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Plant Physiol. (1998) 116: 1431-1441
Extracellular Matrix Assembly in Diatoms
(Bacillariophyceae)1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Achnanthes longipes is
a marine, biofouling diatom that adheres to surfaces via adhesive
polymers extruded during motility or organized into structures called
stalks that contain three distinct regions: the pad, shaft, and collar.
Four monoclonal antibodies (AL.C1-AL.C4) and antibodies from two
uncloned hybridomas (AL.E1 and AL.E2) were raised against the
extracellular adhesives of A. longipes. Antibodies
were screened against a
hot-water-insoluble/hot-bicarbonate-soluble-fraction. The
hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, 
20,000,000 Mr; F2, 
100,000
Mr; and F3, <10,000
Mr relative to dextran standards. The
100,000-Mr fraction consisted of highly
sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and
low-sulfate (approximately 2%) FGGs, whereas F1 was
composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2
recognized carbohydrate complementary regions on FGGs, with
antigenicity dependent on fucosyl-containing side chains. AL.C3 was
unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and
immunocytochemistry indicated that low-sulfate FGGs are expelled from
pores surrounding the raphe terminus, creating the cylindrical outer
layers of the shaft, and that highly sulfated FGGs are extruded from
the raphe, forming the central core. Antibody-labeling patterns and
other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility.
Because of the complexity of the higher-plant ECM, we have only
recently begun to understand the spatial relationships between ECM
components, and know little about the intra- and intermolecular interactions responsible for cell wall organization and assembly (Levy
and Staehelin, 1992 The marine, biofouling diatom Achnanthes longipes relies on
production of highly organized extracellular adhesive biocomposites for
cell motility and permanent adhesion to submerged surfaces (Wang et
al., 1997 The stalks of A. longipes can be described as having three
distinct regions: a surface-associated pad, a collar associated with
the frustule at the raphe terminus, and an intervening shaft that
separates the cell from the surface (Daniel et al., 1987
Puhlmann et al. (1994) Source of Algal Material, Culturing Conditions, and Chemical
Isolation of Diatom ECM
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). McCann et al. (1990) succeeded in visualizing the
intermolecular organization of the onion primary cell wall,
demonstrating cross-links between cellulose and hemicellulose and a
separate surrounding network made up pectic polymers. Taylor and
Haigler (1993) found that cellulose serves as a template mediating the
self-organization of other molecules such as xylan and Gly-rich proteins in the secondary cell walls of tracheary elements. ECM polymers appear to interact initially through short-range forces, most
notably hydrogen binding. Hydrogen binding, hydrophobic interactions, and ionic cross-bridging are noncovalent associations essential to
maintenance of cell wall integrity. Recently,
Ca2+-dependent pectin-binding proteins were
isolated (Penel and Greppin, 1996), including several vitronectin-like
proteins and isoperoxidases. These results indicate that an ion-binding
mechanism may be responsible for the organization of some proteins in
the plant cell wall. Covalent binding may be a final step in the
self-assembly process, cementing together all of the individual
components of the matrix. The most common covalent linkages are: (a)
sugar-sugar glycosidic linkages; (b) sugar-protein glycosidic linkages;
and (c) sugar-sugar, protein-protein, and sugar-protein
peroxidase-mediated phenolic cross-links.
