the central wells. a = 280-kDa glycoprotein; b = 150-kDa glycoprotein; c - 97-kDa polypeptide; d= 66-kDa polypeptide; e = 56-kDa polypeptide; f = 43-kDa polypeptide; g = 38-kDa glycoprotein; h = 35kDa glycoprotein; i=26-kDa component. The antibody loads (40 gl; protein concentration 5 mg.ml-1) in the peripheral wells were: 1 =preimmune IgG; 2=anti-12 kDa IgG; 3=anti-26 kDa IgG; 4=anti-35 kDa IgG: 5=anti-38 kDa IgG; 6=anti-150 kDa IgG
ing capacity for the a n t i b o d y against the 1 5 0 - k D a g l y c o p r o t e i n a n d d e s t r o y e d the antigenic sites for the a n t i b o d y against the 2 6 k D a c o m p o n e n t , whereas the binding o f the o t h e r antibodies was essentially not affected (row c). Only the antibodies raised against the 150 k D a glycoprotein a n d the 3 5 - k D a c o m p o n e n t were able to recognize the chemically deglycosylated cell-wall p o l y p e p t i d e s (row d). T h e binding capacity for b o t h antibodies,
however, was strongly reduced by chemical deglycosylation o f the cell-wall polypeptides.
Specificities o f antibodies saturated with cell-wallderived glycopeptides. Nitrocellulose filters containing m a c r o m o l e c u l e s , which were extracted by a q u e o u s LiC1 f r o m C. reinhardii wild-type cells a n d s e p a r a t e d by S D S - P A G E , were p r o b e d with different antibodies p r e i n c u b a t e d in the presence or ab-
380
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 6. Immunoblot analysis of LiCl-extracted polypeptides from C. reinhardii wild-type strain 137C and the cell-wall-deficient mutant CW-15 using anti-150-kDa antibodies. Macromolecules extracted by treatment with aqueous LiC1 from intact C. reinhardii wild-type or CW-15 cells were separated by SDS-PAGE on slab gels containing 15 % acrylamide and electrophoretically transferred to nitrocellulose filters. One part of each gel (lanes f - k ) was stained with Indian ink, the other part (lanes a-e) was successively treated with anti-150-kDa rabbit IgG, biotinylated anti-rabbit IgG (donkey), and peroxidase-conjugated streptavidine. Finally, the blots were stained by incubation with enzyme substrate solution containing diaminobenzidine and H202. Lanes a, f." extract from 2.10 v synchronized wild-type cells harvested at the end of the growth period; lanes b, g: extract from 2.107 synchronized wild-type cells harvested during cell division; lanes c, h: extract from 2.10 v wild-type cells asynchronously grown under mixotrophic conditions; lanes d, i: extract from 5-107 CW-15 cells asynchronously grown under mixotrophic conditions; lanes e, j : extract from 1.107 wild-type cells asynchronously grown under autotrophic conditions; lane k: mixture of marker proteins (1-3 I~g each) phosphorylase (Mr 97000), bovine serum albumin (Mr 68 000), ovalbumin (Mr 46000), carbonic anhydrase (Mr 29000)
Fig. 7. Dot-immunobinding assays on chemically modified cell-wall polypeptides isolated from LiCl-extracts from intact wild-type C. reinhardii cells by gel exclusion chromatography. Each row was dotted with 3 gl of partially purified cell-wall potypeptides (protein concentration 50 gg.ml 1) which were either untreated (a), incubated for 2 h with sodium metaperiodate (b), treated with 2 mold-1 KOH for 2 h (c) or chemically degtycosylated with trifluoromethane-suifonic acid/anisol (d), Prior to use, the differentially pretreated polypeptides were extensively dialysed against 10 mmo1.1-x Tris-borate, pH 8.3, and adjusted to a protein concentration of 50 gg-ml ~. Each dot in a column was assayed with one of the different antibodies as indicated in the figure; pre = preimmune IgG
s e n c e o f c e l l - w a l l - d e r i v e d g l y c o p e p t i d e s ( F i g . 8). The same components were found to be labelled by the polyclonal antibodies raised against the 150 k D a , t h e 35 k D a a n d t h e 26 k D a c o m p o n e n t s ( F i g . 8). T h e s a m e l a b e l l i n g p a t t e r n w a s a l s o o b s e r v e d w h e n t h e a n t i b o d i e s a g a i n s t t h e 38 k D a a n d the 12kDa components were used (data not shown). Preincubation of the different antibodies
