Báo cáo khoa học: N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface docx

N-Terminal segment of potato virus X coat protein subunits
is glycosylated and mediates formation of a bound water
shell on the virion surface
Lyudmila A. Baratova
1
, Nataliya V. Fedorova
1
, Eugenie N. Dobrov
1
, Elena V. Lukashina
1
,
Andrey N. Kharlanov
2
, Vitaly V. Nasonov
3
, Marina V. Serebryakova
4
, Stanislav V. Kozlovsky
1
,
Olga V. Zayakina
1
and Nina P. Rodionova
1
1
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia;
2
Department of Chemistry, Moscow State
University, Russia;
3

M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Moscow, Russia;
4
V. N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia
The primary structures of N-terminal 19-mer peptides,
released by limited trypsin treatment of coat protein (CP)
subunits in intact virions of three potato virus X (PVX)
isolates, were analyzed. Two wild-type PVX strains, Russian
(Ru) and British (UK3), were used and also the ST mutant of
UK3 in which all 12 serine and threonine residues in the CP
N-terminal segment were replaced by glycine or alanine.
With the help of direct carbohydrate analysis a nd MS, it w as
found that the acetylated N-terminal peptides of both wild-
type strains are glycosylated by a single monosaccharide
residue (galactose or fuco se) at NAcSer in the first position
of the CP sequence, whereas the acetylated N-termin al seg-
ment of the ST mutant CP is unglycosylated. Fourier
transform infrared spectra in the 1000–4000 cm
)1
region
were m easured for fi lms o f the intact and in situ trypsin-
degraded PVX preparations at low a nd high humidity.
These spectra revealed the presence of a broad-ban d in the
region of valent vibrations of OH bonds (3100–3700 cm
)1
),
which can be represented b y superposition of three bands
corresponding to tightly bound, weakly bound, and free OH
groups. On calculating difference (ÔwetÕ minus ÔdryÕ) spectra,
it was f ound that the intact w ild-type PVX virions are

characterized by high water-absorbing capacity and the
ability to o rder a large number of water molecules on the
virus particle. This effect w as much weaker for the ST
mutant and completely absent in the trypsin-treated PVX.
It is proposed that the s urface-located and glycosylated
N-terminal CP segments of intact PVX virions induce the
formation of a columnar-type shell from bound water
molecules around the virions, w hich probably play a major
role in maintaining the virion surface structure.
Keywords: bound water; coat protein; Fourier transform
infrared spectroscopy; glycosylation; potato virus X.
Potato virus X (PVX) is the type member of the potexvirus
group of filamentous plant viruses [1]. Its coat protein (CP)
was extensively studied and it h as been shown that the CP
participation in the PVX infection cycle is not limited to its
role in virion formation. The PVX CP has b een shown to be
involved in processes of ge nomic RNA accumulation and
infection transport in plants [2–4]. It is also r esponsible for
induction of the R
x
resistance system in potato plants [5,6],
and has been recently shown to play a m ajor part in
regulation of virion translational activity at different stages
of the infection process [7–10]. These (and many others)
studies demonstrate the importance of potexvirus CP at all
stages of virus–plant interactions. Thus, the question arises
which features of t he CP structure d etermine the different
kinds of its activity.
It is well known t hat, on SDS/PAGE, i ntact P VX CP
displays anomalously slow electrophoretic mobility. This

