Digestion of monomeric and trimeric SV-IV with trypsin First, 25 nmol monomeric protein concentration 0.015 mgÆmL1 and trimeric protein concentration 1.0 mgÆmL1 SV-IV were digested separ
Trang 1Structural properties of the protein SV-IV
Carlo Caporale1, Carla Caruso1, Giovanni Colonna2,3, Angelo Facchiano4, Pasquale Ferranti4,5,
Gianfranco Mamone4, Gianluca Picariello4, Flavia Colonna3, Salvatore Metafora6and Paola Stiuso2,3
1 Dipartimento di Agrobiologia ed Agrochimica, Universita´ della Tuscia, Viterbo, Italy; 2 Dipartimento di Biochimica e Biofisica, Seconda University Napoli, Italy; 3 Centro di Ricerca Interdipartimentale di Scienze Computazionali e Biotecnologiche, Napoli, Italy;
4 Istituto di Scienze dell’Alimentazione, CNR, Roma, Italy; 5 Dipartimento di Scienza degli Alimenti, Universita` degli Studi
di Napoli ‘Federico II’, Italy; 6 Istituto Internazionale di Genetica e Biofisica, CNR, Napoli, Italy
We have investigated the molecular mechanisms that
produce different structural and functional behavior in the
monomeric and trimeric forms of seminal vesicle protein
no 4, a protein with immunomodulatory,
anti-inflamma-tory, and procoagulant activity secreted from the rat
seminal vesicle epithelium The monomeric and trimeric
forms were characterized in solution by CD Details of
the self-association process and structural changes that
accompany aggregation were investigated by different
experimental approaches: trypsin proteolysis, sequence
analysis, chemical modification, and computer modeling
The self-association process induces conformational
change mainly in the 1–70 region, which appears to be without secondary structure in the monomer but contains a-helix in the trimer In vivo, proteolysis of seminal vesicle protein no 4 generates active peptides and this is affected
by the monomer/trimer state, which is regulated by the concentration of the protein The information obtained shows how conformational changes between the mono-meric and trimono-meric forms represent a crucial aspect of activity modulation
Keywords: monomer; proteolysis; seminal vesicle protein; SV-IV; trimer
SV-IV (seminal vesicle protein no 4, according to its
electrophoretic mobility in SDS/PAGE) is a basic (pI¼
8.9), thermostable, secretory protein of low Mr (9758)
secreted from the rat seminal vesicle epithelium under
strict androgen transcriptional control [1–6] SV-IV has
been purified to homogeneity and characterized
exten-sively [1–7] We have demonstrated that this protein is a
highly flexible molecule behaving in aqueous solution as
a concentration-dependent self-associating system, with
the degree of association (monomer« dimer « trimer
equilibrium) related to its biological activity [7] Its
polypeptide sequence is 90 amino acids long and is
encoded by a gene that has been isolated, sequenced,
and expressed in Escherichia coli [8–11] SV-IV possesses
potent nonspecies-specific immunomodulatory,
anti-inflammatory, and procoagulant activity [12–22] We
have demonstrated recently by electrospray MS that
10% of the native SV-IV molecules are phosphorylated
in vitro by protein kinase C and that this modification
involves only Ser58 [23] Furthermore, we have
unam-biguously demonstrated that a Tyr36-linked phosphate
group is present in 14% of all native SV-IV molecules
[24]
SV-IV possesses a marked ability to inhibit both in vivo and in vitro phospholipase A2 activity and the platelet-activating factor biosynthetic pathway [13–15] The native protein, transformed by transglutaminase (EC 2.3.2.13) into a complex polymer, binds to the surface of epididymal spermatozoa, greatly decreasing their strong immunogenic-ity [25,26] Although many studies have been devoted to the functional aspects of this protein, very little is known about its structural properties and conformational behavior in aqueous solutions Recent studies have shown that its biological activities are modulated by molecular association
of the protein [7] In this paper, we characterize the solution structure of the monomeric and trimeric forms of SV-IV Experimental CD spectra were deconvoluted into secon-dary-structural elements and compared with structural predictions Finally, details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling
Materials and methods
The experiments were all repeated at least four times
Chemicals All chemicals were of reagent grade and purchased from BDH (Milan, Italy) or Sigma-Aldrich (Milan, Italy) HPLC-grade solvents and reagents were obtained from Carlo Erba (Milan, Italy) Endoproteinase Glu-C and trypsin (sequence-grade) were from Boehringer-Mann-heim
Correspondence to P Stiuso, Dipartimento di Biochimica e Biofisica,
Seconda Universita` degli studi di Napoli, Via Costantinopoli 16,
80138-Napoli, Italy Fax: + 39 81 5665869,
E-mail: paola.stiuso@unina2.it
Abbreviation: SV-IV, seminal vesicle protein no 4.
