Identification and characterization of a biomarker of toxicity from the proteome of the paralytic sh

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Identification and characterization of a
biomarker of toxicity from the proteome of the
paralytic shellfish toxin-producing
dinoflagellate Alexandrium tamarense
(Dinophyceae)

• Leo Lai Chan, Wai-Hung Sit, Paul Kwan-sing Lam,
  Dennis Paul Hsientang Hsieh, Ivor John Hodgkiss,
  Jennifer Man-Fan Wan, Alvin Yam-Tat Ho, Nicola
  Man-Chi Choi, Da-Zhi Wang, and David Dudgeon
• Proteomics 2006, 6, 654-666

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BACKGROUND

• Paralytic shellfish toxin illness caused by
  ingesting mollusks containing microalgae which
  produces saxitoxin.
• Filter-feeding mollusks concentrate toxins
  thereby becoming harmful to mammals.
• Symptoms can include tingling of lips and
  tongue, dizziness, nausea and diarrhea,
  respiratory distress, paralysis of the chest and
  abdomen.
• Death can result in 2-12 hours if left untreated.

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SAXITOXIN DERIVATIVES

• STX
• Neosaxitoxin
• Gonyautoxins 2 and 3 (GTX 2, 3)
• Gonyautoxins 1 and 4 (GTX 1, 4)
• Decarbamoyl saxitoxin (dcSTX)
• B-1 (GTX 5)
• C-1 and C-2
• C-3 and C-4
• B-2 (GTX 6)

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MECHANISM OF ACTION

• All saxitoxin derivatives are responsible for
  paralytic shellfish poisoning and respiratory
  paralysis.
• They selectively block sodium ion channels in
  nerves and muscles.

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CURRENT PROBLEMS IN MONITORING FOR PSTs

• Identification and monitoring of toxic microalgae
  is difficult due to morphologically similar
  species.
• Species can exist in toxic and non-toxic
  varieties, and they can co-occur in the same
  population.

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CURRENT MONITORING METHODS

• ribosomal RNA sequences - molecular probe
• FITC-conjugated lectins - cellular probe
• Immunofluorescence - to distinguish toxic and
  non-toxic forms
• HPLC - PST quantification in dinoflagellates

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END GOAL

• Identification and characterization of
  biomarkers of toxicity - those proteins
  specific for the toxin producing species of
  Alexandrium tamarense

http//www.chm.bris.ac.uk/motm/stx/saxi.htm
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EXPERIMENTAL METHODS

• HPLC with fluorescense detection
• 2-dimensional gel electrophoresis
• MALDI-TOF MS
• N-terminal amino acid sequencing by Edman
  degradation
• Western blot analyses

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STRAINS STUDIED

• A. tamarense AT-CI01, Daya Bay, China
• A. tamarense AT-HK9301, Dapeng Bay, China
• A. tamarense AT-Polar, Southern Ocean
• A. tamarense AT-WHOI, USA
• A. tamarense AT-HKJB, Junk Bay, China
• A. minutum AM-TK4, Tung Kang, Taiwan
• A. minutum AM-KS2, Kaohsiung, Taiwan

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HPLC PROFILE OF MAJOR TOXINS ACCORDING TO SPECIES
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• Based on HPLC profiles the species were grouped
  into three classes
• C1-C4 toxin dominated - AT-HK9301
• C1-C2 toxin dominated - AT-CI01, AT-Polar,
  AT-WHOI (C2 gt 90)
• Nontoxic - AT-HKJB
• AM strains were GTX toxin dominated.

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2-D ANALYSIS

• Protein extracts from toxic strains of A.
  tamarense were compared to the non-toxic strain,
  AT-HKJB, in order to identify the expression
  pattern of marker proteins.
• AT-T1 and AT-T2 were found in all toxic strains
  of A. tamarense but not in the non-toxic strain.
• AT-T3 was only detected in AT-CI01, AT-Polar, and
  AT-WHOI strains.

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A. tamarense AT-HK9301 C1-C4 toxic profile AT-T1
and AT-T2 only
A. minutum AM-TK4 AM-T1
A. tamarense AT-HKJB Non-toxic strain
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MALDI-TOF MS

• Tryptic digestion was performed to 2-D gel spots
  for AT-T1, AT-T2, and AT-T3 of the AT-Polar
  strain.
• These were also compared to the tryptic digest
  from AM-T1.

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MALDI-TOF MS

• AT-T1, AT-T2, and AT-T3 were found to have
  identical PMFs (peptide mass fingerprints).
• The shared mass peaks were
• 1200.70
• 1439.78
• 1507.75
• 2615.50

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N-TERMINAL SEQUENCING

• N-terminal sequencing suggests that AT-T1, AT-T2,
  AT-T3, and AM-T1 are isoforms of the same protein
  but their PMFs are species specific.
• They have the same first 30 N-terminal amino acid
  sequence.
• Database searches revealed that these were novel
  proteins.

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N-TERMINAL SEQUENCING CONTD

• The authors speculate that the three proteins are
  derived from expression of the same gene, but
  that they have been proteolytically processed or
  post-translationally modified differentially.
• The proteins had no polymorphic variations and
  were conserved among all analyzed strains.

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WESTERN BLOT ANALYSIS

• murine anti-AT-T1 polyclonal antibody was made by
  excising AT-T1 from a gel spot, emulsifying in
  Freunds complete adjuvant, and injecting into
  BALB/c mice.
• Boosters were administered 7 days prior to serum
  collection.
• Specificity was confirmed by western blot of 2-D
  gel.

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WESTERN BLOT ANALYSIS

• anti-AT-T1 serum recognized all isoforms of AT-T1
  (AT-T1, AT-T2, AT-T3, and AM-T1).
• PCBP was found in all strains of Alexandrium.

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CONCLUSIONS

• A highly conserved biomarker of toxicity for
  the toxin-producing species of A. tamarenses was
  found.
• Eventhough AT-T1, AT-T2, AT-T3, and AM-T1 share
  the same 30 a.a. N-terminal sequence, they have
  different PMFs as seen by MALDI-TOF MS.
• The authors suggest a lower throughput method,
  such as ESI-MS/MS, since few proteins and little
  genomic data are known about dinoflagellates.

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REFERENCES

• Chan, L.L., W. Sit, P.K. Lam, D.P.H. Hsieh, I.J.
  Hodgkiss, J.M. Wan, A.Y. Ho, N.M. Choi, D. Wang,
  and D. Dudgeon. Identification and
  characterization of a biomarker of toxicity
  from the proteome of the paralytic shellfish
  toxin-producing dinolflagellate Alexandrium
  tamarense (Dinophyceae). Proteomics 2006,
  6654-666.
• Molecule of the Month SAXITOXIN
• Neil Edwards, University of Sussex at Brighton
• http//www.chm.bris.ac.uk/motm/stx/saxi.htm