The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors
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J. ALCANTARA, J. S. PADDA, AND A. B. SCHRYVERS Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, A lta., Canada T2N IN4 Received April 1, 1992 Revision received June 18, 1992 Accepted July 7, 1992 ALCANTARA, J., PADDA,J. S., and SCHRYVERS, A. B. 1992. The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors. Can. J. Microbiol. 38 : 1202-1205. The oligosaccharides of human lactoferrin were enzymatically removed with glycopeptidase F, resulting in a preparation containing partial and fully deglycosylated human lactoferrin. The derivatives were separated by Concanavalin A affinity chromatography and compared with native human lactoferrin with respect to their ability to bind t o bacterial receptors. Competitive binding experiments demonstrated that the lactoferrin derivatives were equally capable as native lactoferrin in binding t o receptors of Neisseria rneningitidis, Neisseria gonorrhoeae, and Moraxella catarrhalis. This result indicates that the oligosaccharides on human lactoferrin are not essential for binding to the bacterial receptors. Key words: lactoferrin, oligosaccharides, deglycosylation, receptor, Neisseria. ALCANTARA, J., PADDA,J. S., et SCHRYVERS, A. B. 1992. The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors. Can. J. Microbiol. 38 : 1202-1205. Les oligosaccharides de la lactoferrine humaine ont ete retires par la glycopeptidase F. Le produit de l'action enzymatique contenait de la lactoferrine humaine partiellement et totalement deglycosylee. Les derives ont ete separes par chromatographie d'affinite a la concanavaline A puis compares a la lactoferrine humaine native en ce qui concerne leur capacite de se lier aux recepteurs bacteriens. Les experiences de liaison competitive ont demontre que les derives de lactoferrine sont aussi capables de se lier aux recepteurs de Neisseria rneningitidis, Neisseria gonorrhoeae et Moraxella catarrhalis que la lactoferrine native. Ce resultat indique que les oligosaccharides de la lactoferrine humaine ne sont pas essentiels pour la liaison aux recepteurs bacteriens. Mots cles : bactoferrine, oligosaccharides, deglycosylation, recepteur, Neisseria. [Traduit par la redaction]
The transition metal, iron, is essential for most living organisms. It plays a critical role in biological functions such as DNA synthesis and oxidation-reduction reactions where it serves as a prosthetic group in enzymes and cytochromes (Bergeron 1986). Although there is an abundance of iron in the mammalian host, the majority of iron is found in intracellular pools. The total amount of extracellular iron is sufficient to support the growth of microorganisms, but sequestration by iron binding proteins lowers the effective concentration of free aqueous iron to only 10-l8 M (Finkelstein et al. 1983). The dominant iron binding proteins are transferrin, found in serum, and lactoferrin, found in mucosal secretions (Payne and Finkelstein 1978). The ability of a microorganism to obtain iron in the host is postulated to be an essential requirement for disease causation (Weinberg 1978). Many bacterial species have developed a system for acquisition of extracellular iron involving the synthesis and secretion of highly specific, soluble iron chelating compounds referred to as siderophores (Weinberg 1978). After complexing with extracellular iron, the ironsiderophore complex binds to outer membrane receptor proteins on the bacterial surface and is internalized (Neilands 1981). An alternative mechanism of iron acquisition involving direct removal of iron by the bacterium from the host's iron binding proteins, transferrin and lactoferrin, has been described in a variety of bacterial species (McKenna et al. 1988; Schryvers and Lee 1988; Gonzalez et al. 1990; Ogunnariwo and Schryvers 1990). This mechanism of iron acquisition from transferrin and lactoferrin is poorly understood but involves direct contact with the bacterium and ' ~ u t h o rto whom all correspondence should be addressed. Printed In Canada / lmprime au Canada
probably involves iron removal from the protein at the surface (Archibald and DeVoe 1979; Simonson et al. 1982; McKenna et al. 1988). A characteristic feature of these iron acquisition systems is the specificity for the host's proteins in binding and iron acquisition (Schryvers and Gonzalez 1990; Schryvers and Morris 1988a, 19883; Gonzalez et al. 1990; Ogunnariwo and Schryvers 1990). Three pathogenic bacterial species from the family Neisseriaceae, Neisseria meningitidis, Neisseria gonorrhoeae, and Moraxella (Branhamella) catarrhalis, have been shown to specifically acquire iron for growth from both human transferrin and human lactoferrin (Schryvers and Morris 1988a, 19883; Schryvers and Lee 1988; McKenna et al. 1988; Blanton et al. 1990). Competitive binding assays and isolation of mutants deficient in transferrin binding or lactoferrin binding indicate that there are separate and distinct receptors mediating surface binding of human transferrin and human lactoferrin (Schryvers and Morris 1988a; Lee and Schryvers 1988; Schryvers and Lee 1988; Blanton et al. 1990). Affinity isolation experiments have demonstrated that the transferrin receptor is composed of two iron-repressible outer membrane proteins, TBPl and TBP2, whereas the lactoferrin receptor consists of a single iron-repressible outer membrane protein, LBP (Schryvers and Morris 19883; Schryvers and Lee 1988; Lee and Bryan 1989). Although utilization of transferrin and lactoferrin iron is mediated by different surface receptors, mutant analysis has shown that the two acquisition pathways are linked by a nonreceptor gene product that is not essential for acquisition of iron from other iron sources (Blanton et al. 1990). Preliminary studies have indicated that the transferrin and lactoferrin receptors share some binding characteristics in
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FIG. 1. Analysis of human lactoferrin and the deglycosylated derivatives. Samples of apo-human lactoferrin and the deglycosylated derivatives were applied to duplicate SDS-PAGE gels and then electroblotted onto Immobilon membrane. One membrane was stained with amido black to detect protein (lanes A-C) and the other set was sequentially exposed to Con A, horseradish peroxidase, and perioxidase substrate to detect oligosaccharides (lane A' -C Lanes A and A ' , control human lactoferrin sample without enzyme treatment; lanes B and B' , purified partially deglycosylated human lactoferrin; lanes C and C ' , purified fully deglycosylated human lactoferrin. I ) .
that they are both specific for .the host's protein and are capable of binding both the iron-loaded and apo forms of the proteins (Schryvers and Morris 1988a, 19886; Blanton et al. 1990). However, the differences in properties of transferrin and lactoferrin and the observed differences in receptor protein composition suggest that the structural details of the ligand-receptor interaction may be quite different with these two receptors. Studies directed at delineating the interaction between transferrin and the bacterial receptors have demonstrated that the oligosaccharides of human transferrin are not required for binding to the bacterial transferrin receptors (Padda and Schryvers 1990) and that isolated transferrin N-lobe does not bind the meningococcal receptor (Schryvers et al. 1991). This study was directed at characterization of the interaction between lactoferrin and the bacterial lactoferrin receptors and specifically investigates the role of oligosaccharides on human lactoferrin in binding to the bacterial lactoferrin receptors. Glycopeptidase F was selected for the preparation of deglycosylated derivatives of human lactoferrin because this enzyme is able to cleave off the entire N-linked glycan chain (Tarentino et al 1985). Although preliminary experiments demonstrated that deglycosylation proceeded much more rapidly in the presence of reducing agents such as 2-mercaptoethanol, all subsequent experiments were performed in the absence of reducing agent to preserve the native conformation of human lactoferrin. Since deglyco-
sylation under these conditions required significant quantities of enzyme, we decided to utilize a commercial preparation of endoglycosidase F, which contained 100-150 units of glycopeptidase F for every 6 units of endoglycosidase F. Endoglycosidase F differs from glycopeptidase F in that it leaves a single sugar residue attached to the protein. We selected deglycosylation conditions (pH 8.5) that strongly favored glycopeptidase F activity over endoglycosidase F activity. In deglycosylation experiments performed with human transferrin we observed that there was an apparent loss in the reddish color of the transferrin solution (Padda and Schryvers 1990). In anticipation of a similar phenomenon occurring with human lactoferrin, we chose to perform all of our deglycosylation experiments with apo-human lactoferrin so that .the effect of deglycosylation could be assessed independently from the iron status. Partially and fully deglycosylated forms of human lactoferrin were obtained by enzymatic treatment of human lactoferrin by endoglycosidase F. The two deglycosylated forms of human lactoferrin were then separated by Con A affinity chromatography. Eighty microlitres (4 units) of endoglycosidase F stock solution was added to 12.5 mg of human lactoferrin in 1 mL of 100 mM sodium phosphate buffer, pH 8.5, and the mixture was incubated at 37OC for approximately 20 h. After incubation, the buffer was exchanged by passing the sample through a gel filtration column (Bio-