(Received 20 June 2003, revised 24 September 2003,
accepted 14 November 2003)
Trang 2Purification of SV-IV
SV-IV was purified to homogeneity from adult rat (Wistar–
Fisher strain) seminal vesicle secretion by a previously
published technique [1] The purity of the protein was
assessed by electrophoresis on 15% polyacrylamide gel
in denaturing and non-denaturing conditions, analysis of
amino-acid composition, fingerprint technique, and fast
atom bombardment MS [3,22] The preparations of SV-IV
were completely free of lipopolysaccharide and tumor
necrosis factor as determined by specific biological assays
[27,28] The concentration of the purified protein was
evaluated by its molar absorption at 276 nm
(4100M )1Æcm)1), calculated on the basis of the tyrosine
and phenylalanine residues present in the polypeptide chain
[7]
Spectral measurements
CD measurements were performed at room temperature
with a Jasco J-720 spectropolarimeter, using quartz cells
with a path length of 1 cm and 1 mm Mean residue
ellipticities were calculated from:
½h ¼ MRMhobs=cd where [h] is the mean residue ellipticity in degreesÆcm)2Æ
dmol)1, hobsis the observed ellipticity, MRM is the mean
residue molecular mass calculated from the sequence, d is
the optical path length (cm), and c is the concentration
in gÆmL)1 The CD spectra were analyzed in the region
between 200 and 250 nm to evaluate the amount of
secondary structure by using the instrument computerized
program Spectroscopic analyses were always carried out on
dialyzed samples
Concentration difference spectra
The difference spectra were determined by comparison of
the spectra measured with two different protein
concen-trations in two different cells The CD spectra were
obtained at 25C, using two cells with different light-path
lengths (L1 and L2) and filled with solutions of SV-IV in
NaCl/Pi(0.15MNaCl in 0.05Msodium phosphate buffer,
pH 7.5) The SV-IV concentrations, C1 and C2, were
chosen in such a way that C1· L1 ¼ C2 · L2 In these
conditions, equal numbers of molecules are expected to be
in the light pathway at the two different concentrations
used
Digestion of monomeric and trimeric SV-IV with trypsin
First, 25 nmol monomeric (protein concentration 0.015
mgÆmL)1) and trimeric (protein concentration 1.0 mgÆmL)1)
SV-IV were digested separately with trypsin (enzyme/
substrate, 1 : 50, w/w) at 37C in 0.1% ammonium
bicarbonate buffer, pH 8.0 Aliquots of the incubation
mixtures, corresponding to 5 nmol of the original protein,
were then withdrawn at times ranging from 15 min to 12 h
and freeze-dried The digests were then dissolved in 0.2 mL
aqueous 0.1% trifluoroacetic acid and resolved by
RP-HPLC on a l-Bondapak C18 column Eluent A was
aqueous 0.1% trifluoroacetic acid and eluent B was 0.07% trifluoroacetic acid in acetonitrile The elution was per-formed at a flow rate of 1 mLÆmin)1 using the following program: 10 min 5% B followed by a two-step linear gradient from 5% to 18% B over 50 min and from 18% to 28% B over 70 min Peaks were collected manually and freeze-dried HPLC procedures were carried out on a Beckman GOLD apparatus equipped with a variable-wavelength monitor (model 166) The l-Bondapak C18 column (0.39· 30 cm) was from Waters-Millipore (Mil-ford, MA, USA)
Sequence analyses The purified tryptic peptides of monomeric and trimeric SV-IV were dissolved in aqueous 0.1% trifluoroacetic acid (30–60 lL); aliquots (200–500 pmol) were submitted to sequence analysis using a pulsed liquid-phase automatic sequencer (model 477A) equipped on-line with phenyl-thiohydantoin amino acid analyzer (model 120A) Relevant reagents were from Perkin Elmer/Applied Biosystems Samples were loaded on to a trifluoroacetic acid-treated glass-fiber filter, coated with polybrene, and washed according to the manufacturer’s instructions The average and combined repetitive amino acid yields determined by the instrument software were not lower than 90% for each sequenced peptide The theoretical initial yields were not lower than 50%
Acetylation Appropriate amounts of purified native SV-IV (trimeric form, 4300 gÆmL)1) were acetylated in the presence of excess acetic anhydride (6 : 1, w/v) over total amino groups, and then purified by HPLC
HPLC/electrospray MS HPLC was performed using a C18, 5 lm reverse-phase column (2.1 mm internal diameter· 250 mm; Vydac) with
a flow rate of 0.5 mLÆmin)1 on a Kontron modular system The column effluent was split 1 : 25 with a Valco tee to give a flow rate of about 20 lLÆmin)1 into the electrospray nebuliser The bulk of the flow was run through the detector for peak collection after reading of peptide absorbance at 220 nm Solvent A was 0.03% trifluoroacetic acid in water (v/v); solvent B was 0.02% trifluoroacetic acid in acetonitrile
