Cloning and characterization of hemolymph clottable proteins of kuruma prawn (Marsupenaeus japonicus) and white shrimp (Litopenaeus vannamei)

Winton Cheng, Inn H. Tsai, Chang J. Huang, Pei C. Chiang, Chia Hsiung Cheng, Maw Sheng Yeh

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

The hemolymph clottable protein (CP) of Marsupenaeus japonica (designated as Mj-CP) was purified by a DEAE anion-exchanger and a Sepharose CL-6B gel filtration column. In the presence of Ca2+, it formed stable clots in vitro upon the addition of the hemocytes lysate containing transglutaminase. Results of gel filtration chromatography and SDS-PAGE indicated that Mj-CP mainly existed as disulfide-linked homodimers of 390 kDa. Specific primers were designed; PCR as well as RACE help to clone and sequence Mj-CP cDNA of 5660 bp. The predicted CP-precursor contains a signal peptide followed by a subunit of 1671 amino acids (isoelectric point 5.6), including two RGD motifs and three potential N-glycosylation sites. Mj-CP is structurally 80% and 38% identical to the CPs of tiger shrimp and crayfish, respectively. Likewise, CP cDNA of white shrimp (Litopenaeus vannamei) was also cloned and sequenced; the predicted CP has 1666 amino acid residues and an isoelectric point of 5.2. Both CPs bear potential transglutaminase cross-linking sites, i.e. seven Ser-Lys-Thr repeats near the N-terminus, a Ser- and Gln-rich region in the middle, and polyGln (n=8-11) near the C-terminus. Phylogenetic analyses of crustacean CPs and vitellogenins revealed divergent evolution of the two protein families. By RT-PCR, the sub-cuticular epidermis was identified as one of the major tissues that express CP in M. japonica.

Original languageEnglish
Pages (from-to)265-274
Number of pages10
JournalDevelopmental and Comparative Immunology
Volume32
Issue number3
DOIs
Publication statusPublished - 2008
Externally publishedYes

Fingerprint

Hemolymph
Organism Cloning
Proteins
Transglutaminases
Isoelectric Point
Gel Chromatography
Complementary DNA
Tigers
Vitellogenins
Amino Acids
Hemocytes
Polymerase Chain Reaction
Astacoidea
Protein Precursors
Protein Sorting Signals
Glycosylation
Epidermis
Disulfides
Anions
Polyacrylamide Gel Electrophoresis

Keywords

  • cDNA sequences
  • Clottable protein
  • Hemolymph coagulation
  • Shrimps (Marsupenaeus japonicus and Litopenaeus vannamei)
  • Tissue expression profile

ASJC Scopus subject areas

  • Developmental Biology
  • Immunology

Cite this

Cloning and characterization of hemolymph clottable proteins of kuruma prawn (Marsupenaeus japonicus) and white shrimp (Litopenaeus vannamei). / Cheng, Winton; Tsai, Inn H.; Huang, Chang J.; Chiang, Pei C.; Cheng, Chia Hsiung; Yeh, Maw Sheng.

In: Developmental and Comparative Immunology, Vol. 32, No. 3, 2008, p. 265-274.

Research output: Contribution to journalArticle

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abstract = "The hemolymph clottable protein (CP) of Marsupenaeus japonica (designated as Mj-CP) was purified by a DEAE anion-exchanger and a Sepharose CL-6B gel filtration column. In the presence of Ca2+, it formed stable clots in vitro upon the addition of the hemocytes lysate containing transglutaminase. Results of gel filtration chromatography and SDS-PAGE indicated that Mj-CP mainly existed as disulfide-linked homodimers of 390 kDa. Specific primers were designed; PCR as well as RACE help to clone and sequence Mj-CP cDNA of 5660 bp. The predicted CP-precursor contains a signal peptide followed by a subunit of 1671 amino acids (isoelectric point 5.6), including two RGD motifs and three potential N-glycosylation sites. Mj-CP is structurally 80{\%} and 38{\%} identical to the CPs of tiger shrimp and crayfish, respectively. Likewise, CP cDNA of white shrimp (Litopenaeus vannamei) was also cloned and sequenced; the predicted CP has 1666 amino acid residues and an isoelectric point of 5.2. Both CPs bear potential transglutaminase cross-linking sites, i.e. seven Ser-Lys-Thr repeats near the N-terminus, a Ser- and Gln-rich region in the middle, and polyGln (n=8-11) near the C-terminus. Phylogenetic analyses of crustacean CPs and vitellogenins revealed divergent evolution of the two protein families. By RT-PCR, the sub-cuticular epidermis was identified as one of the major tissues that express CP in M. japonica.",
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AU - Cheng, Winton

AU - Tsai, Inn H.

AU - Huang, Chang J.

AU - Chiang, Pei C.

AU - Cheng, Chia Hsiung

AU - Yeh, Maw Sheng

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KW - Tissue expression profile

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