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Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection
Biochimica et Biophysica Acta 1528 (2001) 107^115
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Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection Fabiana Superti a , Rosa Siciliano b , Barbara Rega b , Francesco Giansanti c , Piera Valenti d , Giovanni Antonini c; * a
c
Department of Ultrastructures, Istituto Superiore di Sanita©, I-00161 Rome, Italy b Institute of Food Science and Technology, CNR, I-83100 Avellino, Italy Department of Basic and Applied Biology, University of L'Aquila, Coppito, I-67010 L'Aquila, Italy d Institute of Microbiology, II University of Naples, I-80135 Naples, Italy Received 18 April 2001; received in revised form 26 June 2001; accepted 26 June 2001
Abstract Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the antirotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86^258) and a small peptide (324^329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Lactoferrin ; Rotavirus; Sialic acid ; Metal saturation; Tryptic fragment; Mass spectrometry
1. Introduction Lactoferrin is a monomeric glycoprotein with a molecular mass of about 80 kDa [1,2] which binds two iron atoms with very high a¤nity ; it is present in various biological £uids and in speci¢c granules of polymorphonuclear leucocytes [3,4]. Lactoferrin possesses a variety of biological functions, such as promotion of iron absorption [5], immunomodulation [6] and inhibiting activity towards bacteria, fungi, protozoans [6] and viruses [7]. Bovine lactoferrin (bLf), which consists of a single polypeptide chain of 689 amino acidic residues, like lactoferrin of other mammalian species, is folded into two symmetric globular lobes each of which is itself folded into two domains (Nlobe: N1 and N2 and C-lobe: C1 and C2), each delimiting a Fe3 binding site [8,9]. The complete structures of lactoferrin glycans have been described only a few years ago
* Corresponding author. Fax: +39-862-43-32-73. E-mail address : [email protected] (G. Antonini).
[10^13], although their role in any biological activity related to this protein remains to be de¢ned. Brie£y, in bLf there are two oligomannosidic type (linked to Asn 233 and Asn 545) and two biantennary N-acetyllactosamine type glycans, partially fucosylated and sialylated (linked to Asn 368 and Asn 476) [14,15]. Sialic acid is present in a ratio of about two residues per molecule of bLf [14,15]. Neither the nature nor the location of the bound carbohydrate chains has any e¡ect on the lactoferrin polypeptide conformation [16,17]. Since 1994, human lactoferrin and bLf have been recognised as potent inhibitors towards di¡erent enveloped human pathogen viruses such as herpes simplex virus (HSV) 1 and 2 [18^21], human cytomegalovirus (HCMV) [18], human immunode¢ciency virus (HIV) [22,23] and human hepatitis C virus (HCV) [24]. The antiviral e¡ect of bLf against two naked pathogen viruses, SA-11 rotavirus and poliovirus type 1, has also been demonstrated [25,26]. For all viruses investigated to date, bLf exerts its antiviral activity towards the early phases of infection. Since lactoferrin is known to bind heparan sulphate containing
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 7 8 - 7
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proteoglycans [27,28], which in turn promote the initial interaction of HSV-1 and HIV with host cells [29,30], its inhibiting activity on these viruses has been ascribed to a competition for cell receptors, even though a direct lactoferrin interaction with viral particles cannot be ruled out. For rotavirus, which interacts with sialic acid on cell surfaces [31^35], it has been suggested that lactoferrin could bind to rotavirus viral particles [25], similarly to that reported for poliovirus [26], HIV-1 [22] and HCV [36]. In the present study, we have attempted to clarify the mechanism of the anti-rotavirus e¡ect of bLf. In particular, as regards the role of metal binding, we have investigated the e¡ect of bLf metal ion saturation, and concerning the carbohydrate and protein moieties we tested the anti-rotavirus activity of desialylated bLf and of peptides originated by the tryptic digestion of bLf. We have found that complete iron saturation of the protein did not modify antiviral activity of bLf towards rotavirus, while zinc or manganese saturation slightly decreased it. On the contrary, desialylation enhanced the anti-rotavirus activity of bLf. A large body of work was then undertaken aimed to achieve a detailed structural characterisation of the high molecular mass fragments deriving from bLf tryptic digestion using advanced mass spectrometric methodologies. We have found that only one large and one small peptide retained activity towards rotavirus infection even if at a lower extent with respect to the undigested bLf. 2. Materials and methods 2.1. Cells The human colon adenocarcinoma cell line, HT-29, was obtained from the American Type Culture Collection (ATCC, Rockville, MA, USA). Cells were grown at 37³C in RPMI 1640 medium (Gibco) containing 1.2 g/l NaHCO3 , and supplemented with 10% inactivated foetal calf serum (FCS, Flow Laboratories), 2 mM glutamine, nonessential amino acids, penicillin (100 IU/ml), and streptomycin (100 Wg/ml). 2.2. Virus Simian rotavirus SA-11 was grown in LLC-MK2 cells (a monkey kidney derived cell line). Virus was pre-activated with 10 Wg/ml trypsin (type IX, Sigma) for 60 min at 37³C, diluted 10-fold in 199 medium (HyClone), and then inoculated onto con£uent monolayers grown in roller bottles at a multiplicity of infection (moi) of 5 plaque forming units (pfu)/cell. After 90 min at 37³C, the inoculum was removed, and the monolayers were washed once in phosphate bu¡ered saline (PBS, pH 7.4) and then incubated at 37³C in 199 medium containing 1 Wg/ml trypsin. When an extensive cytopathic e¡ect (CPE) was observed, infected cultures were frozen and thawed three times, centrifuged
(3000Ug; 10 min), and supernatants were stored at 370³C. 2.3. Cytotoxicity assay To establish the maximal non-cytotoxic dose of the bLf derivative to be tested, 2-fold serial dilutions of each substance in RPMI 1640 medium were incubated at 37³C with con£uent HT-29 cells grown in 96-well tissue culture microplates (Flow Laboratories). After 24 h, the following parameters were evaluated : cell morphology and viability (determined by neutral red staining) were examined by light microscopy and cell proliferation was evaluated quantitatively by microscopic counts after dispersion into individual cells with trypsin. Protein dilutions that did not a¡ect any of these parameters were considered non-cytotoxic concentrations and utilised for antiviral assays. 2.4. Antiviral assays Rotavirus infection was synchronised by a temperature shift: viral adsorption was performed at 4³C for 1 h (attachment step) and, after removal of inoculum, virus infection was allowed to progress by raising the temperature to 37³C. HT-29 cells grown in 96-well tissue culture microplates for 48 h at 37³C in 5% CO2 , were incubated with di¡erent concentrations of proteins during the virus attachment step (1 h, 4³C). As viral inoculum was utilised pre-activated SA-11 virus at a multiplicity of infection of 1 pfu/cell. Then, cells were rinsed thoroughly and incubated with the same concentrations of proteins for 24 h at 37³C in 5% CO2 ^air. The CPE induced by rotavirus was measured by the neutral red uptake assay as previously described [20], and results were expressed as percentage of cytopathic e¡ect inhibition by comparison with untreated control cultures. In some experiments, proteins were added to the cells during (1 h at 4³C), during and after (1 h at 4³C and 24 h at 37³C), or after (24 h at 37³C), the viral attachment step, for details see [26]. Each sample was done in triplicate. 2.5. Lactoferrin Apo-lactoferrin from bovine milk (apo-bLf), purchased from Armour Proteins (France), was dissolved as stock solution in PBS. Lactoferrin purity was checked by SDS^PAGE stained with silver nitrate and judged to be greater than 95%. Protein concentration was determined by UV spectroscopy on the basis of the extinction coe¤cient of 15.1 (280 nm, 1% solution) [37]. Apo-, iron-, manganese- and zinc-saturated bLf (apo-bLf, Fe-bLf, Mn-bLf, and Zn-bLf) were prepared and tested as reported previously [21,26]. Brie£y, protein-bound iron was removed by extensive dialysis against 50 mM pyrophosphate bu¡er, pH 4.0, followed by dialysis against PBS, pH 7.2. Metalsaturated proteins were obtained by the addition of an
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adequate molar ratio of the metal ions, in the form of chloride or sulphate salts, in 0.1 M Tris^HCl, pH 8.0, containing 5 mM NaHCO3 . Binding was performed by stirring overnight at 4³C; then, not bound metal ions were removed by dialysis against iron-free PBS pH 7.2, for 72 h at 4³C. As control, the same metal ions at the identical concentration utilised to saturate at 100% the binding sites of bLf were supplied to the cells as sulphate salts. Lactoferrin fragment 324^329 (YLTTLK) was synthesised by Tecnogen (Italy) and its purity was checked by HPLC and electrospray mass spectrometric (ES/MS) analysis. Before biological assays, all proteins were sterilised by ¢ltration on 0.45 Wm Millex HV at low protein retention (Millipore). 2.6. Desialylation Desialylation was carried out using neuraminidase of Arthrobacter ureafaciens (Sigma). 4 mU of neuraminidase were added to 1 mg of apo-bLf in 0.5 ml of 20 mM phosphate bu¡er pH 6.5. The reaction mixture was incubated at 37³C for 48 h. The residual neuraminidase activity after incubation and dilution ( 6 0.05 mU/ml) did not a¡ect rotavirus infection in control experiments. 2.7. Protein blotting and glycan detection The same quantities of each modi¢ed protein were subjected to SDS^PAGE on 10% gels and then ¢xed and silver stained or transferred to a nitrocellulose membrane. After removal of blocking agent and rinsing, incubation with 25 Wg/ml lectins (concanavalin A, speci¢c for K-man and K-glc, or Maackia amurensis lectin, speci¢c for sialic acid, Sigma) was performed. Lectin binding was revealed by peroxidase reaction with 3,3P-diaminobenzidine as substrate. A Glycotrack kit (detecting carbohydrate moieties by using a speci¢c carbohydrate oxidation reaction, Oxford Glyco System, Abingdon, UK) was also used for glycan detection on the blotted proteins. Ascorbate oxidase from zucchini and cytochrome c from horse heart were used as controls (positive and negative, respectively) of glycan detection. Isoelectric focusing was also used to test the sialic acid removal. No relevant alterations of bLf protein structure and iron binding properties in desialylated protein were observed by means of visible and second derivative UV spectrophotometry or CD spectroscopy [17]. 2.8. Enzymatic hydrolysis and chemical modi¢cations of lactoferrin and its tryptic fragments bLf (4 mg/ml) was dissolved in 50 mM ammonium bicarbonate, pH 8.5 and trypsin (from bovine pancreas TPCK treated, Sigma) digestion was performed at 37³C overnight using an enzyme to substrate ratio of 1:50 w/w.