; Wustman et al., 1997). The adhesive polymers of A. longipes are primarily polysaccharide with a small amount of
protein, and preliminary studies suggest the presence of phenolics (Wustman et al., 1997). Extracellular assembly appears to require both
cationic cross-bridging and a peroxidase-mediated phenolic cross-linking (Wustman et al., 1997). These extracellular adhesives must be extruded through the silicious cell wall, or frustule, before
they can be assembled into permanent attachment structures termed
stalks. Because of the relative immutability of the
frustule, extrusion of polymers is necessarily limited to openings in
the frustule, and protoplasmic mediation of the assembly processes occurring distal to the frustule is restricted. The hydrated, amorphous
silica frustule imposes limitations on protoplasm-mediated control of orientation and on movement of extracellular polymers both
during and after synthesis, and on communication across the protoplasm-plasma membrane-extracellular polymer continuum. For this reason, the assembly of complex polysaccharide-protein stalks with
a high degree of organization and detailed substructure in diatoms
represents a fascinating system in which to study self-assembly phenomena. Moreover, in A. longipes, the adhesion process
can be easily manipulated with various molecular probes and inhibitors, and extrusion and assembly of the biocomposites can be monitored via
time-lapse video microscopy and electron microscopy (Wang et al.,
1997). The extracellular adhesive polymers of A. longipes have been isolated and purified from cultures in quantities large enough to enable detailed chemical analysis (Wustman et al., 1997), as
well as polymer-interaction studies. Thus, the A. longipes attachment process represents a unique model system for investigations into cell-subtratum adhesion and physical characteristics of
extracellular matrices based on polymer composition and polymer-polymer
interactions.
; Wang et al.,
1997) (Fig. 1A). Transmission electron
microscopy, lectin-FITC labeling, and cytochemical staining have
demonstrated that the polymers are organized into several outer layers
parallel to the axis of shaft elongation, and an inner core oriented in a radial pattern perpendicular to the axis of shaft elongation (Daniel
et al., 1987; Wang et al., 1997; Wustman et al., 1997). Distributions
of various polymers within the adhesive structure have not been
determined, although evidence suggests that covalent cross-linking of
polysaccharides by proteins and/or phenolics may be involved (Wustman
et al., 1997). We have used lectin-FITC and cytochemical staining in
correlation with chemical analysis to identify the presence of certain
monosaccharide residues within unique regions of the adhesive
structures (Wustman et al., 1997). The initial adhesive found in pads
and between stacked cells preparing to separate contained substantial
amounts of GlcA and fucosyl residues. Outer layers of the shaft
contained GlcA, t-Fuc, and nonsulfated d-Gal residues,
whereas the inner core was primarily constituted of sulfated galactosyl
residues (Johnson, 1995; Wustman et al., 1997).
View larger version (97K):
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Figure 1.
A. longipes ECM and antibody
localization. A, Differential interference contrast microscopy image of
A. longipes showing the frustules (f), collar (cl),
outer shaft regions (a), and inner shaft core (c). B through H,
Fluorescent antibody labeling (B, C, E, G, and H on 200-nm sections):
B, AL.E1 localization of the outer shaft region and central ribbon (cr)
of the shaft core; C, AL.C1 labeling of the inner shaft core; D, AL.C1
labeling of the raphe region of a cell (r) and material associated with
the frustule; E, AL.E2 localization of the outer shaft regions; F, AL.C3 localization of globular matrix material (m) associated with the
shaft (s). G, Sections incubated with preimmune serum in place of
hybridoma supernatant. H, Sections incubated with ascites fluid in
place of hybridoma supernatant. The same photographic exposure times
were used to image controls and sections incubated with the hybridoma
supernatant. Scale bars = 5 µm.
