Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant ...

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Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii Tetsuya Tanaka, Ichiro Nakamura, Nai-Yuan Lee, Haruto Kumura, and Kei-ichi Shimazaki

Abstract: Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLFrelated protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe. Key words: lactoferrin, lactoferrin N-lobe, baculovirus, antimicrobial activity, Prototheca zopfii. Résumé : La lactoferrine (LF) est une glycoprotéine multifonctionnelle qui lie le fer et se trouve dans les sécrétions des mammifères. Dans ce travail, l’ADN codant la lactoferrine bovine (LFb) ou la moitié N-terminale de la LFb (lobe N de la LFb) a été inséré dans un vecteur de transfert du baculovirus et un virus recombinant exprimant la LFb ou le lobe N de la LFb a été isolé. Une protéine de 80 kDa semblable à la LFb synthétisée par le baculovirus recombinant a été détectée par des anticorps monoclonaux dirigés contre le lobe N de la LFb et contre la moitié C-terminale de la LFb (lobe C de la LFb). Une protéine de 43 kDa semblable au lobe N de la LFb synthétisée par le baculovirus recombinant a été détectée par un anticorps monoclonal dirigé contre le lobe N de la LFb, mais non par un anticorps monoclonal dirigé contre le lobe C de la LFb. Ces protéines sont également sécrétées dans le surnageant des cultures de cellules d’insectes. La LFb recombinante (LFbr) et le lobe N recombinant de la LFb (lobe Nr de la LFb) sont affectés en présence de tunicamycine, ce qui indique que la LFbr et le lobe Nr de la LFb ont un site de N-glycosylation. L’activité antimicrobienne de ces protéines recombinantes envers Prototheca zopfii (un champignon semblable à la levure et qui entraîne une mammite chez la vache) a été évaluée en mesurant la densité optique des microplaques de culture. Prototheca zopfii est sensible à la LFbr et au lobe Nr de la LFb, ainsi qu’à la LFb native. Le lobe Nr de la LFb a la même activité antimicrobienne que le lobe C de la LFb. Mots clés : lactoferrine, lobe N de la lactoferrine, baculovirus, activité antimicrobienne, Prototheca zopfii. [Traduit par la Rédaction]

Introduction

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Lactoferrin (LF) is an iron-binding protein present in milk, saliva, tears, mucus secretions, and secondary granules of neutrophils. Each LF molecule can bind 2 Fe (III) ions tightly but reversibly. The antimicrobial properties of LF are Received 21 May 2003. Revision received 1 August 2003. Accepted 28 August 2003. Published on the NRC Research Press Web site at http://bcb.nrc.ca on 10 October 2003. T. Tanaka,1 I. Nakamura, N.-Y. Lee, H. Kumura, and K. Shimazaki. Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Hokkaido, Japan. 1

Corresponding author (e-mail: [email protected]).

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often dependent upon its iron status. LF is a prominent antimicrobial component of mucosal surfaces prone to be attacked by microbial pathogens. LF is actively secreted by neutrophils in the inflammatory response (Gutteberg et al. 1984; Hansen et al. 1975; Harmon et al. 1976). As an antimicrobial component of colostrum and milk, LF may play a significant role in the protection of neonates from infectious diseases (Sanchez et al. 1992). The ability of LF to inhibit growth of a wide variety of microorganisms in vitro is well documented (Bullen et al. 1972). Moreover, LF acts synergistically with lysozyme (Ellison and Giehl 1991) and IgA (Stephens et al. 1980), and it is frequently found together with one or both of these agents in various physiological fluids. The importance of LF in host defense is underlined by findings indicating that patients with congenital (Boxer et al. 1982) or acquired defects of LF production exhibit an abnor-