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Deglycosylation was performed by treatment with PNGase F (Boehringer) in the same bu¡er at 37³C overnight using 0.1 enzyme unit per 300 Wg of tryptic fragments. Reduction of disulphide bridges and carboxyethylation of cysteine residues of the fragments were performed as already reported [38], except that iodoacetamide instead of iodoacetic acid was used as alkylating agent. In gel digestion of bands excised from gel was carried out using a protocol adapted from Shevchenko et al. [39]. 2.9. HPLC separation of peptides The bLf tryptic peptides were puri¢ed by RP-HPLC on a Vydac C18 column (250U10 mm, 5 Wm) using a Waters HPLC System (Datasystem Millennium, HPLC pumps Waters 510, Detector Waters 486). Eluents were : 0.1% tri£uoroacetic acid (solvent A) and 0.07% tri£uoroacetic acid in 95% acetonitrile (solvent B). The elution was performed by means of a linear gradient from 10% to 55% solvent B over 45 min at a £ow rate of 3.5 ml/min and monitored at 220 nm. The trypsin containing fraction was discharged since it did not co-elute with fractions containing bLf fragments. In addition, control experiments did not show residual tryptic activity in the digested fractions of bLf since the enzyme underwent auto-hydrolysis and inactivation due to denaturing conditions of HPLC. Fractions containing bLf fragments were collected, dried in a Speed-Vac centrifuge (Savant), lyophilised twice and stored at 320³C. The amount of the large fragments was determined according to the method of Bradford [40]. Electrophoretic analysis (SDS^PAGE) of the HPLC fraction was carried out using 12.5% gels stained by Coomassie blue R250. 2.10. Mass spectrometric analyses HPLC fractions were submitted to ES/MS analyses using a Platform single quadrupole mass spectrometer (Micromass). Samples were dissolved in 1% acetic acid in 50% acetonitrile and 2^10 Wl were injected into the mass spectrometer at a £ow rate of 10 Wl/min. The quadrupole was scanned from m/z 300 to 1600 at 10 s/scan and the spectra were acquired and elaborated using the MassLynx software. All mass values are reported as average masses. Matrix-assisted laser desorption ionisation (MALDI) mass spectra were recorded using a Voyager DE-PRO MALDI-TOF (AB Biosystems). The instrument, equipped with a delayed extraction ion source, was operated in the re£ector mode; the monoisotopic mass values were recorded in the mass spectra. To ensure accurate mass assignment, an internal two point calibration of the mass spectra was performed using molecular ions from angiotensin III at 931.5154 Da and from an ACTH fragment (18^39) at 2465.1989 Da. K-Cyano-4-hydroxy-trans-cinnamic acid was used as matrix.
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2.11. Statistical analysis Statistical analysis was performed using Student's t-test for unpaired data. Data were expressed as the mean þ S.D. and P values of 6 0.05 were considered statistically signi¢cant. 3. Results 3.1. E¡ect of apo-, iron-, zinc-, and manganese-saturated lactoferrin on CPE by rotavirus in HT-29 cells A preliminary set of experiments was carried out in order to determine the maximal non-cytotoxic concentrations of apo-, iron-, zinc-, and manganese-lactoferrin. For this purpose, 2-fold serial dilutions of proteins from 20 mg/ml in RPMI 1640 medium were incubated with HT29 cells for 24 h at 37³C. No cytotoxic e¡ect was observed up to a bLf concentration of 2 mg/ml. To establish whether this milk protein could inhibit a rotavirus cytopathic e¡ect in HT-29 cells, 2-fold serial dilutions of each protein, starting from the highest non-cytotoxic concentration, were incubated with the cells through the infection. The concentration^response curves of lactoferrins towards rotavirus infection are shown in Fig. 1. All tested proteins showed a similar dose-dependent inhibitory activity, even though Zn- and Mn-saturated bLf appear to have slightly less activity than unsaturated or Fe-saturated bLf (see Table 1). In appropriate control experiments, in which metal ions were added as sulphate salts at the same concentrations used to saturate bLf, the viral replication was una¡ected (data not shown). To establish whether the anti-rotavirus activity of di¡erently metal-saturated bLf is based on the inhibition of early phases of viral infection
Table 1 Antiviral activity towards rotavirus of di¡erently metal-saturated and desialylated bovine lactoferrin and of its peptidic fragments Compound
EC50 (Wg/ml)
Apo bovine lactoferrin Iron-saturated bovine lactoferrin Zinc-saturated bovine lactoferrin Manganese-saturated bovine lactoferrin Fragment 86^258 Fragment 324^329 Desialylated bovine lactoferrin
50 þ 3.0 47 þ 2.0 62 þ 2.5* 62 þ 2.1* 125 þ 10.0* 1000 þ 25* 12 þ 1.0*
HT-29 cells were infected with SA-11 rotavirus (1 pfu/cell). Compounds were incubated with the cells during the viral adsorption step (1 h at 4³C) and newly added after the removal of virus inoculum. After 24 h incubation at 37³C, the percentage of cytopathic e¡ect was evaluated. EC50 , e¡ective concentration 50%, represents the dose required to inhibit the viral cytopathic e¡ect by 50%. Data represent the mean values ( þ S.D.) of at least three independent experiments and each sample was done in triplicate. *P 6 0.05 with respect to apo-bLf.
or involves the inhibition of intracellular replication, the activity of these derivative lactoferrins (0.20 mg/ml) was tested by the following experimental conditions: (i) the proteins were added together with virus inoculum during the adsorption step (1 h at 4³C) and then removed ; (ii) the proteins were present during the whole experiment ; or (iii) the proteins were present only after the adsorption step for 24 h at 37³C. The results obtained show that lactoferrin in apo-, iron-, zinc-, and manganese-saturated forms exerts high inhibition of viral infection (about 60^75%) not only during the virus adsorption step but in all experimental conditions, including the post-adsorption step (Table 2). However, according to previous data [25], preincubation of HT-29 cells with lactoferrin before infection did not induce a signi¢cant reduction of viral infection.
