Limits...
Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria.

Schnitger AK, Yassine H, Kafatos FC, Osta MA - J. Biol. Chem. (2009)

Bottom Line: Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood.Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria.Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

ABSTRACT
C-type lectins (CTLs) are a family of proteins that share a common structural motif, the carbohydrate recognition domain, and may act as receptors in pathogen recognition. Indeed, some vertebrate CTLs, particularly the collectins, are unequivocally implicated in the innate immune response to certain microbes. Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood. Here we report that two CTLs, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are transcriptionally induced by bacterial challenge. Using in vivo reverse genetic analysis, we show that both CTLs are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Furthermore, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

Show MeSH

Related in: MedlinePlus

CTL4 and CTLMA2 form a disulfide-linked heterodimer. A, alignment of CTL4 and CTLMA2 protein sequences using T-COFFEE align software. The predicted signal peptides are underlined. The N-terminal cysteine residues involved in disulfide linkages between CTL4 and CTLMA2 are in boldface. The four cysteine residues that are diagnostic of the carbohydrate recognition domain are enclosed in rectangles. Amino acid residues that form the putative sugar-binding site were determined by sequence comparison with the rat mannose-binding protein (41) and are shaded in gray. The numbers refer to residues counted from the initiation methionine. Residues identical in both sequences are marked with an asterisk. B and C, Western blots of hemolymph proteins extracted 4 days after injecting naive mosquitoes with the indicated dsRNAs and separated by SDS-PAGE on a 13% gel. Under reducing conditions, CTL4 and CTLMA2 antibodies detect 17- and 19-kDa bands, respectively, and they both detect major bands with similar molecular masses of ∼28 kDa in nonreducing conditions. Note that CTLMA2 exhibits a minor homodimeric form of 30 kDa. Polyclonal rabbit anti-CTL4 and monoclonal mouse anti-CTLMA2 antibodies were used to detect CTL4 and CTLMA2, respectively. A polyclonal antibody against Serpin 3 (SRPN3) was used as loading control. D, affinity purification of CTL4V5His on nickel-Sepharose captures nontagged CTLMA2. CTLMA2 was either singly or co-expressed with CTL4V5His in Sf9 cells. His tag capture from conditioned medium of cells co-expressing CTLMA2 and CTL4V5His co-purifies CTLMA2 (lane 4) is shown. CTLMA2 did not bind to nickel-Sepharose in the absence of CTL4V5His (lane 3). CM, conditioned medium of mock-transfected cells (lane 1) or cells expressing only CTLMA2 (lane 2). E, two-color Western blot analysis of Sf9-conditioned media expressing CTLV5His and CTLMA2V5His either separately or simultaneously and subjected to nonreducing SDS-PAGE. The membrane was probed simultaneously with rabbit anti-CTL4 and mouse anti-CTLMA2 followed by simultaneous incubation with anti-mouse (green) and anti-rabbit (red) antibodies conjugated to infrared fluorescent dyes. CTL4 and CTLMA2 homodimers are in red and green, respectively, while the CTL4-CTLMA2 heterodimer appears yellow. Transfection with an empty pIZT/V5-His plasmid was used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2719400&req=5