doi: 10.1139/O03-062

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mal predisposition to recurrent infections by bacteria, fungi, viruses, and parasites (Baynes et al. 1986; Hanson et al. 1984; Tanaka et al. 1995, 1996; Venge et al. 1984). LF damages the outer membrane of Gram-negative bacteria, causing loss of lipopolysaccharide. This enhances susceptibility to lysozyme and antibiotics such as rifampcin in a manner similar to that of polycationic membrane-active agents such as poly-L-lysine and the peptide antibiotic polymyxin B (Ellison et al. 1988; Ellison and Giehl 1991). Recent studies have demonstrated that enzymatic cleavage of LF generates peptides that have more potent antimicrobial activity than LF (Tomita et al. 1991). Such studies have led to identification of the structural region responsible for the membranedisruptive properties of LF and its lethal effects against various microorganisms (Bellamy et al. 1992). Separation of the N-terminal half of LF (LF N-lobe) from the C-terminal half of LF (LF C-lobe) is a useful approach to studying the structure–function relationship of LF. However, it is very difficult to separate these 2 lobes by digestion with proteolytic enzymes or chemical cleavage, because one of the peptide bonds (K282—S283) in the bovine LF (bLF) N-lobe (bLF N-lobe) is very susceptible to digestion by trypsin (Shimazaki et al. 1993). Nakamura et al. (2001) achieved expression of the bLF N-lobe in cultured insect cells using a baculovirus expression system. In the present study, we used a similar system to express recombinant bLF and bLF N-lobe, and we examined the antimicrobial activity of the these proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis).

Materials and methods Virus and cells Autographa californica nuclear polyhedorosis virus (AcNPV) and recombinant viruses were grown in Spodoptera frugiperda (Sf9) cells in TC-100 insect medium (Life Technologies, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) and 0.26% bactotryptose broth (Difco Laboratories, Detroit, Mich.). Cloning of DNA encoding bLF and bLF N-lobe Total RNA was extracted from bovine mammary cells using Trizol reagent (Life Technologies). mRNA was isolated using the BioMag mRNA purification kit (PerSeptive Biosystems, Framingham, Mass.), and single-strand cDNA was synthesized using SuperScript II reverse transcriptase and a random primer. RT–PCR primers were designed based on the sequence of bLF (GenBank™ accession number NM 180998). The sequence analysis of PCR products was determined using an ABI Prism sequencer (model 310, Foster City, Calif.). The amplified DNA was subjected to 1.5% agarose gel electrophoresis. A DNA band of the expected size (2127 bp) was recovered from the gel and inserted into the BamHI site of the vector pVL 1392 (BD PharMingen, San Diego, Calif.). The resulting plasmid was designated pVL bLF. Preparation of DNA encoding bLF N-lobe was performed according to the methods of Nakamura et al. (2001).

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Transfection and selection of the recombinant baculovirus Sf9 cells were transfected with a mixture of AcNPV DNA and the recombinant transfer vector DNA using lipofectin reagent (Life Technologies) according to the method of Xuan et al. (1996). After 4 days of incubation at 27 °C, the culture supernatants (containing recombinant virus) were harvested and subjected to plaque purification. Following three cycles of purification of polyhedrin-negative plaques, a recombinant baculovirus was selected and designated AcbLF or AcbLF N-lobe. Immunofluorescence test Sf9 cells were infected with AcbLF or AcbLF N-lobe (10 plaque-forming units (PFU) per cell, 96 h), and subjected to an immunofluorescence test (IFAT). The infected Sf9 cells were fixed with acetone and incubated with mouse anti-bLF N-lobe or anti-bLF C-lobe monoclonal antibody prepared by Shimazaki et al. (1998), and the cells were stained with fluorescein-conjugated goat anti-mouse antibody (Southern Biotechnology Associates Inc., Birmingham, Ala.). The slides were examined using fluorescence microscopy. Western blot analysis Sf9 cells infected with AcbLF or AcbLF N-lobe at a multiplicity of 10 PFU/cell were harvested and washed twice with phosphate-buffered saline (PBS). The supernatants were filtered through a 0.22-µm filter (Millipore Corp., Bedford, Mass.). The cell extracts and supernatants were boiled for 5 min in sample buffer (62.5 mmol Tris-HCl/L (pH 6.8), 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue) and subjected to electrophoresis in 10% polyacrylamide gels, as described by Laemmli (1970). The separated proteins were transferred onto PVDF membranes (Osmonics Inc., Westborough, Mass.). The membranes were blocked with 3% bovine serum albumin (BSA) in PBS overnight and incubated with mouse anti-LF antibody at 37 °C for 1 h. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Waco pure Chemical, Osaka, Japan) at 37 °C for 1 h. Binding of secondary antibody to protein bands was visualized by reaction with 0.5 mg/mL diaminobenzidine and 0.005% H2O2. Tunicamycin treatment Because the culture supernatants of transformed Sf9 cells contain not only rbLF or rbLF N-lobe but also other proteins, further purification of the recombinant products is necessary for detection of glycans on the recombinant proteins after treatment with tunicamycin (Sigma Chemicals Co., St. Louis, Mo.), which is a reagent preventing the synthesis of N-linked sugars. Western blot analysis was performed after treatment of cell extracts with tunicamycin, to confirm whether glycan moieties were present on the recombinant proteins. Recombinant baculovirus-infected Sf9 cells (10 PFU/cell) were incubated in medium containing 10 µg/mL tunicamycin from 1 to 96 h post-infection. The cells were then harvested and subjected to Western blot analysis. © 2003 NRC Canada



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Purification of recombinant bLF and bLF N-lobe After Sf9 cells infected with AcbLF or AcbLF N-lobe were incubated in protein-free Sf-900 medium (Life Technologies) for 4 days, the supernatants were purified using a HiTrap Heparin HP column (Amersham Bioscience Corp., Piscataway, N.J.) equilibrated with 10 mmol/L phosphate buffer (pH 7.0). After samples were loaded onto the column, the column was washed with 10 mL of 10 mmol/L phosphate buffer (pH 7.0), and elution was performed stepwise in 5-mL increments with 0.5 mol/L and 1.0 mol/L NaCl in 10 mmol/L phosphate buffer (pH 7.0). After purification of bLF or bLF N-lobe, these samples were concentrated and desalted using Ultrafree-MC Centrifugal FilterUnit (Millipore Corp.). Measurement of antimicrobial activity Antimicrobial activity was measured by 96-well microplate assay (Kimura et al. 2000) using P. zopfii (ATCC15627) as the test strain. The test strain was preincubated in heart infusion broth at 27 °C. In the microplate wells, 2 µL of a diluted suspension of the precultured test strain (7.5 × 104 colony forming units (cfu)/mL) was mixed with 50 µL of tryptone glucose (TG) broth (final concentrations: 1% bacto-tryptone, 2% glucose), and 50 µL of the sample solution was added. The microplates were sealed and incubated at 27 °C for 10 h. After incubation, the microplates were shaken gently with a microplate mixer, and optical density (OD) at 600 nm was then measured using an MPR-A4iII microplate reader (TOSOH, Tokyo, Japan), to estimate the extent of bacterial growth. We confirmed that the elution buffer used in the heparin column had no effect on the growth of P. zopfii. The antimicrobial activity of recombinant bLF (rbLF), rbLF N-lobe, native bLF (Morinaga Milk Industry Co., Zama, Tokyo, Japan), and bLF C-lobe (Shimazaki et al. 1993) was expressed as 2 µmol/L concentration. To eliminate effects of culture supernatants of Sf9 cells, heparin column fractions were examined as negative controls. Data from this experiment were evaluated using Student’s t test. A 95% level of significance was used in the analysis.

Results Expression of recombinant bLF and bLF N-lobe in Sf9 cells In the immunofluorescence analysis, the anti-bLF N-lobe monoclonal antibody reacted only with Sf9 cells infected with AcbLF N-lobe (Fig. 1C). In Western blot analysis of cell extracts prepared 4 days after infection, bands not seen in analysis of the Sf9 cell extracts were detected using bLF N-lobe or bLF C-lobe monoclonal antibody, whereas bands with molecular masses of approximately 80 kDa (Figs. 2A and 2B, lane 5) or 42 and 43 kDa (Fig. 2A, lane 3) were observed in cells infected with AcbLF or AcbLF N-lobe. Also, 42- and 43-kDa bLF N-lobe-related proteins expressed by recombinant baculovirus (Fig. 2A, lane 3) were detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody (Fig. 2B, lane 3). Electrophoretic mobility of the recombinant proteins in culture