Fig. 1. Dose^response curves of apo-lactoferrin (E), Fe3 -lactoferrin (7), Zn2 -lactoferrin (a), and Mn2 -lactoferrin (O) towards SA-11 rotavirus cytopathic e¡ect in cultured HT-29 cells. The percentages of the observed CPE inhibitions are plotted against protein concentrations. HT-29 cells were infected with SA-11 rotavirus (1 pfu/cell). Di¡erently metal-saturated bovine lactoferrins were incubated with the cells during the viral adsorption step (1 h at 4³C) and newly added after the removal of virus inoculum. After 24 h incubation at 37³C, the percentage of cytopathic e¡ect inhibition was evaluated. The data represent the means of at least three independent experiments and each sample was done in triplicate. S.D. never exceed 10%.
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Table 2 CPE inhibition of apo-bLf, Fe-bLf, Zn-bLf, Mn-bLf on di¡erent phases of rotavirus infection in cultured HT-29 cells Addition of metal-saturated bLf
CPE inibition (%)
During viral absorption step During the entire period of viral multiplication After viral adsorption step
Apo-bLf
Fe-bLf
Zn-bLf
Mn-bLf
85 þ 2.5 85 þ 3.0 75 þ 2.5*
85 þ 3.0 85 þ 5.0 75 þ 5.0*
75 þ 2.0* 75 þ 1.0* 75 þ 2.0*
75 þ 0.5* 75 þ 2.0* 75 þ 0.5*
HT-29 cells were infected with SA-11 rotavirus (1 pfu/cell). Protein preparations (0.2 mg/ml) were incubated with the cells during the viral adsorption step (1 h at 4³C), or the proteins were present for the entire period of viral multiplication (1 h at 4³C plus 24 h at 37³C), or the proteins were added after the adsorption step (24 h at 37³C). Data represent the mean values ( þ S.D.) of at least three independent experiments and each sample was done in triplicate. *P 6 0.05 with respect to apo-bLf incubated during viral adsorption step.
3.2. Anti-rotavirus activity of desialylated lactoferrin Desialylated lactoferrin showed reduced cellular toxicity and no cytotoxic e¡ect has been observed up to a bLf concentration of 1 mg/ml. In desialylated lactoferrin, glycan detection was still positive, while in isoelectric focusing the treated protein migrated di¡erently with respect to normal lactoferrin and showed negative reactivity with M. amurensis lectin. These results suggest that the sialic acid residues were almost completely removed in desialylated bLf. In the experiments reported here it is clearly shown that desialylated lactoferrin was a signi¢cantly better inhibitor of SA-11 rotavirus infection than untreated bovine lactoferrin in antiviral activity assays (P values 6 0.05) (see Table 1). 3.3. Mass spectrometric structural analysis of high molecular mass fragments originated from the tryptic digestion of bLf Aimed to investigate whether speci¢c regions of bLf are directly involved in the mechanism of anti-rotavirus activ-
ity, the protein was digested with trypsin and the peptide mixture was analysed by RP-HPLC. The elution pro¢le showed the presence of 31 peaks (Fig. 2). The late eluting fractions (from 28 to 31) contained high molecular mass fragments, as demonstrated by SDS^PAGE analysis, which was also carried out in order to assess the purity of the HPLC fractions (Fig. 3). Moreover, by means of advanced mass spectrometric methodologies, a complete structural characterisation of the large fragments was achieved. The SDS^PAGE analysis of fraction 28 showed that a single component was present having a molecular mass of about 45 kDa (Fig. 3, lane 2). This result suggested that the N- and/or the C-lobes were eluted in this fraction. To determine which lobe was present, analyses were carried out exploiting the mass-mapping strategy [41]. The fraction was subjected to reduction of the disulphide bridges and carboxyamidomethylation of the free SH groups, to deglycosylation by PNGase F and to a further tryptic digestion step. The resulting peptide mixture was directly analysed by MALDI mass spectrometry. The mass signals present in the spectrum were assigned to the correspond-
Fig. 2. RP-HPLC puri¢cation of peptides originated from the tryptic digestion of bLf. Relevant fractions are indicated: fraction 14 exhibited antirotavirus activity; fractions 28, 29, 30 and 31 contained the high molecular mass fragments. The corresponding amino acid position of the fragments in the bLf sequence is also reported.
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ing peptides along the bLf protein sequence on the basis of their molecular masses and of the enzyme speci¢city. All the signals had originated from peptides along the sequence of the C-lobe (fragment 345^689). No peptides originated from the N-lobe were observed. SDS^PAGE analysis of fraction 29 (Fig. 3, lane 3) showed that the main component exhibited a molecular mass of about 60 kDa, although very minor contaminants were intact bLf and a smaller fragment (molecular mass around 30 kDa) due to a partial co-elution of fraction 30. Therefore, in order to de¢ne the region of bLf included in the fragment with a molecular mass of 60 kDa, the main band present in the corresponding lane in the gel was cut and subjected to in situ reduction and alkylation, and to a further tryptic digestion step [39]. The resulting peptide mixture was analysed by MALDI mass spectrometry. Similarly to what was reported for fraction 28, this analysis demonstrated that this fragment had originated from the region 285^689 of bLf. The fragment eluted in fraction 30 showed a molecular mass of 30 kDa (Fig. 3, lane 4). As already reported [42], ES/MS analyses allowed us to assess that this fraction contained the fragment 1^280 (N-lobe) containing a high mannose type glycan chain linked to Asn 233. Finally, a smaller fragment was eluted in fraction 31 (molecular mass about 20 kDa, Fig. 3, lane 5). ES/MS analysis of the fraction showed the presence of two main peaks A and B with a molecular mass of 20.897 Da and 20.735 Da, respectively. The 162 mass di¡erence between them (corresponding to a monosaccharide residue) suggested that the two peaks had originated from di¡erent glycoforms of the same fragment. Therefore, the HPLC fraction was submitted to deglycosylation, reduction and alkylation of the cysteine residues. ES/MS spectra were
recorded after each reaction. Following the strategy already reported for the analysis of fraction 30 [42], we could assess that fraction 31 contained the fragment 86^ 258 of bLf, with a single glycosylation site on Asn 233. Two main glycoforms were present, having high mannose type structures with composition Man9 GlucNAc2 and Man8 GlucNAc2 , respectively. 3.4. Anti-rotavirus activity of large tryptic peptides derived from bovine lactoferrin It is worth noting that the entire peptide mixture showed an antiviral activity similar to that of the native protein (data not shown), indicating that the inhibition of viral infection could not be exclusively linked to undigested bLf (which was estimated to be less than 5% in the puri¢ed large fragments). Antiviral experiments were then carried out to test the antiviral activity of the di¡erent large fractions, obtained from the HPLC separation procedure, towards SA-11 rotavirus. Among the characterised large fractions, only one, the fragment 86^258 (fraction 31), exhibited antiviral activity (Table 1). 3.5. Anti-rotavirus activity of small tryptic peptides derived from bovine lactoferrin Fractions 1^27, mostly containing small tryptic peptides, were tested for anti-rotavirus activity; only fraction 14 (Fig. 2), containing a peptide with a molecular mass of 737.4 Da and attributed to amino acid sequence 324^329 of bLf (YLTTLK), displayed a signi¢cant activity (Table 1). It is worth noting that this peptide showed no antiherpesvirus activity and is not present in hLf which, in turn, is characterised by a much lower anti-rotavirus activity in comparison to bLf when the antiviral activity of these proteins was tested in the experimental conditions described here (data not shown). The chemically synthesised fragment displayed the same antiviral activity as fragments 324^329 (YLTTLK), obtained by HPLC puri¢cation of bLf enzymatic hydrolysis, indicating that no major contaminants are responsible for the observed antiviral activity. 4. Discussion
Fig. 3. SDS^PAGE analysis of the HPLC fractions. Lanes: 1, native bLf; 2, fraction 28; 3, fraction 29; 4, fraction 30; 5, fraction 31; 6, molecular mass markers.
Mammalian lactoferrin is found mainly in external secretions, such as breast milk, tears, saliva, vaginal secretions, bronchoalveolar lavage £uid, and is synthesised by polymorphonuclear leucocytes and glandular epithelial cells, contributing to host defence [6,7]. There is increasing evidence that the defence against viral infections represents one of the most relevant activities among the various biological functions of lactoferrin. As a matter of fact, recently a potent antiviral activity of both human and bovine lactoferrins towards some enveloped viruses such as
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cytomegalovirus [18], HSV-1 and HSV-2 [18^20], HIV [22,23] and HCV [24], and two naked viruses such as rotavirus [25] and poliovirus [26] has been shown. Since bLf is able to bind iron as well as other metal ions and is physiologically partially iron-saturated, we have investigated the e¡ect of metal saturation on bLf antiviral activity towards rotavirus, which is an intestinal pathogen virus. In this paper we report data indicating that zincand manganese-saturated bLf are statistically signi¢cantly less e¤cient inhibitors of rotavirus replication when compared to both apo- or iron-saturated bLf (P 6 0.05). It is relevant to note that bLf saturation with di¡erent metal ions has no e¡ect on the antiviral activity towards herpesvirus [21]. It should also be recalled that Zn-saturated bLf displays higher antiviral activity towards poliovirus with respect to either apo-, iron-saturated or Mn-saturated bLf ; we collected data indicating that the enhancement of the antiviral activity of Zn-saturated bLf towards poliovirus may be attributed to the bLf-induced delivery of zinc ions to the host cell, since a decrease in poliovirus replication was observed upon addition of zinc sulphate to the medium of cultured cells [26]. However, contrary to our observation with poliovirus, in the present case we were not able to observe any e¡ect on rotavirus replication induced by sulphate salts of any metal ions, suggesting that other mechanisms, rather than ion delivery to the host cell, are involved in the decrease in the antiviral activity of Zn- and Mn-saturated bLf. The observed di¡erences in the antiviral activity of di¡erently metal-saturated bLf therefore cannot be attributed to di¡erences in the molecular shape of the protein, since it has been demonstrated that bLf, as other lactoferrins, upon binding to iron or other metal ions undergoes a relevant conformational change which appears to be almost the same independent from the type of metal bound [16]. The in£uence of metal saturation on the antiviral activity of bLf appears therefore a still puzzling question that demands further investigations, being possibly related to the di¡erent viruses towards which bLf displays antiviral activity. Moreover, contrary to what was observed with herpesvirus [20], most of the anti-rotavirus activity is retained when bovine lactoferrin (either in apo- or metal-saturated forms) is added after the virus attachment step, while it has been reported that bLf did not interfere with rotavirus antigen synthesis when incubated with the cells prior to virus addition [25], demonstrating that the anti-rotavirus activity of bLf is not limited to interference with the virus attachment step. We have also analysed the in£uence of sialic acid of bovine lactoferrin on viral infection by testing the antiviral activity of the desialylated bLf towards rotavirus. Desialylated bLf exerts a better anti-rotavirus activity compared to native bLf. It has been reported that most of the animal rotavirus attach to sialic acid on cell surfaces, and this interaction has been shown to be required for the e¤cient infection of virus to susceptible cells both in vitro and in
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vivo [31^35]. It has also been hypothesised that binding of animal rotaviruses to cells in culture might ¢rst occur via a rapid association with a sialic acid containing cell receptor followed by a slower binding step through a sialic-independent receptor [43]. Ludert et al. [43] postulated that the removal of sialic acid from the cell surface would render the putative secondary receptor for rotaviruses more accessible, thereby facilitating the binding of the virion to cells. Moreover, concerning SA-11 rotavirus, it has been demonstrated that both sialic acid and galactose participate in the receptor structure on HT-29 cells [35]. While bLf sialic acid residues are mainly linked to galactose residues in bLf [12], our ¢ndings that desialylated bLf exerts a better anti-rotavirus activity compared to native bLf suggest that the removal of sialic acid unmasked an additional binding site for rotavirus such as galactose [35] which could therefore increase the interaction between the protein and the virus. The interference of bLf with the virus attachment step by a competition for either virus or cell receptors has been generally considered the most probable mechanism of action of bLf towards virus infection [42]. As a matter of fact, bovine lactoferrin has an alkaline isoelectric point (pI about 9) and its strongly cationic nature could be a major factor in its ability to bind to many cell types and to many anionic molecules. bLf possesses three notable concentrations of positive charge: at the N-terminus (residues 1^7), along the outside of the ¢rst helix (residues 13^30) and in the interlobe region, close to the connecting helix (residues 339^344), important for the binding to glycosaminoglycans (GAG) [27,28], which are speci¢c binding sites for some enveloped viruses, amongst others HSV-1 and HSV-2 [29,30]. It has been suggested that most of the antiviral activity of bLf towards HSV arises from bLf interference in the early phases of HSV-1 and HSV-2 infection by binding to GAG and hindering HSV adhesion to host cells [7,20]. Here we have reported that the antirotavirus activity of a large peptide (86^258), not containing clusters of positively charged amino acids and not containing a N-acetyllactosaminic type glycan chain (with either neuraminic acid or galactose residues), was almost the same as that of the whole protein. On the contrary, two large fragments, both containing clusters of positive charges (i.e. fragment 285^689 and 1^280) as well as another large fragment (345^689) containing a Nacetyllactosaminic type glycan chain (with both neuraminic acid and galactose residues), did not display any relevant anti-rotavirus activity. Therefore it can be inferred that the cluster of positive charges present in bLf, considered to be crucial for anti-herpesvirus activity, is not important for anti-rotavirus activity. It can also be inferred that galactose residues unmasked by neuraminidase treatment could eventually increase the interaction between bLf and rotavirus, but they are not the sites mainly responsible for the exploitation of bLf anti-rotavirus activity. Within the same framework, it should be noted that
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the active fragment 86^258 belongs to the N-lobe of bLf while the two other fragments that belong to the C-lobe (345^689 and 285^689) do not have anti-rotavirus activity despite the high internal amino acid sequence homology between the two lobes present in bLf [2], demonstrating the importance of speci¢c recognition events. In conclusion, we have demonstrated here that di¡erent moieties of the bLf molecule, possibly interfering with different steps of virus replication, are involved in the inhibition of rotavirus infections, being also clearly di¡erent from those a¡ecting anti-herpesvirus activity. The importance of speci¢c molecular recognition events is clearly assessed by the reported experiments. The antiviral activity of lactoferrin towards viruses belonging to di¡erent families appears therefore due to speci¢c, although di¡erent, mechanisms, depending on the inhibited virus. Acknowledgements
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Dr. Paola Rossi is gratefully acknowledged for having prepared and characterised desialylated bovine lactoferrin and Dr. M.G. Ammendolia for the help in performing antiviral experiments. This work was supported by grant CNR target project on `Biotechnology', by MURST PRINs `Role of metal ions in intracellular infections', by the Italian Ministry of Health, Project 1% `Caratterizzazione delle basi genomiche di infezioni virali, batteriche e parassitarie ed applicazioni per strategie di controllo' and by contract FERS No. 94.05.09.013, ARINCO No. 94.IT.16.028 ^ `Realizzazione di una rete di spettrometria di massa'.
[16]
[17]
[18]
[19]
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and zinc-saturated lactoferrin, Med. Microbiol. Immunol. 187 (1999) 199^204. D.M. Mann, E. Romm, M. Migliorini, Delineation of the glycosaminoglycan-binding site in the human in£ammatory response protein lactoferrin, J. Biol. Chem. 269 (1994) 23661^23667. H.F. Wu, D.M. Monroe, F.C. Church, Characterization of the glycosaminoglycan-binding region of lactoferrin, Arch. Biochem. Biophys. 317 (1995) 85^92. D. WuDunn, P.G. Spear, Initial interaction of herpes simplex virus with cells is binding to heparan sulfate, J. Virol. 69 (1989) 2233^2239. G. Roderiquez, T. Oravecz, M. Yanagishita, D.C. Bou-Habib, H. Mostowski, M.A. Nocross, Mediation of human immunode¢ciency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41, J. Virol. 357 (1995) 393^399. R.H. Yolken, R.E. Willoughby, S.B. Wee, R. Misku¡, S. Vonderfecht, Sialic acid glycoproteins inhibit in vitro and in vivo replication of rotaviruses, J. Clin. Invest. 79 (1987) 148^154. D.J. Keljo, A.K. Smith, Characterization of binding of simian rotavirus SA-11 to cultured epithelial cells, J. Pediatr. Gastroenterol. Nutr. 7 (1988) 249^256. K. Fukudome, Y. Osamu, T. Konno, Comparison of human, simian, and bovine rotavirus for requirement of sialic acid in hemagglutination and cell adsorption, Virology 172 (1989) 192^205. F. Superti, G. Donelli, Gangliosides as binding sites in SA-11 rotavirus infection of LLC-MK2 cells, J. Gen. Virol. 72 (1991) 2467^ 2474.