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FIG. 2. Competitive dot binding assay illustrating the effects of removal of oligosaccharides from human lactoferrin on binding to the meningococcal receptors. Immobilized Nekseria meningitidk iron-deficient membranes were exposed to mixtures containing 160 ng/mL of HRP-human lactoferrin and the following concentrations of the unconjugated proteins; A, 20.0 pg/mL; B, 10.0 pg/mL; C, 5.0 pg/mL; D, 2.5 pg/mL; E, 1.25 pg/mL; F, 625 ng/mL; G, 313 ng/mL; H , 0. bLf, bovine lactoferrin; hLf, iron-free (apo)-human lactoferrin: p-hLf, partially deglycosylated (apo)-human lactoferrin, f-hLf, fully deglycosylated (apo)-human lactoferrin. Rad, lODG, 10 mL) equilibrated in Con A buffer. The column was then washed with 20 mL of equilibrating buffer to fully collect deglycosylated human lactoferrin. The partially deglycosylated human lactoferrin was then obtained by applying a 20-mL gradient of 0-200 mM methyl-a-Dmannopyranoside in Con A buffer. All chromatographic steps were performed at room temperature. The eluted samples were dialyzed against several changes of 50 mM Tris-HC1 buffer, pH 8, concentrated by ultrafiltration and stored at 4OC. Since the two oligosaccharide chains on human lactoferrin each contribute approximately 2500 to the molecular weight of the glycoprotein, we were able to monitor the deglycosylation of human lactoferrin by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). During the deglycosylation reaction, removal of a single oligosaccharide chain proceeded relatively rapidly. This resulted in a preparation consisting predominantly of partially deglycosylated lactoferrin (p-hLf). Under prolonged incubation the second oligosaccharide chain was gradually removed resulting in a mixed preparation containing p-hLf and fully deglycosylated lactoferrin (f-hLf). Even with prolonged incubation and increased enzyme concentrations we were unable to obtain a preparation of f-hLf that was essentially free of p-hLf. As illustrated in Fig. 1, the combination of enzymatic deglycosylation followed by Con A affinity chromatography allowed us to obtain relatively pure preparations of p-hLf and f-hLf. Samples containing untreated, native human lactoferrin (lanes A and A ' ) and derivatives isolated after enzymatic deglycosylation (lanes, B, B ' , C, and C ' ) were ran in duplicate SDS-PAGE gels. The gels were subsequently electroblotted and developed with either amido black stain to detect protein (lanes A-C) or exposed sequentially to Con A, peroxidase, and peroxidase substrate to detect the oligosaccharides (lanes A ' -C '). The sample that eluted from the Con A column upon the addition of methyla-D-mannopyrannoside contained p-hLf. The amido black
stain (lane B) and the Con A binding assay (lane B ' ) revealed that the p-hLf had a lower molecular weight than the native human lactoferrin. The sample that did not bind to the Con A column contained f-hLf. As seen in the amido black stain, f-hLf had an apparent molecular weight lower than p-hLf (lane C), and the lack of Con A binding indicated that it was fully deglycosylated (lane C'). To assess the ability of the deglycosylated derivatives of human lactoferrin to bind to and compete for the human lactoferrin receptors, solid-phase competitive binding dot assays were performed. In these assays, crude membrane preparations from iron-deficient cells were immobilized and exposed to mixtures containing a HRP-human lactoferrin conjugate and varying concentrations of native human lactoferrin or the deglycosylated derivatives. After the binding step, the filter-bound membranes were washed and the amount of bound HRP-human lactoferrin conjugate was determined by developing with a substrate mixture for HRP. Figure 2 illustrates that partially deglycosylated human lactoferrin (p-hLf) and fully deglycosylated human lactoferrin (f-hLf) are equally as effective as native human lactoferrin at blocking binding of the HRP-human lactoferrin conjugate to the meningococcal lactoferrin receptors. Similar experiments were performed with representative strains of N. gonorrhoeae and M. catarrhalis to determine the role of lactoferrin oligosaccharides in binding to .the receptors in these pathogens. The results of the two human pathogens were essentially identical in that the deglycosylated forms of human lactoferrin were as effective at binding to the bacterial receptors as native lactoferrin (data not shown). In this study we demonstrate that the lactoferrin oligosaccharides are not required for binding to the lactoferrin receptor in N. meningitidis, N. gon orrh oeae, or M. catarrhalis (Figs. 1-2). Therefore, further studies attempting to delineate the detailed structural interaction between bactoferrin and the bacterial receptors can take advantage of the
NOTES
detailed structural information available on t h e protein portion o f t h e lactoferrin glycoprotein (Anderson et al. 1987; Baker et al. 1987).
