N-Terminal segment of potato virus X coat protein subunitsis glycosylated and mediates formation of a bound water shell on the virion surface Lyudmila A.. Orekhovich Institute of Biomedi
Trang 1N-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 Baratova1, Nataliya V Fedorova1, Eugenie N Dobrov1, Elena V Lukashina1,
Andrey N Kharlanov2, Vitaly V Nasonov3, Marina V Serebryakova4, Stanislav V Kozlovsky1,
Olga V Zayakina1and Nina P Rodionova1
1
A N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia;2Department of Chemistry, Moscow State University, Russia;3M M Shemyakin and Yu A Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;4V 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 and MS, it was
found that the acetylated N-terminal peptides of both
wild-type strains are glycosylated by a single monosaccharide
residue (galactose or fucose) at NAcSer in the first position
of the CP sequence, whereas the acetylated N-terminal
seg-ment of the ST mutant CP is unglycosylated Fourier
transform infrared spectra in the 1000–4000 cm)1 region
were measured for films of the intact and in situ
trypsin-degraded PVX preparations at low and high humidity
These spectra revealed the presence of a broad-band in the
region of valent vibrations of OH bonds (3100–3700 cm)1), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups On calculating difference (wet minus dry) spectra,
it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX
It is proposed that the surface-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, which 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 has been shown that the CP
participation in the PVX infection cycle is not limited to its
role in virion formation The PVX CP has been shown to be
involved in processes of genomic RNA accumulation and
infection transport in plants [2–4] It is also responsible for
induction of the Rxresistance system in potato plants [5,6],
and has been recently shown to play a major 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 the CP structure determine the different kinds of its activity
It is well known that, on SDS/PAGE, intact PVX 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 glycosyl 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 significant 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 PVX CP structure in the virion (based on tritium planigraphy and secondary-structure prediction data) has been suggested [15]
The N-terminal region of potexvirus CP is surface-located [15,16], highly sensitive to the action of 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: baratova@belozersky.msu.ru
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)
Trang 2elucidate 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 sites) in the
N-terminal segment are substituted by glycine or alanine
residues [7]
The aim of this work was to 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 strains (Russian and British) and the ST
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 intact and trypsin-treated PVX virions, we used
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 virions 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, Russia)
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 freshly 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 calcmagnes-ium
oxide Pyridine was distilled twice over sodium hydroxide
and once over barium oxide HCl and trifluoroacetic acid
were additionally purified by distillation in borosilicate
glassware
Virus preparations
The Russian (Ru) and British (UK3) strains of PVX were
purified from systemically infected leaves of Nicotiana
benthamiana 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 lg virus preparation in 0.5–1 mL 0.025MTris/HCl buffer, pH 8.0) was carried out at an enzyme/substrate ratio of 1 : 500 (w/w) for 2 h at 37C in 0.2MTris/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, 4C, 1.5 h), and pellets and peptide-containing supernatants were used for further analysis
SDS/PAGE SDS/PAGE (8–20% gels) was carried out essentially as 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 particles of Nucleosil C18, pore size 120 A˚ (Macherey-Nagel, Duren, Germany) Separations were performed at 25C; 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 80 lLÆmin)1 Fractions were collected for subse-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 method for determining the monosaccharide compo-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 fol-lowing monosaccharides in the presence of NaCNBH3:
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) were used AMC-sugars were separated
at 25C 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-Terminal amino-acid sequences of the coat proteins of Russian
(Ru) and British (UK3) PVX strains and the ST mutant of UK3 PVX.