mobility corresponds for different PVX strains to molecular
masses of 27–29 kDa, instead of 25 kDa as determined
from the primary structure [11,12]. In 1994 Tozzini et al.
[13] found that PVX CP contains O-linked carbohydrates. It
was the first report on the presence of glycosyl residues in
coat proteins of plant viruses. However, the exact nature of
the g lycosyl residues, their location in the PVX CP sequence,
and their functional importance remain unknown. Thus, a
more detailed analysis is needed, all the more so, as it is now
generally accepted that glycosylation can induce s ignificant
alterations in biopolymer structure and flexibility [14].
High-resolution X-ray (fiber) diffraction data for potex-
viruses are not available and only one, more or less detailed,
model of P VX CP structure in the virion (based on tritium
planigraphy and s econdary-structure prediction data) has
been suggested [15].
The N-terminal region of potexvirus CP is surface-
located [15,16], highly sensitive to the action o f plant sap
proteases [12], and can be easily removed by mild trypsin
treatment without disruption of the virion structure [17]. To
Correspondence to L. A. Baratova, Department of Chromatography,
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow
State University, Moscow 119992, Russia. Fax: + 7095 9393181,
Tel.: + 7095 9395408, E-mail:
Abbreviations: CP, coat protein; CPY, carboxypeptidase Y;
FTIR, Fourier transform infrared; PVX, potato virus X.
(Received 24 January 2004, revised 15 May 2004,
accepted 3 June 2004)
Eur. J. Biochem. 271, 3136–3145 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04243.x
elucidate the role of the PVX CP N-terminal region in

determining the physicochemical properties of the virion
surface, preservation of the virion integrity, and the
anomalous electrophoretic mobility, we used a special
PVX CP mutant. In this mutant (designated ST) all serine
and threonine residues (potential glycosylation s ites) in the
N-terminal segment are substituted by glycine or alanine
residues [7].
The aim of this work was t o determine the presence and
nature of carbohydrate residues in the peptides removed by
a limited trypsin hydrolysis from the intact PVX virions of
two wild-type s trains (Russian and British) a nd the S T
mutant. The N-terminal amino-acid sequences of these
three PVX isolate CPs are shown in Fig. 1 [7,18,19]. It is
widely known that many polyoxy molecules (including
proteins) exist in water solution in a hydrated state, and the
presence of carbohydrate residues may greatly increase the
bound water content [20]. To estimate bound water content
in the i ntact a nd trypsin-treated P VX virions, w e u sed
Fourier transform infrared (FTIR) spectroscopy. A possible
role for the surface-located and glycosylated PVX CP
N-terminal segment in preserving the structural and func-
tional integrity of the PVX virion s is discussed.
Materials and methods
Reagents and chemicals
Trypsin treated with 1-chloro-4-phenyl-3-
L
-toluene-p-sulfo-
namidobutan-2-one (TPCK-trypsin) was obtained from
Sigma. Trifluoroacetic acid was from Perkin-Elmer. Aceto-
nitrile was from Criochrom (St Petersburg, Russ ia). Rea-

gents for preparing gels were from Bio-Rad Laboratories.
Water was obtained using a Milli-Q System (Millipore). All
other chemicals were analytical grade.
For carbohydrate analysis, only f reshly prepared reagents
thrice-distilled in quartz glassware water were used. Meth-
anol was additionally purified by distillation over magnes-
ium methylate, and ethanol by distillation over calcium
oxide. Pyridine was d istilled twice over sodium hydroxide
and once over barium oxide. HCl and trifluoroacetic acid
were additionally purified by distillation in borosilicate
glassware.
Virus preparations
The R ussian (Ru) and British (UK3) strains of PVX were
purified from systemically infected leaves of Nicotiana
bentham iana and Datura stramonium as described previ-
ously [9]. The ST mutant of the UK3 strain with the wild-
type N-terminal sequence, SAPASTTQPIGSTTSTTTKT,
changed to AAPAGGAQPIGAGGAAGAKA was obtained
as described by Kozlovsky et al.[7].
Limited tryptic digestion of the virus preparations
Limited tryptic digestion of PVX virions (500 lgvirus
preparation in 0.5–1 mL 0.025
M
Tris/HCl buffer, pH 8.0)
was carried out at an enzyme/substrate ratio of 1 : 500
(w/w) for 2 h at 37 °Cin0.2
M
Tris/HCl buffer, pH 8.0. In
the case of the ST mutant, the tryptic digestion was carried
out at an enzyme/substrate ratio of 1 : 2000 (w/w). Then the

