Interaction of beta-1,3-glucan with its recognition protein activates hemolymph proteinase 14, an initiation enzyme of the prophenoloxidase activation system in Manduca sexta

Abstract

A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101-34106). Here, we report the purification and characterization of pro-HP14 from the hemolymph of bacteria-injected M. sexta larvae. The zymogen, consisting of a single polypeptide with a molecular mass of 68.5 kDa, is truncated at the amino terminus. It is converted to a two-chain active form in the presence of beta-1,3-glucan (a fungal cell wall component) and beta-1,3-glucan recognition protein-2. The 45-kDa heavy chain contains four low-density lipoprotein receptor A repeats, one Sushi domain, and one unique cysteine-rich region, whereas the 30-kDa light chain contains a serine proteinase domain, which was labeled by [(3)H]diisopropyl fluorophosphate. Pro-HP14 in the plasma strongly binds curdlan, zymosan, and yeast and interacts with peptidoglycan and Micrococcus luteus. Addition of autoactivated HP14 elevated phenoloxidase activity level in the larval plasma. Recombinant M. sexta serpin-1I reduced prophenoloxidase activation by inhibiting HP14. These data are consistent with the current model on initiation and regulation of the prophenoloxidase activation cascade upon recognition of pathogen-associated molecular patterns by specific pattern recognition proteins.

FIGURE 1.. SDS-PAGE analysis of pro-HP14 isolated from the hemolymph of M. sexta larvae injected with bacteria

A, electrophoretic separation and immunoblot detection of pro-HP14 in 12 µl of flow-through fraction 18 (left) and bound fraction 54 (right) from the hydroxylapatite column. B, SDS-PAGE and silver staining of the 75-kDa pro-HP14 eluted from the dextran sulfate column. The fractions 9–13 (12 µl each, lanes 1–5) were treated with SDS sample buffer containing dithiothreitol, separated on a 12% SDS-PAGE gel, and stained with silver.

FIGURE 2.. Autoactivation of pro-HP14 upon exposure to β-1,3-glucan and βGRP2

A, the purified pro-HP14 (200 ng) was incubated with 10 µg of curdlan and 40 ng of βGRP2 at 37 °C for 2 h. After being treated with SDS sample buffer containing dithiothreitol, the reaction mixture and controls were subjected to 12% SDS-PAGE and silver staining. B, a duplicate reaction mixture separated by SDS-PAGE under nonreducing condition. C, to label the active site Ser residue, the reaction mixture was incubated at 37 °C in the presence of [³H]DFP (1 µl, 0.1 mm, and 10 mCi/mmol). The labeling mixture was resolved by 12% SDS-PAGE under reducing condition and subjected to fluorography (24). a, 75-kDa pro-HP14; b, βGRP2;, c, 45-kDa HP14 heavy chain; and d, 30-kDa HP14 light chain.

FIGURE 3.. Optimization of the reaction conditions for pro-HP14 autoprocessing

The general conditions for pro-HP14 autoactivation were: pro-HP14 (10 µl and 20 ng/µl), curdlan (1 µl and 10 µg/µl), and βGRP2 (2 µl and 20 ng/µl) incubated with 20 mm Tris-HCl, pH 7.8, 20 mm NaCl, 5 mm CaCl₂ at 37 °C for 90 min. Incubation time (A), βGRP2 amount (B), NaCl concentration (C), or curdlan amount (D) was changed in each experiment. The reaction mixtures were separated by 12% SDS-PAGE under reducing condition and visualized by silver staining. a–d, see Fig. 2 legend.