The electrospray device was a Platform single-quadrupole mass spectrometer (Micromass, Manchester, UK) The source temperature was 120C Mass scale calibration was carried out using myoglobin as the reference compound Quantitative analysis of components was performed by integration of the multiple charged ions of the single species For protein analysis, the separation was attained with a linear gradient of 20–40% solvent B over 40 min and mass spectra were acquired in the range 1800–500 m/z at a scan cycle of 5 s/scan For peptide analysis, the separation was carried out with a linear gradient of 8–40% solvent B over 60 min, and mass spectra were acquired in the range 1600–400 m/z at a scan cycle of 5 s/scan
Trang 3Endoproteinase Glu-C digestion
Endoproteinase Glu-C (Boehringer-Mannheim Italia)
hydrolytic digestion was carried out in 0.4% ammonium
bicarbonate, pH 8, at 40C for 18 h at a substrate/enzyme
ratio of 50 : 1 (w/w) The reaction was stopped by
freeze-drying
MALDI-TOF MS
a-Cyano-4-hydroxycinnamic acid (Fluka, Buchs,
Switzer-land) was used as matrix The protein or peptide samples
(1 lL from a solution 1 gÆL)1 in water) were loaded on
the target and dried Afterwards, 1 lL of a 10 mgÆmL)1
solution of matrix in a mixture of 0.1% trifluoroacetic
acid in water and acetonitrile The samples were analysed
with a Voyager DE-Pro (PerSeptive Biosystem,
Framing-ham, MA, USA) mass spectrometer operating either in
linear or in reflector mode for post source decay tandem
MS
Structure predictions and modeling
Software and databases publicly available on the net were
used for the sequence analyses and structure predictions
BLAST [29] was used to search for amino-acid sequence
similarities between the SV-IV sequence and proteins
collected in databases 3D-PSSM [30], genetTHREAD
[31], andTOPITS[32] were used to apply the fold recognition
strategy, searching for known protein folds compatible with
the SV-IV sequence PHD [33], JPRED [34], and
PSI-PRED [35] web services were used to predict the secondary
structure
The 3D model of the peptide corresponding to the
segment 70–90 of SV-IV was created by using theINSIGHTII
package (Accelrys, San Diego, CA, USA) The Biopolymer
module was used to build the chain of amino acids, folded
as an a-helix in agreement with the secondary-structure
prediction and CD spectra results The initial model was
geometrically optimized by energy minimization according
to the standard settings of the Optimize option
Results
Conformational study of SV-IV Structural modifications between the monomeric and trimeric form of the SV-IV protein are evident on CD spectra (Fig 1) The far-UV CD spectra, as characterized
by an isodichroic point located at about 208 nm indicate the presence of two-state equilibria between the monomeric and trimeric forms The self-association process was accompan-ied by structural changes in the protein The secondary-structure analysis program estimated that the a-helix content is 24% in the monomeric form and 45% in the trimeric form, and b-structure is absent from both forms The CD spectra of the 1–70 and 70–90 regions of the protein are reported in the inset of Fig 1 The secondary-structure analysis program estimated about 100% a-helix for the 70–90 fragment and 2–3% for the 1–70 segment This suggests that the C-terminal segment of the protein is organized as a-helix in the whole protein, and the 1–70 region is poorly structured
SDS increases the a-helix content of proteins revealing the helical potential The a-helix content of the monomeric form of SV-IV is approximately doubled by the addition of 5.4 mMSDS (Fig 1), whereas in the 1–70 region, the a-helix content increases from 2–3% to 23% in the presence of SDS (data not shown) This suggests that the effect of SDS
on the whole protein is exerted in the 1–70 region, which probably plays a fundamental role in the self-association process, with secondary-structure reorganization occurring
in going from the monomeric to the trimeric form Predictive methods have been applied to obtain a theoretical model of the structural organization of SV-IV protein The amino-acid sequence was analyzed using the BLASTprogram to find similar proteins in the nr database (nonredundant database consisting of all protein sequences
Fig 1 Structural characterization of the
monomeric (0.01 lgÆlL)1) and trimeric
(0.1 lgÆlL)1) form of SV-IV protein Far-UV
CD spectra at different concentrations of the
protein (A, 0.01 lgÆlL)1monomeric form;
B, 0.05 lgÆlL)1monomeric/trimeric mixture;
C, 0.1 lgÆlL)1trimeric form) Monomeric
form in SDS (j, 5.4 m M ) The CD spectra of
the 1–70 and 70–90 fragment of SV-IV are
reported in the inset Each spectrum represents
an average of five scans The SV-IV samples
are in 50 m Tris/HCl, pH 7.2.