virus particles were pelleted by high-speed centrifugation
(50.2Ti rotor, Beckman L5-50; 105 000 g,4°C, 1.5 h), and
pellets and p eptide-containing supernatants were used for
further analysis.
SDS/PAGE
SDS/PAGE (8–20% gels) was carried out essentially a s
described by Laemmli [21]. The protein bands were visual-
ized by staining with Coomassie Brilliant Blue (G-250).
HPLC equipment and conditions
HPLC analyses were performed on a narrow-bore column
(Milichrom A-02; EnviroChrom LC, Chromatography
Institute ECONOVA, Novosibirsk, Russia; 75 · 2 mm)
packed with 5-lm p articles of Nucleosil C
18
, pore size 120 A
˚
(Macherey-Nagel, Duren, Germany). Separations were
performed at 25 °C; a dual wavelength (214 nm and
280 nm) detector was used. The elution gradient profile
was as follows. The elution solvents were A (0.1%
trifluoroacetic acid in water, pH 2.2) and B (acetonitrile
with 0.1% trifluoroacetic acid). The linear gradient was
0–60% B in 60 min and then 60–80% B in 10 min; the flow
rate was 8 0 lLÆmin
)1
. Fractions were collected for s ubse-
quent analysis using a Gilson 201 fraction collector. Peptide
yields were 30–50%. The conditions used allowed separ-
ation of all full-size PVX CP tryptic peptides.
Identification of carbohydrates in tryptic peptides

The m ethod for d etermining the monosaccharide c ompo-
sition of glycoconjugates (glycopeptides and glycoproteins)
involved derivatization of the monosaccharides, released on
acid hydrolysis, into N-(4-methylcoumarin-7-yl)glycamines
(AMC-sugars) and their subsequent analysis by reverse-
phase (RP) HPLC with fluorimetric detection [22]. The
authentic AMC-sugars were prepared by reductive
N-alkylation of 7-amino-4-methylcoumarin with the f ol-
lowing monosaccharides in the presence o f N aCNBH
3
:
D
-Glc,
D
-Gal,
D
-Man,
L
-Fuc,
D
-GlcNAc,
D
-GalNAc,
D
-ManNAc.
HPLC analyses were performed on a Du Pont 8800
chromatograph equipped with fluorescence detector .
Columns with Ultrasphere ODS (Beckman; 250 · 4.6 mm
internal diameter) w ere used. AMC-sugars were separated
at 25 °C using 17.5% ethanol in water with 0.1% trifluoro-

acetic acid, pH 2.5–2.6. The flow rate was 0.75 mLÆmin
)1
.
Carboxypeptidase Y (CPY) hydrolysis of N-terminal
PVX CP tryptic peptide
For CPY hydrolysis, peptide-containing solutions were
dried to eliminate trifluoroacetic acid and dissolved in water
Fig. 1. N-Ter minal amino-acid sequences of the coat proteins of Russian
(Ru) and British (UK3) PVX strains and the ST m utant of UK3 PVX.
Ó FEBS 2004 N-Terminal glycosylation of potato virus X protein (Eur. J. Biochem. 271) 3137
to a concentration of  0.1 mgÆmL
)1
.CPY(Sigma)was
added to this solution to a n enzyme/peptide ratio of 1 : 5
(w/w). Hydrolysis was c arried out at 22 °C. To obtain
MALDImassspectra,0.5-lL samples were removed at
different times from the hydrolysis start.
Mass spectrometry
Mass spectra were obtained using a MALDI-TOF mass
spectrometer (Reflex III model; Bruker Analytic GmbH,
Bremen, Germany) with 337 nm UV laser. A study sample
solution (0.5 lL) was mixed with an equal volume of
2,5-dihydroxybenzoic acid (Aldrich; 10 mgÆmL
)1
in 20%
acetonitrile in water with 0.1% trifluoroacetic acid), and the
mixture was dried in the air.
Mass spectra of material from HPLC fractions and CPY
hydrolysates of NAcSAPAS-peptide were obtained in
reflectron mode with positive ion detection (mass peak