generated monoclonal antibodies to several cell
wall components of suspension-cultured sycamore maple cells, and
Freshour et al. (1996) used them to help discern the pattern of
organization within the ECM and to determine how these patterns changed
during different stages of root development in Arabidopsis
thaliana seedlings. ECM assembly and physical characteristics can
also be investigated by discerning which epitopes are directly involved
in polymer-polymer or polymer-surface interactions. We report here the
characterization of several monoclonal antibodies generated against
extracellular adhesives of A. longipes. Based in part on the
affinities and labeling patterns of these antibodies, we have created a
model that predicts how polymers are organized into supramolecular
complexes (adhesive stalks) by A. longipes and identifies
chemically unique regions within the stalk substructure. Based on these
results, we can begin to correlate polymer structure and polymer
interactions with the overall physical characteristics of the
extracellular adhesive.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
. Stauroneis decipiens was isolated and
cultured as described by Lind et al. (1997). WIBS, WIBS/CPC-soluble ECM
fractions of A. longipes and A. coffeaeformis,
and WIBS/CPC-insoluble ECM fractions of A. longipes and
A. coffeaeformis were isolated as described by Wustman et al. (1997).
Generation of Hybridoma Cell Lines
A. longipes cells and ECM were scraped from 3-week-old culture flasks and fixed with 2% glutaraldehyde. Fixed cells and ECM (2 mg) were mixed with an equal amount of methylated BSA in PBS (20 mm PO4, 0.15 m NaCl, pH 7.2), an equal volume of Titremax adjuvant (CytRx, Norcross, GA) was added, and 200 µL was injected (intraperitoneally) into female Balb/c mice. A second injection of 200 µL was administered 1 month after the first, and a final 200-µL boost, consisting of A. longipes WIBS material mixed with an equal volume of methylated BSA in PBS without adjuvant, was injected 2 d before fusion.. Hybridoma
supernatant was screened by ELISA against A. longipes WIBS
material with detection of bound antibodies by anti-mouse whole IgG
conjugated to alkaline phosphatase (Sigma) and by antibody-FITC cell
staining of unfixed cells as described below. During the screening
process we selected against hybridomas producing antibodies that bound
to bacteria. Positive cell lines were cloned by limiting dilution.
Isotyping of Monoclonal Antibodies
Isotyping of monoclonal antibodies and antibodies from uncloned, extended hybridoma cell lines was done using the Amersham Mouse Monoclonal Antibody Isotyping Kit (RPN 29, Amersham).ELISA
A. longipes WIBS material was dissolved in PBS (20 mm PO4, 0.15 m NaCl, pH 7.2) (50 µg/mL) and incubated in microtiter trays (Probind Falcon, Becton Dickinson) at 37°C for 1 h. Wells were washed twice with PBS containing 1% (w/v) BSA, incubated at 37°C for 1 h with PBS containing 1% (w/v) BSA, and washed once with PBS. Hybridoma supernatant or mouse serum (diluted 1:500 in PBS) was incubated at 37°C for 1 h and washed five times with PBS containing 0.5% Tween 20 (v/v). Antibody binding was detected by a secondary antibody (goat anti-mouse IgG, IgM, or polyvalent immunoglobulin labeled with FITC, diluted 1:1000 in PBS with 4% [w/v] PEG, 0.5% [v/v] Tween 20, and 1% [w/v] ovalbumin). The wells were incubated with secondary antibody for 1 h at 37°C, washed four times with PBS containing 0.5% (v/v) Tween 20, and two times with double-distilled water. One hundred microliters of phosphate substrate solution (1 mg/mL p-nitrophenylphosphate in 10 mm diethanolamine, 0.5 m MgCl2, pH 9.5) was added to each well and incubated for 20 min at 24°C, and the A405 was read.Antibody-FITC Cell Staining
Diatom cells and ECM were isolated from culture, washed in microcentrifuge tubes with marine PBS (20 mm PO4, 0.5 m NaCl, pH 7.2) for 10 min, followed by a stepwise marine PBS:PBS gradient (3:1, 1:1, 1:3, 100% PBS, 5 min/wash), and incubated for 30 min with 0.05% (w/v) Gly and for 1 h in FGB-PBS (PBS containing 1% [v/v] fish skin gelatin and 0.02% [v/v] Tween 20). Cells were centrifuged at 500g for 2 min, the supernatant was discarded, and the pellet was resuspended in mouse serum or hybridoma supernatant (diluted 1:25 in FGB-PBS). After incubation for 3 h at 24°C, cells were washed four times with FGB-PBS (5 min each), resuspended in 10 µL of secondary antibody (goat anti-mouse IgG, IgM, or polyvalent immunoglobulins [IgG, IgA, IgM] labeled with FITC, diluted 1:25 in FGB-PBS), incubated at 24°C for 1 h in the dark, and washed five times with FGB-PBS. The cells were resuspended in 7 µL of p-phenylenediamine (1 mg/mL in PBS with 30% [v/v] glycerol) and viewed with differential interference contrast microscopy and epifluorescence microscopy, as described by Wustman et al. (1997). Controls were treated as described above with either preimmune serum or
mouse ascites fluid in place of the hybridoma supernatant and by
incubating with secondary antibody alone.
