{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T00:08:25Z","timestamp":1766966905257,"version":"3.48.0"},"reference-count":39,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:00:00Z","timestamp":1766707200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxin serotype A (BoNT\/A) is the most potent known neurotoxin. While its light chain (LC) catalytic domain is a prime target for next-generation vaccines and therapeutics, the functional differences among BoNT\/A subtype LCs (A1, A2, A3) remain to be definitively characterized, despite notable sequence variation. This work aimed to systematically compare the proteolytic activity and immunoprotective efficacy of recombinant BoNT\/A1-LC, A2-LC, and A3-LC. Recombinant A1-LC-His, A2-LC-His, A3-LC-His, and A3-LC-Twin-Strep proteins were expressed in Escherichia coli (E. coli) and purified with affinity chromatography. Their proteolytic activity was assessed via in vitro SNAP-25 cleavage assays. The protective potency of these antigens was evaluated in a mouse model. In vitro cleavage assays revealed a substrate cleavage efficiency order of A2-LC &gt; A1-LC &gt; A3-LC. In vivo, both A1-LC and A2-LC immunization conferred robust, broad protection against high-dose challenges with all three toxin subtypes. In stark contrast, A3-LC provided only minimal protection against its homologous toxin and none against heterologous subtypes. Crucially, the functional deficit of A3-LC was confirmed to be an intrinsic property, as the A3-LC-TS variant, designed to exclude tag-specific interference, exhibited comparable low efficacy. According to structural research, A3-LC\u2019s compromised function may be caused by a four-amino-acid loss. The inferior performance of A3-LC is inherent to its primary structure. This work identified A1-LC or A2-LC as the potential proteolytic activity molecule and vaccine antigen by demonstrating functional differences among BoNT\/A subtype LCs. These findings provide crucial insights for developing subtype-specific countermeasures against botulism.<\/jats:p>","DOI":"10.3390\/toxins18010016","type":"journal-article","created":{"date-parts":[[2025,12,28]],"date-time":"2025-12-28T23:54:36Z","timestamp":1766966076000},"page":"16","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Proteolytic Activities and Immunological Effects of Light Chains of Botulinum Neurotoxin A1, A2 and A3 Subtypes"],"prefix":"10.3390","volume":"18","author":[{"given":"Yiying","family":"Liao","sequence":"first","affiliation":[{"name":"College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China"},{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Hu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1613-2019","authenticated-orcid":false,"given":"Jingrong","family":"Wang","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiansheng","family":"Lu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuo","family":"Yu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yunzhou","family":"Yu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1710-8582","authenticated-orcid":false,"given":"Wenhui","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Tereshko, Y., Dal Bello, S., Lettieri, C., Belgrado, E., Gigli, G.L., Merlino, G., and Valente, M. 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