AvKTI is <i>O</i>-glycosylated and inhibits trypsin and chymotrypsin.

<p>(A) SDS-PAGE (left) and western blot analysis (right) of purified recombinant AvKTI expressed in baculovirus-infected Sf9 insect cells. Recombinant AvKTI was identified using a His-tag antibody. (B) Glycoprotein staining of AvKTI. Purified AvKTI and control protein samples were subjected to 12% SDS-PAGE (left) and then analyzed by glycoprotein staining (right). Horseradish peroxidase (5 µg), a glycosylated protein, was used as a positive control. Soybean trypsin inhibitor (5 µg), a non-glycosylated protein, was used as a negative control. (C) Enzyme inhibition by AvKTI. Trypsin or chymotrypsin was incubated with increasing amounts of AvKTI, and the residual enzyme activity was then determined (<i>n</i> = 3).</p

AvKTI inhibits plasmin and elastase, but not factor Xa, thrombin, or tPA.

<p>(A) Inhibitory activity of AvKTI against several enzymes associated with the hemostatic system. Factor Xa, thrombin, or tPA was incubated with increasing amounts of AvKTI, and the residual enzyme activity was determined (<i>n</i> = 3). (B, C) The inhibitory activities of AvKTI against plasmin (B) and neutrophil elastase (C). Plasmin or neutrophil elastase was incubated with increasing amounts of AvKTI, and the residual enzyme activity was determined (<i>n</i> = 3).</p

AvKTI is a Kunitz-type serine protease inhibitor.

<p>(A) The nucleotide and deduced amino acid sequences of <i>AvKTI</i> cDNA (GenBank accession no. JX844659). The start codon (ATG) is boxed, and the termination codon is indicated with an asterisk. The putative polyadenylation signal is underlined. The predicted signal sequence, a pro-peptide, and the mature peptide are indicated. The characteristic cysteine residues are indicated by squares. The P1 position is marked with a circle. (B) The alignment of the amino acid sequences for mature AvKTI with other known Kunitz-type serine protease inhibitors. The characteristic cysteine residues are shown in bold. The P1 position is marked with an asterisk. The sources of the aligned sequences were <i>A. ventricosus</i> (this study, GenBank accession no. JX844659), <i>Sarcophaga bullata</i> SBPI (P26228), <i>Bombyx mori</i> BmSPI1 (NP_001037044), <i>Anemonia sulcata</i> AsKC1 (Q9TWG0), <i>Haematobia irritans irritans</i> HiTI (AAL87009), <i>Anthopleura aff. xanthogrammica</i> AXPI-I (P81547), <i>Pseudonaja textilis textilis</i> Txln-1 (Q90WA1), <i>Hadrurus gertschi</i> Hg1 (P0C8W3), <i>Bos taurus</i> BPTI (P00974), <i>Pseudonaja textilis textilis</i> Txln-4 (Q90W98), <i>Haplopelma schmidti</i> HWTX-XI (P68425), and <i>Bombus ignitus</i> Bi-KTI (AEM68408). The AvKTI sequence was used as a reference for the identity/similarity (Id/Si) values. (C) Expression of <i>AvKTI</i> in <i>A. ventricosus</i>. Total RNA was isolated from the epidermis, fat body, silk gland, and venom gland of <i>A. ventricosus</i>. RNA was separated by 1.2% formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane, and hybridized with radiolabeled <i>AvKTI</i> cDNA (lower panel). <i>AvKTI</i> transcripts are indicated with an arrow. The ethidium bromide-stained RNA gel shows uniform loading (upper panel).</p

AvKTI forms complexes with plasmin.

<p>Western blot analysis of the formation of plasmin-AvKTI complex via native gel electrophoresis was performed. Three micrograms of plasmin were incubated with 1 µg of AvKTI, and the samples (plasmin, AvKTI, or plasmin-AvKTI mixture) were resolved on a 10% polyacrylamide gel. After electrophoresis, the protein samples were incubated with antiserum against plasmin or His-tag. The plasmin, AvKTI, and plasmin-AvKTI complex are indicated with arrows.</p

The inhibitory activities of AvKTI against serine proteases.

a<p>The concentration of enzyme used in this experiment.</p>b<p>The molar ratio of IC₅₀ to the enzyme concentration.</p