Cloning, expression and characterization of four serpin-1 cDNA variants from the spruce budworm, Choristoneura fumiferana

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2009-10, Vol.154 (2), p.165-173
• Main Authors: Zheng, Y.-P., He, W.-Y., Béliveau, C., Nisole, A., Stewart, D., Zheng, S.-C., Doucet, D., Cusson, M., Feng, Q.-L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Blood Coagulation; Choristoneura fumiferana; Cloning, Molecular; Cuticle; Degradation; DNA, Complementary - genetics; Gene Expression Profiling; Genes, Insect - genetics; Immunity, Innate; Lepidoptera - enzymology; Lepidoptera - genetics; Lepidoptera - growth & development; Lepidoptera - immunology; Melanins - metabolism; Metamorphosis; Molecular Sequence Data; Molt; Molting; Mutation; Pancreatic Elastase - metabolism; Peptide Hydrolases - metabolism; Phylogeny; Protein Isoforms - biosynthesis; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment; Serpin; Serpins - biosynthesis; Serpins - chemistry; Serpins - genetics; Serpins - metabolism
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2009.05.016

Four cDNAs ( Cfserpin-1a, Cfserpin-1b, Cfserpin-1c and Cfserpin-1d) of the Choristoneura fumiferana serpin-1 gene were cloned from an epidermis cDNA library. Analysis of the deduced amino acid sequences indicated that the cloned cDNAs encode four different proteins displaying identical N- but distinct C-termini, the latter region containing the inhibitory loop. The entire CfSerpin-1 gene is transcribed while the variants are generated. Antibodies generated against the purified recombinant serpins cross-reacted with the other three. Each of the four Cfserpin-1 cDNA variants was transcribed throughout larval development, from the 4th to the 6th instar, but transcript levels during the intermolt phases were generally higher than during the molting phase. The epidermis and fat body had higher levels of Cfserpin-1 transcripts than the midgut. Cfserpin-1 proteins, detected with the Cfserpin-1a antibody, were found in the epidermis, midgut, fat body, plasma and molting fluid of 6th instar larvae and pre-pupae. Prepupal and pupal insects had higher levels of the proteins than the 6th instar feeding larvae, despite a drop in transcript levels. Cfserpin-1a could bind with the serine proteinase elastase and form a complex in vitro. We hypothesize that the cloned serpins could be involved in the regulation of cuticle degradation during the insect molting cycle.