Planta (1984)162:506--517
P l a n t a 9 Springer-Verlag 1984
Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga, Fucus V. Vreeland, M. Slomich* and W.M. Laetsch Department of Botany, University of California, Berkeley, CA 94720, USA
Abstract. Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immuno9 globulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function. Key words: Carbohydrate antigens (alginate, fucan) - Cell wall - Immunofluorescence - Fucus Monoclonal antibody - Phaeophyta.
Introduction
There is a growing awareness that carbohydrates carry specific information in their structures (for a U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA
* Present address:
EDTA = ethylenediaminetetraacetic acid; EIA = enzyme immunoassay; Ig = immunoglobulin (IgG, IgM and IgA are immunoglobulin types) Abbreviations:
review, see Preston 1977), and that the functions of carbohydrates depend on their conformations (Preston 1979; Rees et al. 1982). Interactions between, and modifications of, tertiary structures cause functional changes in complex carbohydrates from both plants and animals (Preston 1979; Rees et al. 1982). Carbohydrates of brown algae are complex and variable in structure. Alginate and fucan have no regularly repeating subunits. These acidic polysaccharides consist of populations of molecules varying in size, composition and sugar arrangement. Fucans are a family of branched carbohydrates containing sulfated fucose, xylose, glucuronate, a small amount of protein and often galactose, mannose and glucose (Percival 1979). Alginate is a linear polymer with a block substructure, having mannuronate and guluronate units arranged in homopolymeric and mixed sequences (Haug etal. 1967). The proportions of mannuronate and guluronate vary with species and part of the plant, as do the proportions of the three types of blocks (Haug et al. 1974). Physical properties of alginate such as solubility, cation affinity and formation of gels and fibers are correlated with block composition (Smidsrod 1974). Antibodies can serve as highly specific markers for important structural details and conformations of complex carbohydrates (Feizi 1982). An immunological approach has proved valuable in studies of Fucus cell wall carbohydrates. Alginate and fucan antigens were labelled in tissues from a range of brown algae by antisera (Vreeland 1974, 1975, 1981), and the extracellular distribution of alginate in Fucus tissues was described (Vreeland 1970, 1971, 1972). Crossed immunoelectrophoresis w a s used to investigate carbohydrate complexity, and to study carbohydrate immunochemical relationships within the brown algae (Vreeland and Chapman 1978 a,b). Recently, changes in cell wall epitopes during early development in Fucus em-
V. Vreeland et al.: Monoclonal antibodies to Fucus cell wall carbohydrates
bryos were studied with antisera (Vreeland and Laetsch 1981). Immunoelectrophoretic patterns were complex for both alginate and fucan, and changed during early development. These results demonstrated the need for monospecific antibodies in order to investigate individual epitopes separately. We report here the preparation of monoclonal antibodies to carbohydrates extracted from Fucus plants. Abstracts of preliminary work have been published (Vreeland etal. 1981, 1982). Carbohydrate preparation, monoclonal antibody production, and localization data for antibodies from a single fusion are presented. These antibodies are specific for a variety of cell wall epitopes by immunofluorescence. Consequences of variable carbohydrate properties and extracellular interactions for labelling are discussed. Specificity studies for individual antibodies will be reported separately. Material and methods
507
0.2M HCI (Ih,55~
residue discarded
5% Na2CO3 (Ih, 55~ I carbonatesoluble material I
soluble 0.67% CaC~2 ;soluble Carbonate] Fucan
] ~.2 M NaC'2,PH 2. l insoluble1 0.67% CeCI2
insoluble I [ Acid Alginate
I soluble insoluble1 (no material recovered)
[Carbonate Alginate
Fig. 1. Flow diagram of carbohydrate extraction and fractionation procedures. See text for detaits
Antigen preparation Carbohydrate extraction and fractionation procedures are outlined in Fig. 1. Carbohydrates extracted by acid and by carbonate were included in the immunizing preparation in an effort to obtain a wide range of antibody specificities.
