8.B.13 The Kunitz-type Serine Protease Inhibitor (HAI) Family

Peptides have been isolated from several species of sea anemones and shown to block currents through various potassium ion channels, particularly in excitable cells. The toxins can be grouped into four structural classes: type 1 with 35-37 amino acid residues and three disulphide bridges; type 2 with 58-59 residues and three disulphide bridges; type 3 with 41-42 residues and three disulphide bridges; and type 4 with 28 residues and two disulphide bridges (Castañeda & Harvey, 2009). Examples from the first class are BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS (or kaliseptine) from Anemonia sulcata (now A. viridis). These interfere with binding of radiolabelled dendrotoxin to synaptosomal membranes and block currents through channels with various Kv1 subunits and also intermediate conductance K(Ca) channels. Toxins in the second class are homologous to Kunitz-type inhibitors of serine proteases; these toxins include kalicludines (AsKC 1-3) from A. sulcata and SHTXIII from S. haddoni; they block Kv1.2 channels. The third structural group (8.B.11) includes proteins from A. sulcata and Anthropleura elegantissima. Their pharmacological specificities differs: BDS-I and -II toxins block currents involving Kv3 subunits while APETx1 blocks ERG channels. The fourth group comprises the SHTX I and II toxins from S. haddoni. Their channel blocking specificity is not yet known, but they displace dendrotoxin binding from synaptosomal membranes (Castañeda & Harvey, 2009).  These small toxins (e.g., 8.B.13.1.1) are homologous throughout their lengths to a central part of amyloid-β protein precursors such as the one under TC# 1.C.50.1.2.

The integral membrane, Kunitz-type serine protease inhibitors HAI-1 and HAI-2, can suppress the proteolytic activities of the type 2 transmembrane serine protease, matriptase (TC# 8.A,131,1,4), with high specificity and potency (Chiu et al. 2022). High levels of extracellular matriptase proteolytic activity have been observed in some neoplastic B-cells with high levels of endogenous HAI-2, indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level. Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells, which naturally express matriptase with very low levels of HAI-2 and no HAI-1, nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme. With increasing HAI-1 expression, cellular matriptase-HAI-1 complex increased, and extracellular active matriptase decreased proportionally. Increasing HAI-2 expression resulted in cellular matriptase-HAI-2 complex levels reaching a plateau, while extracellular active matriptase remained high. In contrast to this differential effect, both HAI-1 and HAI-2, even at very low levels, were shown to promote the expression and cell-surface translocation of endogenous matriptase. The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2. The HAIs, therefore, resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity (Chiu et al. 2022).