Chan Leo Lai, Sit Wai-Hung, Lam Paul Kwan-Sing, Hsieh Dennis Paul Hsientang, Hodgkiss Ivor John, Wan Jennifer Man-Fan, Ho Alvin Yam-Tat, Choi Nicola Man-Chi, Wang Da-Zhi, Dudgeon David
TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin, PR China.
Proteomics. 2006 Jan;6(2):654-66. doi: 10.1002/pmic.200401350.
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.
本研究的目的是从塔玛亚历山大藻（一种产生麻痹性贝类毒素的甲藻）的蛋白质组中鉴定并表征一种“毒性生物标志物”。采用二维电泳（2-DE）和质谱（MS）相结合的方法，在几种毒素组成不同、来自不同地理位置的塔玛亚历山大藻菌株的营养细胞中鉴定感兴趣的蛋白质。通过二维电泳对这些有毒菌株的总水溶性蛋白质进行电泳分析，结果表明，几种丰度较高的蛋白质，即AT-T1、AT-T2和AT-T3，其表观分子量和等电点略有不同，在所有塔玛亚历山大藻有毒菌株中均一致存在。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和N端氨基酸测序进一步分析表明，它们是同一蛋白质的同工型。更有趣的是，塔玛亚历山大藻中的这些蛋白质具有相似的氨基酸序列，并且与先前在微小亚历山大藻中报道的一种“毒性生物标志物”密切相关。通过比较这两个物种中蛋白质的肽质量指纹图谱（PMF），后来实现了明确且高度物种特异性的鉴定。通过制备针对AT-T1蛋白的鼠多克隆抗体对这些蛋白质进行表征的初步尝试取得了成功。使用鼠源AT-T1多克隆抗体进行的蛋白质印迹分析鉴定出了所有塔玛亚历山大藻和微小亚历山大藻的有毒菌株，但未鉴定出塔玛亚历山大藻的无毒菌株。这些结果表明，这些有毒菌株的蛋白质特征具有物种特异性，并且它们是受试藻类的稳定特性，无论地理位置和毒素组成如何都能清晰区分。据我们所知，这是第一项证明使用针对从二维电泳凝胶中纯化的标记蛋白的多克隆抗体来区分产生麻痹性贝类毒素的甲藻亚历山大藻不同菌株和物种的研究。它为针对那些基因组特征不完全明确的甲藻，生产针对“毒性生物标志物”的单克隆抗体探针提供了基础。潜在地，可以开发免疫测定法来在常规监测计划中检测有毒藻类的存在，以及预测藻华的发展和移动。
