Plant Physiol. (1998) 116: 477-483
Purification and Characterization of a Galactose-Rich Basic
Glycoprotein in Tobacco1
Tetsuo Takeichi*,
Junko Takeuchi,
Takako Kaneko, and
Shinji Kawasaki
National Institute of Agrobiological Resources, Tsukuba, Ibaraki
305, Japan (T.T., S.K.); and Japan Women's University, Faculty of
Science II, Bunkyo-ku, Tokyo 112, Japan (J.T., T.K.)
 |
ABSTRACT |
We found a galactose-rich basic
glycoprotein (GBGP) in the cell walls of cultured tobacco
(Nicotiana tabacum) cells. GBGP and extensin were
isolated as the major components of basic, salt-extracted cell wall
glycoproteins. GBGP and extensin were separated by gel filtration in 6 m guanidine hydrochloride as 49- and 90-kD peaks, respectively, and further purified with reverse-phase chromatography. The protein moiety of GBGP constitutes about one-half of the molecule (w/w) and contains lysine (16%), proline (12%), hydroxyproline (10%), tyrosine (4%), alanine (7%), leucine (6%), and cystine (1.4%). Galactose accounted for 72% of the sugar moiety, arabinose content was low (17%), and a significant amount of mannose (7%) was
found. No immunological cross-reaction was detected between GBGP and
extensin. The antibody against native GBGP with sugar chains reacted
with other glycoproteins on the gel blots, whereas the antibodies
against deglycosylated GBGP and native extensin were highly specific.
Immunolocalization analysis in tobacco stems showed that GBGP is
specific to parenchyma tissue and that extensin localizes in the
epidermis. This tissue-specific and exclusive distribution suggests
important functions of these basic glycoproteins.
| |
INTRODUCTION |
Primary cell walls of higher plants contain various structural
(glyco)proteins: extensins, arabinogalactan-proteins, repetitive Pro-rich proteins, and Gly-rich proteins (Showalter and Varner, 1989
;
Showalter, 1993; Kieliszewski and Lamport, 1994). The functions of
these proteins have yet to be elucidated, although they are thought to
relate to tissue hardening. Gly-rich protein is mainly expressed in
stem protoxylem cells (Ye and Varner, 1991; Ye et al., 1991), where
secondary wall thickening occurs (Keller et al., 1989). Repetitive
Pro-rich protein is also expressed in stem xylem tissue (Ye et al.,
1991). Extensin is expressed in the seed coat (Cassab and Varner, 1987)
or epidermis (Ye and Varner, 1991), although it is also expressed in
response to wounding or pathogen infection (Esquerré-Tugayé
et al., 1979; Showalter et al., 1985) and in soft tissues such as the
meristem (Keller et al., 1989) and cultured cells (Kawasaki, 1989). In
contrast, arabinogalactan-protein seems to be ubiquitous in the
intercellular spaces of higher plant tissues and cultured cells
(Fincher et al., 1983). Therefore, it has been of interest to find wall
glycoproteins specific to the parenchyma, which is relatively soft and
is universally distributed in vegetative tissues.
Extensin appears to be unique and is of interest because it contains a
large amount of Lys and is therefore highly basic. Extensin may
interact with the cell surface, which has a negative charge. In fact,
the cortical microtubule, closely related to cell shapes, has been
reported to be stabilized by artificially added extensin or poly-Lys
(Akashi et al., 1990). These facts indicate that there may be
some type of interaction between cell wall glycoproteins and cortex
cytoplasm and raise the question of whether extensin is unique as a
basic component in cell wall glycoproteins.
In this study we analyzed basic glycoproteins isolated from cell walls
of cultured tobacco (Nicotiana tabacum) cells and found GBGP, which was purified, characterized, and compared with extensin. Immunohistochemistry showed tissue-specific and exclusive distributions of these two basic glycoproteins in the tobacco stem: GBGP was specific
to the parenchyma and extensin in the epidermis.
| |
MATERIALS AND METHODS |
Tobacco (Nicotiana tabacum cv Xanthi, cell line XD-6S;
cv Bright Yellow, cell line BY-2) cells were cultured for 5 to 7 d after every being subcultured every 7 d in Murashige-Skoog medium, as described previously (Kawasaki, 1989). At these times, the cells
were at the logarithmic phase and were used for preparation. Tobacco
plants (cv Xanthi) were grown in a greenhouse or growth chamber for
60 d in field soils in pots. Suspension cultures of rice
(Oryza sativa cv Nipponbare), pinto bean (Phaseolus
vulgaris cv pinto VI 111), and catiang (Vigna catiang
cv Kurodane-sanjaku) were cultured as described above.