. Sections (200 nm thick) mounted on slides were stained
and viewed as described above.
Column Chromatography
Size-exclusion-gel Sepharose 2B and Sephadex G-100 (Sigma) column dimensions were 20 cm × 1.4 cm and 30 cm × 1.4 cm, respectively. The mobile phase was distilled water or 0.1 or 0.5 m imidazole HCl with a flow rate of 0.8 mL/min. The sample (0.5 mL of 2 mg/mL solution) was loaded onto the columns and the effluent was monitored at 280 nm and collected in 2-mL fractions. Each fraction was screened for carbohydrate using the phenol-sulfuric acid assay, as described below. Identified peaks were collected and screened for antigenicity by dot-blot analysis as described below. Blue dextran 20,000,000 Mr, dextran 500,000 Mr, and Glc (Sigma) were used to calibrate the columns.Dot-Blot Assay
Samples were solubilized in TBS (20 mm Tris and 0.5 m NaCl, pH 7.5) to 0.2 mg/mL, and 200 µL was added to each well of a dot-blot apparatus (Bio-Rad) and allowed to flow by gravity through the nitrocellulose membrane. Each well was washed with 200 µL of TBS and incubated with 200 µL of FGB-TBS for 1 h, followed by two washes with TBS. Hybridoma culture supernatant (200 µL; diluted 1:10 in TTBS [20 mm Tris, 0.5 m NaCl, 0.05% Tween 20, and 1% fish skin gelatin, pH 7.5]) was added and incubated for 1 h. The membranes were then removed and washed twice with TTBS for 5 min with gentle agitation, transferred to goat anti-mouse IgG, IgM, or polyvalent immunoglobulin labeled with horseradish peroxidase (diluted 1:1000 in TTBS), and incubated for 1 h with gentle agitation. Blots were then washed twice with TTBS and once with TBS as described above, developed in substrate solution (0.015 g of 4-chloro-1-naphthol in 5 mL of cold methanol added to 25 mL of TBS with 30 µL of 30% H2O2) for 1 h in the dark, and washed five times with double-distilled water. Blots were then imaged using a video camera (DXC-930 3-CCD, Sony, Tokyo, Japan) with output to a digitizer board (SNP-24, Active Imaging, Berkshire, UK). Image Tool version 1.25 (The University of Texas Health Science Center, San Antonio) was used to determine the density of bands with background subtraction of adjacent areas, providing averaged, background-subtracted values for each band. For each antibody, the background-subtracted value of A. longipes WIBS was designated as 100 and other values were scaled appropriately.Chemical Modification of A. longipes WIBS ECM
Periodate oxidation and 0.5 m NaOH treatment of A. longipes WIBS material were carried out according to Sledjeski and Weiner (1993). WIBS was desulfated by the methods of
Falshaw and Furneaux (1994). The pyridinium salt was formed by
dissolving 5 mg of purified WIBS in 1 mL of distilled water, passing
the solution through a 5-mL column of Dowex 50W-X12
(H+ form), adding excess pyridine, and
freeze-drying.
. All modified samples were assayed by
dot-blot analysis, and NaOH-,
Na2CO3-, and
NaClO3-treated samples were also analyzed with
size-exclusion chromatography to determine if the respective treatment
reduced the size of the native polymers.
) in a modified procedure as described below.
A. longipes bicarbonate-soluble ECM (5 mg) was dissolved in
2 mL of 0.01 m NaAc (pH 4.75), CMC (0.5 mL of 250 mg CMC/mL
of 0.01 m NaAc buffer, pH 4.75) was added, and the mixture was stirred on ice for 2 h. Ice-cold 4 m imidazole HCl
(0.5 mL), pH 7.0, was added, followed by 300 mg of
NaB2H4 powder, and stirred for 1 to
2 h (until effervescence ceased). The solution was then dialyzed
against distilled water for 36 h, lyophilized, and assayed by
dot-blot analysis as described previously.
with modifications as described by Lau et al. (1987). Reducing sugar
assay and size-exclusion chromatography were used to verify polymer
cleavage, and the samples were assayed by dot-blot analysis.