Figure 5: CTL4 and CTLMA2 form a disulfide-linked heterodimer. A, alignment of CTL4 and CTLMA2 protein sequences using T-COFFEE align software. The predicted signal peptides are underlined. The N-terminal cysteine residues involved in disulfide linkages between CTL4 and CTLMA2 are in boldface. The four cysteine residues that are diagnostic of the carbohydrate recognition domain are enclosed in rectangles. Amino acid residues that form the putative sugar-binding site were determined by sequence comparison with the rat mannose-binding protein (41) and are shaded in gray. The numbers refer to residues counted from the initiation methionine. Residues identical in both sequences are marked with an asterisk. B and C, Western blots of hemolymph proteins extracted 4 days after injecting naive mosquitoes with the indicated dsRNAs and separated by SDS-PAGE on a 13% gel. Under reducing conditions, CTL4 and CTLMA2 antibodies detect 17- and 19-kDa bands, respectively, and they both detect major bands with similar molecular masses of ∼28 kDa in nonreducing conditions. Note that CTLMA2 exhibits a minor homodimeric form of 30 kDa. Polyclonal rabbit anti-CTL4 and monoclonal mouse anti-CTLMA2 antibodies were used to detect CTL4 and CTLMA2, respectively. A polyclonal antibody against Serpin 3 (SRPN3) was used as loading control. D, affinity purification of CTL4V5His on nickel-Sepharose captures nontagged CTLMA2. CTLMA2 was either singly or co-expressed with CTL4V5His in Sf9 cells. His tag capture from conditioned medium of cells co-expressing CTLMA2 and CTL4V5His co-purifies CTLMA2 (lane 4) is shown. CTLMA2 did not bind to nickel-Sepharose in the absence of CTL4V5His (lane 3). CM, conditioned medium of mock-transfected cells (lane 1) or cells expressing only CTLMA2 (lane 2). E, two-color Western blot analysis of Sf9-conditioned media expressing CTLV5His and CTLMA2V5His either separately or simultaneously and subjected to nonreducing SDS-PAGE. The membrane was probed simultaneously with rabbit anti-CTL4 and mouse anti-CTLMA2 followed by simultaneous incubation with anti-mouse (green) and anti-rabbit (red) antibodies conjugated to infrared fluorescent dyes. CTL4 and CTLMA2 homodimers are in red and green, respectively, while the CTL4-CTLMA2 heterodimer appears yellow. Transfection with an empty pIZT/V5-His plasmid was used as a negative control.

Mentions: Hemolymph from naive mosquitoes, extracted in reducing SDS protein sample buffer and immunoblotted with CTL4 and CTLMA2 antibodies, revealed two bands with apparent molecular masses of 17 (Fig. 5B) and 19 kDa (Fig. 5C) corresponding to CTL4 and CTLMA2 monomers, respectively. The theoretical molecular masses of CTL4 and CTLMA2 are 17.2 and 17.8 kDa, respectively, excluding the predicted N-terminal signal peptide (Fig. 5A). As we could not detect potential N-glycosylation sites in CTLMA2 by bioinformatic analysis, its low electrophoretic mobility in SDS-PAGE (19 kDa) as compared with its predicted molecular mass (17.8 kDa) is most convincingly attributed to lesser SDS binding due its predicted low isoelectric point (pI = 4.2). Anomalous migration of proteins rich in acidic amino acids on SDS-PAGE has been reported previously in the literature (38). Interestingly, in nonreducing conditions, both antibodies detected major bands of similar molecular masses (∼28 kDa). The fact that these CTLs have different molecular masses under reducing conditions but display an identical molecular mass under nonreducing conditions suggests that they either form a disulfide-linked heterodimer or are part of two different complexes with fortuitously identical molecular masses. Interestingly, when hemolymph was extracted from dsCTLMA2 mosquitoes and immunoblotted with CTL4 antibody, both the CTL4 monomer (17 kDa) and the 28-kDa heterodimer were almost undetectable (Fig. 5B). Similarly, in hemolymph extracted from dsCTL4 mosquitoes, the CTLMA2 monomer was barely detectable, and the 28-kDa heterodimer was completely absent (Fig. 5C). These results suggest that heterodimerization is required for efficient secretion of these CTLs in vivo. The apparent molecular mass of the CTL4-CTLMA2 heterodimer is lower than its expected 35-kDa value. This aberrant migration is probably because of a compact structure resulting from disulfide bonds within the CTLDs (two bonds in each domain) as well as between the N-terminal domains of CTL4 and CTLMA2. Under nonreducing conditions, a faint CTLMA2 band migrating slower than the heterodimer was present in dslacZ control mosquitoes but absent from dsCTLMA2 mosquitoes. This 30-kDa band is most convincingly interpreted as a minor CTLMA2 homodimer; it was slightly enhanced in the absence of the CTL4 partner (Fig. 5C), possibly because surplus CTLMA2 monomers associated to form homodimers. No CTL4 homodimer was detected in the mosquito hemolymph.


Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria.

Schnitger AK, Yassine H, Kafatos FC, Osta MA - J. Biol. Chem. (2009)

CTL4 and CTLMA2 form a disulfide-linked heterodimer. A, alignment of CTL4 and CTLMA2 protein sequences using T-COFFEE align software. The predicted signal peptides are underlined. The N-terminal cysteine residues involved in disulfide linkages between CTL4 and CTLMA2 are in boldface. The four cysteine residues that are diagnostic of the carbohydrate recognition domain are enclosed in rectangles. Amino acid residues that form the putative sugar-binding site were determined by sequence comparison with the rat mannose-binding protein (41) and are shaded in gray. The numbers refer to residues counted from the initiation methionine. Residues identical in both sequences are marked with an asterisk. B and C, Western blots of hemolymph proteins extracted 4 days after injecting naive mosquitoes with the indicated dsRNAs and separated by SDS-PAGE on a 13% gel. Under reducing conditions, CTL4 and CTLMA2 antibodies detect 17- and 19-kDa bands, respectively, and they both detect major bands with similar molecular masses of ∼28 kDa in nonreducing conditions. Note that CTLMA2 exhibits a minor homodimeric form of 30 kDa. Polyclonal rabbit anti-CTL4 and monoclonal mouse anti-CTLMA2 antibodies were used to detect CTL4 and CTLMA2, respectively. A polyclonal antibody against Serpin 3 (SRPN3) was used as loading control. D, affinity purification of CTL4V5His on nickel-Sepharose captures nontagged CTLMA2. CTLMA2 was either singly or co-expressed with CTL4V5His in Sf9 cells. His tag capture from conditioned medium of cells co-expressing CTLMA2 and CTL4V5His co-purifies CTLMA2 (lane 4) is shown. CTLMA2 did not bind to nickel-Sepharose in the absence of CTL4V5His (lane 3). CM, conditioned medium of mock-transfected cells (lane 1) or cells expressing only CTLMA2 (lane 2). E, two-color Western blot analysis of Sf9-conditioned media expressing CTLV5His and CTLMA2V5His either separately or simultaneously and subjected to nonreducing SDS-PAGE. The membrane was probed simultaneously with rabbit anti-CTL4 and mouse anti-CTLMA2 followed by simultaneous incubation with anti-mouse (green) and anti-rabbit (red) antibodies conjugated to infrared fluorescent dyes. CTL4 and CTLMA2 homodimers are in red and green, respectively, while the CTL4-CTLMA2 heterodimer appears yellow. Transfection with an empty pIZT/V5-His plasmid was used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719400&req=5