Purification of Basic Glycoproteins
Cultured tobacco cells of about 1300 g wet weight were
harvested on filter paper from 9 L of suspension culture, washed with culture medium, and gently stirred in 2 L of 75 mm
CaCl2 and 50 mm Tris-HCl, pH 7.5, for 1 h
at room temperature. The salt extract was precipitated with 70%
ethanol at 
20°C, extracted with 50 mm sodium acetate,
pH 5.0, and loaded onto a cation-exchange column (CM-Toyopearl 650S,
Toso, Tokyo, Japan). The main peak of basic proteins, eluted with a
linear gradient of NaCl in the same buffer, was fractionated by CsCl
density-gradient ultracentrifugation for 48 h at 20°C. The main
fraction of glycoproteins, containing about 50% (w/w) of sugar chains,
was further purified by HPLC gel filtration with two tandemly connected
size-exclusion columns (TSK-GEL G4000SW and TSK-GEL G3000SW, Toso) in 6 m guanidine HCl and reverse-phase chromatography using a
5C8 column (Wakosil, Wako Chemicals, Richmond, VA). In a typical case,
3.7 mg of GBGP and 1.9 mg of extensin were obtained from the 1300 g of cells.
Chemical Analysis
Sugar and protein contents were determined by the phenol-sulfuric
acid method (Dubois et al., 1956) and by the method of Lowry et al.
(1951), using Gal and BSA, respectively, as standards. The amino acid
composition was determined using an amino acid analyzer (model 2850, Hitachi, Tokyo, Japan) after hydrolysis with 6 m HCl at
110°C for 20 h or with performic acid at 100°C for 20 h
to quantify the Cys content. The composition of neutral sugars was
analyzed by the method of Albersheim et al. (1967) using a gas
chromatograph (model 663, Hitachi) after the conversion of the sugars
into the respective alditol acetates. Deglycosylation of the
glycoproteins was performed using trifluoromethanesulfonic acid (Edge
et al., 1981). The N-terminal amino acid sequence was analyzed with a
protein sequencer (model PPSQ-10, Shimadzu, Tokyo, Japan) after direct
application or after blotting onto a PVDF membrane after SDS-PAGE using
dg-GBGP.
Immunology
Rabbits were immunized with two injections of about 1 mg of native
GBGP, native extensin, or dg-GBGP at 2-week intervals using Freund's
complete (first injection) or incomplete (second injection) adjuvant.
Each serum was stored at 80°C after testing the titer. Proteins on
SDS-PAGE gels were electrically transferred onto nitrocellulose filters. Young stems and petioles were cut with a razor blade and
pressed carefully onto nitrocellulose filters. These filters were
treated with 3% BSA in PBS for 3 h at 37°C and incubated with
the serum diluted to 1:1000 with PBS containing 1% BSA for about
12 h at 37°C. After the filters were washed three times with
0.05% Tween 20 in PBS, they were incubated with secondary goat
anti-rabbit IgG conjugated with peroxidase (Wako) in PBS containing 1% BSA for about 6 h at 37°C. After washing,
antigens on the filters were detected as black color development by
incubating in PBS containing 0.05%
H2O2 and 0.05%
4-chloro-1-naphthol.
| |
RESULTS |
Purification of Basic Glycoproteins
The early steps of the purification were intended to isolate the
major components of basic glycoproteins containing extensin. Salt-extractable wall proteins were obtained from cultured tobacco cells by stirring gently in a buffer containing 75 mm
CaCl2. They were precipitated with ethanol, extracted with
an acidic buffer, and loaded onto a cation-exchange column to isolate
basic proteins. A major peak was eluted with about 250 mm
NaCl (Fig. 1) and then subjected to CsCl
density-gradient centrifugation (Fig. 2)
for the purpose of isolating glycoproteins with a high content of sugar. Glycoproteins of with a density of about 1.4 g/cm3
were pooled as major peaks.
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| Figure 1.
Cation-exchange chromatography of salt-extractable
cell wall proteins from cultured tobacco cells. Extracts from the cell wall surface were precipitated with ethanol, dissolved in acidic buffer, and loaded onto a 20-mL column. Basic proteins were retained on
the column, eluted with a linear gradient of NaCl, and pooled (5 mL/each fraction). A main peak at about 250 mm NaCl
(fractions 15-23), which proved to contain GBGP and extensin, was
advanced to the next step.
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| Figure 2.