accuracy 0.015%). Mass spectra of CPY h ydrolysates of the
N-terminal PVX C P t ryptic peptide and its C-terminal
fragment were obtained in reflectron mode with negative ion
detection (peak accuracy 0.02%).
FTIR spectroscopy
IR spectra were acquired with a FTIR spectromete r
Equinox 55/S (Bruker Analytic GmbH, Bremen, Germany)
in the wavenumber range 1000–4000 cm
)1
. To obtain thin
films, 10 lL of t he virus s uspensions ( 6mgÆmL
)1
)in
20 m
M
Tris/HCl buffer, pH 7.8, were applied to B aF
2
plates
and dried in a vacuum desiccator (at 0.13 Pa) over P
2
O
5
at
20 °C, with visual control o f the film homogeneity. T o
create 100% humidity, distilled water was placed at the
bottom of the desiccator.
Analytical methods
Tryptic peptides were hydrolyzed as described by Tsugita &
Scheffler [23], and amino-acid analysis was carried out on a
Hitachi-835 analyzer (Tokyo, Japan) in the standard mode

for protein hydrolysate analysis with cation-exchange
separation and ninhydrin postcolumn derivatization. The
short N-terminal amino-acid sequences of the tryptic
peptides were determined by Edman degradation on the
automated Procise cLC Protein Sequencing System (model
491; PE Applied Biosystems). Phenylthiohydantoin deriva-
tives of amino acids were identified with the PTH Analyzer
(model 120A; PE Applied Biosystems).
Results
Electrophoretic analysis of wild-type and mutant PVX CP
On SDS/PAGE of PVX preparations, it was found that
the intact wild-type and ST mutant CPs differ i n their
electrophoretic mobility, and this difference disappears after
mild trypsin treatment resulting in r emoval of the
N-terminal CP peptide (Fig. 2). T his indicates t hat the
anomalous PVX CP electrophoretic mobility is determined
by the N-terminal segment.
The relatively large difference in electrophoretic mobili-
ties between t he intact wild-type a nd ST mutant CPs can
hardly be explained by minor differences in m olecular mass
and may be due to the absence of certain post-translational
modifications in the mutant protein (see below).
HPLC analysis of the PVX CP peptides released
on limited trypsin hydrolysis
After the trypsin treatment, PVX virions were pelleted by
high-speed centrifugation, and the peptide-containing sup-
ernatants were subjected to RP-HPLC. The surface location
of the N-terminal CP peptide in PVX virions [15,16]
suggests that the peptides released from the PVX virions on
mild trypsin hydrolysis correspond to the N-terminal

regions of the PVX CP subunits. As can be seen in Fig. 3,
the supernatant f rom t he ST mutant contains one major
peptide fraction and the supernatants from the UK3 and Ru
strains contain two. [The differences in peak mobilities for
the UK3 and Ru peptides are probably due to differences
in their amino-acid seque nces: the former has p roline and
isoleucine at positions 9 and 10, respectively, and the latter
has alanine and threon ine (Fig. 1).] The elution of the ST
mutant peptide a t h igher a cetonitrile concentrations than
the UK3 peptide may be due to the substitution of 11 serine
and threonine residues with g lycine and alanine, which
would increase the peptide hydrophobicity. The minor
amounts of other peptides observed in the chromatographic
profiles in Fig. 3 were not analysed further.
Determination of primary structure of trypsin-cleaved
peptides
The HPLC-purified peptide fractions were used for a mino-
acid analysis, microsequencing and MALDI MS. The only
peptide released on mild trypsinolysis of the ST mutant
(Fig. 3 C) was the acetylated N-terminal peptide w ith a
molecular mass of 1535 Da (Table 1), corresponding
exactly t o the calculat ed mass of the first 19 amino acids
of the ST mutant CP with a n acetyl group. The trypsin-
cleaved peptides o f the wild-type (Ru and UK3) PVX
strains each gave two peaks (1 and 2) on RP-HPLC
(Fig. 3 A,B). For both viruses, the amino-acid compositions
of the material in the two peaks were identical and coincided
with that predicted for the corresponding N-terminal (19
residues) sequence (Fig. 1). Microsequencing results were
negative for all four peaks, confirming that material in all