Trang 4present in the databases) No protein of known 3D structure
was found to have sequence similarity suitable to apply the
homology modeling strategy, i.e at least 20–30% sequence
identity As an alternative, the fold-recognition approach
was applied by using three independent servers on the net:
3D-PSSM, GenetTHREAD, TOPITS None of the
meth-ods identified a known fold suitable for modeling the SV-IV
protein or the 1–70 region Therefore, in conclusion, the two
most reliable strategies for predicting the 3D model of a
protein, i.e homology modeling and fold-recognition
strat-egies, were unable to create a model for either the whole
SV-IV protein or the 1–70 region, and this suggests that this
protein assumes a global structure that is not similar to any
protein of known 3D structure
Secondary-structure predictions were performed by
dif-ferent methods, i.e JPRED, PHD, PSI-PRED (Fig 2) A
consensus prediction based on the agreement between
different methods can be considered more successful than
the single method used The consensus prediction suggests a
few helical regions (48–53 and the C-terminal region)
covering 20% of the protein, which is in good agreement
with the secondary-structure content revealed by CD studies
of 24% a-helix for the monomeric form
Protein digestion and fragment characterization
SV-IV is a 90-amino-acid protein lacking disulfide bridges
and possessing nine lysine and seven arginine residues,
which represents a large number of potential hydrolysis sites
for trypsin For this reason, we selected this protease to
investigate the different accessibility of crucial sites
sup-porting molecule aggregation and characterizing the
mono-meric and trimono-meric forms Both forms of SV-IV were
digested separately using the same enzyme/substrate ratio
Aliquots of the incubation mixtures were withdrawn at
various times, and the formation of fragments was
moni-tored by RP-HPLC Figure 3 shows chromatograms of the
digestion mixtures of the trimeric (Fig 3A) and monomeric
(Fig 3B) forms after 12 h incubation Lower amounts of all
the peptides were also produced after 15 min incubation,
indicating that both monomeric and trimeric forms were
readily digested by trypsin (not shown) Each fragment
collected was submitted to automatic sequence analysis The
corresponding start-end position in the protein sequence is indicated in the figure Some differences in the hydrolytic pathways were found The protein is hydrolyzed at Arg57 only in the monomeric form In fact, whereas fragments 40–56 and 60–63 are common to both chromatograms, fragments 40–57 and 58–63 arose only from the monomeric form (Fig 3B) The tripeptide 57–59 complementing frag-ments 40–56 and 60–63 which originated from digestion
of the trimer was not identified in the chromatogram (Fig 3A) Furthermore, fragment 80–83, complementary to fragments 64–79 and 84–90, was generated only from hydrolysis of the trimeric form (Fig 3A), whereas fragment 82–90 arose only from the monomeric form (Fig 3B), indicating further digestion at the level of Arg81 The dipeptide Lys80–Arg81, complementary to fragments 64–79 and 82–90, was not identified in the chromatogram (Fig 3B) Both peptide bonds Lys80–Arg81 and Arg81– Ser82 seem to be hidden in the trimer, as the whole fragment Lys80-Arg81-Ser82-Arg83 was found (Fig 3A), whereas no fragment ending at Lys80 or starting at Arg81 arose from digestion of the monomer (Fig 3B) As a consequence, Arg81 should be accessible only in the monomeric form, whereas Lys80 also seems to be quite hidden in the monomeric form
SV-IV is not fully acetylated by acetic anhydride
An aliquot was directly analysed by HPLC/electrospray MS
to characterize the acetylated form of SV-IV protein As shown by its transformed spectrum (Fig 4), several com-ponents were present, differing with respect to the number
of acetyl groups (mass increase of 42 for each acetyl group incorporated), and indicating that the reaction was not complete, but generated a mixture of incompletely acetyl-ated forms of the protein These contained from three to eight acetyl groups (the maximum expected was 10, considering nine lysine residues and the N-terminal amino group), the most abundant ranging from four to six
To identify the acetylated residues, another aliquot of protein was first digested with endoproteinase Glu-C and then analysed by HPLC/electrospray MS to obtain the relevant peptide map The peptides identified are shown in Table 1 We were therefore able to screen the whole protein sequence to identify the acetylated peptides
Peptides were identified by their molecular mass on the basis of the known protein sequence and the endoproteinase Glu-C specificity In most cases, a mixture of the native and acetylated peptides was observed and identified by the mass increase of 42 mass units The relative level of acetylation of
a peptide was estimated on the basis of the intensity ratio of the native and acetylated species From the data summar-ized in Table 1, it can be seen that some of the peptides were almost completely acetylated whereas some showed low or minimal acetylation To locate acetylated Lys residues on peptides containing more than one Lys, the fractions collected from the HPLC separation (Fig 6) were analysed
by tandem MS using MALDI-TOF PSD-MS As an example, peptide 6–12, containing Lys6, was acetylated only
to 5%; peptide 72–90, containing Lys78, Lys79, and Lys80, showed partial acetylation at one of the three residues, because the signal of the triacetylated species was less intense than that of the diacetylated species The MS/MS
Fig 2 Secondary-structure prediction Amino-acid sequence and
sec-ondary-structure predictions performed with PHD, JPRED, and
PSI-PRED (see Materials and methods) a-Helix is indicated by H and
b-strand conformation is indicated by E.
Trang 5analysis of the chromatographic fraction showed that the
two Lys residues at positions 78 and 79 were both
acetylated, whereas Lys80 was not (Fig 5)
Molecular modeling of 70–90 region
CD spectra of SV-IV fragment 70–90 and the
secondary-structure prediction suggested that the region 70–90 should
have a high a-helix content We created a computer model
of the 70–90 peptide The initial conformation of the
backbone was imposed as a-helix, and energy minimization was performed in order to optimize the peptide structure As
a consequence of such optimization, the initial backbone conformation was approximately conserved only in the region corresponding to the 70–81 segment (Fig 6) In the 82–90 segment, a helical conformation was conserved, but it was not consistent with a-helix features, as the Kabsch and Sander assignment of secondary structure did not define helix in this segment of the peptide The initial conformation
of side chains was also modified under energy minimization,
Fig 3 Protein digestion Chromatograms of digestion mixtures of trimeric (A) and monomeric (B) forms of SV-IV after 12 h incubation with trypsin Each peak is labelled with the corresponding protein fragment Peaks present in both chromatograms refer to the protein segments 60–63, 33–39, 64–78, 64–79, 84–90, 40–56, 5–32 + 7–32, 5–39 + 7–39 Peaks present in chromatogram A but not in B refer to segment 80–83 Peaks present in chromatogram B but not in A refer to the protein segments 50–63, 82–90, 40–57.