FIGURE 4.. Association of microbes and their cell wall components with pro-HP14 in the fractionated plasma

Aliquots of 0–35% ammonium sulfate fraction of the induced hemolymph were incubated with M. luteus (lane 1), peptidoglycan (lane 2), E. coli (lane 3), zymosan (lane 4), yeast (lane 5), or curdlan (lane 6) at room temperature for 30 min. After centrifugation at 13,000 ✕ g for 1 min, the supernatants (A) were removed for SDS-PAGE under reducing condition. The pellets were washed five times with 50 mm Tris-HCl, 5mm CaCl₂, pH 7.8. Bound proteins (B) were eluted from the pellet by SDS sample buffer at 100 °C for 5 min and resolved by SDS-PAGE. Electrotransfer and immunoblot analysis were performed to detect pro-HP14 using its antibodies. Some of the signal (panel B, lane 1) came from the cross-reactivity with a co-migrating molecule from M. luteus.

FIGURE 5.. Binding of purified pro-HP14 to microbes and their surface molecules

Purified pro-HP14 (1 µg) was incubated with 1 mg of heat-treated microbial cells in the presence or absence of their binding proteins (200 ng) in 70 µl, 20 mm Tris-HCl, 20 mm NaCl, 5 mm CaCl₂, pH 7.8, at 37 °C for 60 min. After centrifugation at 13,000 ✕ g for 1 min, the supernatants (S) were recovered for SDS-PAGE analysis. The pellets were washed five times with 20 mm Tris-HCl, 5 mm CaCl₂, pH 7.8, and the bound proteins (P) were eluted by treating the pellets with SDS sample buffer at 100 °C for 5 min. A, detection of pro-HP14 or HP14 binding by immunoblot analysis. For background reduction, 1:1000 diluted HP14 antiserum was preincubated with lysates of E. coli, M. luteus, and S. cerevisae.*, unknown; note that the HP14 polyclonal antibodies hardly recognize the 30-kDa catalytic domain (16). B, association of pro-HP14 with curdlan examined by SDS-PAGE and silver staining. a–d, see Fig. 2 legend.

FIGURE 6.. Activation of pro-PO by autocleaved HP14

Purified pro-HP14 (1 µg), 1 mg of curdlan, and 200 ng of βGRP2 were incubated at 37 °C for 90 min in 20 mm Tris-HCl, 20 mm NaCl, 5 mm CaCl₂, pH 8.0. After centrifugation, 2 µl of the supernatant was incubated on ice with 17.5 µl of buffer and 0.5 µl of plasma samples from naïve (A) or bacteria-injected (B) larvae. Plasma alone, plasma and pro-HP14, as well as a mixture of plasma, curdlan, and βGRP2 were included as controls. After 20 min, PO activity was determined using dopamine as a substrate (25) and plotted as mean ± S.E. (n = 3) in the bar graph.

FIGURE 7.. Down-regulation of pro-PO activation by serpin-1I, an HP14 inhibitor

A, to detect the covalent complex of HP14 and serpin-1I, 200 ng of purified pro-HP14, 10 µg of curdlan, and 40 ng of βGRP2 were incubated with 3 µg of serpin-1I in pH 8.0, 20 mm Tris-HCl, 5 mm CaCl₂ at 37 °C for 90 min. The reaction mixture (lane 3) was analyzed by immunoblot analysis using 1:1000 diluted serpin-1 antiserum as the first antibody. The activation mixture (lane 1) and inhibitor alone (lane 2) were run as negative controls. B, to examine the effect of serpin-1I on pro-PO activation triggered by HP14, 1 µg of pro-HP14 was first activated by 1 mg of curdlan, and 200 ng of βGRP2 in the presence of 5 mm CaCl₂ at 37 °C for 90 min. After centrifugation, the supernatant (2 µl) and serpin-1I (3 µg and 1 µl) were reacted on ice for 10 min in 20 mm Tris-HCl, 5 mm CaCl₂, pH 8.0. Plasma (0.5 µl) from naïve larvae was then added to the HP14-serpin-1I mixture or HP14 (positive control). Plasma alone and a mixture of plasma and serpin-1I were used as negative controls. After 20 min on ice, PO activities in the reaction and controls were measured and plotted as mean ± S.E. (n = 3).