Fig. 2. Anal ysis of wild-type (UK3) PVX and ST mutant preparations
by SDS/PAGE (8–20% gels). Lanes1and2,UK3PVX;lanes3and4,
ST mutant; lanes 1 and 3, CPs from intact virions; lanes 2 and 4, CPs
from trypsin-treated virions.
3138 L. A. Baratova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the peaks was N-blocked, in a ccordance with our p revious
results [15]. Possible reasons for the differences in c hroma-
tographic mobility of peak 1 and peak 2 material were
revealed by MS (Table 1). The molecular m asses for both
Ru and UK3 peptides turned ou t to be significantly higher
than expected. In peak 1 (of both strains) this difference was
162 Da, corresponding exactly to the addition of a hexose
residue. In peak 2 (again for both viruses), a more complex
picture was observed. Some material had the same mass as
in peak 1, but there was also material with a molecular mass
that differed from that expected by 146 Da (Table 1). This
may correspond to the addition of a deoxyhexose residue.
No peaks corresponding to more than a singly glycosylated
peptide (i.e. for instance, 2041 + 162 or 2041 + 146 in the
case of the UK3 strain peak 1) were detected in the mass
spectrograms. These r esults forced us to turn to direct
carbohydrate analysis.
Chromatographic analysis of the PVX CP N-terminal
peptide carbohydrates
Direct determination of carbohydrates in the P VX CP
N-terminal peptides involved acid hydrolysis, derivatization
of released monosaccharides to AMC-sugars, and identifi-
cation of derivatives by RP-HPLC with fluorescence
detection [22]. This analysis re vealed (for both Ru a nd
UK3 strains) the presence of a galactose residue (the

additional 162 Da) in t he material of peak 1 (Fig. 3A,B)
and both galactose and fucose (the a dditional 1 46 Da)
residues in peak 2 (th e data for the Ru strain are shown in
Fig. 4). These results correlated closely with the r esults of
MS (Table 1). No carbohydrates were found in the CP
N-terminal peptide of t he ST mutant, also in accordance
with the MS.
Thus, from the results of both MS and direct carbo-
hydrate determination it follows that the surface-located
N-terminal CP segments of the two wild-type PVX strains
studied are glycosylated and contain a sugar residue
(galactose or fucose) O-linked to serine or threonine
residues, which are absent from the N-terminal CP peptide
of the ST mutant.
The presence of galactose in both peak 1 and peak 2 may
be explained by the glycoconjugate stereochemistry. It is
known that carbohydrate diastereomers differ in their
physicochemical properties, and therefore they may have
Fig. 3. RP-HPLC separation of PVX CP preparations after mild
trypsin hydrolysis of intact virions. (A) Russian strain (Ru); (B) British
strain (UK3); (C) ST m utant.
Table 1. R esults of MS analysis of trypsin-cleaved PVX CP N-terminal
fragments.
PVX strain
Peptide
fraction
number (Fig. 3)
Peptide molecular mass (Da)
Observed Calculated Difference
Russian (Ru) 1 2003 1841 162

2 1987 146
2003 162
British (UK3) 1 2041 1879 162
2 2025 146
2041 162
ST mutant 1 1535 1535 –
Ó FEBS 2004 N-Terminal glycosylation of potato virus X protein (Eur. J. Biochem. 271) 3139
different chromatographic mobility on highly selective
sorbents.
The question a rises: are a ll CP subunits in the P VX
virions N-terminally glycosylated? On one hand, we
found n o o ther major peaks on HPLC other than peaks
1 and 2 (Fig. 3A,B), which indicates that the vast
majority of the 1 300 CP molecu les in t he PVX v irion
are glycosylated. On the other, on m ass spectrograms of
tryptic h ydrolysates of t he isolate d full-size UK 3 a nd Ru
CPs some material with molecular masses corresponding
to unglycosylated peptides (1879 and 1841 Da, respect-
ively) could be s een (data not shown). T his may mean
that PVX v irions contain a proportion (not more than
10%) of CP molecules that a re not N-terminally glycosy-
lated, assum ing that it is not the r esult of partial peptide
deglycosylation in t he course of MS . We d id not observe
any peaks corresponding to deacetylated PVX CP
N-terminal peptides.
Identification of glycosylation site(s) in the PVX CP
N-terminal segment
To locate glycosylation site(s) in the N-terminal segment of
PVX CP, we obtained spectra of MS fragmentation of
material from chromatographic peaks 1 and 2, prepared by