Trang 6and some interesting results were obtained In particular, the
initial extended conformation of the Tyr76 and Lys79 side
chains were modified and assumed an orientation suitable
for hydrogen-bond formation (Fig 6) This finding is in
good agreement with other experimental results and will be
discussed below
Discussion
SV-IV is a protein with immunomodulatory,
anti-inflam-matory, and procoagulant activity Its physiological
concentration ranges from 2 to 48 lM, i.e from 0.019 to 0.47 lgÆlL)1, in different conditions and organs [19] We have recently demonstrated that, in the same concentra-tion range, the protein shows a monomer fi dimer
fi trimer quaternary organization, and the equilibrium of self-association appears to control the biological properties
of the protein [7] Moreover, the immunomodulatory activity is related to the structural integrity of the whole molecule, whereas the anti-inflammatory and procoagu-lant activity is located in the unstructured 1–70 region of the molecule In this work, structural differences between the monomeric and trimeric form of SV-IV have been confirmed from CD spectra, which revealed double the content of a-helix in the trimeric form compared with the monomeric form As suggested by CD spectra of the 1–70 and 71–90 fragments, as well as by prediction methods, the C-terminal region has high propensity to form a-helix,
so this region may be responsible for the a-helix observed
by CD in the monomer On the other hand, the increase
in a-helix in the trimeric form may result from rearrange-ment of the 1–70 region, where some predictive methods assign a-helix conformation This region is poorly struc-tured, but the addition of SDS revealed a hidden ability to form helical structure
To find functional differences related to the structural modifications occurring in the monomer–trimer transition,
we investigated how proteolysis and post-translational modifications could be affected by self-association Limited proteolysis showed that both monomeric and trimeric forms are very sensitive to trypsin hydrolysis, Lys80 being the only putative proteolytic site not hydrolyzed in both forms It is interesting to note that Arg57 and Arg81 are hydrolyzed in the monomeric but not the trimeric form These differences
Fig 4 Electrospray mass spectrum of the protein SV-IV acetylated with
acetic anhydride SV-IV, purified by gel filtration and ion-exchange
chromatography, was incubated with acetic anhydride under the
conditions described in Materials and methods, desalted, and then
analysed by electrospray MS.
Table 1 Analysis of the endoproteinase Glu-C digest of acetylated SV-IV by HPLC/electrospray MS Acetylated SV-IV was digested with endo-proteinase Glu-C The resulting peptide mixture was analyzed using a Vydac C 18 column (250 · 2.1 mm, 5 lm) on-line with a Platform mass spectrometer The experimental details are given in Materials and methods The measured mass is the mean ± SD molecular mass calculated by integrating the multiple peaks corresponding to each molecular species and differing only in the total number of charges measured by electrospray
MS Theoretical mass is the mass calculated on the basis of the protein amino-acid sequence The relative abundance refers to the ratio of the acetylated/unacetylated SV-IV forms ID, identification number of peaks.
HPLC
peak ID
Measured mass
(Da)
Theoretical mass (Da) Peptidea Acetylated residues
Relative abundance (% of acetylation)
1 432.5 ± 0.1 432.5 13–16
2 786.5 ± 0.4 786.5 1–5 Lys 2 and 4; N-term 100
435.1 ± 0.2 435.4 49–52
3 2075.5 ± 0.4 2076.3 53–71
2117.9 ± 0.5 2118.3 53–71 Lys59 24
4 1214.7 ± 0.1 1214.1 17–29
5 2261.6 ± 0.9 2262.4 53–73
2303.5 ± 0.2 2304.4 53–73 Lys59 24
6 2022.4 ± 0.9 2022.3 30–48
7 2064.4 ± 0.9 2064.3 30–48 Lys 34 or 39 45
2106.3 ± 0.9 2106.3 30–48 Lys 34 and 39 5
8 2436.9 ± 0.5 2437.9 30–52
2479.0 ± 0.5 2479.9 30–52 Lys 34 or 39 45 2521.5 ± 0.5 2521.9 30–52 Lys 34 and 39 5
9 2080.6 ± 0.4 2081.2 74–90 Lys 78 or 79 or 80 45
10 2123 6 ± 0.4 2123.2 74–90 Lys 78, 79 and 80 55
a
Numbers refer to the N-terminus and C-terminus of each peptide.