with cell-wall-derived glycopeptides reduced the a m o u n t s o f I g G m o l e c u l e s r e a c t i n g w i t h t h e LiC1extracted macromolecules. Staining of the bands was observed after a longer incubation period than in t h e c a s e o f u n t r e a t e d a n t i b o d i e s . L a b e l l i n g o f the various macromolecules by the antibodies a g a i n s t t h e 150 k D a a n d t h e 35 k D a c o m p o n e n t s w a s m u c h less a f f e c t e d b y t h e p r e i n c u b a t i o n w i t h
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
Fig. 8. Effect of preincubation with cell-wall-derived glycopeptides on the reactivity of different antibodies with polypeptides present in the LiC1 extract of C. reinhardii wild-type cells. Macromolecules extracted by aqueous LiC1 from synchronously growing wild-type cells, which were harvested in the middle of the ceil-enlargement period, were separated by SDS-PAGE on slab gels containing 10% acrylamide and electrophoretically transferred to nitrocellulose paper. The strips were treated with different antibodies which were preincubated in the presence (+) or absence ( - ) of cell-wall-derived glycopeptides. Bound rabbit IgGs were detected as described in the legend of Fig. 6
cell-wall-derived glycopeptides than labelling by the a n t i b o d y against the 26 k D a c o m p o n e n t (Fig. 8). Labelling o f the LiCl-extracted polypeptides with the antibodies against the 38 k D a and the 12 k D a c o m p o n e n t s was suppressed a h n o s t completely when these antibodies were saturated with cell-wall-derived glycopeptides (data not shown). Therefore, the antibodies raised against the 38 kDa, the 26 k D a and the 12 k D a c o m p o nents, seem to be p r e d o m i n a n t l y reactive against the c a r b o h y d r a t e side chains, whereas the polyclonal antibodies against the 150 k D a and the 35 k D a c o m p o n e n t s are also reactive against the polypeptide backbone. F u r t h e r m o r e , these results indicate that all the immunologically related polypeptides present in the LiC1 extracts contain b o t h similar c a r b o h y d r a t e side chains a n d similar structures within the non-glycosylated stretches o f the polypeptide chains, because the labelling pattern by the antibodies against the 150 k D a and the 35 k D a c o m p o n e n t s was not changed by the prein-
381
Fig. 9. Immunoblot analysis of polypeptides present in purified cell walls from growing tobacco pollen tubes with antibodies to the 150-kDa cell-wall glycoprotein from C. reinhardii. Polypeptides extracted from two cell-wall preparation (lanes a, c and b, d) of growing tobacco pollen tubes were separated by SDS-PAGE on slab gels containing 15% (w/v) acrylamide and subsequently transferred electrophoretically to nitrocellulose filters. One part of each filter blot was stained with Indian ink (lanes a, b), the other part was probed using anti-150-kDa IgGs (lanes c, cO. Lane e. marker proteins (1 3 g each) phosphorylase (M~ 97000), bovine serum albumin (Mr 68000), ovalburain (Mr 46000), carbonic anhydrase (Mr 29000)
cubation o f the antibodies with the cell-wall-derived glycopeptides. Cross-reactivity o f antibodies against cell-wall glycoproteins f r o m C. reinhardii with a potypeptide present in the cell wall o f growing tobacco pollen tubes. Isolated cell walls f r o m growing tobacco pollen tubes were extracted with urea-SDS buffer and the extracted macromolecules submitted to Western-blot analysis using the a n t i b o d y against the 150 k D a cell-wall glycoprotein o f C. reinhardii (Fig. 9). Only one o f the different polypeptides (Fig. 9, slots a, b) was f o u n d to be reactive against the a n t i b o d y (Fig. 9, slots c, d). This cross-reacting polypeptide was estimated to have an Mr o f 75 000. Discussion Antibodies were raised against five putative cellwall glycoproteins which were isolated f r o m LiC1 extracts o f intact wild-type cells o f C. reinhardii.