mild trypsin treatment of UK3 PVX virions (Fig. 3).
Standard MS fragmentation methods should lead to
formation o f i ons of the p eptide C-terminal fragments,
because the dominant protonation site (Lys19) is located at
the C-terminus. In MALDI postsource decay spectra [24],
we ob served only masses corresponding to ions of
C-terminal fragments (y-ions) of our peptide without
carbohydrate residues. On the basis of these results, i t
may be suggested that the glycosylation site is located on the
N-terminal serine of the peptide. However, it is well known
that, on MALDI fragmentati on, intensive deglycosyla-
tion takes place [25], making unequivocal conclusions
Fig. 4. RP-H PLC analysis of carbohydrate content of the Ru strain CP N-terminal peptide. Monosaccharides were analyzed as AMC derivatives.
(A) Analysis of a blank sample ( eluate fraction between peaks in chromatograp hic profile shown in Fig. 3). (B) Analysis of a stan dard mixture
containing Glc, Gal, Man, Fuc, GlcNAc, GalNAc, ManNAc. (C) Analysis of peak 1 (Fig. 3A). (D) A nalysis of peak 2 (Fig. 3A).
3140 L. A. Baratova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
impossible. MS s equencing using electr ospray ionization
also resulted in deglycosylation.
Therefore we decided to obtain a series of MALDI
spectra of PVX CP N -terminal tryptic peptides shortened
from the C-terminus by CPY hydrolysis [26]. For these
experiments, we modified the procedure for preparing the
N-terminal PVX CP segment: time, temperature and
trypsin/protein r atio were kept the same, but the PVX
virion (and enzyme) concentration w as increased about
fivefold. On HPLC of the p eptide-containing sample, three
major p eaks were eluted before peaks 1 and 2 (Fig. 5A).
Previously, we sometimes obtained similar chromato-
graphic profiles but did not analyze them further supposing
that they resulted from overhydrolysis. However, this time

we performed MALDI MS analysis of all five peaks eluted
at the beginning of the chromatogram (peaks 1*, 2*, 3*, 1
and 2 in Fig. 5A).
For peaks 1 and 2, molecular masses of 2025 and
2041 Da were obtained as before (Table 1). The molecular
mass of peak 3* was 1424 Da, corresponding to the
unglycosylated C-terminal part of the PVX CP N-terminal
tryptic peptide (Thr6–Lys19; Fig. 1). Figure 5B shows a
combined MALDI mass spectrum of peptides from p eaks
3*, 1 and 2 after partial CPY hydrolysis. The observed mass
values of fragments p roduced by seq uential removal of the
C-terminal residues (up to Ile10) from the initial tryptic
peptide confirm the localization of a carbohydrate residue
in the nonfragmented N-terminal part of the analyzed CP
peptide. Moreover, the mass spectrum of peak 3* material
treated with CPY confirmed our suggestion about its
Fig. 5. Stage s of glycosylation site identification. (A) Initial part of RP-HPLC profile of PVX preparation after modification of the trypsin treatment
procedure (see text). (B) Combined MALDI mass spectrum of material from peaks 3*, 1 and 2 after partial CPY hydrolysis. (C) M ALDI mass
spectra of p eak 2* mater ial before (upper part) and after (lower part) CPY treatment (642 Da, sodium salt of deoxyhexose-containing peptide;
658 Da, potassium salt of the same peptide and/or sodium salt of hexose-containing peptide; 674 Da, potassium salt of hexose-containing peptide;
additional peaks in lower spectrum correspond to salts from CPY solution).
Ó FEBS 2004 N-Terminal glycosylation of potato virus X protein (Eur. J. Biochem. 271) 3141
primary structure. Thus, carbohydrate residues could be
linked only to S er1 o r Ser5 of o ur peptide N-terminal
pentameric part NAcSAPAS
Peaks 1* and 2* were shown to contain only these
modified N -terminal p entapeptides: in peak 1 *, hexose was
linked to the peptide, and in peak 2* (just as in peak 2) either
hexose or deoxyhexose. With the help of CPY hydrolysis,
we managed to unambiguously localize t he glycosylation