Trang 7can be compared with structural predictions and structural
features of both forms
Lysine acetylation gave us further information about the
structural environment of the lysines, as acetylated lysines
can be considered to be exposed to the surface of the
protein, whereas non-acetylated lysines are probably not
Some of the data appear to contradict the results of limited
proteolysis In particular, Lys6 appears not to be acetylated
and therefore not exposed to the surface, but proteolytic
cleavage occurs at this residue These contrasting data may
be explained by the possibility that Lys6 becomes exposed
only after the hydrolysis of Lys2 and Lys4 Moreover,
Lys34 is partially acetylated (5%) but is not hydrolysed by trypsin in both monomeric and trimeric forms The fact that Lys34 is followed by Pro35 and the poor efficiency of trypsin in cleaving Lys–Pro bonds may explain why Lys34 is not hydrolyzed in both monomeric and trimeric forms Moreover, it may be possible that this Lys is exposed in the monomeric but not the trimeric form In fact, at the protein concentration used for acetylation, the trimeric form is predominant, so the low acetylation observed may be related to the low amount of the monomeric form always present in equilibrium with the trimeric form
Most of the peptide bonds hydrolyzed by trypsin are located in regions without secondary-structure elements such as helices or b-strands, which may confer protease resistance on the backbone [36–39]
A long helix is predicted in the 75–88 region It is interesting to note that Lys78, Lys79, and Arg81, located in such a helical region, are hydrolyzed by trypsin, while the enzyme does not hydrolyze Lys80 Secondary-structure predictions allow us to hypothesize that Lys80 could not be hydrolyzed because the amino group of its side chain might
be hydrogen-bonded to the -OH group of the Tyr76 side chain It is known that helical residues in position i and
i+ 3/i + 4 expose their side chains on the same side of the helical surface and may interact by forming salt bridges or hydrogen bonds Therefore, our hypothesis is supported by two experimental observations: (a) tyrosine titration does not act on all three tyrosines of SV-IV protein [7]; (b) the peptide containing Lys78, Lys79, and Lys80 is only partially acetylated The partial titration of tyrosine may be explained by the formation of tyrosinate The modeling of
a peptide corresponding to the 70–90 region of SV-IV suggests that Tyr76 may form a hydrogen-bond with Lys79, supporting this hypothesis As tyrosinate formation is not evident in the trimeric form, the Tyr76 side chain should be suitable to form a transient hydrogen-bond with Lys79 or Lys80 in the monomeric form, whereas, in the trimeric form, the region that includes Lys79, Lys80 and Arg81 may
be involved in the oligomerization, as demonstrated by the change in sensitivity to trypsin hydrolysis, in agreement with
Fig 6 Molecular model of the peptide corresponding to the 70–90
region of SV-IV Top, initial model, with the imposed a-helix backbone
conformation Bottom, conformation reached after energy
minimiza-tion The loss of a-helix conformation on the C-terminal side is evident.
A dashed line indicates the hydrogen-bond between the Tyr76 and
Lys79 side chains It can be seen how, after minimization, the
back-bone is modified in the middle, and the helix is interrupted.
Fig 5 MALDI-TOF mass spectrum in
post-source decay mode of the peptide at 2081.7 m/z.
The peptide at m/z 2081.7 corresponded to the
diacetylated peptide 74–90 from the
endo-proteinase Glu-C digest of acetylated protein
SV-IV Signal diagnostics of peptide structure
are indicated in the figure.
Trang 8the absence of tyrosinate In fact, Arg81 is hydrolyzed only
in the monomeric form of SV-IV There are two different
explanations for these data In the first, the resistance of
Arg81, as well as Arg57, to attack by trypsin in the trimeric
form is due to subunit association and the consequent loss
of exposed surface Arg57 and Arg81 may be located in the
region of interaction, and therefore would be exposed in the
monomeric form and buried in the trimeric form The
second hypothesis is based on the observation of a higher
a-helix content in the trimeric form of SV-IV The long
helices predicted in segments 48–60 and 75–88 may be
responsible for a rigid conformation, which is resistant to
proteases, thus preventing hydrolysis at the level of Arg57
and Arg81 However, the three prediction methods do not
agree in predicting these two long helices It may be possible
that such differences in secondary-structure predictions are
caused by regions being able to adopt different secondary
structures under different quaternary structure conditions
JPRED predicted in the 75–88 region two short helices,
connected by a short nonhelical segment, which includes
Arg81 and Ser82 It is possible that this region is folded
differently in the monomeric and trimeric forms of SV-IV: a
long helix is formed in the trimeric protein, whereas two
short helices are present in the monomeric form Such
conformations are compatible with the different responses
to trypsin hydrolysis; Arg81 may be in a loop region when
the protein is in the monomeric form, and therefore sensitive
to hydrolysis, whereas it might be in a long a-helix when the
protein is in the trimeric form, making it resistant to
protease attack Similarly, the 48–60 region is only predicted
to be a-helix by PSI-PRED, the other two methods
predicting a shorter helix, leaving Arg57 in a loop region
Finally, we note that the differences in proteolytic
sensi-tivity of the monomeric and trimeric forms of SV-IV are
located at residues Arg57 and Arg81, coinciding with
peptide bonds proteolysed in vivo We have previously
demonstrated that the partially purified SV-IV fraction
includes detectable amounts of SV-IV peptides, i.e 1–16,
42–90, 81–88, 58–90, 1–80 The hypothesis that such
peptides play a functional role is in good agreement with
the opportunity to control proteolysis via the monomer–
trimer equilibrium
Conclusions
The aim of this work was to understand the molecular
mechanisms that produce different structural and functional
behavior in the monomeric and trimeric forms of SV-IV
We have previously demonstrated that SV-IV is active in
different biological assays as three different functional
states: monomeric, trimeric, and proteolytically cleaved In
this paper, we show that self-association induces a
con-formational change mainly in the 1–70 region, which
appears to be partially a-helix in the trimer but without
secondary structure in the monomer This conformational
change may modulate the proteolysis of SV-IV, which
in vivogenerates active peptides The different physiological
levels of the protein in different conditions and organs may
activate SV-IV by shifting the structure between monomeric
and trimeric forms, producing two forms with different
activities and different sensitivities to proteolysis, which
generates active peptides In conclusion, this study indicates
that conformational changes between the monomeric and trimeric forms is an important aspect of the activity modulation
Acknowledgements
This work was supported by a grant from Regione Campania.