Carbohydrate extraction Mature, reproductive Fucus distichus plants were collected at Duxbury Reef, Bolinas, Cal., USA, on 27 Dec 1978. After overnight treatment in 10% formalin in seawater, the plants were ground in a Waring blendor in 75% ethanol. Algal residue (50 g wet weight) was preextracted in 250 mt 0.2 M HC1 each of three times for 1 h at 55~C. The first Acid Extract was neutralized with NaOH, filtered to 1.2 gM, precipitated from four volumes 95% ethanol, washed in acetone and ether, and dried at 40~ The residue was extracted in 250 ml of 3% sodium carbonate for 1 h at 55~ The viscous carbonate extract was brought to pH 8 with HC1, and filtered. Carbohydrate was precipitated by two volumes of 95% ethanol, and dried as above. Cream-colored material (2.14 g) was recovered.
material, while a stiff gel formed from the acid-insoluble material. The calcium supernatants were dialyzed extensively against distilled water, filtered, brought to 0.05 M NaC1 and precipitated with three volumes of 95% ethanol. Calcium precipitates were dissolved by dialysis against 0.33 M ethylenetiaminotetraacetic acid (EDTA) at pH 7.2, and then dialyzed against distilled water before precipitation by one volume of 95% ethanol. Carbonate Fucan (71 mg) was recovered from the acid-soluble calcium supernatant; no precipitate formed in the acid-insolublecalcium supernatant. Acid Alginate (690 mg) was recovered from the fibrous acid-soluble calcium precipitate, and Carbonate Alginate (970 rag) was recovered from the getled acid-insolublecalcium precipitate. Carbonate Alginate and Acid Alginate aliquots were partially hydrolyzed to reduce molecular weight (Haug et al. 1967): alginates were suspended in 0.3 M HC1 and heated at 100~ for 20 min, immediately cooled, and then centrifuged. Pellets were suspended in distilled water, neutralized with NaOH, brought to 0.1 M in NaC1 and precipitated with four volumes of 95% ethanol.
Carbohydrate characterization Carbohydrate fractionations The carbonate-extracted material was first fractionated in acid (Haug et al. 1966). It was dissolved in distilled water at 5 mg ml-1, brought to 0.2 M NaC1 by the addition of 5 M NaC1, and 0.2 M HC1 was then added to bring the pH to 2.1. The precipitate was washed in 0.2 M NaC1 (pH 2.1), resuspended in 100 ml distilled water, and dissolved by addition of 1 M NaOH to bring the pH to 7. Carbohydrate was precipitated by addition of one volume of 95% ethanol and dried as above. Carbohydrate in the acid supernatant was precipitated by two volumes of 95% ethanol and dried. The acid-soluble and acidinsoluble fractions were further fractionated with calcium (Haug and Smidsred 1965). They were dissolved in distilled water at 5 mg m l - I, and 0.5 volume of 2% CaCI2 was added slowly with stirring. A fibrous precipitate formed from the acid-soluble
Carbazole analysis. Relative amounts of mannuronic, guluronic and glucuronic acids in immunizing alginate preparations (Table 1) were determined by the procedure of Knutson and Jeanes (1968). Sugar standards were I>glucuronic acid (Sigma Chemical Company, St. Louis, Mo., USA), and chromatographically purified D-mannuronicand L-guluronic acids (Haug and Larsen 1962) supplied by Bj~rn Larsen (University of Trondheim, Norway). Pure monosaccharides were necessary as standards; when low-molecular-weight alginate fractions (supplied by B. Larsen, with mannuronic and guluronic acid content determined by nuclear magnetic resonance) were used as standards, no reliable results could be calculated. A small amount of glucuronic acid is present in many alginate samples; this is not accounted for by presently available nuclear magnetic resonance analysis and possibly caused this discrepancy.
508
V. Vreeland et al.: Monoclonal antibodies to Fucus cell wall carbohydrates
Table 1. Carbazole determination of uronic acid composition of immunizing Fucus alginate fractions (in %) Alginate sample
Mann- GulGluc- Ratio uronic uronic uronic mannuronic/ acid acid acid guluronic acid
Carbonate Alginate Acid Alginate Acid Alginate, partially hydrolyzed
71 52
23 34
6 14
3.1 1.6
54
46
0
1.2
Table 2. Fucose, sulfate and protein content of Yucus carbohydrate fractions as weight percent
Fucose Sulfate Protein
Acid extract
Carbonate fucan
Carbonate alginate
Acid alginate
Acid alginate partially hydrolyzed
29 23.3 1.3
22 13.3 4.4
ND a 0.8 0.6
P l a n t a 9 Springer-Verlag 1984
Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga, Fucus V. Vreeland, M. Slomich* and W.M. Laetsch Department of Botany, University of California, Berkeley, CA 94720, USA
Abstract. Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immuno9 globulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function. Key words: Carbohydrate antigens (alginate, fucan) - Cell wall - Immunofluorescence - Fucus Monoclonal antibody - Phaeophyta.