CsCl density-gradient centrifugation of basic
proteins. The cation-exchange column eluent was fractionated with CsCl
equilibrium density-gradient ultracentrifugation (0.5 mL/each
fraction). The A280 was monitored, and both
the sugar content and the density of each fraction were measured. The
main peak of glycoproteins (fractions 14-23), which had nearly equal
contents of protein and sugar, were pooled.
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The isolated basic glycoproteins were analyzed by HPLC gel filtration
in 6 m guanidine HCl. Two major peaks were found at the
positions of 49 and 94 kD (Fig. 3). They
were pooled separately and further purified by the same
chromatography. Finally, they were subjected to reverse-phase
chromatography (Fig. 4). The 49- and
94-kD proteins showed clear, single peaks at different positions.
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| Figure 3.
Gel filtration in 6 m guanidine HCl of
basic glycoproteins. The obtained basic glycoproteins were loaded onto
tandemly connected size-exclusion columns and fractionated (1 mL/fraction). Two major peaks (a and b) were found and separately
pooled. The molecular masses of peaks a and b were determined as 94- and 49-kD, respectively, using the calibration curve of standard
proteins. The 49- and 94-kD glycoproteins, which were identified later
as GBGP and extensin, were further purified with one more gel
filtration and with reverse-phase chromatography (Fig. 4).
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| Figure 4.
Reverse-phase chromatography of 49- and 94-kD
proteins. The 49- and 94-kD proteins were finally loaded onto a 5C8
column in 0.05% trifluoroacetic acid and eluted with a gradient
of acetonitrile. They migrated as clear, single peaks. We identified
them as GBGP and extensin, by analyzing their amino acid and sugar
composition.
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Amino Acid and Sugar Composition of 49- and 94-kD Basic
Glycoproteins
The contents of sugars and protein of the purified samples were
determined by the phenol-sulfuric acid method and by the Lowry method,
respectively. The sugar contents of the 49- and 94-kD proteins are
shown in Table I by percentage, assuming
that the sum of the sugar content and the protein content is 100% of
the glycoproteins.
The amino acid and sugar compositions of the 49- and 94-kD proteins
were analyzed (Table I). The 94-kD protein, which consists of about the
same quantity of protein and sugar and is rich in Ara, Hyp, and Lys,
was identified as extensin, since its composition was close to that of
extensin purified from a culture medium of tobacco cells (Kawasaki,
1989). The 49-kD protein also consists of about the same quantity of
protein and sugar and is so rich in Lys that it is very basic. In these
characteristics it is similar to extensin but quite different from
arabinogalactan-proteins, which are acidic and have a higher sugar
content. However, in contrast to extensin, the 49-kD protein is very
rich in Gal instead of Ara. Moreover, it contains less Hyp and Tyr than
extensin. Therefore, the 49-kD protein is clearly different from
extensin, and we called it GBGP.
The amino acid composition of GBGP was similar to that of GaRSGP, which
was recently isolated from styles of Nicotiana alata (Sommer-Knudsen et al., 1996). However, GBGP and GaRSGP were
distinguishable in sugar content (48 and 75% [w/v], respectively),
as well as in sugar composition: GBGP contains 72.4 mol% Gal, 16.5 mol% Ara, and 7.0 mol% Man, whereas GaRSGP has been reported to
contain 83.0 mol% Gal, 6.8 mol% Ara, and 3.5 mol% Man. Moreover, the
N-terminal amino acid sequence of GBGP is different from that of
GaRSGP. Immunological analysis showed that in some other aspects the
two proteins are also distinguishable (see ``Discussion''). In
conclusion, GBGP isolated from cultured cells differs from style
GaRSGP, as well as from any other glycoprotein reported to date.
Immunology and Tissue Localization Analysis
In SDS-PAGE, purified GBGP and extensin migrated as broad single
bands, which are characteristic of glycoproteins with high sugar
content (Fig. 5A, a). No immunological
cross-reaction between GBGP and extensin was detected (Fig. 5A, b
and c). GBGP was dg with trifluoromethanesulfonic
acid and purified by gel filtration and reverse-phase chromatography.
dg-GBGP showed a single 28-kD band upon SDS-PAGE (Fig. 5B).
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| Figure 5.
SDS-PAGE of GBGP and extensin. A, The purified
GBGP (lanes G) and extensin (lanes E) were electrophoresed on 10%
acrylamide gel. Then they were stained with Coomassie brilliant blue
(a) or blotted onto nitrocellulose membranes and detected with the antibodies against native GBGP (b) or extensin (c). GBGP and extensin appeared as single-smear bands of 50 to 120 and 90 to 200 kD, respectively, because of their high content of sugar. No immunological cross-reaction between GBGP and extensin was detected. B, The purified
native GBGP (lane G) and its dg peptide backbone (lane dG) were
electrophoresed on 12.5% acrylamide gel and stained with Coomassie
brilliant blue. dg-GBGP appeared as a 29-kD band. Lanes M, Marker
proteins.