References
1 Ostrowski, M.C., Kistler, M.K & Kistler, W.S (1979) Purifica-tion and cell-free synthesis of a major protein from rat seminal vesicle secretion A potential marker for androgen action J Biol Chem 254, 383–390.
2 Mansson, P.E., Sugino, A & Harris, S.E (1981) Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression Nucleic Acids Res 9, 935–946.
3 Abrescia, P., Corbo, L & Metafora (1986) S Maturation in dif-ferent translational systems of the protein RSV-IV secreted from the rat seminal vesicle epithelium Bull Mol Biol Med 11, 19–33.
4 Metafora, S., Facchiano, F., Facchiano, A., Esposito, C., Peluso,
G & Porta, R (1987) Homology between rabbit uteroglobin and the rat seminal vesicle sperm binding protein: prediction of structural features of glutamine substrates for transglutaminase.
J Protein Chem 6, 353–359.
5 Metafora, S., Lombardi, G., De Rosa, M., Quagliozzi, L., Rav-agnan, G., Peluso, G & Abrescia, P (1987) A protein family immunorelated to a sperm-binding protein and its regulation in human semen Gamete Res 16, 229–241.
6 Abrescia, P., Lombardi, G., De Rosa, M., Quagliozzi, L., Guardiola, J & Metafora, S (1985) Identification and preliminary characterization of a sperm-binding protein in normal human semen J Reprod Fertil 73, 71–77.
7 Stiuso, P., Metafora, S., Facchiano, A.M., Colonna, G & Ragone, R (1999) The self association of protein SV-IV and its possibile functional implications Eur J Biochem 266, 1029–1035.
8 Harris, S.E., Mansson, P.-E., Tully, D.B & Burkhart, B (1983) Seminal vesicle secretion IV gene: allelic difference due to a series
of 20-base-pair direct tandem repeats within an intron Proc Natl Acad Sci USA 80, 6460–6464.
9 Kandala, C., Kistler, M.K., Lawther, R.P & Kistler, W.S (1983) Characterization of a genomic clone for rat seminal vesicle secre-tory protein IV Nucleic Acids Res 11, 3169–3186.
10 McDonald, C., Williams, L., McTurck, P., Fuller, F., McIntosh,
E & Higgins, S (1983) Isolation and characterisation of genes for androgen-responsive secretory proteins of rat seminal vesicles Nucleic Acids Res 11, 917–930.
11 D’Ambrosio, E., Del Grosso, N., Ravagnan, G., Peluso, G & Metafora, S (1993) Cloning and expression of the rat genomic DNA sequence coding for the secreted form of the protein SV-IV Bull Mol Biol Med 18, 215–223.
12 Galdiero, F., Tufano, M.A., De Martino, L., Capasso, C., Porta, R., Ravagnan, G., Peluso, G & Metafora, S (1989) Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium J Reprod Immunol 16, 269–284,.
13 Metafora, S., Peluso, G., Persico, P., Ravagnan, G., Esposito, C.
& Porta, R (1989) Immunosuppressive and anti-inflammatory properties of a major protein secreted from the epithelium of the rat seminal vesicles Biochem Pharmacol 38, 121–131.
14 Metafora, S., Porta, R., Ravagnan, G., Peluso, G., Tufano, M.A.,
De Martino, L., Ianniello, R & Galdiero, F (1989) Inhibitory effect of SV-IV, a major protein secreted from the rat seminal
Trang 9vesicle epithelium, on phagocytosis and chemotaxis of human
polymorphonuclear leukocytes J Leukoc Biol 46, 409–416.
15 Camussi, G., Tetta, C., Bussolino, F., Metafora, S., Peluso, G.,
Esposito, C & Porta, R (1990) An anti-inflammatory protein
secreted from rat seminal vescicle epithelium inhibits the synthesis
of platelet: activating factor and the release of arachidonic acid
and prostacyclin Eur J Biochem 192, 481–485.
16 Vuotto, M.L., Peluso, G., Mancino, D., Colonna, G., Facchiano,
A., Ielpo, M.T., Ravagnan, G & Metafora, S (1993) Inhibition of
interleukin-1 release and activity by the rat seminal vesicle protein
SV-IV J Leukoc Biol 53, 214–222.
17 Peluso, G., Porta, R., Esposito, C., Tufano, M.A., Toraldo, R.,
Vuotto, M.L., Ravagnan, G & Metafora, S (1994) Suppression
of rat epididymal sperm immunogenicity by a seminal vesicle
secretory protein (SV-IV) and transglutaminase both in vivo and
in vitro Biol Reprod 50, 593–602.
18 Romano-Carratelli, C., Galdiero, M., Nuzzo, I., Bentivoglio, C.,
Porta, R., Peluso, G., Ravagnan, G & Metafora, S (1995) In vivo
inhibition of cell-mediated and humoral immune responses to
cellular antigens by SV-IV, a major protein secreted from the rat
seminal vesicle epithelium J Reprod Immunol 28, 15–30.