Acknowledgment
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This w o r k was supported b y t h e Medical Research Council, g r a n t N o . MA-10350. Anderson, B.F., Baker, H.M., Dodson, E.J., Norris, G.E., Rumball, S.V., Waters, J.M., and Baker, E.N. 1987. Structure of human lactoferrin at 3.2-Angstrom resolution. Proc. Natl. Acad. Sci. U.S.A. 84: 1769-1773. Archibald, F.S., and DeVoe, I. W. 1979. Removal of iron from human transferrin by Neisseria meningitidis. Microbiol. Lett. 6: 159-161. Baker, E.N., Rumball, S.V., and Anderson, B.F. 1987. Transferrins: insights into structure and function from studies on lactoferrin. Trends Biochem. Sci. 12: 350-355. Bergeron, R.J. 1986. Iron: a controlling nutrient in proliferative processes. Trends Biochem. Sci. 11: 133-136. Blanton, K.J., Biswas, G.D., Tsai, J., Adams, J., Dyer, D.W., Davis, S.M., Koch, G.G., Sen, P.K., and Sparling, P.F. 1990. Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin. J. Bacteriol. 172: 5225-5235. Finkelstein, R.A., Sciortino, C.V., and McIntosh, M.A. 1983. Role of iron in microbe-host interactions. Rev. Infect. Dis. 5: s759-s777. Gonzalez, G.C., Caamano, D.L., and Schryvers, A.B. 1990. Identification and characterization of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae. Mol. Microbiol. 4: 1173-1179. Lee, B.C., and Bryan, L.E. 1989. Identification and comparative analysis of the lactoferrin and transferrin receptors among clinical isolates of gonococci. J. Med. Microbiol. 28: 199-204. Lee, B.C., and Schryvers, A.B. 1988. Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae. Mol. Microbiol. 2: 827-829.
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McKenna, W.R. Mickelsen, P.A., Sparling, P.F., and Dyer, D.W. 1988. Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae. Infect. Immun. 56: 785-791. Neilands, J.B. 1981. Microbial iron compounds. Annu. Rev. Microbiol. 50: 7 15-73 1. Ogunnariwo, J.A., and Schryvers, A.B. 1990. Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor. Infect. Immun. 58: 2091-2097. Padda, J.S., and Schryvers, A.B. 1990. N-linked oligosaccharides of human transferrin are not required for binding t o bacterial transferrin receptors. Infect. Immun. 58: 2972-2976. Payne, S.M., and Finkelstein, R.A. 1978. The critical role of iron in host-bacterial interactions. J. Clin. Invest. 61: 1428- 1440. Schryvers, A.B., and Gonzalez, G.C. 1990. Receptors for transferrin in pathogenic bacteria are specific for the host's protein. Can. J . Microbiol. 36: 145-147. Schryvers, A.B., and Lee, B.C. 1988. Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae. Can. J . Microbiol. 35: 409-41 5. Schryvers, A.B., and Morris, L. J. 1988a. Identification and characterization of the transferrin receptor from Neisseria meningitidis. Mol. Microbiol. 2: 281-288. Schryvers, A.B., and Morris, L.J. 1988b. Identification and characterization of the human lactoferrin-binding from Neisseria meningitidis. Infect. Immun. 56: 1144- 1149. Schryvers, A.B., Irwin, S.W., Middelveen, M.J., Ogunnariwo, J.A., and Alcantara, J., 1991. Iron acquisition in Neisseria: bacterial receptors for human transferrin and human lactoferrin in Neisseria meningitidis. In Neisseriae 1990. Edited by M. Achtman, P. Kohl, C. Marchal, G. Morelli, A. Seiler, and B. Thiesen. Walter de Gruyter, Berlin. p.p. 481-486. Simonson, C., Brener, D., and Devoe, I. W. 1982. Expression of a high-affinity mechanism for acquisition of transferrin iron by Neisseria meningitidis. Infect. Immun. 36: 107- 113. Tarentino, A.L, Gomez, C.M., and Plummer, T.H. 1985. Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry, 24: 4665-467 1. Weinberg, E.D. 1978. Iron and infection. Microbiol. Rev. 42: 45-66.