Introduction
There is a growing awareness that carbohydrates carry specific information in their structures (for a U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA
* Present address:
EDTA = ethylenediaminetetraacetic acid; EIA = enzyme immunoassay; Ig = immunoglobulin (IgG, IgM and IgA are immunoglobulin types) Abbreviations:
review, see Preston 1977), and that the functions of carbohydrates depend on their conformations (Preston 1979; Rees et al. 1982). Interactions between, and modifications of, tertiary structures cause functional changes in complex carbohydrates from both plants and animals (Preston 1979; Rees et al. 1982). Carbohydrates of brown algae are complex and variable in structure. Alginate and fucan have no regularly repeating subunits. These acidic polysaccharides consist of populations of molecules varying in size, composition and sugar arrangement. Fucans are a family of branched carbohydrates containing sulfated fucose, xylose, glucuronate, a small amount of protein and often galactose, mannose and glucose (Percival 1979). Alginate is a linear polymer with a block substructure, having mannuronate and guluronate units arranged in homopolymeric and mixed sequences (Haug etal. 1967). The proportions of mannuronate and guluronate vary with species and part of the plant, as do the proportions of the three types of blocks (Haug et al. 1974). Physical properties of alginate such as solubility, cation affinity and formation of gels and fibers are correlated with block composition (Smidsrod 1974). Antibodies can serve as highly specific markers for important structural details and conformations of complex carbohydrates (Feizi 1982). An immunological approach has proved valuable in studies of Fucus cell wall carbohydrates. Alginate and fucan antigens were labelled in tissues from a range of brown algae by antisera (Vreeland 1974, 1975, 1981), and the extracellular distribution of alginate in Fucus tissues was described (Vreeland 1970, 1971, 1972). Crossed immunoelectrophoresis w a s used to investigate carbohydrate complexity, and to study carbohydrate immunochemical relationships within the brown algae (Vreeland and Chapman 1978 a,b). Recently, changes in cell wall epitopes during early development in Fucus em-
V. Vreeland et al.: Monoclonal antibodies to Fucus cell wall carbohydrates
bryos were studied with antisera (Vreeland and Laetsch 1981). Immunoelectrophoretic patterns were complex for both alginate and fucan, and changed during early development. These results demonstrated the need for monospecific antibodies in order to investigate individual epitopes separately. We report here the preparation of monoclonal antibodies to carbohydrates extracted from Fucus plants. Abstracts of preliminary work have been published (Vreeland etal. 1981, 1982). Carbohydrate preparation, monoclonal antibody production, and localization data for antibodies from a single fusion are presented. These antibodies are specific for a variety of cell wall epitopes by immunofluorescence. Consequences of variable carbohydrate properties and extracellular interactions for labelling are discussed. Specificity studies for individual antibodies will be reported separately. Material and methods
507
0.2M HCI (Ih,55~
residue discarded
5% Na2CO3 (Ih, 55~ I carbonatesoluble material I
soluble 0.67% CaC~2 ;soluble Carbonate] Fucan
] ~.2 M NaC'2,PH 2. l insoluble1 0.67% CeCI2
insoluble I [ Acid Alginate
I soluble insoluble1 (no material recovered)
[Carbonate Alginate
Fig. 1. Flow diagram of carbohydrate extraction and fractionation procedures. See text for detaits
Antigen preparation Carbohydrate extraction and fractionation procedures are outlined in Fig. 1. Carbohydrates extracted by acid and by carbonate were included in the immunizing preparation in an effort to obtain a wide range of antibody specificities.