Monosaccharide and Linkage/Substitution-Site Determination
Monosaccharide composition and linkage/substitution sites were determined by GC-MS of alditol acetates and partially methylated alditol acetates, respectively, as described previously (Wustman et al., 1997, and refs. therein). Samples were routinely hydrolyzed for
3 h at 121°C in 2 m TFA. Per-O-methylated
alditol acetate mole percentages were calculated using effective
carbon-response factors, as described by Sweet et al. (1975).
Colorimetric Assays
Total carbohydrate and reducing sugar content were estimated by the phenol-sulfuric acid assay (Dubois et al., 1956) and the 3,5-dinitrosalicylic acid reagent assay (Miller, 1959), respectively, using a Gal:Fuc (1:1) standard curve.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Production of Hybridomas
Four hybridoma colonies producing antibodies with a high affinity for A. longipes WIBS material were cloned, and two other colonies producing only one class of antibody each were extended for possible future cloning. All hybridomas produced antibodies with
light chains. Monoclonal antibodies AL.C1 and AL.C2 and uncloned
hybridoma AL.E2 produced IgG1s, AL.C3 produced an IgG3, and AL.E1
produced IgMs.
Cell Staining
FITC-conjugated anti-A. longipes antibodies differentially labeled A. longipes extracellular adhesives. The localization patterns for each antibody are summarized in Table I. All of the antibodies demonstrated the same specific labeling patterns of live cells, with similar titer, when PBS was replaced with seawater. AL.C1 and AL.E1 localized mucilage located between stacked cells and pads, collars, and portions of the shaft. AL.E1 labeling was most intense in the outer perimeter and central ribbon of the shaft core (Fig. 1B). AL.C1 uniformly labeled the shaft core (Fig. 1C), with a lower affinity for the outer layers, and also labeled material within the raphe (Fig. 1D), mucilage in the form of rings surrounding the terminal nodules, and amorphous mucilage associated with the cells and stalks. AL.C2, AL.C4, and AL.E2 (Fig. 1E) exhibited identical labeling patterns of shafts, pads, and collars, with binding restricted to the outermost layer of the shaft (Table I). AL.C3 labeled mucilage between cells (Table I) and amorphous mucilage loosely associated with the shafts (Table I; Fig. 1F) and frustules. Controls revealed no nonspecific antibody interactions (Fig. 1, G and H).
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Characteristics of Antigen Epitopes
A. longipes WIBS material was separated into three size ranges by Sepharose 2B and Sephadex G-100 size-exclusion chromatography (Fig. 2, A and B): F1,20,000,000 Mr;
F2, 100,000 Mr; and
F3, <10,000 Mr, relative
to dextran standards. F1 and
F2 were the major fractions and consisted mostly
of carbohydrate with a small amount of protein
(F1 approximately 5%, F2 < 1%), whereas F3 was almost entirely protein.
F3 produced a smear when separated by SDS-PAGE,
indicating that carbohydrate was most likely still associated with the
protein. WIBS material treated with 0.5 m NaOH (100 or 5°C) or trypsin and separated by size-exclusion chromatography yielded a decrease in F1 and an increase in
F2. F3 yield also increased
after the cold NaOH treatment, whereas trypsin degraded this fraction.
Treatment with cold 0.5 m
Na2CO3 or 0.6 m
NaClO3 had no effect on the relative amounts of
each fraction, indicating a lack of modification by cleavage of ester
bonds or isodityrosine cross-links.
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A. longipes WIBS and chemically modified WIBS were blotted
onto nitrocellulose and screened against supernatant from four cloned
(AL.C1-AL.C4) and two uncloned (AL.E1 and AL.E2) hybridoma colonies.