site in the PVX CP N-terminal segment. In Fig. 5C,
MALDI mass spectra of peak 2* material before and after
CPY treatment are s hown (upper and lower parts, respect-
ively). Mass values in the upper p art correspond to ions of
sodium and po tassium salts of the glycosylated peptide
NAcSAPAS. After CPY treatment, the unglycosylated
C-terminal serine was r eleased (lower p art of F ig. 5C),
confirming our suggestion that a hexose or deoxyhexose
residue is linked to the acetylated N-terminal serine residue
(NAcSer1) of the PVX C P.
As far as w e know, t his type o f glycosylation has not
previously been observed in plant virus proteins.
FTIR spectroscopy of wild-type and mutant virus
preparations
Figure 6 shows FTIR spectra in the 1000–4000 cm
)1
region
for films of t he intact and trypsin-treated Ru PVX and the
intact ST mutant preparations. To compare the water-
absorbing capacity of the different PVX variants, we
measured their FTIR spectra in dry films and after
saturation of the film with water at 100% relative humidity.
Infrared band assignments were taken from Parker’s book
[27]. In t he FTIR spectra, peaks cor responding t o valent
CH-bond vibrations ( 2900 cm
)1
), valent C¼O bond
vibrations (amide I,  1650 cm
)1
), defo rmational amide

group vibrations (amide II,  1550 cm
)1
, and amide III,
 1300 cm
)1
) can be seen (Fig. 6). A broad-band in the
3100–3700 cm
)1
region corresponds to superposition of
several absorption p eaks. Here a complex band o f valent
water OH-bond vibrations is overlapped with p eaks of
valent OH-bond and NH-bond vibr ations of PVX CP.
Thus, to estimate the state of water molecules in the virus
preparations, we calculated FTIR difference spectra (the
samples at 100% humidity minus dry samples).
The broad 3100–3700 cm
)1
band in the difference spectra
(Fig. 7 ) can be represented by s uperposition o f three bands
corresponding to absorption of OH bonds in tightly bound
OH groups (the band a t  3240 cm
)1
), in weakly bound
OH groups ( 3440 cm
)1
) and in free OH groups of
absorbedwatermolecules( 3600 cm
)1
)[27].Fromthe
data in Fig. 7, it can be seen that the intact wild-type P VX

differs from the other virus preparations by a greatly
increased water-absorbing capacity. W hat is e ven more
important, on s aturation of the wild-type PVX film with
water, an intense 3240 cm
)1
band was observed in the
difference spectrum, supporting the o rdering o f a large
number of water mole cules on the virus particle. This effe ct
was much weaker for the ST mutant and completely absent
in the case of the trypsin-treated P VX.
Fig. 6. FTI R absorbance spectra of the intact and trypsin-treated Ru PVX and of the intact ST mutant preparations in the 1000–4000 cm
-1
region, for
dry films and after saturation of the film with water at 100% relative humidity.
3142 L. A. Baratova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
The role of CP glycosylation in plant virus life cycles
remains unclear, although data o n the effects of glycosyla-
tion on the conformation and dynamics of O-linked
glycoproteins are accumulating [28–30].
First, we were interested in the presence of glycosyl
modification(s) in the t rypsin-cleaved PVX CP N-terminal
segment. Structural analysis of the peptides cleaved from the
wild-type protein revealed the presence of two monosac-
charides, galactose and fucose, alternatively linked to
NAcSer in the first position of the CP sequence.
It was also shown that almost all PVX CP subunits in the
wild-type PVX virion contain sugar residues in their
N-terminal peptides. Small amounts of unglycosylated CP
in the virus samples may originate from the virio n ends,