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[35] F. Superti, G. Donelli, Characterization of SA-11 rotavirus receptorial structures on human colon carcinoma cell line HT-29, J. Med. Virol. 47 (1995) 421^428. [36] M. Yi, S. Kaneko, D.Y. Yu, S. Murakami, Hepatitis C virus envelope proteins bind lactoferrin, J. Virol. 71 (1997) 5997^6002. [37] M.L. Groves, The isolation of a red protein from milk, J. Am. Chem. Soc. 82 (1960) 3345^3350. [38] G. Nitti, S. Orru', C. Bloch, L. Mohry, G. Marino, P. Pucci, Amino acid sequence and disulphide-bridge pattern of three gamma-thionins from Sorghum bicolor, Eur. J. Biochem. 228 (1995) 250^256. [39] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels, Anal. Chem. 68 (1996) 850^858. [40] M.M. Bradford, Anal. Biochem. 72 (1976) 248^251. [41] H.R. Morris, M. Panico, G.W. Taylor, FAB-mapping of recombinant-DNA protein products, Biochem. Biophys. Res. Commun. 117 (1983) 299^305. [42] R. Siciliano, B. Rega, M. Marchetti, L. Seganti, G. Antonini, P. Valenti, Bovine lactoferrin peptidic fragments involved in inhibition of herpes simplex virus type-1 infection, Biochem. Biophys. Res. Commun. 264 (1999) 19^23. [43] J.E. Ludert, N. Feng, J.H. Yu, R.L. Broome, Y. Hoshino, H.B. Greenberg, Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo, J. Virol. 70 (1996) 487^ 493.
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Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection Fabiana Superti a , Rosa Siciliano b , Barbara Rega b , Francesco Giansanti c , Piera Valenti d , Giovanni Antonini c; * a
c
Department of Ultrastructures, Istituto Superiore di Sanita©, I-00161 Rome, Italy b Institute of Food Science and Technology, CNR, I-83100 Avellino, Italy Department of Basic and Applied Biology, University of L'Aquila, Coppito, I-67010 L'Aquila, Italy d Institute of Microbiology, II University of Naples, I-80135 Naples, Italy Received 18 April 2001; received in revised form 26 June 2001; accepted 26 June 2001
Abstract Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the antirotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86^258) and a small peptide (324^329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Lactoferrin ; Rotavirus; Sialic acid ; Metal saturation; Tryptic fragment; Mass spectrometry
1. Introduction Lactoferrin is a monomeric glycoprotein with a molecular mass of about 80 kDa [1,2] which binds two iron atoms with very high a¤nity ; it is present in various biological £uids and in speci¢c granules of polymorphonuclear leucocytes [3,4]. Lactoferrin possesses a variety of biological functions, such as promotion of iron absorption [5], immunomodulation [6] and inhibiting activity towards bacteria, fungi, protozoans [6] and viruses [7]. Bovine lactoferrin (bLf), which consists of a single polypeptide chain of 689 amino acidic residues, like lactoferrin of other mammalian species, is folded into two symmetric globular lobes each of which is itself folded into two domains (Nlobe: N1 and N2 and C-lobe: C1 and C2), each delimiting a Fe3 binding site [8,9]. The complete structures of lactoferrin glycans have been described only a few years ago
* Corresponding author. Fax: +39-862-43-32-73. E-mail address : [email protected] (G. Antonini).
[10^13], although their role in any biological activity related to this protein remains to be de¢ned. Brie£y, in bLf there are two oligomannosidic type (linked to Asn 233 and Asn 545) and two biantennary N-acetyllactosamine type glycans, partially fucosylated and sialylated (linked to Asn 368 and Asn 476) [14,15]. Sialic acid is present in a ratio of about two residues per molecule of bLf [14,15]. Neither the nature nor the location of the bound carbohydrate chains has any e¡ect on the lactoferrin polypeptide conformation [16,17]. Since 1994, human lactoferrin and bLf have been recognised as potent inhibitors towards di¡erent enveloped human pathogen viruses such as herpes simplex virus (HSV) 1 and 2 [18^21], human cytomegalovirus (HCMV) [18], human immunode¢ciency virus (HIV) [22,23] and human hepatitis C virus (HCV) [24]. The antiviral e¡ect of bLf against two naked pathogen viruses, SA-11 rotavirus and poliovirus type 1, has also been demonstrated [25,26]. For all viruses investigated to date, bLf exerts its antiviral activity towards the early phases of infection. Since lactoferrin is known to bind heparan sulphate containing
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 7 8 - 7
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proteoglycans [27,28], which in turn promote the initial interaction of HSV-1 and HIV with host cells [29,30], its inhibiting activity on these viruses has been ascribed to a competition for cell receptors, even though a direct lactoferrin interaction with viral particles cannot be ruled out. For rotavirus, which interacts with sialic acid on cell surfaces [31^35], it has been suggested that lactoferrin could bind to rotavirus viral particles [25], similarly to that reported for poliovirus [26], HIV-1 [22] and HCV [36]. In the present study, we have attempted to clarify the mechanism of the anti-rotavirus e¡ect of bLf. In particular, as regards the role of metal binding, we have investigated the e¡ect of bLf metal ion saturation, and concerning the carbohydrate and protein moieties we tested the anti-rotavirus activity of desialylated bLf and of peptides originated by the tryptic digestion of bLf. We have found that complete iron saturation of the protein did not modify antiviral activity of bLf towards rotavirus, while zinc or manganese saturation slightly decreased it. On the contrary, desialylation enhanced the anti-rotavirus activity of bLf. A large body of work was then undertaken aimed to achieve a detailed structural characterisation of the high molecular mass fragments deriving from bLf tryptic digestion using advanced mass spectrometric methodologies. We have found that only one large and one small peptide retained activity towards rotavirus infection even if at a lower extent with respect to the undigested bLf. 2. Materials and methods 2.1. Cells The human colon adenocarcinoma cell line, HT-29, was obtained from the American Type Culture Collection (ATCC, Rockville, MA, USA). Cells were grown at 37³C in RPMI 1640 medium (Gibco) containing 1.2 g/l NaHCO3 , and supplemented with 10% inactivated foetal calf serum (FCS, Flow Laboratories), 2 mM glutamine, nonessential amino acids, penicillin (100 IU/ml), and streptomycin (100 Wg/ml). 2.2. Virus Simian rotavirus SA-11 was grown in LLC-MK2 cells (a monkey kidney derived cell line). Virus was pre-activated with 10 Wg/ml trypsin (type IX, Sigma) for 60 min at 37³C, diluted 10-fold in 199 medium (HyClone), and then inoculated onto con£uent monolayers grown in roller bottles at a multiplicity of infection (moi) of 5 plaque forming units (pfu)/cell. After 90 min at 37³C, the inoculum was removed, and the monolayers were washed once in phosphate bu¡ered saline (PBS, pH 7.4) and then incubated at 37³C in 199 medium containing 1 Wg/ml trypsin. When an extensive cytopathic e¡ect (CPE) was observed, infected cultures were frozen and thawed three times, centrifuged
(3000Ug; 10 min), and supernatants were stored at 370³C. 2.3. Cytotoxicity assay To establish the maximal non-cytotoxic dose of the bLf derivative to be tested, 2-fold serial dilutions of each substance in RPMI 1640 medium were incubated at 37³C with con£uent HT-29 cells grown in 96-well tissue culture microplates (Flow Laboratories). After 24 h, the following parameters were evaluated : cell morphology and viability (determined by neutral red staining) were examined by light microscopy and cell proliferation was evaluated quantitatively by microscopic counts after dispersion into individual cells with trypsin. Protein dilutions that did not a¡ect any of these parameters were considered non-cytotoxic concentrations and utilised for antiviral assays. 2.4. Antiviral assays Rotavirus infection was synchronised by a temperature shift: viral adsorption was performed at 4³C for 1 h (attachment step) and, after removal of inoculum, virus infection was allowed to progress by raising the temperature to 37³C. HT-29 cells grown in 96-well tissue culture microplates for 48 h at 37³C in 5% CO2 , were incubated with di¡erent concentrations of proteins during the virus attachment step (1 h, 4³C). As viral inoculum was utilised pre-activated SA-11 virus at a multiplicity of infection of 1 pfu/cell. Then, cells were rinsed thoroughly and incubated with the same concentrations of proteins for 24 h at 37³C in 5% CO2 ^air. The CPE induced by rotavirus was measured by the neutral red uptake assay as previously described [20], and results were expressed as percentage of cytopathic e¡ect inhibition by comparison with untreated control cultures. In some experiments, proteins were added to the cells during (1 h at 4³C), during and after (1 h at 4³C and 24 h at 37³C), or after (24 h at 37³C), the viral attachment step, for details see [26]. Each sample was done in triplicate. 2.5. Lactoferrin Apo-lactoferrin from bovine milk (apo-bLf), purchased from Armour Proteins (France), was dissolved as stock solution in PBS. Lactoferrin purity was checked by SDS^PAGE stained with silver nitrate and judged to be greater than 95%. Protein concentration was determined by UV spectroscopy on the basis of the extinction coe¤cient of 15.1 (280 nm, 1% solution) [37]. Apo-, iron-, manganese- and zinc-saturated bLf (apo-bLf, Fe-bLf, Mn-bLf, and Zn-bLf) were prepared and tested as reported previously [21,26]. Brie£y, protein-bound iron was removed by extensive dialysis against 50 mM pyrophosphate bu¡er, pH 4.0, followed by dialysis against PBS, pH 7.2. Metalsaturated proteins were obtained by the addition of an