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J. ALCANTARA, J. S. PADDA, AND A. B. SCHRYVERS Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, A lta., Canada T2N IN4 Received April 1, 1992 Revision received June 18, 1992 Accepted July 7, 1992 ALCANTARA, J., PADDA,J. S., and SCHRYVERS, A. B. 1992. The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors. Can. J. Microbiol. 38 : 1202-1205. The oligosaccharides of human lactoferrin were enzymatically removed with glycopeptidase F, resulting in a preparation containing partial and fully deglycosylated human lactoferrin. The derivatives were separated by Concanavalin A affinity chromatography and compared with native human lactoferrin with respect to their ability to bind t o bacterial receptors. Competitive binding experiments demonstrated that the lactoferrin derivatives were equally capable as native lactoferrin in binding t o receptors of Neisseria rneningitidis, Neisseria gonorrhoeae, and Moraxella catarrhalis. This result indicates that the oligosaccharides on human lactoferrin are not essential for binding to the bacterial receptors. Key words: lactoferrin, oligosaccharides, deglycosylation, receptor, Neisseria. ALCANTARA, J., PADDA,J. S., et SCHRYVERS, A. B. 1992. The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors. Can. J. Microbiol. 38 : 1202-1205. Les oligosaccharides de la lactoferrine humaine ont ete retires par la glycopeptidase F. Le produit de l'action enzymatique contenait de la lactoferrine humaine partiellement et totalement deglycosylee. Les derives ont ete separes par chromatographie d'affinite a la concanavaline A puis compares a la lactoferrine humaine native en ce qui concerne leur capacite de se lier aux recepteurs bacteriens. Les experiences de liaison competitive ont demontre que les derives de lactoferrine sont aussi capables de se lier aux recepteurs de Neisseria rneningitidis, Neisseria gonorrhoeae et Moraxella catarrhalis que la lactoferrine native. Ce resultat indique que les oligosaccharides de la lactoferrine humaine ne sont pas essentiels pour la liaison aux recepteurs bacteriens. Mots cles : bactoferrine, oligosaccharides, deglycosylation, recepteur, Neisseria. [Traduit par la redaction]
The transition metal, iron, is essential for most living organisms. It plays a critical role in biological functions such as DNA synthesis and oxidation-reduction reactions where it serves as a prosthetic group in enzymes and cytochromes (Bergeron 1986). Although there is an abundance of iron in the mammalian host, the majority of iron is found in intracellular pools. The total amount of extracellular iron is sufficient to support the growth of microorganisms, but sequestration by iron binding proteins lowers the effective concentration of free aqueous iron to only 10-l8 M (Finkelstein et al. 1983). The dominant iron binding proteins are transferrin, found in serum, and lactoferrin, found in mucosal secretions (Payne and Finkelstein 1978). The ability of a microorganism to obtain iron in the host is postulated to be an essential requirement for disease causation (Weinberg 1978). Many bacterial species have developed a system for acquisition of extracellular iron involving the synthesis and secretion of highly specific, soluble iron chelating compounds referred to