A. coffeaeformis WIBS was also blotted and screened against
the antibodies for cross-reactivity. Detection was by peroxidase
secondary anti-mouse antibody with digital imaging of the developed
blot. Numbers represent averaged, background-subtracted density values
that have been scaled to WIBS.
Daniel et al. (1987) High- and Low-Sulfate FGGs
Relationship of Shaft Polymers to Those Involved in Cell
Motility
), and methylation
analysis disclosed a large decrease in the relative abundance of
2,3-Gal and 3,6-Gal, and an increase in t-Man and 4-Man residues. All
antibodies except AL.C3 showed a high affinity for
F1 and F2 and a reduced
affinity for F3 (Table
II). AL.C3 had a much lower affinity for
WIBS than the other five antibodies, although antibody-FITC cell
staining revealed that AL.C3 had a high affinity for the pads, collars, globular matrices associated with stalks and cells, and mucilage between stacked cells.
View this table:
Table II.
Interaction of antibodies with WIBS and fractions
or modifications
1 corresponding to sulfate
ester (Craigie and Leigh, 1978) for both fractions. This absorbance was
significantly reduced for peak 2, providing additional evidence of
reduced sulfate content. Linkage/substitution-site profiles for peaks 1 and 2 were indistinguishable from those reported for WIBS material
(Wustman et al., 1997).
View larger version (15K):
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Figure 3.
Monosacchrides released from A. longipes WIBS ECM by time-course hydrolysis (1 m
TFA at 80°C). Fuc was the only neutral monosaccharide detected after
0.5 h of hydrolysis. Increasing amounts of Gal and Xyl were
released after 1 and 5 h of hydrolysis. Neutral monosaccharide composition was determined by GC-MS of alditol acetates and is presented as a percentage of the total detected. 
, Fuc; 
, Xyl; and 
, Gal.
View this table:
Table III.
Linkage/substitution-site profiles for A. longipes
WIBS after time-course hydrolysis with 1 m TFA (80°C)
Glycosyl linkage/substitution sites of acid-resistant material were
determined by GC-MS of partially methylated alditol acetates. Uronosyl
residues were not determined. ND, Not detected.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
and Wang et al. (1997) demonstrated that the
shafts of A. longipes consist of several outer layers
oriented parallel to the axis of shaft elongation, and a core that has fibrous material organized in layered radial arrays perpendicular to
the axis of shaft elongation and with a central ribbon. The uniqueness
of these regions is supported by the successful generation of
antibodies with differing affinities for each region. The six antibodies can be divided into three categories (Table I; Fig. 1) based
on differing affinities for (a) regions other than the shaft, including
pads, collars, and amorphous material loosely associated with the
cells; (b) the outermost layers of the shaft; and (c) the central core.
From these results it can be assumed that the antibodies recognize
different epitopes that are distributed nonuniformly throughout the
ECM. Spatial organization and induced conformational changes of
antigenic molecules during the extracellular adhesive assembly process
may account for the multiple localization patterns observed. Because
all six antibodies showed a high affinity for A. longipes
WIBS ECM, we fractionated WIBS by anion-exchange chromatography,
size-exclusion chromatography, and CPC precipitation, and screened
fractions for antigenicity.
and Daniel et al. (1987).
Johnson (1995) used x-ray microanalysis of sectioned A. longipes stalks to show that sulfate was located mainly in the
stalk core, excluding the central ribbon, and Daniel et al. (1987)
provided cytochemical evidence for a higher concentration of
less-sulfated carboxylated polymers in the pads, collars, and outer
layers of the shafts. Chemical and immunocytochemical evidence has now
revealed that although these regions differ in polymer organization,
they consist of related polymers (FGGs) that vary in their sulfate
content. We hope to use this model system to investigate how such
modifications affect polymer interactions, organization, and overall
physical characteristics.
, in which labeling by
Fuc-binding lectin in the raphe correlated with periods of cell
motility. This Fuc-binding lectin also labeled the stalk shaft cores.