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adequate molar ratio of the metal ions, in the form of chloride or sulphate salts, in 0.1 M Tris^HCl, pH 8.0, containing 5 mM NaHCO3 . Binding was performed by stirring overnight at 4³C; then, not bound metal ions were removed by dialysis against iron-free PBS pH 7.2, for 72 h at 4³C. As control, the same metal ions at the identical concentration utilised to saturate at 100% the binding sites of bLf were supplied to the cells as sulphate salts. Lactoferrin fragment 324^329 (YLTTLK) was synthesised by Tecnogen (Italy) and its purity was checked by HPLC and electrospray mass spectrometric (ES/MS) analysis. Before biological assays, all proteins were sterilised by ¢ltration on 0.45 Wm Millex HV at low protein retention (Millipore). 2.6. Desialylation Desialylation was carried out using neuraminidase of Arthrobacter ureafaciens (Sigma). 4 mU of neuraminidase were added to 1 mg of apo-bLf in 0.5 ml of 20 mM phosphate bu¡er pH 6.5. The reaction mixture was incubated at 37³C for 48 h. The residual neuraminidase activity after incubation and dilution ( 6 0.05 mU/ml) did not a¡ect rotavirus infection in control experiments. 2.7. Protein blotting and glycan detection The same quantities of each modi¢ed protein were subjected to SDS^PAGE on 10% gels and then ¢xed and silver stained or transferred to a nitrocellulose membrane. After removal of blocking agent and rinsing, incubation with 25 Wg/ml lectins (concanavalin A, speci¢c for K-man and K-glc, or Maackia amurensis lectin, speci¢c for sialic acid, Sigma) was performed. Lectin binding was revealed by peroxidase reaction with 3,3P-diaminobenzidine as substrate. A Glycotrack kit (detecting carbohydrate moieties by using a speci¢c carbohydrate oxidation reaction, Oxford Glyco System, Abingdon, UK) was also used for glycan detection on the blotted proteins. Ascorbate oxidase from zucchini and cytochrome c from horse heart were used as controls (positive and negative, respectively) of glycan detection. Isoelectric focusing was also used to test the sialic acid removal. No relevant alterations of bLf protein structure and iron binding properties in desialylated protein were observed by means of visible and second derivative UV spectrophotometry or CD spectroscopy [17]. 2.8. Enzymatic hydrolysis and chemical modi¢cations of lactoferrin and its tryptic fragments bLf (4 mg/ml) was dissolved in 50 mM ammonium bicarbonate, pH 8.5 and trypsin (from bovine pancreas TPCK treated, Sigma) digestion was performed at 37³C overnight using an enzyme to substrate ratio of 1:50 w/w.
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Deglycosylation was performed by treatment with PNGase F (Boehringer) in the same bu¡er at 37³C overnight using 0.1 enzyme unit per 300 Wg of tryptic fragments. Reduction of disulphide bridges and carboxyethylation of cysteine residues of the fragments were performed as already reported [38], except that iodoacetamide instead of iodoacetic acid was used as alkylating agent. In gel digestion of bands excised from gel was carried out using a protocol adapted from Shevchenko et al. [39]. 2.9. HPLC separation of peptides The bLf tryptic peptides were puri¢ed by RP-HPLC on a Vydac C18 column (250U10 mm, 5 Wm) using a Waters HPLC System (Datasystem Millennium, HPLC pumps Waters 510, Detector Waters 486). Eluents were : 0.1% tri£uoroacetic acid (solvent A) and 0.07% tri£uoroacetic acid in 95% acetonitrile (solvent B). The elution was performed by means of a linear gradient from 10% to 55% solvent B over 45 min at a £ow rate of 3.5 ml/min and monitored at 220 nm. The trypsin containing fraction was discharged since it did not co-elute with fractions containing bLf fragments. In addition, control experiments did not show residual tryptic activity in the digested fractions of bLf since the enzyme underwent auto-hydrolysis and inactivation due to denaturing conditions of HPLC. Fractions containing bLf fragments were collected, dried in a Speed-Vac centrifuge (Savant), lyophilised twice and stored at 320³C. The amount of the large fragments was determined according to the method of Bradford [40]. Electrophoretic analysis (SDS^PAGE) of the HPLC fraction was carried out using 12.5% gels stained by Coomassie blue R250. 2.10. Mass spectrometric analyses HPLC fractions were submitted to ES/MS analyses using a Platform single quadrupole mass spectrometer (Micromass). Samples were dissolved in 1% acetic acid in 50% acetonitrile and 2^10 Wl were injected into the mass spectrometer at a £ow rate of 10 Wl/min. The quadrupole was scanned from m/z 300 to 1600 at 10 s/scan and the spectra were acquired and elaborated using the MassLynx software. All mass values are reported as average masses. Matrix-assisted laser desorption ionisation (MALDI) mass spectra were recorded using a Voyager DE-PRO MALDI-TOF (AB Biosystems). The instrument, equipped with a delayed extraction ion source, was operated in the re£ector mode; the monoisotopic mass values were recorded in the mass spectra. To ensure accurate mass assignment, an internal two point calibration of the mass spectra was performed using molecular ions from angiotensin III at 931.5154 Da and from an ACTH fragment (18^39) at 2465.1989 Da. K-Cyano-4-hydroxy-trans-cinnamic acid was used as matrix.
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2.11. Statistical analysis Statistical analysis was performed using Student's t-test for unpaired data. Data were expressed as the mean þ S.D. and P values of 6 0.05 were considered statistically signi¢cant. 3. Results 3.1. E¡ect of apo-, iron-, zinc-, and manganese-saturated lactoferrin on CPE by rotavirus in HT-29 cells A preliminary set of experiments was carried out in order to determine the maximal non-cytotoxic concentrations of apo-, iron-, zinc-, and manganese-lactoferrin. For this purpose, 2-fold serial dilutions of proteins from 20 mg/ml in RPMI 1640 medium were incubated with HT29 cells for 24 h at 37³C. No cytotoxic e¡ect was observed up to a bLf concentration of 2 mg/ml. To establish whether this milk protein could inhibit a rotavirus cytopathic e¡ect in HT-29 cells, 2-fold serial dilutions of each protein, starting from the highest non-cytotoxic concentration, were incubated with the cells through the infection. The concentration^response curves of lactoferrins towards rotavirus infection are shown in Fig. 1. All tested proteins showed a similar dose-dependent inhibitory activity, even though Zn- and Mn-saturated bLf appear to have slightly less activity than unsaturated or Fe-saturated bLf (see Table 1). In appropriate control experiments, in which metal ions were added as sulphate salts at the same concentrations used to saturate bLf, the viral replication was una¡ected (data not shown). To establish whether the anti-rotavirus activity of di¡erently metal-saturated bLf is based on the inhibition of early phases of viral infection
Table 1 Antiviral activity towards rotavirus of di¡erently metal-saturated and desialylated bovine lactoferrin and of its peptidic fragments Compound
EC50 (Wg/ml)
Apo bovine lactoferrin Iron-saturated bovine lactoferrin Zinc-saturated bovine lactoferrin Manganese-saturated bovine lactoferrin Fragment 86^258 Fragment 324^329 Desialylated bovine lactoferrin
50 þ 3.0 47 þ 2.0 62 þ 2.5* 62 þ 2.1* 125 þ 10.0* 1000 þ 25* 12 þ 1.0*
HT-29 cells were infected with SA-11 rotavirus (1 pfu/cell). Compounds were incubated with the cells during the viral adsorption step (1 h at 4³C) and newly added after the removal of virus inoculum. After 24 h incubation at 37³C, the percentage of cytopathic e¡ect was evaluated. EC50 , e¡ective concentration 50%, represents the dose required to inhibit the viral cytopathic e¡ect by 50%. Data represent the mean values ( þ S.D.) of at least three independent experiments and each sample was done in triplicate. *P 6 0.05 with respect to apo-bLf.