as siderophores (Weinberg 1978). After complexing with extracellular iron, the ironsiderophore complex binds to outer membrane receptor proteins on the bacterial surface and is internalized (Neilands 1981). An alternative mechanism of iron acquisition involving direct removal of iron by the bacterium from the host's iron binding proteins, transferrin and lactoferrin, has been described in a variety of bacterial species (McKenna et al. 1988; Schryvers and Lee 1988; Gonzalez et al. 1990; Ogunnariwo and Schryvers 1990). This mechanism of iron acquisition from transferrin and lactoferrin is poorly understood but involves direct contact with the bacterium and ' ~ u t h o rto whom all correspondence should be addressed. Printed In Canada / lmprime au Canada
probably involves iron removal from the protein at the surface (Archibald and DeVoe 1979; Simonson et al. 1982; McKenna et al. 1988). A characteristic feature of these iron acquisition systems is the specificity for the host's proteins in binding and iron acquisition (Schryvers and Gonzalez 1990; Schryvers and Morris 1988a, 19883; Gonzalez et al. 1990; Ogunnariwo and Schryvers 1990). Three pathogenic bacterial species from the family Neisseriaceae, Neisseria meningitidis, Neisseria gonorrhoeae, and Moraxella (Branhamella) catarrhalis, have been shown to specifically acquire iron for growth from both human transferrin and human lactoferrin (Schryvers and Morris 1988a, 19883; Schryvers and Lee 1988; McKenna et al. 1988; Blanton et al. 1990). Competitive binding assays and isolation of mutants deficient in transferrin binding or lactoferrin binding indicate that there are separate and distinct receptors mediating surface binding of human transferrin and human lactoferrin (Schryvers and Morris 1988a; Lee and Schryvers 1988; Schryvers and Lee 1988; Blanton et al. 1990). Affinity isolation experiments have demonstrated that the transferrin receptor is composed of two iron-repressible outer membrane proteins, TBPl and TBP2, whereas the lactoferrin receptor consists of a single iron-repressible outer membrane protein, LBP (Schryvers and Morris 19883; Schryvers and Lee 1988; Lee and Bryan 1989). Although utilization of transferrin and lactoferrin iron is mediated by different surface receptors, mutant analysis has shown that the two acquisition pathways are linked by a nonreceptor gene product that is not essential for acquisition of iron from other iron sources (Blanton et al. 1990). Preliminary studies have indicated that the transferrin and lactoferrin receptors share some binding characteristics in
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NOTES
FIG. 1. Analysis of human lactoferrin and the deglycosylated derivatives. Samples of apo-human lactoferrin and the deglycosylated derivatives were applied to duplicate SDS-PAGE gels and then electroblotted onto Immobilon membrane. One membrane was stained with amido black to detect protein (lanes A-C) and the other set was sequentially exposed to Con A, horseradish peroxidase, and perioxidase substrate to detect oligosaccharides (lane A' -C Lanes A and A ' , control human lactoferrin sample without enzyme treatment; lanes B and B' , purified partially deglycosylated human lactoferrin; lanes C and C ' , purified fully deglycosylated human lactoferrin. I ) .