351 Fig. 1. Immunofluorescence analysis of bovine lactoferrin (bLF) and the N-terminal half of bLF (bLF N-lobe) expressed in insect cells, using (A, C) monoclonal antibodies against bLF N-lobe or (B, D) bLF C-lobe. Panels A and B show Sf9 cells infected with Autographica californica nuclear polyhedrosis virus bLF (AcbLF); panels C and D show Sf9 cells infected with bLF N-lobe (AcbLF N-lobe) from recombinant baculovirus.

supernatants was less than that of the proteins in cell extracts (data not shown). Glycosylation of recombinant bLF or bLF N-lobe Treatment of cell extracts with tunicamycin resulted in changes in mobility of the recombinant proteins, as determined by Western blot analysis (Figs. 2A and 2B, lanes 4 and 6). Thus, the molecular masses of the recombinant proteins treated with tunicamycin were less than those of the untreated proteins. Purification and antibacterial activity of recombinant bLF or bLF N-lobe The rbLF or rbLF N-lobe was purified from the culture supernatants obtained 4 days after infection by means of a HiTrap Heparin HP column. After loading the culture supernatants onto the column, the column was washed with 10 mmol/L sodium phosphate buffer (pH 7.0), and the adsorbed constituents were eluted with 10 mmol/L sodium phosphate buffer containing NaCl. After eluting with 0.5 mol NaCl/L, the recombinant proteins were eluted with 1.0 mol NaCl/L. The results of recombinant protein purification are shown in Fig. 3 (lanes 2 and 3). Both the recombinant proteins and native bLF exhibited strong antimicrobial activity against P. zoffii (P < 0.05, Student’s t test vs. control group) after 10 h (Fig. 4). There were no differences in antimicrobial activity among these proteins.

Discussion In previous structural and functional studies of bLF and human LF (hLF), recombinant proteins have been expressed in mammalian baby hamster kidney (BHK) cells (Kamerling © 2003 NRC Canada



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Fig. 2. Western blot analysis of bovine lactoferrin (bLF) and the N-terminal half of bLF (bLF N-lobe) expressed in insect cells, using (A) anti-bLF N-lobe or (B) anti-bLF C-lobe monoclonal antibody. Lane 1, Sf9 cells; lane 2, Sf9 cells treated with tunicamycin; lane 3, Autographa californica nuclear polyhedorosis virus bLF N-lobe (AcbLF N-lobe) infected Sf9 cells; lane 4, AcbLF N-lobe-infected Sf9 cells treated with tunicamycin; lane 5, AcbLF-infected Sf9 cells; lane 6, AcbLF-infected Sf9 cells treated with tunicamycin; lane 7, bLF (2 µg). Molecular masses of marker proteins are given in kilodaltons.

Fig. 3. SDS–PAGE electrophoresis of recombinant bovine lactoferrin (rbLF) and the N-terminal half of rbLF (rbLF N-lobe) purified using a heparin column. At 4 days after infection, 50-mL aliquots of culture supernatant of Autographa californica nuclear polyhedorosis virus bLF (AcbLF)-infected or AcbLF N-lobe-infected cells were applied to a HiTrap-heparin column. Lane 1, molecular standards marker; lane 2, rbLF; lane 3, rbLF N-lobe; lane 4, bovine lactoferrin (bLF) (2 µg). Molecular masses of marker proteins are given in kilodaltons.

Fig. 4. Evaluation of the antimicrobial effects of recombinant bovine lactoferrin (rbLF), the N-terminal half of rbLF (rbLF Nlobe), bovine lactoferrin (bLF), and the C-terminal half of bLF (bLF C-lobe) against Prototheca zopfii by 96-well microplate assay. Inoculum, 5.0 × 106 cfu/mL; medium, 50 µL of tryptone glucose (TG) broth (final concentrations: 1% bacto-tryptone, 2% glucose); culture incubation time, 10 h; temperature, 27 °C. Each value is the mean ± SD of triplicate samples. OD600nm, optical density at 600 nm.

