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用烟酰胺腺嘌呤二核苷酸对百日咳毒素S-1亚基的Glu-129进行光标记。

Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD.

作者信息

Barbieri J T, Mende-Mueller L M, Rappuoli R, Collier R J

机构信息

Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.

出版信息

Infect Immun. 1989 Nov;57(11):3549-54. doi: 10.1128/iai.57.11.3549-3554.1989.

DOI:10.1128/iai.57.11.3549-3554.1989
PMID:2807535
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC259866/
Abstract

UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from [3H-nicotinamide]NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from [32P-adenylate]NAD (0.2 mol/mol of protein). Label from [3H-nicotinamide]NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with [3H-nicotinamide]NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.

摘要

紫外线照射显示能促使放射性标记从烟酰胺标记的NAD高效转移至一种包含百日咳毒素S-1亚基催化区域的重组蛋白（C180肽）。相对于从[32P-腺苷酸]NAD掺入（0.2摩尔/摩尔蛋白），[3H-烟酰胺]NAD的标记掺入效率很高（0.5至0.6摩尔/摩尔蛋白）。[3H-烟酰胺]NAD的标记特异性地与Glu-129相关联。用甘氨酸或天冬氨酸取代Glu-129使该蛋白对[3H-烟酰胺]NAD的光标记产生抗性，而用丝氨酸取代附近的谷氨酸Glu-139则不会。C180肽用NAD进行光标记与用白喉毒素和铜绿假单胞菌外毒素A观察到的情况相似，在这两种毒素中，NAD的烟酰胺部分分别转移至Glu-148和Glu-553。这些结果表明S-1亚基的Glu-129是一个活性位点残基，也是百日咳毒素基因改造以开发抗百日咳博德特氏菌无细胞疫苗的潜在重要位点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/d302ca8fd248/iai00071-0305-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/54d68caeb65d/iai00071-0302-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/bfd355c1c895/iai00071-0303-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/d302ca8fd248/iai00071-0305-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/54d68caeb65d/iai00071-0302-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/bfd355c1c895/iai00071-0303-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7dec/259866/d302ca8fd248/iai00071-0305-a.jpg

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