Figure 5: CTL4 and CTLMA2 form a disulfide-linked heterodimer. A, alignment of CTL4 and CTLMA2 protein sequences using T-COFFEE align software. The predicted signal peptides are underlined. The N-terminal cysteine residues involved in disulfide linkages between CTL4 and CTLMA2 are in boldface. The four cysteine residues that are diagnostic of the carbohydrate recognition domain are enclosed in rectangles. Amino acid residues that form the putative sugar-binding site were determined by sequence comparison with the rat mannose-binding protein (41) and are shaded in gray. The numbers refer to residues counted from the initiation methionine. Residues identical in both sequences are marked with an asterisk. B and C, Western blots of hemolymph proteins extracted 4 days after injecting naive mosquitoes with the indicated dsRNAs and separated by SDS-PAGE on a 13% gel. Under reducing conditions, CTL4 and CTLMA2 antibodies detect 17- and 19-kDa bands, respectively, and they both detect major bands with similar molecular masses of ∼28 kDa in nonreducing conditions. Note that CTLMA2 exhibits a minor homodimeric form of 30 kDa. Polyclonal rabbit anti-CTL4 and monoclonal mouse anti-CTLMA2 antibodies were used to detect CTL4 and CTLMA2, respectively. A polyclonal antibody against Serpin 3 (SRPN3) was used as loading control. D, affinity purification of CTL4V5His on nickel-Sepharose captures nontagged CTLMA2. CTLMA2 was either singly or co-expressed with CTL4V5His in Sf9 cells. His tag capture from conditioned medium of cells co-expressing CTLMA2 and CTL4V5His co-purifies CTLMA2 (lane 4) is shown. CTLMA2 did not bind to nickel-Sepharose in the absence of CTL4V5His (lane 3). CM, conditioned medium of mock-transfected cells (lane 1) or cells expressing only CTLMA2 (lane 2). E, two-color Western blot analysis of Sf9-conditioned media expressing CTLV5His and CTLMA2V5His either separately or simultaneously and subjected to nonreducing SDS-PAGE. The membrane was probed simultaneously with rabbit anti-CTL4 and mouse anti-CTLMA2 followed by simultaneous incubation with anti-mouse (green) and anti-rabbit (red) antibodies conjugated to infrared fluorescent dyes. CTL4 and CTLMA2 homodimers are in red and green, respectively, while the CTL4-CTLMA2 heterodimer appears yellow. Transfection with an empty pIZT/V5-His plasmid was used as a negative control.
Mentions: Hemolymph from naive mosquitoes, extracted in reducing SDS protein sample buffer and immunoblotted with CTL4 and CTLMA2 antibodies, revealed two bands with apparent molecular masses of 17 (Fig. 5B) and 19 kDa (Fig. 5C) corresponding to CTL4 and CTLMA2 monomers, respectively. The theoretical molecular masses of CTL4 and CTLMA2 are 17.2 and 17.8 kDa, respectively, excluding the predicted N-terminal signal peptide (Fig. 5A). As we could not detect potential N-glycosylation sites in CTLMA2 by bioinformatic analysis, its low electrophoretic mobility in SDS-PAGE (19 kDa) as compared with its predicted molecular mass (17.8 kDa) is most convincingly attributed to lesser SDS binding due its predicted low isoelectric point (pI = 4.2). Anomalous migration of proteins rich in acidic amino acids on SDS-PAGE has been reported previously in the literature (38). Interestingly, in nonreducing conditions, both antibodies detected major bands of similar molecular masses (∼28 kDa). The fact that these CTLs have different molecular masses under reducing conditions but display an identical molecular mass under nonreducing conditions suggests that they either form a disulfide-linked heterodimer or are part of two different complexes with fortuitously identical molecular masses. Interestingly, when hemolymph was extracted from dsCTLMA2 mosquitoes and immunoblotted with CTL4 antibody, both the CTL4 monomer (17 kDa) and the 28-kDa heterodimer were almost undetectable (Fig. 5B). Similarly, in hemolymph extracted from dsCTL4 mosquitoes, the CTLMA2 monomer was barely detectable, and the 28-kDa heterodimer was completely absent (Fig. 5C). These results suggest that heterodimerization is required for efficient secretion of these CTLs in vivo. The apparent molecular mass of the CTL4-CTLMA2 heterodimer is lower than its expected 35-kDa value. This aberrant migration is probably because of a compact structure resulting from disulfide bonds within the CTLDs (two bonds in each domain) as well as between the N-terminal domains of CTL4 and CTLMA2. Under nonreducing conditions, a faint CTLMA2 band migrating slower than the heterodimer was present in dslacZ control mosquitoes but absent from dsCTLMA2 mosquitoes. This 30-kDa band is most convincingly interpreted as a minor CTLMA2 homodimer; it was slightly enhanced in the absence of the CTL4 partner (Fig. 5C), possibly because surplus CTLMA2 monomers associated to form homodimers. No CTL4 homodimer was detected in the mosquito hemolymph.

Bottom Line: Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood.Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria.Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

ABSTRACT
C-type lectins (CTLs) are a family of proteins that share a common structural motif, the carbohydrate recognition domain, and may act as receptors in pathogen recognition. Indeed, some vertebrate CTLs, particularly the collectins, are unequivocally implicated in the innate immune response to certain microbes. Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood. Here we report that two CTLs, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are transcriptionally induced by bacterial challenge. Using in vivo reverse genetic analysis, we show that both CTLs are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Furthermore, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

Show MeSH
Related in: MedlinePlus