19 Tufano, M.A., Porta, R., Farzati, B., Di Pierro, P., Rossano, F.,
Catalanotti, P., Baroni, A & Metafora, S (1996) Rat seminal
vesicle protein SV-IV and its transglutaminase-synthesized
poly-aminated derivative Spd 2 -SV-IV induce cytokine release from
human resting lymphocytes and monocytes in vitro Cell Immunol.
168, 148–157.
20 Di Micco, B., Colonna, G., Porta, R & Metafora, S (1994) Rat
protein SV-IV accelerates human blood coagulation in vitro by
selective inhibition of antithrombin III Biochem Pharmacol 48,
345–352.
21 Di Micco, B., Stiuso, P., Colonna, C., Porta R., Marchese, M.,
Schinina`, M.E., Macalello, M.A & Metafora, S (1997) A peptide
derivative (1–70 fragment) of protein SV-IV accelerates human
blood coagulation in vitro by selective inhibition of the
heparin-induced antithrombin III acivation J Peptide Res 49, 179–182.
22 Di Micco, B., Caen, J., Colonna, C., Macalello, M.A., Marchese,
M., Stiuso, P., Di Micco, P., Morelli, F & Metafora, S (2000)
Inhibition of antithrombin by protein SV-IV normalizes the
coa-gulation of hemophilic blood Eur J Pharmacol 391, 1–9.
23 Metafora, V., Franco, P., Massa, O., Morelli, F., Stiuso, P.,
Ferranti, P., Mamone, G., Malori, A., Stoppelli, M.P &
Meta-fora, S (2001) Phosphorilation of seminal vesicle protein IV on
Ser58 enhances its peroxidase-stimulating activity Eur J
Bio-chem 268, 3858–3869.
24 Ferrant, P., Mamone, G., Malorni, A., Guardiola, J., Stiuso, P &
Metafora, S (1997) Structural heterogeneity, post-traslational
modification, and biological activities of SV-IV, a major protein
secreted from that rat seminal vesicle epthelium Rapid Commun.
Mass Spectrom 11, 1007–1014.
25 Porta, R., Esposito, C., Gentile, V., Mariniello, L., Peluso, G &
Metafora, S (1990) Transglutaminase-catalyzed modifications of
SV-IV, a major protein secreted from the rat seminal vesicle
epi-thelium Int J Peptide Protein Res 35, 117–122.
26 Porta, R., Esposito, C., Metafora, S., Malorni, A., Pucci, P., Siciliano, R & Mari (1991) Mass spectrometric identification of the amino donor receptor sites in a transglutaminase protein substrate secreted from rat seminal vesicles Biochemistry 30, 3114–3120.
27 Yin, E.T., Galanos, C., Kinsky, S., Bradshaw, R.A., Wessler, S., Luderig, O & Sarmiento, M.F (1972) Picogram-sensitive assay for endotoxin: gelation of Limulus polyphemus blood cell lysate induced by purified lipopolysaccharides and lipid A from Gram-negative bacteria Biochim Biophys Acta 261, 284–289.
28 Rubin, B.Y., Anderson, S.L., Sullivan, S.A., Williamson, B.D., Carswell, E.A & Old, L (1985) Purification and characterization
of a human tumor necrosis factor from the LuKII cell line Proc Natl Acad Sci USA 82, 6637–6641.
29 Altschul, S.F., Madden, T.L., Scha¨ffer, A.A., Zhang, J., Zhang, Z., Miller, W & Lipman, D.J (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 25, 3389–3402.
30 Kelley, L.A., MacCallum, R.M & Sternberg, M.J.E (2000) Enhanced genome annotation using structural profiles in the program 3D-PSSM J Mol Biol 299, 499–520.
31 Jones, D.T (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences J Mol Biol 287, 797–815.
32 Rost, B (1995) TOPITS: threading one-dimensional predictions into three-dimensional structures In Third International Confer-ence on Intelligent Systems for Molecular Biology (ISMB); Cambridge, UK (July 16–19, 1995) (Rawlings, C., Clark, D., Altman, R., Hunter, L., Lengauer, T & Wodak, S., eds), pp 314–
321 AAAI Press, Menlo Park, CA.
33 Rost, B & Sander, C (1993) Improved prediction of protein secondary structure by use of sequence profiles and neural net-works Proc N atl Acad Sci USA 90, 7558–7562.
34 Cuff, J.A., Clamp, M.E., Siddiqui, A.S., Finlay, M & Barton, G.J (1998) JPred: a consensus secondary structure prediction server Bioinformatics 14, 892–893.
35 Jones, D.T (1999) Protein secondary structure prediction based
on position-specific scoring matrices J Mol Biol 292, 195–202.
36 Caporale, C., Caruso Facchiano, C., Nobile, A., Leonardi, M., Bertini, L., Colonna, L & Buonocore, G (1999) Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures Proteins
36, 192–204.
37 Hubbard, S.J., Beynon, R.J & Thornton, J.M (1998) Assessment of conformational parameters as predictors of limited proteolytic sites in native protein structures Protein Eng 11, 349–359.
38 Hubbard, S.J., Eisenmenger, F & Thornton, J.M (1994) Mod-eling studies of the change in conformation required for cleavage
of limited proteolytic sites Protein Sci 3, 757–768.
39 Hubbard, S.J., Campbell, S.F & Thornton, J.M (1991) Mole-cular recognition Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors J Mol Biol 220, 507–530.