A Cell Adhesion Protein from the Crayfish Pacifastacus leniusculus, a Serine Proteinase Homologue Similar toDrosophila Masquerade

Abstract

A cDNA encoding a protein resembling masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster, was identified in hemocytes of the adult freshwater crayfish, Pacifastacus leniusculus. The crayfish protein is similar to Drosophila masquerade in the following aspects: (a) overall sequence of the serine proteinase domain, such as the position of three putative disulfide bridges, glycine in the place of the catalytic serine residue, and the presence of a substrate-lining pocket typical for trypsins; (b) the presence of several copies of a disulfide-knotted motif in the putative propeptide. This masquerade-like protein is cleaved into a 27-kDa fragment, which could be detected by immunoblot analysis using an affinity-purified antibody against a synthetic peptide in the C-terminal domain of the protein. The 27-kDa protein could be immunoaffinity-purified from hemocyte lysate supernatant and exhibited cell adhesion activity in vitro, indicating that the C-terminal domain of the crayfish masquerade-like protein mediates cell adhesion.

Characterization of a Pattern Recognition Protein, a Masquerade-Like Protein, in the Freshwater Crayfish Pacifastacus leniusculus

“…All the amino acid sequences could be matched exactly to the previously cloned and characterized crayfish mas-like protein ( Fig. 2A) (17). The N-terminal amino acid sequence of the 65-and 63-kDa proteins had identical amino acid sequences from T102 to A106.…”

“…An antiserum against the crayfish mas-like protein was made by first preparing a synthetic peptide, CFTPQDLRVRWVSGRSTS, corresponding to a portion of the 33-kDa band (the serine proteinase-like domain) of the crayfish mas-like protein as described by Huang et al (17). Briefly, the synthetic peptide was coupled to OVA (Sigma) using sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Calbiochem, La Jolla, CA) as a coupling agent and then was used for production of a rabbit antiserum.…”

“…Interestingly, several serine proteinase homologues are present in animals, and they have a variety of biological functions, such as a cell adhesion activity, e.g., Pacifastacus masquerade (mas)-like protein (17), Drosophila mas (18), glutactin (19), and neurotactin (20); antimicrobial and LPS binding activity, e.g., human azurocidin (21)(22)(23)(24) and horseshoe crab factor D (25); to function as a growth factor, e.g., human hepatocyte growth factor (HGF) (26); a component of proPO system, e.g., 45-kDa serine proteinase homologue protein of coleopteran insect, H. diomphalia (27); and an immune molecule, e.g., mosquito infection-responsive serine protease-like protein (ispl5) (28). These molecules show homology to serine proteinases, except for a substitution(s) within the catalytic triad that will render them without proteinase activity.…”

Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously

“…Through interactions with PAP and proPO, these proteinase-like molecules may anchor proPO activation to the surface of microorganisms. A masquerade-like, clip-domain SPH from the crayfish Pacifastacus leniusculus may serve as an opsonin by associating with microbial cells and host hemocytes (Huang et al, 2000;Lee and Söderhäll, 2001). We have isolated three clip-domain serine proteinases from M. sexta, which cleave proPO at Arg 51 but require the SPHs to generate active PO.…”

Manduca sexta prophenoloxidase activating proteinase-1 (PAP-1) gene: Organization, expression, and regulation by immune and hormonal signals

“…There is at least one regulatory clip domain at the amino terminus of these enzymes. Clip domains, initially identified in the horseshoe crab proclotting enzyme (Muta et al, 1990), are frequently found in arthropod SPs and serine proteinase homologs (SPHs) (Jiang and Kanost, 2000;Huang et al, 2000;Ross et al, 2003;Christophides et al, 2002). We have purified three PAPs from M. sexta prepupae, which cleave proPO at Arg 51 and require two clip-domain SPHs to generate active PO.…”