The importance of polysaccharide synthesis in stalk synthesis and cell
motility is further indicated by the inhibition of both processes by
the specific extracellular polysaccharide synthesis inhibitor
dichlorobenzonitrile and related compounds (Wang et al., 1997). We
conclude that the central core region of the shaft is raphe derived and
is related to material exuded from the raphe during cell motility.
Cross-Linking Proteins/Glycoproteins
Size-exclusion chromatography separated WIBS into three fractions: (a) F1,20,000,000 Mr;
(b) F2, 100,000 Mr; and
(c) F3, <10,000 Mr (Fig.
2). F1 and F2 appeared to
be mostly carbohydrate and their monosaccharide profiles were
indistinguishable, whereas F3 contained mainly
protein/glycoprotein. The F3 fraction could be
concentrated by precipitating out CPC-insoluble material.
Monosaccharide profiles of the resulting CPC-soluble material revealed
an increase in mannosyl residues (Wustman et al., 1997). The
F3 fraction is water insoluble and can be
separated from F1 and F2
only with relatively high concentrations of imidazole buffer. A
plausible explanation for this may be that F3
contains mannoproteins that have a high affinity for other adhesive
components found in F1 and
F2. Mannoproteins of Candida albicans
fungal cell walls are involved in both covalent and noncovalent
interactions, and have been shown to self-assemble in vitro after
removal of the solubilizing agents by dialysis against double-distilled
water (Sentandreu et al., 1995). Imidazole may prevent aggregation of
polymers by interrupting ionic interactions. It has been shown that
high concentrations of imidazole (0.5 m) can be used in
place of
trans-1,2-diaminocyclohexane-N,N,N
,N-tetraacetic acid and EDTA to solubilize pectins, presumably by competing with carboxyl groups for Ca2+ and thereby disrupting
the "egg-box" formations (Mort et al., 1991). However, because
neither chelating agents nor imidazole buffers solubilized intact
A. longipes stalks, it appears that F3
protein does not cross-link native stalks via ionic interactions.
), and such cross-linking components may be present in the F3 fraction.
F1 was also converted to F2
by treatment with trypsin, providing further evidence for the
cross-linking of F2 polymers by
proteins/glycoproteins into large F1 complexes.
Because NaClO2 and cold
Na2CO3 treatments did not
degrade F1, ester-linked phenolics or
isodityrosine linkages are most likely not important in bridging the
F2 molecules. Because trypsin and NaOH treatments
of WIBS did not affect antigenicity, it appears that all of the
antibodies recognized carbohydrate epitopes of the FGGs or of a
possible glycoprotein described above. It is also possible that such
glycoproteins from A. longipes play a more direct role in
adhesion and motility. In the marine diatom S. decipiens,
antibodies raised against extracellular proteoglycans localized
material associated with the raphe and inhibited both cell motility and
adhesion (Lind et al., 1997).
Characterization of the Antibody Epitopes
Wustman et al. (1997) identified t-Fuc residues throughout the
entire stalk and between stacked cells preparing to separate. It was
found that Fuc made up 42% of the WIBS ECM, one-half of which was
t-Fuc residues, as estimated by linkage analysis (Wustman et al.,
1997). Thus, it would be reasonable to assume that these residues may
be important in inducing antibody formation because so many t-Fuc
residues are available, and terminal nonreducing sugars are commonly
recognized as the immunodominant group of branched polysaccharides
(Kabat, 1976). This may be why some of the antibodies showed a small
amount of cross-reactivity with A. coffeaeformis WIBS ECM,
which also contains a substantial amount of t-Fuc (Wustman et al.,
1997). C. cistula, which contains mostly Gal and Xyl and
lacks Fuc (Wustman et al., 1997), showed no detectable cross-reactivity.
1:1 (Table III). Loss of antigenicity by removal of ester sulfate during hydrolysis can be ruled
out for all of the antibodies except AL.C4, for which desulfation of
WIBS reduced antigenicity by 50% (Table II). However, because
antigenicity was not completely lost after desulfation, but was below
detection limits after 1 h of TFA hydrolysis, it would seem that
TFA hydrolysis does not affect AL.C4 affinity for WIBS by
desulfation alone. Because removal of fucosyl residues greatly reduced antigenicity in comparison with desulfation, NaOH and trypsin treatments, CMC-activated reduction, and polymer cleavage by lithium at uronic acid sites in ethylenediamine, five antibodies appeared to recognize fucosyl-containing side chains, regions near
these side chains, or regions that depend on these side chains to
maintain conformation of the epitope. Furthermore, antigenicity for
WIBS was significantly reduced by periodate oxidation for AL.C2, AL.E1,
and AL.E2, as would be expected if t-Fuc units played a role in
antibody-antigen recognition. Fucosyl-containing side chains are most
likely responsible for the hydrophobic nature of the stalks (Wang et
al., 1997; Wustman et al., 1997) involved in initial passive attachment
(Wang et al., 1997), and further studies with the antibodies
characterized here may help to understand how these side chains
interact and contribute to such overall characteristics as flexibility,
tensile strength, resistance to microbial degradation, and cell
adhesion.
).
Adhesive Model and Conclusions
Anion-exchange and size-exclusion column chromatography revealed highly sulfated (11%) and low-sulfate (2%) FGGs of100,000 Mr (relative to dextran). Five of the antibodies
raised against A. longipes adhesive structures appear to
recognize a family of related epitopes with antigenicity dependent on
fucosyl-containing side chains present on both the highly sulfated and
the low-sulfate FGGs. These polysaccharides are cross-linked via
Oglycosidic sugar-protein linkages into large
(>20,000,000 Mr) proteoglycan assemblages. The
antibody AL.C1, which localized the shaft core, had a higher affinity
for the highly sulfated FGGs, and the antibodies AL.C2, AL.C4, AL.E1,
and AL.E2, which localized the outer layers of the shaft, had higher
affinities for the low-sulfate FGGs. AL.C3 appears to recognize a
unique epitope in pads, collars, globular material surrounding and
between cells, and in the F3 protein fraction.
Received September 19, 1997;
accepted December 19, 1997.
Abbreviations:
CMC, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate.
CPC, cetylpyridinium chloride.
ECM, extracellular matrix.
FGG, fucoglucuronogalactan.
FITC, fluorescein isothiocyanate.
TFA, trifluoroacetic acid.
WIBS, hot-water-insoluble/hot-bicarbonate-soluble
fraction.
The authors thank Tony Chiovitti and Yan Wang for helpful
comments and discussions.
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View larger version (17K):
[in a new window]
Figure 4.
Model of the stalk of A. longipes
detailing the location of components characterized here and putative
sites of origin from the frustule. A hollow cylinder made up of
polymers oriented parallel to the length of the stalk is created when
low-sulfate FGGs are expelled through pores surrounding the terminal
region of the raphe. Simultaneously, highly sulfated FGGs are expelled
through the raphe terminus, expanding into the hollow cylinder and
forming a fibrillar array of polymers oriented perpendicular to the
length of the shaft. The FGGs are then cross-linked extracellularly by protein and phenolic components. ×, Protein or phenolic cross-link; 
, highly sulfated FGG; and , low-sulfate FGG.
1
This research was supported by the Office of
Naval Research (grant nos. N00014-94-1-0273 and N00014-94-1-0766) to
M.R.G and Kyle D. Hoagland and a Michigan Technological University
Fellowship Award to B.A.W.
FOOTNOTES
*
Corresponding author; e-mail mrgretz{at}mtu.edu; fax
1-906-487-3167.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
2)-linked fucosyl-containing epitope.
Plant Physiol
104:
699-710
[Abstract]
Copyright Clearance Center: 0032-0889/98/116/1431/11
© 1998 American Society of Plant Physiologists
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, Affinity below detectable limits;
+/
, protein; and dotted line, NaCl.