Carbohydrate extraction Mature, reproductive Fucus distichus plants were collected at Duxbury Reef, Bolinas, Cal., USA, on 27 Dec 1978. After overnight treatment in 10% formalin in seawater, the plants were ground in a Waring blendor in 75% ethanol. Algal residue (50 g wet weight) was preextracted in 250 mt 0.2 M HC1 each of three times for 1 h at 55~C. The first Acid Extract was neutralized with NaOH, filtered to 1.2 gM, precipitated from four volumes 95% ethanol, washed in acetone and ether, and dried at 40~ The residue was extracted in 250 ml of 3% sodium carbonate for 1 h at 55~ The viscous carbonate extract was brought to pH 8 with HC1, and filtered. Carbohydrate was precipitated by two volumes of 95% ethanol, and dried as above. Cream-colored material (2.14 g) was recovered.
material, while a stiff gel formed from the acid-insoluble material. The calcium supernatants were dialyzed extensively against distilled water, filtered, brought to 0.05 M NaC1 and precipitated with three volumes of 95% ethanol. Calcium precipitates were dissolved by dialysis against 0.33 M ethylenetiaminotetraacetic acid (EDTA) at pH 7.2, and then dialyzed against distilled water before precipitation by one volume of 95% ethanol. Carbonate Fucan (71 mg) was recovered from the acid-soluble calcium supernatant; no precipitate formed in the acid-insolublecalcium supernatant. Acid Alginate (690 mg) was recovered from the fibrous acid-soluble calcium precipitate, and Carbonate Alginate (970 rag) was recovered from the getled acid-insolublecalcium precipitate. Carbonate Alginate and Acid Alginate aliquots were partially hydrolyzed to reduce molecular weight (Haug et al. 1967): alginates were suspended in 0.3 M HC1 and heated at 100~ for 20 min, immediately cooled, and then centrifuged. Pellets were suspended in distilled water, neutralized with NaOH, brought to 0.1 M in NaC1 and precipitated with four volumes of 95% ethanol.
Carbohydrate characterization Carbohydrate fractionations The carbonate-extracted material was first fractionated in acid (Haug et al. 1966). It was dissolved in distilled water at 5 mg ml-1, brought to 0.2 M NaC1 by the addition of 5 M NaC1, and 0.2 M HC1 was then added to bring the pH to 2.1. The precipitate was washed in 0.2 M NaC1 (pH 2.1), resuspended in 100 ml distilled water, and dissolved by addition of 1 M NaOH to bring the pH to 7. Carbohydrate was precipitated by addition of one volume of 95% ethanol and dried as above. Carbohydrate in the acid supernatant was precipitated by two volumes of 95% ethanol and dried. The acid-soluble and acidinsoluble fractions were further fractionated with calcium (Haug and Smidsred 1965). They were dissolved in distilled water at 5 mg m l - I, and 0.5 volume of 2% CaCI2 was added slowly with stirring. A fibrous precipitate formed from the acid-soluble
Carbazole analysis. Relative amounts of mannuronic, guluronic and glucuronic acids in immunizing alginate preparations (Table 1) were determined by the procedure of Knutson and Jeanes (1968). Sugar standards were I>glucuronic acid (Sigma Chemical Company, St. Louis, Mo., USA), and chromatographically purified D-mannuronicand L-guluronic acids (Haug and Larsen 1962) supplied by Bj~rn Larsen (University of Trondheim, Norway). Pure monosaccharides were necessary as standards; when low-molecular-weight alginate fractions (supplied by B. Larsen, with mannuronic and guluronic acid content determined by nuclear magnetic resonance) were used as standards, no reliable results could be calculated. A small amount of glucuronic acid is present in many alginate samples; this is not accounted for by presently available nuclear magnetic resonance analysis and possibly caused this discrepancy.
508
V. Vreeland et al.: Monoclonal antibodies to Fucus cell wall carbohydrates
Table 1. Carbazole determination of uronic acid composition of immunizing Fucus alginate fractions (in %) Alginate sample
Mann- GulGluc- Ratio uronic uronic uronic mannuronic/ acid acid acid guluronic acid
Carbonate Alginate Acid Alginate Acid Alginate, partially hydrolyzed
71 52
23 34
6 14
3.1 1.6
54
46
0
1.2
Table 2. Fucose, sulfate and protein content of Yucus carbohydrate fractions as weight percent
Fucose Sulfate Protein
Acid extract
Carbonate fucan
Carbonate alginate
Acid alginate
Acid alginate partially hydrolyzed
29 23.3 1.3
22 13.3 4.4
ND a 0.8 0.6