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Salt-extractable proteins from the cell wall, protoplast, and culture
medium were examined using western analysis (Fig.
6A). Anti-native GBGP antibody reacted
with many kinds of wall and medium glycoproteins, although it
recognized neither protoplast proteins nor extensin. In contrast,
anti-dg-GBGP antibody and anti-extensin antibody were concluded to be
highly specific, since only one signal characteristic of GBGP or
extensin was found. It is also noticeable that GBGP was detected only
in wall extracts, whereas extensin was found both in wall extracts and
in the medium.
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| Figure 6.
Immunoblot analysis of GBGP and extensin. A,
Salt-extractable proteins from the cell wall (lanes W), protoplast
(lanes P), and the culture medium (lanes M) were separated by SDS-PAGE
(5-15% gradient gel). Gels were stained with Coomassie brilliant blue (a) or blotted onto nitrocellulose filters. Antigens were detected with
native GBGP antibody (b), dg-GBGP antibody (c), or extensin antibody
(d). Although the native GBGP antibody reacted with various glycoproteins from the cell wall and from the medium, the antibodies against dg-GBGP and extensin were found to be highly specific. GBGP was
detected only in wall extracts (c), whereas extensin was found in
proteins from both the cell wall and the medium (d). B, Salt extracts
from the cell walls of cultured cells of pinto beans (lane 1), catiang
(lane 2), and rice (lane 3) were separated by SDS-PAGE (5-15%
gradient gel) and blotted onto nitrocellulose filters. Antigens were
detected with dg-GBGP antibody. GBGP homologs were found in pinto beans
and catiang.
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Salt-extractable wall proteins from cultured cells of bean and rice
were examined with the specific anti-dg-GBGP antibody (Fig. 6B).
Proteins from the two species of legume reacted with anti-dg-GBGP
antibody at similar and rather high-molecular-mass ranges,
respectively, compared with tobacco GBGP. Therefore, a GBGP homolog
seems to exist in legumes. The signal in pinto beans, at about twice
the molecular mass of GBGP, may result from dimer formation. In catiang
the position of the signal was almost the same as in tobacco. In rice,
a monocotyledon, no signal was detected; therefore, either no GBGP
homolog exists or if it does it reacts immunologically with
tobacco dg-GBGP antibody.
Tissue distributions of antigens were examined by tissue printing (Fig.
7). The anti-native GBGP antibody, which
has rather low specificity, showed a virtually homogeneous distribution
of antigen (Fig. 7E), which suggests universal occurrence of galactan epitopes and verifies uniform transfer of materials by printing. In
contrast, the antibodies against dg-GBGP and extensin showed tissue-specific distributions of both proteins. GBGP was localized in
the parenchyma of the stele and cortex of young stems (Fig. 7, A and
B). At high magnification (Fig. 7B), a concentrated reaction of GBGP
was visible in the collenchyma at the contact point of three cells. The
weak reaction of GBGP is universal in parenchyma, since the outline of
each cell, shown by black signal development in Figure 7B, is clear
(compare with that in Fig. 7D). In a blotting of a petiole (Fig. 7F),
essentially the same distribution of GBGP was shown, although GBGP was
also found in the vascular bundle. In contrast to GBGP, extensin
localized specifically in the epidermis of young stems (Fig. 7, C and
D). These distribution patterns of GBGP and extensin were also
confirmed by direct staining of sections (data not shown), although
extensin was also found in the cambium. In conclusion, the expression
patterns of the two basic glycoproteins are tissue specific and
mutually exclusive.
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| Figure 7.
Tissue-print analysis of GBGP and extensin. The
stem (A-E) or the petiole (F) of tobacco was printed onto
nitrocellulose filters. The antigens were detected with anti-dg-GBGP
(A, B, and F), anti-extensin (C and D), and anti-native-GBGP (E) as
black color development. Green areas are the chloroplasts transferred
to the membrane. Scale bars: A, C, E, and F, 1 mm; B and D, 0.5 mm. B and D are magnified photographs of the upper region of A and C,
respectively.
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N-Terminal Amino Acid Sequence of GBGP
The N-terminal 20-amino acid sequence of GBGP is shown in Figure
8. At a few positions, two kinds of amino
acids appeared in all trials. The second peak was consistently smaller
than the first (about 80 mol%). We concluded that this was due to the
presence of two isoforms, since dg-GBGP always migrated as a single
peak in reverse-phase chromatography and SDS-PAGE.