Trang 3to a concentration of 0.1 mgÆmL)1 CPY (Sigma) was
added to this solution to an enzyme/peptide ratio of 1 : 5
(w/w) Hydrolysis was carried out at 22C To obtain
MALDI mass spectra, 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)1in 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 hydrolysates of the
N-terminal PVX CP tryptic 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 spectrometer
Equinox 55/S (Bruker Analytic GmbH, Bremen, Germany)
in the wavenumber range 1000–4000 cm)1 To obtain thin
films, 10 lL of the virus suspensions ( 6 mgÆmL)1) in
20 mMTris/HCl buffer, pH 7.8, were applied to BaF2plates
and dried in a vacuum desiccator (at 0.13 Pa) over P2O5at
20C, with visual control of the film homogeneity To
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 in their
electrophoretic mobility, and this difference disappears after
mild trypsin treatment resulting in removal of the
N-terminal CP peptide (Fig 2) This indicates that the
anomalous PVX CP electrophoretic mobility is determined
by the N-terminal segment
The relatively large difference in electrophoretic
mobili-ties between the intact wild-type and ST mutant CPs can
hardly be explained by minor differences in molecular 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 from the 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 sequences: the former has proline and isoleucine at positions 9 and 10, respectively, and the latter has alanine and threonine (Fig 1).] The elution of the ST mutant peptide at higher acetonitrile concentrations than the UK3 peptide may be due to the substitution of 11 serine and threonine residues with glycine 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 amino-acid analysis, microsequencing and MALDI MS The only peptide released on mild trypsinolysis of the ST mutant (Fig 3C) was the acetylated N-terminal peptide with a molecular mass of 1535 Da (Table 1), corresponding exactly to the calculated mass of the first 19 amino acids
of the ST mutant CP with an acetyl group The trypsin-cleaved peptides of the wild-type (Ru and UK3) PVX strains each gave two peaks (1 and 2) on RP-HPLC (Fig 3A,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 Analysis of wild-type (UK3) PVX and ST mutant preparations
by SDS/PAGE (8–20% gels) Lanes 1 and 2, UK3 PVX; lanes 3 and 4,
ST mutant; lanes 1 and 3, CPs from intact virions; lanes 2 and 4, CPs from trypsin-treated virions.
Trang 4the peaks was N-blocked, in accordance with our previous results [15] Possible reasons for the differences in chroma-tographic mobility of peak 1 and peak 2 material were revealed by MS (Table 1) The molecular masses for both
Ru and UK3 peptides turned out 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 results forced us to turn to direct carbohydrate analysis
Chromatographic analysis of the PVX CP N-terminal peptide carbohydrates
Direct determination of carbohydrates in the PVX 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 revealed (for both Ru and UK3 strains) the presence of a galactose residue (the additional 162 Da) in the material of peak 1 (Fig 3A,B) and both galactose and fucose (the additional 146 Da) residues in peak 2 (the data for the Ru strain are shown in Fig 4) These results correlated closely with the results of
MS (Table 1) No carbohydrates were found in the CP N-terminal peptide of the 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 mutant.
Table 1 Results 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
Trang 5different chromatographic mobility on highly selective
sorbents
The question arises: are all CP subunits in the PVX
virions N-terminally glycosylated? On one hand, we
found no other major peaks on HPLC other than peaks
1 and 2 (Fig 3A,B), which indicates that the vast
majority of the 1300 CP molecules in the PVX virion
are glycosylated On the other, on mass spectrograms of
tryptic hydrolysates of the isolated full-size UK3 and Ru
CPs some material with molecular masses corresponding
to unglycosylated peptides (1879 and 1841 Da,
respect-ively) could be seen (data not shown) This may mean
that PVX virions contain a proportion (not more than
10%) of CP molecules that are not N-terminally
glycosy-lated, assuming that it is not the result of partial peptide
deglycosylation in the course of MS We did 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 of ions of the peptide C-terminal fragments, because the dominant protonation site (Lys19) is located at the C-terminus In MALDI postsource decay spectra [24],
we observed only masses corresponding to ions of C-terminal fragments (y-ions) of our peptide without carbohydrate residues On the basis of these results, it 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 fragmentation, intensive deglycosyla-tion takes place [25], making unequivocal conclusions
Fig 4 RP-HPLC 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 chromatographic profile shown in Fig 3) (B) Analysis of a standard mixture containing Glc, Gal, Man, Fuc, GlcNAc, GalNAc, ManNAc (C) Analysis of peak 1 (Fig 3A) (D) Analysis of peak 2 (Fig 3A).
Trang 6impossible MS sequencing using electrospray 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 ratio were kept the same, but the PVX
virion (and enzyme) concentration was increased about
fivefold On HPLC of the peptide-containing sample, three
major peaks 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 peaks 3*, 1 and 2 after partial CPY hydrolysis The observed mass values of fragments produced by sequential 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 Stages 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) MALDI mass spectra of peak 2* material 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).
Trang 7primary structure Thus, carbohydrate residues could be
linked only to Ser1 or Ser5 of our peptide N-terminal
pentameric part NAcSAPAS-
Peaks 1* and 2* were shown to contain only these
modified N-terminal pentapeptides: 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 the 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 shown (upper and lower parts,
respect-ively) Mass values in the upper part correspond to ions of
sodium and potassium salts of the glycosylated peptide
NAcSAPAS After CPY treatment, the unglycosylated
C-terminal serine was released (lower part of Fig 5C),
confirming our suggestion that a hexose or deoxyhexose
residue is linked to the acetylated N-terminal serine residue
(NAcSer1) of the PVX CP
As far as we know, this type of 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)1region
for films of the 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 the FTIR spectra, peaks corresponding to valent CH-bond vibrations ( 2900 cm)1), valent C¼O bond vibrations (amide I, 1650 cm)1), deformational 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 peaks Here a complex band of valent water OH-bond vibrations is overlapped with peaks of valent OH-bond and NH-bond vibrations 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)1band in the difference spectra (Fig 7) can be represented by superposition of three bands corresponding to absorption of OH bonds in tightly bound
OH groups (the band at 3240 cm)1), in weakly bound
OH groups ( 3440 cm)1) and in free OH groups of absorbed water molecules ( 3600 cm)1) [27] From the data in Fig 7, it can be seen that the intact wild-type PVX differs from the other virus preparations by a greatly increased water-absorbing capacity What is even more important, on saturation of the wild-type PVX film with water, an intense 3240 cm)1 band was observed in the difference spectrum, supporting the ordering of a large number of water molecules on the virus particle This effect was much weaker for the ST mutant and completely absent
in the case of the trypsin-treated PVX
Fig 6 FTIR absorbance spectra of the intact and trypsin-treated Ru PVX and of the intact ST mutant preparations in the 1000–4000 cm-1region, for dry films and after saturation of the film with water at 100% relative humidity.
Trang 8The role of CP glycosylation in plant virus life cycles
remains unclear, although data on the effects of
glycosyla-tion on the conformaglycosyla-tion and dynamics of O-linked
glycoproteins are accumulating [28–30]
First, we were interested in the presence of glycosyl
modification(s) in the trypsin-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 virion ends,
where the subunit conformation may be unsuitable for
glycosylation
What is the importance of this CP N-terminal
glycosy-lation for PVX virion structure and function? According to
our model [15], the PVX CP N-terminal segment is located
on the virion 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 folding, 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 structures are known to have different
types of hydration shell: 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 virions Thus, a PVX virion may be considered as an electric cable with several layers of insulation The virus RNA is packed into the 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 surface 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 high content of hydroxyl-containing amino acids (11 serine and threonine residues in the 19-residue sequence) This, as well as the predicted existence of a three-stranded b structure in this 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], and, as 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)1region.
Trang 9The 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 surface We propose that the absence of the
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 of the externally located regions
of the virus protein coat However, it cannot be excluded
that the structure of internally located parts of the virion
protein subunits and the structure of the virion itself are
affected by changes in the state of the virion water shell
and this state may be altered by changes in glycosylation,
phosphorylation and other types of modification of the
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 of the wild-type PVX, is accessible to ribosomes in the
intact virions and can be effectively translated 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 cleavage
Acknowledgements
We thank Professor J G Atabekov for valuable discussions The work
was partially supported by Russian Foundation for Basic Research
(grants 02-04-48651 and 03-04-48833).
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Baulcombe, D.C (1993) A feature of the coat protein of potato
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