Molecular Cloning and Functional Studies of Two Kazal-Type Serine Protease Inhibitors Specifically Expressed by Nasonia vitripennis Venom Apparatus

Abstract

Two cDNA sequences of Kazal-type serine protease inhibitors (KSPIs) in Nasonia vitripennis, NvKSPI-1 and NvKSPI-2, were characterized and their open reading frames (ORFs) were 198 and 264 bp, respectively. Both NvKSPI-1 and NvKSPI-2 contained a typical Kazal-type domain. Real-time quantitative PCR (RT-qPCR) results revealed that NvKSPI-1 and NvKSPI-2 mRNAs were mostly detected specifically in the venom apparatus, while they were expressed at lower levels in the ovary and much lower levels in other tissues tested. In the venom apparatus, both NvKSPI-1 and NvKSPI-2 transcripts were highly expressed on the fourth day post eclosion and then declined gradually. The NvKSPI-1 and NvKSPI-2 genes were recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvKSPI-1 and GST-NvKSPI-2 to three serine protease inhibitors (trypsin, chymotrypsin, and proteinase K) were tested and results showed that only NvKSPI-1 could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvKSPI-1 and GST-NvKSPI-2 on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by both recombinant proteins; however, there was no significant inhibition on the PO activity. Our results suggested that NvKSPI-1 and NvKSPI-2 could inhibit PPO activation in host hemolymph and trypsin activity in vitro.

Keywords: Kazal-type; Nasonia vitripennis; humoral immunity; serine protease inhibitors.

Figure 1

Nucleotide and deduced amino sequences of NvKSPI-1 (A) and NvKSPI-2 (B) cDNAs from N. vitripennis. Signal peptides are underlined; the initiator codon “ATG” and terminator codon “TAA” are bolded and highlighted.

Figure 2

Sequence analysis of NvKSPI-1 and NvKSPI-2. (A) Multiple sequences alignment and amino acid identity analysis of Kazal domains (KDs) between NvKSPI-1, NvKSPI-2, and other KSPIs. (B) Predicted tertiary structure of NvKSPI-1 and NvKSPI-2 KDs by phyre² on line. Yellow spheres represent cysteines forming three intra-domain disulfide bridges between cysteine numbers 1–5, 2–4, and 3–6. The corresponding species of abbreviations and their GenBank accession numbers are as follows: PMKD-1,2,3,4,5,6,7: Panstrongylus megistuss (ADF97836); SCKD: Stomoxys calcitrans (AAY98015); GMKD: Glossina morsitans (AFG28187); NCKD: Neospora caninum (AAM29188); BMKD: Bombyx mori (NP_001037047); DPKD-1,2,3: Danaus plexippus (EHJ76238); NVKVD1 and NVKVD2: Nasonia vitripennis (NM_001161523 and NM_001170879).

Figure 2

Sequence analysis of NvKSPI-1 and NvKSPI-2. (A) Multiple sequences alignment and amino acid identity analysis of Kazal domains (KDs) between NvKSPI-1, NvKSPI-2, and other KSPIs. (B) Predicted tertiary structure of NvKSPI-1 and NvKSPI-2 KDs by phyre² on line. Yellow spheres represent cysteines forming three intra-domain disulfide bridges between cysteine numbers 1–5, 2–4, and 3–6. The corresponding species of abbreviations and their GenBank accession numbers are as follows: PMKD-1,2,3,4,5,6,7: Panstrongylus megistuss (ADF97836); SCKD: Stomoxys calcitrans (AAY98015); GMKD: Glossina morsitans (AFG28187); NCKD: Neospora caninum (AAM29188); BMKD: Bombyx mori (NP_001037047); DPKD-1,2,3: Danaus plexippus (EHJ76238); NVKVD1 and NVKVD2: Nasonia vitripennis (NM_001161523 and NM_001170879).

Figure 3

Phylogenetic analysis of NvKSPI-1, NvKSPI-2, and other KSPIs’ amino acid sequences based on the neighbor-joining method. The origin of amino acid sequences and their GenBank accession numbers are as follows: FcKSPI: Fenneropenaeus chinensis (ABC33915); LvKSPI: Litopenaeus vannamei (AAT09421); BmKSPI: Bombyx mori (NP_001037047); NcKSPI: Neospora caninum (AAM29188); GmKSPI: Glossina morsitans (AFG28187); ScKSPI: Stomoxys calcitrans (AAY98015); PmKSPI: Panstrongylus megistus (ADF97836); HsKSPI: Homo sapiens (NP_115955); DpKSPI: Danaus plexippus (EHJ76238); CppKSPI: Culex pipiens pallens (AFN41343); LlKSPI: Lutzomyia longipalpis (ABV60319); PpKSPI: Phlebotomus papatasi (ABV44739); NvKSPI-1 and NvKSPI-2: Nasonia vitripennis (NM_001161523 and NM_001170879).

Figure 4

SDS-PAGE analysis of NvKSPI-1 (A) and NvKSPI-2 (B) recombinant proteins. M: Protein molecular weight marker; 1: Not induced by IPTG; 2: Precipitation after IPTG induction. 3: Supernatant after IPTG induction; 4: Purified proteins.

Figure 5

Tissue distribution of transcript levels of NvKSPI-1 (A) and NvKSPI-2 (B) in N. vitripennis. Abdomen carcass represents the rest of abdomen post dissection. Venom apparatus contains venom reservoir and gland, and venom released. All values in the figure are represented as mean ± standard deviation. Bars labeled with different letters are significantly different (one-way ANOVA followed by LSD test, p < 0.05).

Figure 6

Developmental stages of transcript levels of NvKSPI-1 (A) and NvKSPI-2 (B) in venom apparatus of N. vitripennis. Female adults were sampled on days 0 to 7 post eclosion. All values are presented as mean ± standard deviation. Bars labeled with different letters are significantly different (one-way ANOVA followed by LSD test, p < 0.05).

Figure 7

Effects of recombinant NvKSPI-1 and NvKSPI-2 on the activity of serine proteases. GST: GST-tag protein expressed by pGEX-4T-2; BSA and Buffer: treated by BSA and reaction buffer respectively; NvKSPI-1 and NvKSPI-2: treated by recombination proteins respectively. All values in the figure are represented as mean ± standard deviation. Bars labeled with different letters are significantly different (one-way ANOVA followed by LSD test, p < 0.05).

Figure 8

Effects of recombinant NvKSPI-1 and NvKSPI-2 on the PPO activation (A) and PO activity (B) of hemolymph of M. domestica pupae. (A) Screened hemolymph was incubated with TBS alone, or TBS/M. luteus, NvKSPI-1/M. luteus, NvKSPI-2/M. luteus, BSA/M. luteus, GST/M. luteus, PTU/M. luteus in TBS for 20 min at 25 °C. PPO activation was assayed using l-dopamine as a substrate, as described in Materials and Methods. (B) Screened hemolymph was incubated with M. luteus in TBS for 10 min at 25 °C, and then incubated with TBS, NvKSPI-1, NvKSPI-2, BSA, GST, or PTU for 10 min at 25 °C. PO activity was assayed using l-dopamine as a substrate, as described in Materials and Methods. All values in the figure are represented as mean ± standard deviation. Bars labeled with different letters are significantly different (one-way ANOVA followed by LSD test, p < 0.05).