Molecular cloning and characterisation of a thioester-containing alpha2-macroglobulin (alpha2-M) from the haemocytes of mud crab Scylla serrata.
Molecular approaches were used to clone thioester-containing alpha2-macroglobulin (alpha2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of alpha2-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 alpha2-M cDNA clone consists of 5491bp with an open reading frame (ORF) of 4986bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2kDa with an estimated pI of 8.41. The S. serrata alpha2-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 alpha2-Ms. Sequence comparison showed that alpha2-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 alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24h- and 48h-post lipopolysaccharide (LPS) injection.
Vaseeharan
Baskaralingam
B
Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan.
Lin
Yong-Chin
YC
Ko
Chi-Fong
CF
Chiou
Tzu-Ting
TT
Chen
Jiann-Chu
JC
D000199:Actins / Q000032:analysis / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression / Q000276:immunology*; D006434:Hemocytes / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000276:immunology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D016415:Sequence Alignment; D000511:alpha-Macroglobulins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D000511:alpha-Macroglobulins