et al. 1975; Legrand et al. 1992), Asperigillus (Ward et al. 1992), yeast (Liang and Richardson 1993), and insect cells (Lopez et al. 1997; Salmon et al. 1997; Nakamura et al. 2001). Expression systems involving yeast or bacteria are commonly used in industrial production. However, yeast systems do not allow correct processing of recombinant proteins, essentially in regard to glycan moieties. Although bacterial expression systems have been widely used for low-

molecular-mass recombinant proteins, they are not always successful: in Escherichia coli, correct disulfides and glycosylation are not formed, and the recombinant protein is often located in inclusion bodies. Previously, we attempted to express recombinant bLF and bLF N-lobe in E. coli. However, we were unable to recover these proteins from E. coli, indicating that they were contained in inclusion bodies and insoluble proteins. In a eukaryotic cell expression © 2003 NRC Canada



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system using BHK cells, hLF exhibited the same degree of complexity of glycosylation and iron-binding ability as native hLF (Legrand et al. 1995). However, the BHK system is time consuming and requires clonal selection and complex culture conditions. Thus, it does not allow fast and easy production of mutated proteins. Recently, we developed a method for producing bLF N-lobe using a baculovirus expression system, which allows high-level expression of biologically active bLF and hLF. In the present study, insect cells were infected with DNA encoding bLF or bLF N-lobe, and we characterized posttranslation modification and antimicrobial activity of the resultant product. bLF produced in Sf9 cells reacted with antibLF N-lobe and anti-bLF C-lobe monoclonal antibody. bLF N-lobe produced in insect cells migrated as 2 bands (42 and 43 kDa) in Western blot analyses (Fig. 2A, lane 3). The smaller band (42 kDa) apparently represents the unglycosylated primary translation product, because the molecular mass of this band was similar to that produced following tunicamycin treatment (Fig. 2A, lane 4). The molecular mass of rbLF was also changed by tunicamycin treatment. These results indicate that rbLF and rbLF N-lobe contain N-linked glycosylation sites. The purification level of rbLF (68 µg/mL) and rbLF Nlobe (34 µg/mL) in 50 mL of Sf9 cell culture supernatant was estimated by sandwich enzyme-linked immunosorbent assay (ELISA). rbLF and rbLF N-lobe were secreted into the culture medium, and we succeeded in purifying the protein from the culture supernatants using heparin chromatography. Both the recombinant proteins and native bLF were shown to have bacteriostatic activity against P. zopfii. Prototheca infection of the mammary gland induces a serious mastitic condition (Frank et al. 1969). This microorganism shows resistance to most antimicrobial agents used in mastitis treatment, and such infection is very difficult to cure. The present results indicate that P. zopfii is highly sensitive to rbLF and rbLF N-lobe, as well as native bLF and bLF Clobe, in vitro. These results suggest that bLF, bLF N-lobe, and bLF C-lobe could be useful for treatment of bovine mastitis due to P. zopfii if administered with other agents. However, the mechanism by which bLF inhibits growth of P. zopfii is unknown. Iron-free bovine transferrin and ethylenediamine-tetra acetate (EDTA) had no effect on growth of P. zopfii (data not shown); thus, the antimicrobial activity of bLF may not be related to its iron-binding activity. There was no difference in antibacterial activity between rbLF N-lobe and bLF C-lobe. It has been reported that pepsin fragments of bLF and hLF (lactoferricin B and H) exhibit strong antimicrobial activity (Bellamy et al. 1992), indicating that the conformation of the native protein is not always necessary for antimicrobial activity. Furthermore, a fragment (residues 473–538) corresponding to the C-lobe was shown to have a strong inhibitory effect on the salivainduced aggregation of Streptococcus mutans (Mitoma et al. 2001). Therefore, lactoferricin region of bLF N-lobe and the fragment (residues 473–538) of bLF C-lobe may be responsible for the interaction of bLF with P. zopfii. We intend to conduct further studies of the antimicrobial activity of bLF against P. zopfii. In conclusion, the baculovirus expression system allowed high-level expression of biologically active bLF. Such a con-

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venient system for production of LF and its fragments may be useful in future studies of antimicrobial mechanisms of LF and its structure–function relationships.

Acknowledgment This work was supported by grant from the Clark Foundation.

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