where t he subunit conformation may be unsuitable f or
glycosylation.
What is the importance of this CP N-terminal g lycosy-
lation for PVX virion structure and function? According to
our model [15], the PVX CP N-terminal segment is located
on the v irion surface and forms a super-secondary structure,
a three-stranded b-sheet. We presume that removal of this
structure leads to drastic changes in the physicochemical
properties of the PVX virion surface.
It is widely known that water in close proximity to the
protein surface is fundamental to protein f olding, stability,
recognition and activity, and thus understanding protein
hydration is crucial in unraveling protein functions [31].
Virus particles with their unique highly regular morphology
may be especially interesting in this respect. Different types
of macromolecule s tructures a re known to have different
types of hydration she ll: columnar or sheet-like. Cylindrical
structures (which helical viruses have) usually induce the
columnar type of hydration [20].
The results of our comparative FTIR spectroscopy study
of the intact wild-type and ST mutant PVX virions and the
trypsin-treated wild-type virus particles suggest the pres-
ence of a large number of ordered water molecules ( water
shell) around intact wild-type v irions. Thus, a PVX virion
may be considered as an electric c able with several layers of
insulation. The virus RNA is packed into t he ordered
helical protein shell. The surface-located CP subunit
N-terminal segments with their fixed super-secondary
structure of three b-strands and glycosyl residues linked
to the N-terminal serines form the next ordered layer of the

cable structure. The su rface layer of the cable is formed by
a shell consisting of ordered water molecules. NMR and
ESR data show [32] that two water molecules are usually
bound per sugar ring, i.e. about 2600 molecules per PVX
virion.
Besides the presence of sugar residues, the wild-type PVX
CP N-terminal segment is also characterized by exception-
ally h igh content of hydroxyl-containing amino acids
(11 serine and threonine residues in the 19-residue
sequence). T his, as well as the predicted existence of a
three-stranded b structure in t his segment [15], may facilitate
tightly bound water shell formation. Moreover the presence
of deep grooves on the surface of helical PVX particles was
recently demonstrated by fiber X-ray diffraction analysis
[33], an d, a s shown by Falconi et al. [31], water hydration
sites are mainly located around protein cavities and clefts.
Raman optical activity spectra also indicate a high degree of
hydration in PVX virions [34].
Fig. 7. FTIR difference (‘wet’ minus ‘dry’) spectra of the intact and trypsin-treated Ru PVX and the intact ST mutant preparations in the 2500–
4000 cm
)1
region.
Ó FEBS 2004 N-Terminal glycosylation of potato virus X protein (Eur. J. Biochem. 271) 3143
The PVX virions without N-terminal CP peptide do not
simply lose the surface layer of water molecules; this loss
would lead to drastic changes in physicochemical properties
of the virion s urface. We propose that the absence of t he
outer ordered water layer explains the greater sensitivity to
trypsin of the ST mutant virions compared with the wild-
type PVX particles [7].

Other data [2,17] support our suggestion of critical
changes in PVX virions on cleavage (or changing) of the CP
N-terminal peptide. In their 1972 electron microscopy
study, Tremaine & Agrawal [17] observed unusual twisting
of trypsin-treated PVX particles. In 1992 Chapman et al.[2]
reported that a PVX CP deletion mutant, devoid of 19
N-terminal amino-acid residues, produces virions with
abnormal morphology.
We suggest that the presence of an ordered water
column around PVX virions, by itself and/or through
formation of a water-mediated net of hydrogen bonds
between (or inside) CP subunits, strongly affects the
structure and properties o f the externally lo cated region s
of the virus protein coat. However, it cannot be excluded
that the structu re of internally located parts of the virion
protein subunits and the structure of t he vir ion its elf are
affected by changes i n the state of the virion water shell.
and this state may be altered by changes in glycosylation,
phosphorylation and other t ypes of modification of t he
externally located virus CP regions. In this way the
structure of a whole virion may be changed by a signal
molecule binding to the virion surface. Recent observations
[7–10] that the ST mutant intravirus RNA, in contrast with
that o f the wild-type PVX, is accessible to ribosomes in the
intact virions and can be effectively t ranslated in cell-free
systems may be an example of such a structural transition.
We plan to continue to study structural alterations in the
PVX CP subunits in virions induced by N-terminal
segment modification or c leavage.
Acknowledgements

We thank Professor J. G. Atab ekov for valuable discussions. The work
was partially supported by Russian Foundation for Basic Rese arch
(grants 02-04-48651 and 03-04-48833).
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