Person: Zhang, Sicai
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Zhang
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Sicai
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Zhang, Sicai
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Publication Identification and characterization of a novel botulinum neurotoxin(Nature Publishing Group, 2017) Zhang, Sicai; Masuyer, Geoffrey; Zhang, Jie; Shen, Yi; Lundin, Daniel; Henriksson, Linda; Miyashita, Shin-Ichiro; Martínez-Carranza, Markel; Dong, Min; Stenmark, PålBotulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.Publication Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin(Nature Publishing Group UK, 2017) Huang, Nai-Jia; Pishesha, Novalia; Mukherjee, Jean; Zhang, Sicai; Deshycka, Rhogerry; Sudaryo, Valentino; Dong, Min; Shoemaker, Charles B.; Lodish, Harvey F.A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.Publication Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding(MDPI, 2018) Gustafsson, Robert; Zhang, Sicai; Masuyer, Geoffrey; Dong, Min; Stenmark, PålBotulinum neurotoxins (BoNTs) are a family of highly dangerous bacterial toxins, with seven major serotypes (BoNT/A-G). Members of BoNTs, BoNT/A1 and BoNT/B1, have been utilized to treat an increasing number of medical conditions. The clinical trials are ongoing for BoNT/A2, another subtype of BoNT/A, which showed promising therapeutic properties. Both BoNT/A1 and BoNT/A2 utilize three isoforms of synaptic vesicle protein SV2 (SV2A, B, and C) as their protein receptors. We here present a high resolution (2.0 Å) co-crystal structure of the BoNT/A2 receptor-binding domain in complex with the human SV2C luminal domain. The structure is similar to previously reported BoNT/A-SV2C complexes, but a shift of the receptor-binding segment in BoNT/A2 rotates SV2C in two dimensions giving insight into the dynamic behavior of the interaction. Small differences in key residues at the binding interface may influence the binding to different SV2 isoforms, which may contribute to the differences between BoNT/A1 and BoNT/A2 observed in the clinic.Publication N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A(2016) Yao, Guorui; Zhang, Sicai; Mahrhold, Stefan; Lam, Kwok-ho; Stern, Daniel; Bagramyan, Karine; Perry, Kay; Kalkum, Markus; Rummel, Andreas; Dong, Min; Jin, RongshengBotulinum neurotoxin serotype A1 (BoNT/A1) is one of the most dangerous potential bioterrorism agents, and exerts its action by invading motoneurons. It is also a licensed drug widely used for medical and cosmetic applications. Here we report a 2.0 Å resolution crystal structure of BoNT/A1 receptor-binding domain in complex with its neuronal receptor, the glycosylated human SV2C. We find that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an anti-botulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications to achieve highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.Publication Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC(Nature Publishing Group UK, 2017) Zhang, Sicai; Berntsson, Ronnie P.-A.; Tepp, William H.; Tao, Liang; Johnson, Eric A.; Stenmark, Pål; Dong, MinBotulinum neurotoxins (BoNTs), the most potent toxins known, are potential bioterrorism agents. It is well established that all seven serotypes of BoNTs (BoNT/A–G) require complex gangliosides as co-receptors. Here, we report that BoNT/DC, a presumed mosaic toxin between BoNT/D and BoNT/C1, binds and enters efficiently into neurons lacking complex gangliosides and shows no reduction in toxicity in mice deficient in complex gangliosides. The co-crystal structure of BoNT/DC with sialyl-Thomsen-Friedenreich antigen (Sialyl-T) suggests that BoNT/DC recognizes only the sialic acid, but not other moieties in gangliosides. Using liposome flotation assays, we demonstrate that an extended loop in BoNT/DC directly interacts with lipid membranes, and the co-occurring sialic acid binding and loop–membrane interactions mediate the recognition of gangliosides in membranes by BoNT/DC. These findings reveal a unique mechanism for cell membrane recognition and demonstrate that BoNT/DC can use a broad range of sialic acid-containing moieties as co-receptors.Publication Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity(Nature Publishing Group UK, 2018) Masuyer, Geoffrey; Zhang, Sicai; Barkho, Sulyman; Shen, Yi; Henriksson, Linda; Košenina, Sara; Dong, Min; Stenmark, PålBotulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 Å X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells.