Isolation and characterization of SAP and CRP, two pentraxins from Pangasianodon (Pangasius) hypophthalmus

Giang Duong Thi Huong, Edilbert Van Driessche, Isabel Vandenberghe, Bart Devreese, Sonia Beeckmans

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine-Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 µg SAP and 56 µg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1mM) and by phosphorylcholine (MIC = 1-2mM), respectively. Circular dichroism studies revealed that antiparallel beta-pleated sheets are dominating the secondary structure. Upon removing the Ca2+ ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.
Original languageEnglish
Pages (from-to)743-753
Number of pages11
JournalFish Shellfish Immunology
Volume28
Publication statusPublished - May 2010

Keywords

  • Pangasianodon (Pangasius) hypophthalmus
  • Fish
  • Serum amyloid P component (SAP)
  • C-reactive protein (CRP)
  • Pentraxin
  • Affinity chromatography purification
  • Haemagglutination
  • Aeromonas hydrophila agglutination
  • Edwardsiella ictaluri agglutination
  • Circular dichroism

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