that they are both specific for .the host's protein and are capable of binding both the iron-loaded and apo forms of the proteins (Schryvers and Morris 1988a, 19886; Blanton et al. 1990). However, the differences in properties of transferrin and lactoferrin and the observed differences in receptor protein composition suggest that the structural details of the ligand-receptor interaction may be quite different with these two receptors. Studies directed at delineating the interaction between transferrin and the bacterial receptors have demonstrated that the oligosaccharides of human transferrin are not required for binding to the bacterial transferrin receptors (Padda and Schryvers 1990) and that isolated transferrin N-lobe does not bind the meningococcal receptor (Schryvers et al. 1991). This study was directed at characterization of the interaction between lactoferrin and the bacterial lactoferrin receptors and specifically investigates the role of oligosaccharides on human lactoferrin in binding to the bacterial lactoferrin receptors. Glycopeptidase F was selected for the preparation of deglycosylated derivatives of human lactoferrin because this enzyme is able to cleave off the entire N-linked glycan chain (Tarentino et al 1985). Although preliminary experiments demonstrated that deglycosylation proceeded much more rapidly in the presence of reducing agents such as 2-mercaptoethanol, all subsequent experiments were performed in the absence of reducing agent to preserve the native conformation of human lactoferrin. Since deglyco-
sylation under these conditions required significant quantities of enzyme, we decided to utilize a commercial preparation of endoglycosidase F, which contained 100-150 units of glycopeptidase F for every 6 units of endoglycosidase F. Endoglycosidase F differs from glycopeptidase F in that it leaves a single sugar residue attached to the protein. We selected deglycosylation conditions (pH 8.5) that strongly favored glycopeptidase F activity over endoglycosidase F activity. In deglycosylation experiments performed with human transferrin we observed that there was an apparent loss in the reddish color of the transferrin solution (Padda and Schryvers 1990). In anticipation of a similar phenomenon occurring with human lactoferrin, we chose to perform all of our deglycosylation experiments with apo-human lactoferrin so that .the effect of deglycosylation could be assessed independently from the iron status. Partially and fully deglycosylated forms of human lactoferrin were obtained by enzymatic treatment of human lactoferrin by endoglycosidase F. The two deglycosylated forms of human lactoferrin were then separated by Con A affinity chromatography. Eighty microlitres (4 units) of endoglycosidase F stock solution was added to 12.5 mg of human lactoferrin in 1 mL of 100 mM sodium phosphate buffer, pH 8.5, and the mixture was incubated at 37OC for approximately 20 h. After incubation, the buffer was exchanged by passing the sample through a gel filtration column (Bio-
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e
FIG. 2. Competitive dot binding assay illustrating the effects of removal of oligosaccharides from human lactoferrin on binding to the meningococcal receptors. Immobilized Nekseria meningitidk iron-deficient membranes were exposed to mixtures containing 160 ng/mL of HRP-human lactoferrin and the following concentrations of the unconjugated proteins; A, 20.0 pg/mL; B, 10.0 pg/mL; C, 5.0 pg/mL; D, 2.5 pg/mL; E, 1.25 pg/mL; F, 625 ng/mL; G, 313 ng/mL; H , 0. bLf, bovine lactoferrin; hLf, iron-free (apo)-human lactoferrin: p-hLf, partially deglycosylated (apo)-human lactoferrin, f-hLf, fully deglycosylated (apo)-human lactoferrin. Rad, lODG, 10 mL) equilibrated in Con A buffer. The column was then washed with 20 mL of equilibrating buffer to fully collect deglycosylated human lactoferrin. The partially deglycosylated human lactoferrin was then obtained by applying a 20-mL gradient of 0-200 mM methyl-a-Dmannopyranoside in Con A buffer. All chromatographic steps were performed at room temperature. The eluted samples were dialyzed against several changes of 50 mM Tris-HC1 buffer, pH 8, concentrated by ultrafiltration and stored at 4OC. Since the two oligosaccharide chains on human lactoferrin each contribute approximately 2500 to the molecular weight of the glycoprotein, we were able to monitor the deglycosylation of human lactoferrin by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). During the deglycosylation reaction, removal of a single oligosaccharide chain proceeded relatively rapidly. This resulted in a preparation consisting predominantly of partially deglycosylated lactoferrin (p-hLf). Under prolonged incubation the second oligosaccharide chain was gradually removed resulting in a mixed preparation containing p-hLf and fully