Molecular cloning and characterisation of a thioester-containing α2-macroglobulin (α2-M) from the haemocytes of mud crab Scylla serrata

Abstract

Molecular approaches were used to clone thioester-containing α2-macroglobulin (α2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of α2-M was determined 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 clone consists of 5491 bp with an open reading frame (ORF) of 4986 bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2 kDa with an estimated pI of 8.41. The S. serrata 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 S. serrata has an overall similarity of 68% and 48% to that of kuruma shrimp Marsupenaeus japonicus and American horseshoe crab Limulus polyphemus, respectively. Phylogentic analysis revealed that S. serrata 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. Quantitative real-time RT-PCR analysis showed that α2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24 h- and 48 h-post lipopolysaccharide (LPS) injection.