Purification and characterization of a small cationic protein from the tobacco hornworm Manduca sexta

Abstract

The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 microg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 microg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.

Fig. 1. Purification and characterization of M. sexta cationic protein CP8

(A) Far western blot analysis of M. sexta larval plasma proteins that interact with recombinant IML-3 (rIML-3). Cell-free plasma (2 μl) from M. sexta naïve larvae was analyzed on SDS-PAGE, and plasma proteins were stained with Coomassie Brilliant Blue (lane 1) or transferred to a nitrocellulose membrane. Then the membrane was probed with the His-tagged rIML-3 (lane 2). Binding of rIML-3 to plasma proteins was detected by monoclonal antibody to the His-tag. Lane M is protein ladder. The arrowhead and arrow indicate CP8 and the 14-kDa protein (lysozyme) respectively, which interact with rIML-3. (B) Purification scheme for CP8. 50% AS = 50% ammonium sulfate. Far western blot was used to monitor the desired proteins at each step of purification. (C) Analysis of native and recombinant CP8 by SDS-PAGE. Recombinant (lane 1) and native CP8 (lane 2) (each at 0.5 μg) were analyzed on 15% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. Lane M is protein ladder, and the arrowhead indicates CP8. (D) MALDI-TOFF analysis of CP8 purified from plasma. The main peak of CP8 has a molecular weight of 9094±2 kDa. Insulin was used as an internal molecular weight standard.

Fig. 2. The nucleotide and deduced amino acid sequences of CP8 and alignment of CP8 with homologous proteins

(A) The nucleotide and deduced amino acid sequences of CP8. The deduced amino acid sequence of CP8 is shown below the cDNA sequence. The amino-terminal sequence of the mature CP8 was determined by Edman degradation and is underlined. Cysteine residues are circled. The polyadenylation sequence AATAAA is double-underlined. (B) Alignment of CP8 with homologous proteins. The deduced amino acid sequence of CP8 was aligned with A. mylitta AmFPI-1 (accession number: B0JFB8), G. mellonella ISPI-1 (accession number: P81905), and homologous proteins from P. sitchensis (accession number: ABK25652), B. mori (accession number: AY655143) and B. mandarina (accession number: EF126182). Residues conserved in all five proteins but G. mellonella ISPI-1 are marked with ‘*’ above the alignment. Conserved cysteine residues are indicated by boxes. Identities between CP8 and other proteins are included in the parentheses.

Fig. 3. Expression of CP8 in M. sexta larvae

(A) Real-time PCR analysis of CP8 mRNA in tissues of naïve larvae. Real-time PCR was performed as described in the Materials and Methods. Expression level in the naïve hemocytes was used as the calibrator. (B) Real-time PCR analysis of CP8 mRNA in fat body of immune-challenged larvae. Day 2 fifth instar M. sexta naïve larvae were injected with saline, formaldehyde-killed E. coli (10⁸ cells per larva), M. luteus (100 μg per larva), or yeast (S. cerevisiae, 10⁷ cells per larva), and fat body was collected at 24 h post-injection for preparation of total RNAs. Real-time PCR was performed the same as in (A). Expression level in fat body of M. luteus-injected larvae was used as the calibrator. The significance of difference was calculated using an unpaired t-test program (GraphPad, San Diego, CA). (C) Expression of CP8 protein in hemolymph of immune-challenged larvae. Day 2 fifth instar M. sexta naïve larvae were injected with saline, E. coli, M. luteus, or yeast as described in (B), and hemolymph was collected at 0 h (naïve hemolymph) and 24 h post-injection. Aliquots of plasma (1 μl, a mixture from at least 4 larvae) were analyzed on 15% SDS-PAGE, and CP8 was identified by immunoblotting using rabbit polyclonal antibody to recombinant CP8. The arrow indicates CP8 in hemolymph.

Fig. 4. CP8 does not have an effect on serine proteinase activity

Native CP8 purified from plasma, renatured recombinant CP8, or BSA (each at 0.5 μg) was mixed with a serine proteinase (50ng trypsin, 100ng chymotrypsin, 200ng elastase, 200ng subtilisin A, 200ng proteinase K, 200ng S. griseus proteinase, or 10ng PAP-3) in a total of 20μl assay buffer (100mM Tris-HCl, 100mM NaCl, 1mM CaCl₂, pH7.5) and incubated at room temperature for 30 min. Then a specific peptide substrate (200μl, 50μM) was added and proteinase activity was monitored every min for 30 min. The bars represent the means of three individual measurements ± SEM.

Fig. 5. CP8 stimulates proPO activation in plasma

Aliquots of naïve plasma (#31) (2 μl each) were incubated with heat-treated (65°C, 30 min) native CP8 purified from plasma or BSA (0.1 or 0.5 μg) in a total of 10μl TBS for 20 min (A) or 30 min (B) at room temperature, or with TBS alone (Control), CP8/M. luteus mixture (0.5 μg each) or BSA/M. luteus mixture (0.5 μg each) for 10 min at room temperature (C), or pre-activated with M. luteus (0.5 μg) for 5 min at room temperature and then incubated with CP8 or BSA (0.5 μg each) for another 5 min (totally 10 min) at room temperature (C). Finally, L-dopamine substrate (200 μl, 2mM) was added to each sample and PO activity was monitored with time at 470nm for 30 min. The bars represent the means of four individual measurements ± SEM. The significance of difference was calculated using an unpaired t-test program (GraphPad, San Diego, CA). Assays in the same panel (A, B or C) were performed at the same time, while assays in different panels were performed at different times. Purified CP8 (lanes 1–2, 0.1 and 0.2 μg, respectively), aliquots of naïve plasma #31 (lanes 3–4, 0.5 and 1 μl, respectively), naïve plasma #2 (lane 5, 1 μl) and naïve plasma #38 (lane 6, 1 μl) were also analyzed by SDS-PAGE, and CP8 was identified by immunoblotting using rabbit polyclonal antibody to recombinant CP8 (D). The band intensities of the Western blot in (D) were measured using Kodak Digital Science 1D gel analysis software and normalized to the amount of CP8 (0.2 μg) in lane 2 (E).