deglycosylated lactoferrin (f-hLf). Even with prolonged incubation and increased enzyme concentrations we were unable to obtain a preparation of f-hLf that was essentially free of p-hLf. As illustrated in Fig. 1, the combination of enzymatic deglycosylation followed by Con A affinity chromatography allowed us to obtain relatively pure preparations of p-hLf and f-hLf. Samples containing untreated, native human lactoferrin (lanes A and A ' ) and derivatives isolated after enzymatic deglycosylation (lanes, B, B ' , C, and C ' ) were ran in duplicate SDS-PAGE gels. The gels were subsequently electroblotted and developed with either amido black stain to detect protein (lanes A-C) or exposed sequentially to Con A, peroxidase, and peroxidase substrate to detect the oligosaccharides (lanes A ' -C '). The sample that eluted from the Con A column upon the addition of methyla-D-mannopyrannoside contained p-hLf. The amido black
stain (lane B) and the Con A binding assay (lane B ' ) revealed that the p-hLf had a lower molecular weight than the native human lactoferrin. The sample that did not bind to the Con A column contained f-hLf. As seen in the amido black stain, f-hLf had an apparent molecular weight lower than p-hLf (lane C), and the lack of Con A binding indicated that it was fully deglycosylated (lane C'). To assess the ability of the deglycosylated derivatives of human lactoferrin to bind to and compete for the human lactoferrin receptors, solid-phase competitive binding dot assays were performed. In these assays, crude membrane preparations from iron-deficient cells were immobilized and exposed to mixtures containing a HRP-human lactoferrin conjugate and varying concentrations of native human lactoferrin or the deglycosylated derivatives. After the binding step, the filter-bound membranes were washed and the amount of bound HRP-human lactoferrin conjugate was determined by developing with a substrate mixture for HRP. Figure 2 illustrates that partially deglycosylated human lactoferrin (p-hLf) and fully deglycosylated human lactoferrin (f-hLf) are equally as effective as native human lactoferrin at blocking binding of the HRP-human lactoferrin conjugate to the meningococcal lactoferrin receptors. Similar experiments were performed with representative strains of N. gonorrhoeae and M. catarrhalis to determine the role of lactoferrin oligosaccharides in binding to .the receptors in these pathogens. The results of the two human pathogens were essentially identical in that the deglycosylated forms of human lactoferrin were as effective at binding to the bacterial receptors as native lactoferrin (data not shown). In this study we demonstrate that the lactoferrin oligosaccharides are not required for binding to the lactoferrin receptor in N. meningitidis, N. gon orrh oeae, or M. catarrhalis (Figs. 1-2). Therefore, further studies attempting to delineate the detailed structural interaction between bactoferrin and the bacterial receptors can take advantage of the
NOTES
detailed structural information available on t h e protein portion o f t h e lactoferrin glycoprotein (Anderson et al. 1987; Baker et al. 1987).
Acknowledgment
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/20/14 For personal use only.
This w o r k was supported b y t h e Medical Research Council, g r a n t N o . MA-10350. Anderson, B.F., Baker, H.M., Dodson, E.J., Norris, G.E., Rumball, S.V., Waters, J.M., and Baker, E.N. 1987. Structure of human lactoferrin at 3.2-Angstrom resolution. Proc. Natl. Acad. Sci. U.S.A. 84: 1769-1773. Archibald, F.S., and DeVoe, I. W. 1979. Removal of iron from human transferrin by Neisseria meningitidis. Microbiol. Lett. 6: 159-161. Baker, E.N., Rumball, S.V., and Anderson, B.F. 1987. Transferrins: insights into structure and function from studies on lactoferrin. Trends Biochem. Sci. 12: 350-355. Bergeron, R.J. 1986. Iron: a controlling nutrient in proliferative processes. Trends Biochem. Sci. 11: 133-136. Blanton, K.J., Biswas, G.D., Tsai, J., Adams, J., Dyer, D.W., Davis, S.M., Koch, G.G., Sen, P.K., and Sparling, P.F. 1990. Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin. J. Bacteriol. 172: 5225-5235. Finkelstein, R.A., Sciortino, C.V., and McIntosh, M.A. 1983. Role of iron in microbe-host interactions. Rev. Infect. Dis. 5: s759-s777. Gonzalez, G.C., Caamano, D.L., and Schryvers, A.B. 1990. Identification and characterization of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae. Mol. Microbiol. 4: 1173-1179. Lee, B.C., and Bryan, L.E. 1989. Identification and comparative analysis of the lactoferrin and transferrin receptors among clinical isolates of gonococci. J. Med. Microbiol. 28: 199-204. Lee, B.C., and Schryvers, A.B. 1988. Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae. Mol. Microbiol. 2: 827-829.
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