or involves the inhibition of intracellular replication, the activity of these derivative lactoferrins (0.20 mg/ml) was tested by the following experimental conditions: (i) the proteins were added together with virus inoculum during the adsorption step (1 h at 4³C) and then removed ; (ii) the proteins were present during the whole experiment ; or (iii) the proteins were present only after the adsorption step for 24 h at 37³C. The results obtained show that lactoferrin in apo-, iron-, zinc-, and manganese-saturated forms exerts high inhibition of viral infection (about 60^75%) not only during the virus adsorption step but in all experimental conditions, including the post-adsorption step (Table 2). However, according to previous data [25], preincubation of HT-29 cells with lactoferrin before infection did not induce a signi¢cant reduction of viral infection.
Fig. 1. Dose^response curves of apo-lactoferrin (E), Fe3 -lactoferrin (7), Zn2 -lactoferrin (a), and Mn2 -lactoferrin (O) towards SA-11 rotavirus cytopathic e¡ect in cultured HT-29 cells. The percentages of the observed CPE inhibitions are plotted against protein concentrations. HT-29 cells were infected with SA-11 rotavirus (1 pfu/cell). Di¡erently metal-saturated bovine lactoferrins were incubated with the cells during the viral adsorption step (1 h at 4³C) and newly added after the removal of virus inoculum. After 24 h incubation at 37³C, the percentage of cytopathic e¡ect inhibition was evaluated. The data represent the means of at least three independent experiments and each sample was done in triplicate. S.D. never exceed 10%.
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Table 2 CPE inhibition of apo-bLf, Fe-bLf, Zn-bLf, Mn-bLf on di¡erent phases of rotavirus infection in cultured HT-29 cells Addition of metal-saturated bLf
CPE inibition (%)
During viral absorption step During the entire period of viral multiplication After viral adsorption step
Apo-bLf
Fe-bLf
Zn-bLf
Mn-bLf
85 þ 2.5 85 þ 3.0 75 þ 2.5*
85 þ 3.0 85 þ 5.0 75 þ 5.0*
75 þ 2.0* 75 þ 1.0* 75 þ 2.0*
75 þ 0.5* 75 þ 2.0* 75 þ 0.5*
HT-29 cells were infected with SA-11 rotavirus (1 pfu/cell). Protein preparations (0.2 mg/ml) were incubated with the cells during the viral adsorption step (1 h at 4³C), or the proteins were present for the entire period of viral multiplication (1 h at 4³C plus 24 h at 37³C), or the proteins were added after the adsorption step (24 h at 37³C). Data represent the mean values ( þ S.D.) of at least three independent experiments and each sample was done in triplicate. *P 6 0.05 with respect to apo-bLf incubated during viral adsorption step.
3.2. Anti-rotavirus activity of desialylated lactoferrin Desialylated lactoferrin showed reduced cellular toxicity and no cytotoxic e¡ect has been observed up to a bLf concentration of 1 mg/ml. In desialylated lactoferrin, glycan detection was still positive, while in isoelectric focusing the treated protein migrated di¡erently with respect to normal lactoferrin and showed negative reactivity with M. amurensis lectin. These results suggest that the sialic acid residues were almost completely removed in desialylated bLf. In the experiments reported here it is clearly shown that desialylated lactoferrin was a signi¢cantly better inhibitor of SA-11 rotavirus infection than untreated bovine lactoferrin in antiviral activity assays (P values 6 0.05) (see Table 1). 3.3. Mass spectrometric structural analysis of high molecular mass fragments originated from the tryptic digestion of bLf Aimed to investigate whether speci¢c regions of bLf are directly involved in the mechanism of anti-rotavirus activ-
ity, the protein was digested with trypsin and the peptide mixture was analysed by RP-HPLC. The elution pro¢le showed the presence of 31 peaks (Fig. 2). The late eluting fractions (from 28 to 31) contained high molecular mass fragments, as demonstrated by SDS^PAGE analysis, which was also carried out in order to assess the purity of the HPLC fractions (Fig. 3). Moreover, by means of advanced mass spectrometric methodologies, a complete structural characterisation of the large fragments was achieved. The SDS^PAGE analysis of fraction 28 showed that a single component was present having a molecular mass of about 45 kDa (Fig. 3, lane 2). This result suggested that the N- and/or the C-lobes were eluted in this fraction. To determine which lobe was present, analyses were carried out exploiting the mass-mapping strategy [41]. The fraction was subjected to reduction of the disulphide bridges and carboxyamidomethylation of the free SH groups, to deglycosylation by PNGase F and to a further tryptic digestion step. The resulting peptide mixture was directly analysed by MALDI mass spectrometry. The mass signals present in the spectrum were assigned to the correspond-
Fig. 2. RP-HPLC puri¢cation of peptides originated from the tryptic digestion of bLf. Relevant fractions are indicated: fraction 14 exhibited antirotavirus activity; fractions 28, 29, 30 and 31 contained the high molecular mass fragments. The corresponding amino acid position of the fragments in the bLf sequence is also reported.
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ing peptides along the bLf protein sequence on the basis of their molecular masses and of the enzyme speci¢city. All the signals had originated from peptides along the sequence of the C-lobe (fragment 345^689). No peptides originated from the N-lobe were observed. SDS^PAGE analysis of fraction 29 (Fig. 3, lane 3) showed that the main component exhibited a molecular mass of about 60 kDa, although very minor contaminants were intact bLf and a smaller fragment (molecular mass around 30 kDa) due to a partial co-elution of fraction 30. Therefore, in order to de¢ne the region of bLf included in the fragment with a molecular mass of 60 kDa, the main band present in the corresponding lane in the gel was cut and subjected to in situ reduction and alkylation, and to a further tryptic digestion step [39]. The resulting peptide mixture was analysed by MALDI mass spectrometry. Similarly to what was reported for fraction 28, this analysis demonstrated that this fragment had originated from the region 285^689 of bLf. The fragment eluted in fraction 30 showed a molecular mass of 30 kDa (Fig. 3, lane 4). As already reported [42], ES/MS analyses allowed us to assess that this fraction contained the fragment 1^280 (N-lobe) containing a high mannose type glycan chain linked to Asn 233. Finally, a smaller fragment was eluted in fraction 31 (molecular mass about 20 kDa, Fig. 3, lane 5). ES/MS analysis of the fraction showed the presence of two main peaks A and B with a molecular mass of 20.897 Da and 20.735 Da, respectively. The 162 mass di¡erence between them (corresponding to a monosaccharide residue) suggested that the two peaks had originated from di¡erent glycoforms of the same fragment. Therefore, the HPLC fraction was submitted to deglycosylation, reduction and alkylation of the cysteine residues. ES/MS spectra were
recorded after each reaction. Following the strategy already reported for the analysis of fraction 30 [42], we could assess that fraction 31 contained the fragment 86^ 258 of bLf, with a single glycosylation site on Asn 233. Two main glycoforms were present, having high mannose type structures with composition Man9 GlucNAc2 and Man8 GlucNAc2 , respectively. 3.4. Anti-rotavirus activity of large tryptic peptides derived from bovine lactoferrin It is worth noting that the entire peptide mixture showed an antiviral activity similar to that of the native protein (data not shown), indicating that the inhibition of viral infection could not be exclusively linked to undigested bLf (which was estimated to be less than 5% in the puri¢ed large fragments). Antiviral experiments were then carried out to test the antiviral activity of the di¡erent large fractions, obtained from the HPLC separation procedure, towards SA-11 rotavirus. Among the characterised large fractions, only one, the fragment 86^258 (fraction 31), exhibited antiviral activity (Table 1). 3.5. Anti-rotavirus activity of small tryptic peptides derived from bovine lactoferrin Fractions 1^27, mostly containing small tryptic peptides, were tested for anti-rotavirus activity; only fraction 14 (Fig. 2), containing a peptide with a molecular mass of 737.4 Da and attributed to amino acid sequence 324^329 of bLf (YLTTLK), displayed a signi¢cant activity (Table 1). It is worth noting that this peptide showed no antiherpesvirus activity and is not present in hLf which, in turn, is characterised by a much lower anti-rotavirus activity in comparison to bLf when the antiviral activity of these proteins was tested in the experimental conditions described here (data not shown). The chemically synthesised fragment displayed the same antiviral activity as fragments 324^329 (YLTTLK), obtained by HPLC puri¢cation of bLf enzymatic hydrolysis, indicating that no major contaminants are responsible for the observed antiviral activity. 4. Discussion
Fig. 3. SDS^PAGE analysis of the HPLC fractions. Lanes: 1, native bLf; 2, fraction 28; 3, fraction 29; 4, fraction 30; 5, fraction 31; 6, molecular mass markers.
