Serpin-1 splicing isoform J inhibits the proSpätzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta

Abstract

The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3×10(4)M(-1)s(-1), with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta.

Fig.1

(A-B) SDS-PAGE analysis of purified recombinant serpin-1J and proHP8_Xa. (A) Purified serpin-1J (1.0 μg) was analyzed by SDS-PAGE under reducing conditions followed by coomassie blue staining. (B) Purified proHP8_Xa (75 ng) was analyzed by SDS-PAGE under reducing conditions followed by silver staining. (C-D) SDS-stable complex formation between serpin-1J and HP8. ProHP8_Xa (50 ng) was activated by factor Xa and then incubated for 10 min at room temperature with a 10-fold molar excess of serpin-1J. In control reactions, proHP8 or factor Xa were omitted. The samples were analyzed by SDS-PAGE and immunoblotting using antibodies to HP8 (C) or serpin-1 (D). Serpin-1 antibodies recognized intact serpin-1J (~43 kDa) and a lower band (~42 kDa) that is a serpin-1J degradation product (cleaved serpin). HP8 antibody recognized the 42 kDa proHP8_Xa zymogen, a 34 kDa catalytic domain produced upon activation, and an inactive, truncated proHP8_Xa that is present in the preparation [9]. Both antibodies recognized a band at ~66 kDa, representing a serpin-HP8 complex. Size and positions of molecular mass standards are indicated to the left of each blot.

Fig.2

Analysis of serpin-1J and HP8 interactions. (A) Stoichiometry of inhibition of HP8 by serpin-1J. Recombinant serpin-1J was incubated with Factor Xa-activated HP8_Xa at different molar ratios for 10 min at room temperature. The residual amidase activity was measured using IEARpNA as substrate. The intersection of a line generated by linear regression extrapolated to the X axis occurred at a molar ratio of 5.4. (B) Kinetic analysis of serpin-1J and HP8 interactions. Activated HP8_Xa (6.5 nM) was mixed with 300 μM IEARpNa and serpin-1J at 0 (■), 16.3 (□), 32.5 (▼), 65 (▽), 195 (◆), 325 (◇), and 530 (▲) nM. The progress of enzyme inactivation at each concentration of serpin-1J was followed by measuring the ΔA₄₀₅ of the reaction every 49 sec (inset). Pseudo-first-order rate constants of inhibition of HP8_Xa (k) and the second-order association rate constant (k_a) was calculated as described in Experimental Procedures.

Fig.3

Rate of formation of an SDS-stable complex between HP8 and serpin-1J. Activated HP8_Xa (0.17 μM) was incubated with serpin-1J (1.7 μM) at room temperature for 0, 1, 3, 5, 8, and 10 min. The reaction mixtures were separated by SDS-PAGE, followed by immunoblot analysis with antiserum against M. sexta HP8. Positions of molecular weight standards are indicated on the right.

Fig.4

Serpin-1J inhibits the activation of purified proSpätzle by HP8_Xa. ProHP8_Xa (25 ng) was activated by Factor Xa (50 ng), and then incubated for 10 min at room temperature with serpin-1J at molar ratios indicated at the bottom, then incubated with proSpätzle (100 ng) at 37°C for 1 h. The mixtures were subjected to SDS-PAGE and immunoblotting using Spätzle antibodies. The sizes and positions of molecular weight standards are indicated on the left. Bands representing proSpätzle and the active cysteine-knot domain (Spätzle-C108) are marked by arrows.

Fig.5

Effect of injection of serpin-1J on the expression of bacteria-induced hemolymph proteins. Fifth instar day 0 larvae were injected with 200 μg serpin-1J or BSA. After 30 min, a subset of these larvae was injected with 100 ng of M. luteus. After 20 h, hemolymph was collected and fat body RNA samples were prepared from each insect, for assay of plasma antimicrobial activity and mRNA levels for bacteria-induced hemolymph proteins. (A) Antimicrobial activity of plasma assayed against E. coli, (B) Identification of plasma antimicrobial peptides by SDS-PAGE and MALDI-TOF peptide mass fingerprinting, (C) Quantitative analysis of attacin and cecropin band intensity by densitometry, (D) mRNA levels for indicated genes assayed by quantitative RT-PCR relative to ribosomal protein S3 mRNA level as described in Experimental Procedures. In A, C, and D, bars represent mean ± S.D. (n=3). Bars labeled with different letters are significantly different (one-way ANOVA followed by the Newman-Keuls test, P < 0.05).