Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Abstract

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.

Molecular and expression analysis of a family of the Amblyomma americanum tick Lospins

“…The possibility of AS as a source of diversity among Lospins is not unique to ticks in that this phenomenon, first observed in M. sexta (Jiang et al, 1996), has been observed in serpins from C. felis (Brandt et al, 2004), D. melanogaster and C. elegans (Kruger et al, 2002). Experiments are currently underway to investigate if Lospin diversity could be attributed to AS of mutually spliced exons.…”

Differential expansion and evolution of the exon family encoding the Serpin-1 reactive centre loop has resulted in divergent serpin repertoires among the Lepidoptera

“…These evolutionary models also suggest that the tendency to generate Serpin-1 diversity through expansion and alternate splicing of the 9th exons encoding the inhibitor loop was a property of the ancestral Serpin-1 gene. This mechanism to generate serpin diversity may be commonplace among Insecta as it has been described in Diptera (Bruning et al, 2007) and Siphonaptera (Brandt et al, 2004).…”

“…The intermediate class included a highly conserved group of putative chymotrypsin inhibitors related to MsSerpin-1Z and was found in all four lepidopterans suggesting that the function of its cognate protease is also highly conserved. When the 15 serpin RCL variants encoded by the Ctenophalides felis serpin gene (Brandt et al, 2004) were included in the analysis, they clustered separately from the lepidopteran Serpin-1 RCL variants.…”

“…Each of the Serpin-2, -3, -4, -5 and -6 genes encodes a single serpin protein; however, the Serpin-1 gene encodes 12 isoforms that are generated by alternate splicing of exons encoding carboxy termini with different RCLs onto a common serpin scaffold (Jiang et al, 1994). While this phenomenon appears to be commonplace among invertebrates (Kruger et al, 2002;Brandt et al, 2004;Bruning et al, 2007) and the biochemical specificities of many of the M. sexta Serpin-1 variants have been characterized, their respective biological functions remain largely unknown (Jiang and Kanost, 1997). The expression of the Serpin-1 gene has been studied in two lepidopterans, M. sexta (Kanost et al,1995) and the bertha armyworm, Mamestra configurata (Chamankhah et al, 2003).…”

Functional diversification of a protease inhibitor gene in the genus Drosophila and its molecular basis

“…In some animal taxons, including insects (Sasaki, 1991;Jiang et al, 1996;Krüger et al, 2002;Danielli et al, 2003;Brandt et al, 2004), nematodes (Krüger et al, 2002;Pak et al, 2004), and urochordates (Ragg, 2007), a thrifty mechanism allows generation of multiple serpin isoforms from a single gene, each with different protease specificity. Based on alternative splicing, mutually exclusive RSL sequences are chosen from a set of up to 15 RSL cassette exons and hooked individually to a single serpin core.…”