Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (α2-M) from the haemocytes of tiger shrimp Penaeus monodon

Abstract

An alpha 2-macroglobulin (α2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the α2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5′-untranslated region, and a 327 bp 3′-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated p I of 5.30. The P. monodon α2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate α2-Ms. Sequence comparison showed that α2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species α2-M showed that the overall structure is evolutionarily conserved and phylogentic analysis revealed that P. monodon α2-M is closely related to other arthropod α2-M, and displays the highest similarity to M. japonicus α2-M. The α2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that α2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.