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| Figure 8.
N-terminal amino acid sequence of GBGP. At a few
positions, second amino acid peaks smaller than the first (about 80 mol%) were always found, suggesting the presence of two isoforms. The homologous sequences found in a database search are also shown (TTS1,
NaPRP4, PvPRP, and pMG15). They were deduced from the cDNAs of tobacco
style tissue (TTS1, Cheung et al., 1993; NaPRP4, Chen et al., 1993; and
PMG15, Goldman et al., 1992) and from cultured bean cells (PvPRP, Sheng
et al., 1991) and located near the putative C terminus, not the N
terminus. Identical amino acids are indicated with bold letters.
Sequential numbers from the N terminus (top) and the C terminus
(bottom, C-) are also indicated.
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The same or nearly the same sequences were found in a database (Fig.
8). They were deduced from the cDNAs of tobacco styles (TTS1, Cheung et
al., 1993; NaPRP4, Chen et al., 1993; and PMG15, Goldman et al., 1992)
and cultured bean cells (PvPRP, Sheng et al., 1991). GBGP is clearly
different from these proteins since their homologous sequences locate
near the putative C terminus, not the N terminus, and their predicted
N-terminal sequences are quite different from that of GBGP. GaRSGP,
with an amino acid composition similar to that of GBGP, is encoded by
NaPRP4, one of the above-mentioned cDNAs (Sommer-Knudsen et al., 1996).
This reconfirms that GBGP and GaRSGP are different glycoproteins.
| |
DISCUSSION |
The N-terminal amino acid sequence of GBGP showed close homology
to the sequences of some reported cDNAs, although they locate near the
C terminus. The proteins deduced from these cDNA sequences have a
common characteristic in that they consist of two domains, the first
half with repeated sequences rich in Pro (and/or Hyp?) and the second
half with no special repeats and significant amounts of Leu
(approximately 10%) and Cys (approximately 4%), which are rare or
absent in typical Hyp-rich wall proteins such as extensin. Although the
complete amino acid sequence of GBGP has yet to be determined, our
preliminary experiment by circular dichroism spectroscopy suggested the
possibility that GBGP contains a domain of poly-Pro-II helix and
another domain of random coil (data not shown).
In the above-mentioned cDNAs, which have GBGP homologous sequences near
the C terminus, only NaPRP4 has been characterized as a protein
(GaRSGP; Sommer-Knudsen et al., 1996). GBGP and GaRSGP are similar in
amino acid composition but differ in sugar content, sugar composition,
and N-terminal amino acid sequence, as described in ``Results''.
Moreover, in our preliminary experiments GBGP did not localize in style
tissue and was not recognized by the antibody against dg GaRSGP (a gift
from J. Sommer-Knudsen). GBGP, GaRSGP, and the proteins encoded by the
above-mentioned cDNAs TTS1, PMG15, and PvPRP may belong to a new
family: They may contain two domains of repeated Pro helix and random
coil and may also have sugar residues rich in Gal.
In this study GBGP was revealed to be the first example to our
knowledge of parenchyma-specific glycoprotein, and extensin was
verified to localize in the stem epidermis of tobacco as was reported
for soybeans (Ye and Varner, 1991). It is worth noting that, like GBGP
and extensin, the style-specific GaRSGP is reported to be basic
(Sommer-Knudsen et al., 1996). Although the function of these
tissue-specific basic glycoproteins is not clear, one attractive
hypothesis is that they interact with the cortical microtubule
through the cell membrane (Akashi and Shibaoka, 1991). We
suspect that these basic glycoproteins have important roles in tissue
differentiation and plan to study their distribution in more detail to
elucidate their functions.
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FOOTNOTES |
1
This work was supported by research grants from
the Ministry of Agriculture, Forestry, and Fisheries (Tokyo, Japan).
*
Corresponding author; e-mail takeichi{at}abr.affrc.go.jp; fax
81-298-38-8347.
Received June 23, 1997;
accepted November 6, 1997.
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ABBREVIATIONS |
Abbreviations:
dg, deglycosylated.
GaRSGP, Gal-rich style
glycoprotein.
GBGP, Gal-rich basic glycoprotein.
| |
ACKNOWLEDGMENTS |
We thank Dr. Jens Sommer-Knudsen for his kind gift of native and
dg-GaRSGP antibodies and Drs. Ohashi and Nakasone of our institute for
providing bean and rice cells, respectively.
| |
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Abstract
Introduction