382
All these antibodies specifically reacted with the cell surface of wild-type cells. They showed crossreactivity with the different antigens and were also found to be reactive against some other polypeptides present in the LiC1 extracts from intact wildtype cells, as shown by double-diffusion assays and immunoblot analyses. The corresponding antigens were largely missing in LiC1 extracts from the cellwall-deficient mutant CW-15. These findings indicate that the immunologically related polypeptides present in the LiC1 extracts from intact wild-type cells are intrinsic constituents of the cell wall of C. reinhardii. Investigating the cell-surface polypeptides of C. reinhardii by in-vivo vectoral labelling by glucose-oxidase-coupled lactoperoxidase-dependent 125I-iodination, M o n k et al. (1983) have also identified those polypeptides which are associated with the cell wall. Seventeen cell-wall-associated polypeptides with Mr values between 26000 and 280000 have been identified by these authors. Essentially the same components were found in cell walls shed into the culture medium by the C. reinhardii mutant imp-I (Matsuda et al. 1985) and in cell walls mechanically isolated from gametes of C. reinhardii (Imam et al. 1985). All these polypeptides were also found in the LiC1 extracts from intact wild-type cells (Voigt 1985a, 1986). The main components of the cell-wall-associated macromolecules described by M o n k et al. (1983), Matsuda et al. (1985) and Imam et al. (1985) were found to be more strongly labelled with [3H] proline than with [3s S] methionine (Voigt 1985 a, 1986) and were therefore assumed to be structural polypeptides of the wall. The pattern of these polypeptides which are more strongly labelled with [3H] proline than with [35S] methionine agrees very well with the pattern of immunologically related polypeptides present in the LiC1 extracts from C. reinhardii wild-type cells (this paper). All the obtained polyclonal antibodies predominantly reacted with the carbohydrate side chains of the analyzed glycoproteins. Therefore, the results of the present investigation primarily indicate that the glycosylation patterns of the immunologically related polypeptides are very similar. Only two of the five antibody preparations were found to be reactive against chemically deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides, they still recognized the same polypeptides as did the untreated antibodies. Reactivity of the other antibody preparations, on the contrary, was largely suppressed by preincubation with cell wall-derived glycopeptides. These findings indicate that the an-
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
tibodies against the 35kDa and the 150 kDa components also recognize non-glycosylated stretches of the polypeptide backbone, which are present in all the immunologically related cell-wall polypeptides. Therefore, the question arises whether all the immunologically related cell-wall glycoproteins are derived from a single polypeptide chain by differential post-translational modification or whether they contain different polypeptide chains which are encoded by a gene family. Goodenough etal. (1986) have isolated and characterized the high-molecular-weight cell-wall glycoproteins of Chlamydomonas reinhardii. The amino-acid compositions of these wall constituents were found to be rather similar. However, considerable differences were observed with respect to the proportions of histidine and hydroxyproline, indicating that the different cell-wall polypeptides of C. reinhardii are also encoded by different members of a gene family. This interpretation is supported by the observation that the pattern of immunologically related cell-wall glycoproteins is dependent on cell cycle and growth conditions (Figs. 6, 8). A relatively large number of different, but immunologically related salt-soluble cell-wall glycoproteins have been found in C. reinhardii (this paper). Analyses by SDS-PAGE of the polypeptides released by chemical glycosylation from the insoluble cell-wall component of C. reinhardii yielded a polypeptide pattern of very similar complexity (Vogeler et al. 1986). In higher plants, the number of salt-soluble cell-wall glycoproteins ( = extension precursors) is considerably lower (Smith 1981; Smith et al. 1984b, 1986). The extensins of carrot and tomato contain polypeptides whose amino-acid sequences show some similarities (Epstein and Lamport 1984; Smith et al. 1984b, 1986; Chen and Varner 1985 a, b). The molecular weights of the polypeptide chains, however, were found to be rather different. The cell wall of C. reinhardii contains both a glycoprotein whose molecular weight (35000) corresponds to that of the carrot extensin precursor (Smith 1981; Chen and Varner 1985a) and polypeptides whose molecular weights are comparable to those of the tomato extensin precursors (Smith et al. 1984b, 1986). Therefore, the question arises whether or not the cell-wall polypeptides of C. reinhardii are related to the extensins of higher plants. Recently, it has been reported that the prolyl hydroxylase of C. reinhardii which is required for the posttranslational processing of the cell-wall polypeptides has the same substrate specificity as the corresponding enzyme of higher plants (Tanaka
J. Voigt: Cell-wall glycoproteins of Chlamydomonas
eta1. 1981; Erickson etal. 1984; Blankenstein et al. 1986). This enzyme only hydroxylates prolyl residues which are localized in oligo prolyl clusters. This finding is a first indication that the cell-wall glycoproteins of C. reinhardii contain amino-acid sequences which are similar to the extensins of higher plants. It is, therefore, an important observation that the cell wall of growing tobacco pollen tubes contains a single polypeptide ( M r 7 5 0 0 0 ) which cross-reacts with the antibodies raised against one of the cell-wall glycoproteins of C. reinhardii (Fig. 9). Efforts are presently being made to clarify the question of whether or not this polypeptide is a precursor of tobacco extensin. Furthermore, it has to be clarified whether the cross-reactivity with the antibodies against C. reinhardii cell-wall glycoproteins depends on a similar glycosylation pattern or whether this cross-reactivity is caused by homologous amino-acid sequences. I wish to thank Mrs. Petra Mfinzner for her excellent technical assistance and Mr. D. Janke for his generous gift of purified cell walls from growing tobacco pollen tubes. I am also indepted to Hans-Peter Vogeler for chemical deglycosylation of cell-wall glycoproteins and Dr. M. Meyberg and R. Kappler for their help during the immunofluorescence studies. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
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