Mammalian lactoferrin is found mainly in external secretions, such as breast milk, tears, saliva, vaginal secretions, bronchoalveolar lavage £uid, and is synthesised by polymorphonuclear leucocytes and glandular epithelial cells, contributing to host defence [6,7]. There is increasing evidence that the defence against viral infections represents one of the most relevant activities among the various biological functions of lactoferrin. As a matter of fact, recently a potent antiviral activity of both human and bovine lactoferrins towards some enveloped viruses such as
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cytomegalovirus [18], HSV-1 and HSV-2 [18^20], HIV [22,23] and HCV [24], and two naked viruses such as rotavirus [25] and poliovirus [26] has been shown. Since bLf is able to bind iron as well as other metal ions and is physiologically partially iron-saturated, we have investigated the e¡ect of metal saturation on bLf antiviral activity towards rotavirus, which is an intestinal pathogen virus. In this paper we report data indicating that zincand manganese-saturated bLf are statistically signi¢cantly less e¤cient inhibitors of rotavirus replication when compared to both apo- or iron-saturated bLf (P 6 0.05). It is relevant to note that bLf saturation with di¡erent metal ions has no e¡ect on the antiviral activity towards herpesvirus [21]. It should also be recalled that Zn-saturated bLf displays higher antiviral activity towards poliovirus with respect to either apo-, iron-saturated or Mn-saturated bLf ; we collected data indicating that the enhancement of the antiviral activity of Zn-saturated bLf towards poliovirus may be attributed to the bLf-induced delivery of zinc ions to the host cell, since a decrease in poliovirus replication was observed upon addition of zinc sulphate to the medium of cultured cells [26]. However, contrary to our observation with poliovirus, in the present case we were not able to observe any e¡ect on rotavirus replication induced by sulphate salts of any metal ions, suggesting that other mechanisms, rather than ion delivery to the host cell, are involved in the decrease in the antiviral activity of Zn- and Mn-saturated bLf. The observed di¡erences in the antiviral activity of di¡erently metal-saturated bLf therefore cannot be attributed to di¡erences in the molecular shape of the protein, since it has been demonstrated that bLf, as other lactoferrins, upon binding to iron or other metal ions undergoes a relevant conformational change which appears to be almost the same independent from the type of metal bound [16]. The in£uence of metal saturation on the antiviral activity of bLf appears therefore a still puzzling question that demands further investigations, being possibly related to the di¡erent viruses towards which bLf displays antiviral activity. Moreover, contrary to what was observed with herpesvirus [20], most of the anti-rotavirus activity is retained when bovine lactoferrin (either in apo- or metal-saturated forms) is added after the virus attachment step, while it has been reported that bLf did not interfere with rotavirus antigen synthesis when incubated with the cells prior to virus addition [25], demonstrating that the anti-rotavirus activity of bLf is not limited to interference with the virus attachment step. We have also analysed the in£uence of sialic acid of bovine lactoferrin on viral infection by testing the antiviral activity of the desialylated bLf towards rotavirus. Desialylated bLf exerts a better anti-rotavirus activity compared to native bLf. It has been reported that most of the animal rotavirus attach to sialic acid on cell surfaces, and this interaction has been shown to be required for the e¤cient infection of virus to susceptible cells both in vitro and in
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vivo [31^35]. It has also been hypothesised that binding of animal rotaviruses to cells in culture might ¢rst occur via a rapid association with a sialic acid containing cell receptor followed by a slower binding step through a sialic-independent receptor [43]. Ludert et al. [43] postulated that the removal of sialic acid from the cell surface would render the putative secondary receptor for rotaviruses more accessible, thereby facilitating the binding of the virion to cells. Moreover, concerning SA-11 rotavirus, it has been demonstrated that both sialic acid and galactose participate in the receptor structure on HT-29 cells [35]. While bLf sialic acid residues are mainly linked to galactose residues in bLf [12], our ¢ndings that desialylated bLf exerts a better anti-rotavirus activity compared to native bLf suggest that the removal of sialic acid unmasked an additional binding site for rotavirus such as galactose [35] which could therefore increase the interaction between the protein and the virus. The interference of bLf with the virus attachment step by a competition for either virus or cell receptors has been generally considered the most probable mechanism of action of bLf towards virus infection [42]. As a matter of fact, bovine lactoferrin has an alkaline isoelectric point (pI about 9) and its strongly cationic nature could be a major factor in its ability to bind to many cell types and to many anionic molecules. bLf possesses three notable concentrations of positive charge: at the N-terminus (residues 1^7), along the outside of the ¢rst helix (residues 13^30) and in the interlobe region, close to the connecting helix (residues 339^344), important for the binding to glycosaminoglycans (GAG) [27,28], which are speci¢c binding sites for some enveloped viruses, amongst others HSV-1 and HSV-2 [29,30]. It has been suggested that most of the antiviral activity of bLf towards HSV arises from bLf interference in the early phases of HSV-1 and HSV-2 infection by binding to GAG and hindering HSV adhesion to host cells [7,20]. Here we have reported that the antirotavirus activity of a large peptide (86^258), not containing clusters of positively charged amino acids and not containing a N-acetyllactosaminic type glycan chain (with either neuraminic acid or galactose residues), was almost the same as that of the whole protein. On the contrary, two large fragments, both containing clusters of positive charges (i.e. fragment 285^689 and 1^280) as well as another large fragment (345^689) containing a Nacetyllactosaminic type glycan chain (with both neuraminic acid and galactose residues), did not display any relevant anti-rotavirus activity. Therefore it can be inferred that the cluster of positive charges present in bLf, considered to be crucial for anti-herpesvirus activity, is not important for anti-rotavirus activity. It can also be inferred that galactose residues unmasked by neuraminidase treatment could eventually increase the interaction between bLf and rotavirus, but they are not the sites mainly responsible for the exploitation of bLf anti-rotavirus activity. Within the same framework, it should be noted that
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the active fragment 86^258 belongs to the N-lobe of bLf while the two other fragments that belong to the C-lobe (345^689 and 285^689) do not have anti-rotavirus activity despite the high internal amino acid sequence homology between the two lobes present in bLf [2], demonstrating the importance of speci¢c recognition events. In conclusion, we have demonstrated here that di¡erent moieties of the bLf molecule, possibly interfering with different steps of virus replication, are involved in the inhibition of rotavirus infections, being also clearly di¡erent from those a¡ecting anti-herpesvirus activity. The importance of speci¢c molecular recognition events is clearly assessed by the reported experiments. The antiviral activity of lactoferrin towards viruses belonging to di¡erent families appears therefore due to speci¢c, although di¡erent, mechanisms, depending on the inhibited virus. Acknowledgements
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Dr. Paola Rossi is gratefully acknowledged for having prepared and characterised desialylated bovine lactoferrin and Dr. M.G. Ammendolia for the help in performing antiviral experiments. This work was supported by grant CNR target project on `Biotechnology', by MURST PRINs `Role of metal ions in intracellular infections', by the Italian Ministry of Health, Project 1% `Caratterizzazione delle basi genomiche di infezioni virali, batteriche e parassitarie ed applicazioni per strategie di controllo' and by contract FERS No. 94.05.09.013, ARINCO No. 94.IT.16.028 ^ `Realizzazione di una rete di spettrometria di massa'.
[16]
[17]
[18]
[19]
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