Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid

Abstract

Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (β subunits) from several vertebrates (47-52% and 50-60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa.

Molecular physiology of digestion in arachnida: functional and comparative-evolutionary approaches.

“…The opisthosomal midgut with their midgut glands will be referred in this work as MMG (Figure 1.3B) as a matter of simplifying, unless when previously specified. (51,52). In our group we identified two metallo peptidases from the astacin family acting at basic pHs (Fuzita et al, unpublished) and at least two cysteine peptidases with acidic optimum pH in the digestive system of the spider Nephilengys cruentata (Chapter 4).…”

“…Serine peptidase inhibitors in the spider DJ were previously reported in the literature (52,142 Kunitz-like and one alpha-1 inhibitor3 (Table 5.4). In our group, serine endopeptidase inhibitors were also identified by biochemical assays (Lopes et al, unpublished) which as the ones from Argiope aurantia are resistant to boiling (52).…”

“…In our group, serine endopeptidase inhibitors were also identified by biochemical assays (Lopes et al, unpublished) which as the ones from Argiope aurantia are resistant to boiling (52). In a pooled .…”

“…Astacins 2 and 5a sequences are also incomplete. Nevertheless, these enzymes are normally active on alkaline pH and were found as secreted enzymes in the digestive juice of the spiders Argiope aurantia (52) and Nephilengys cruentata (chapter 5). Thereby Tityus serrulatus astacins will be considered as secreted enzymes.…”

“…Collagenase is a metallopeptidase from M10A subfamily and such type of protein was identified in the present work only at the mRNA level (data not shown). In fact, the spider digestive enzymes named collagenase in literature was merely a hydrolase which activity was tested using collagen as substrate without further characterization (50)(51)(52). Kavanagh and Tillinghast (49) and 18 kDa (respectively p16 and p18) had their amino-terminal sequences determined and an internal fragment of p16 was also obtained.…”