Identification and characterization of a "biomarker of toxicity" from the proteome of the paralytic shellfish toxin-producing dinoflagellate Alexandrium tamarense (Dinophyceae).
The objective of this study was to identify and characterize a "biomarker of toxicity" from the proteome of Alexandrium tamarense, a paralytic shellfish toxin (PST)-producing dinoflagellate. A combination of 2-DE and MS approaches was employed to identify proteins of interest in the vegetative cells of several strains of A. tamarense with different toxin compositions and from different geographical locations. The electrophoretic analysis of the total water-soluble proteins from these toxic strains by 2-DE showed that several abundant proteins, namely AT-T1, AT-T2 and AT-T3, differing slightly in apparent Mr and pIs, were consistently present in all toxic strains of A. tamarense. Further analysis by MALDI-TOF MS and N-terminal amino acid sequencing revealed that they are isoforms of the same protein. Even more intriguing is that these proteins in A. tamarense have similar amino acid sequences and are closely related to a "biomarker of toxicity" previously reported in A. minutum. Unambiguous and highly species-specific identification was later achieved by comparing the PMFs of proteins in these two species. An initial attempt to characterize these proteins by generation of murine polyclonal antibodies against the AT-T1 protein was successful. Western blot analysis using the murine AT-T1-polycolonal antibodies identified all the toxic strains of A. tamarense and A. minutum, but not the nontoxic strain of A. tamarense. These results indicate that these protein characteristics for toxic strains are species-specific and that they are stable properties of the tested algae which are clearly distinguishable irrespective of geographical location and toxin composition. To our knowledge, this is the first study to demonstrate the use of polyclonal antibodies against marker proteins purified from 2-DE gels to distinguish different strains and species of the PST-producing dinoflagellate Alexandrium. It provides the basis for the production of monoclonal antibody probes against the "biomarkers of toxicity" for those dinoflagellates whose genome is incompletely characterized. Potentially, immunoassays could be developed to detect the presence of toxic algae in routine monitoring programs as well as to predict bloom development and movement.
Chan
Leo Lai
LL
TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, PR China.
Sit
Wai-Hung
WH
Lam
Paul Kwan-Sing
PK
Hsieh
Dennis Paul Hsientang
DP
Hodgkiss
Ivor John
IJ
Wan
Jennifer Man-Fan
JM
Ho
Alvin Yam-Tat
AY
Choi
Nicola Man-Chi
NM
Wang
Da-Zhi
DZ
Dudgeon
David
D
D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D004141:Dinoflagellida / Q000145:classification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005843:Geography; D007074:Immunoglobulin G / Q000276:immunology; D008387:Marine Toxins / Q000032:analysis* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D020543:Proteome / Q000276:immunology / Q000378:metabolism*; D012758:Shellfish / Q000469:parasitology / Q000633:toxicity*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015415:Biomarkers; D007074:Immunoglobulin G; D008387:Marine Toxins; D020543:Proteome