Manduca sexta Serpin-7, a Putative Regulator of Hemolymph Prophenoloxidase Activation.

Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Insect biochemistry and molecular biology (Impact Factor: 3.25). 04/2013; DOI: 10.1016/j.ibmb.2013.03.015
Source: PubMed

ABSTRACT Serpins regulate various physiological reactions in humans and insects, including certain immune responses, primarily through inhibition of serine proteases. Six serpins have previously been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a full-length cDNA sequence of another Manduca serpin, named serpin-7. The open reading frame of serpin-7 encodes a polypeptide of 400 amino acid residues with a predicted signal peptide of the first 15 residues. Multiple protein sequence alignment of the reactive center loop region of the M. sexta serpins indicated that serpin-7 contains Arg-Ile at the position of the predicted scissile bond cleaved by protease in the serpin inhibition mechanism. The same residues occur in the scissile bond of the reactive center loop in M sexta serpin-4 and serpin-5, which are protease inhibitors that can block prophenoloxidase activation in plasma. Serpin-7 transcript was detected in hemocytes and fat body, and its expression increased in fat body after injection of larvae with Micrococcus luteus. Recombinant serpin-7 added to larval plasma inhibited spontaneous melanization and decreased prophenoloxidase activation stimulated by bacteria. Serpin-7 inhibited prophenoloxidase-activating protease-3 (PAP3), forming a stable serpin-protease complex. Considering that serpin-3 and serpin-6 are also efficient inhibitors of PAP3, it appears that multiple serpins present in plasma may have redundant or overlapping functions. We conclude that serpin-7 has serine protease inhibitory activity and is likely involved in regulation of proPO activation or other protease-mediated aspects of innate immunity in M. sexta.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Serine protease inhibitors (serpins) are widely known to its inhibitory role on proteases involved in the immune responses. Herein, a novel serine protease inhibitor (Lvserpin7), encoding for 411 amino acids with calculated molecular mass of 46.29 kDa and isoelectric point of 6.98 was characterized from the Pacific white shrimp Litopenaeus vannamei. Lvserpin7 shared 92.9% identities to Penaeus monodon serpin7. Among the tested tissues, Lvserpin7 was mainly expressed in hemocytes and gill. The expression profiles analysis indicated that Lvserpin7 was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Fusion protein expression was induced by IPTG, and the purified recombinant Lvserpin7 protein (rLvserpin7) binds to both the Gram-positive and Gram-negative bacteria. Also rLvserpin7 exhibited inhibitory activity against the proteases secreted by Bacillus subtilis. Moreover, rLvserpin7 showed inhibition role on prophenoloxidase activation. To recap, we proposed that Lvserpin7 was implicated in the shrimp immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Fish &amp Shellfish Immunology 11/2014; 42(2). DOI:10.1016/j.fsi.2014.11.001 · 2.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P < 0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Fish &amp Shellfish Immunology 10/2014; 42(1):79-87. DOI:10.1016/j.fsi.2014.10.028 · 2.96 Impact Factor
  • Source