微生物学报

Subtype	A1	A2	A3	A4	A5	A6	A7	A8
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/A isoforms. For H_C alignment, residues 870−1 296 of the BoNT/A1 sequence were used. The sequence numbers of BoNT/A1−BoNTA8 in NCBI were CAL82360.1^[45], CAA51824.1^[46], ACA57525.1^[47], ACQ51417.1^[47], ACG50065.1^[48], ACW83608.1^[49], AFV13854.1^[50], and AJA05787.1^[51].
A1	−	87.29	86.82	91.55	93.90	90.61	91.78	87.79
A2	89.97	−	98.83	88.47	89.65	90.12	90.35	93.43
A3	84.65	93.13	−	88.24	88.94	89.65	89.88	92.72
A4	89.35	83.35	84.49	−	86.85	88.94	85.92	90.14
A5	97.15	90.35	85.11	87.50	−	95.43	92.72	89.91
A6	95.68	91.67	86.27	87.89	95.83	−	91.08	87.32
A7	93.75	89.74	84.88	86.81	94.37	92.98	−	89.67
A8	93.51	93.52	87.81	89.34	93.59	93.20	91.36	−

Subtype	A1	A2	A3	A4	A5	A6	A7	A8
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/A isoforms. For H_C alignment, residues 870−1 296 of the BoNT/A1 sequence were used. The sequence numbers of BoNT/A1−BoNTA8 in NCBI were CAL82360.1^[45], CAA51824.1^[46], ACA57525.1^[47], ACQ51417.1^[47], ACG50065.1^[48], ACW83608.1^[49], AFV13854.1^[50], and AJA05787.1^[51].
A1	−	87.29	86.82	91.55	93.90	90.61	91.78	87.79
A2	89.97	−	98.83	88.47	89.65	90.12	90.35	93.43
A3	84.65	93.13	−	88.24	88.94	89.65	89.88	92.72
A4	89.35	83.35	84.49	−	86.85	88.94	85.92	90.14
A5	97.15	90.35	85.11	87.50	−	95.43	92.72	89.91
A6	95.68	91.67	86.27	87.89	95.83	−	91.08	87.32
A7	93.75	89.74	84.88	86.81	94.37	92.98	−	89.67
A8	93.51	93.52	87.81	89.34	93.59	93.20	91.36	−

Monosaccharide	H_C/A1: GD1a	H_C/A1: GT1b	H_C/A2: GD1a	H_C/A3: GD1a	H_C/A4: GD1a	H_C/A5: GM1b	H_C/A6: GD1a
Hydrogen bond distances for ganglioside binding in the structure H_C/A1: GD1a, H_C/A1: GT1b, H_C/A2: GD1a, H_C/A3: GD1a, H_C/A4: GD1a, H_C/A5: GM1b, and H_C/A6: GD1a. Water-mediated interactions are indicated in italics as “-H₂O molecules (n1, n2)”, where n1 is the distance between amino acid residues and water and n2 is the distance between water and monosaccharides.
Sia⁶	Trp 1 266 (3.5)	Trp 1 266 (3.1) Gln 1 270-H₂O (2.6, 2.5) Arg 1 276 (3.1)	Trp 1 266 (3.5)	Unmodelled	Unmodelled	Unmodelled	Unmodelled
Sia⁵	Tyr 1 117 (2.9) Tyr 1 267-H₂O (2.5, 3.5) Gly 1 279-H₂O (2.6, 2.8) Arg 1 276-H₂O (2.8, 2.8)	Tyr 1 117 (2.8, 3.0) Ser 1 275 (3.2) Arg 1 276-H₂O (3.1, 2.7) Gly 1 279-H₂O (2.7, 2.7)	Tyr 1 267-H₂O (2.5) Leu 1 250-H₂O (3.1, 2.8)	Tyr 1 263 (2.7) Gly 1 275 (2.9) Leu 1 250-H₂O (2.9, 2.8)	Tyr 1 123 (2.8) Tyr 1 273 (2.5) Gly 1 285 (3.1) Arg 1 282 (3.8)	Tyr 1 117 (2.8) Tyr 1 267 (2.7) Gly 1 279 (3.2)	Tyr 1 267 (2.6) Gly 1 279 (2.9)
Gal⁴	Glu 1 203 (2.8) Phe 1 252 (2.7) His 1 253 (2.7) Ser 1 264 (2.8)	Glu 1 203 (2.7) Phe 1 252 (2.6) His 1 253 (2.8) Gln 1 254-H₂O (2.6, 2.5) Ser 1 264 (2.7)	Glu 1 203 (2.6) Phe 1 252 (2.5) His 1 253 (2.9) Leu 1 254-H₂O (2.9, 3.1) Ser 1 264 (2.7)	Glu 1 199 (2.7) Phe 1 248 (2.5) His 1 249 (3.1) Ser 1 260 (2.7) Leu 1 250-H₂O (2.9, 3.0)	Glu 1 209 (2.4) Phe 1 258 (2.8) His 1 259 (2.7) Ser 1 270 (2.5)	Glu 1 203 (2.6) Phe 1 252 (2.8) His 1 253 (3.1) Ser 1 264 (2.9)	Glu 1 203 (2.7) Phe 1 252 (2.7) His 1 253 (3.0) Ser 1 264 (2.7)
GalNAc³	Glu 1 203 (2.5)	Glu 1 203 (2.6) Arg 1 269-H₂O (2.9, 3.1)	Glu 1 203 (2.7)	Glu 1 199 (2.5)	Glu 1 209 (2.6)		Glu 1 203 (2.5)

Monosaccharide	H_C/A1: GD1a	H_C/A1: GT1b	H_C/A2: GD1a	H_C/A3: GD1a	H_C/A4: GD1a	H_C/A5: GM1b	H_C/A6: GD1a
Hydrogen bond distances for ganglioside binding in the structure H_C/A1: GD1a, H_C/A1: GT1b, H_C/A2: GD1a, H_C/A3: GD1a, H_C/A4: GD1a, H_C/A5: GM1b, and H_C/A6: GD1a. Water-mediated interactions are indicated in italics as “-H₂O molecules (n1, n2)”, where n1 is the distance between amino acid residues and water and n2 is the distance between water and monosaccharides.
Sia⁶	Trp 1 266 (3.5)	Trp 1 266 (3.1) Gln 1 270-H₂O (2.6, 2.5) Arg 1 276 (3.1)	Trp 1 266 (3.5)	Unmodelled	Unmodelled	Unmodelled	Unmodelled
Sia⁵	Tyr 1 117 (2.9) Tyr 1 267-H₂O (2.5, 3.5) Gly 1 279-H₂O (2.6, 2.8) Arg 1 276-H₂O (2.8, 2.8)	Tyr 1 117 (2.8, 3.0) Ser 1 275 (3.2) Arg 1 276-H₂O (3.1, 2.7) Gly 1 279-H₂O (2.7, 2.7)	Tyr 1 267-H₂O (2.5) Leu 1 250-H₂O (3.1, 2.8)	Tyr 1 263 (2.7) Gly 1 275 (2.9) Leu 1 250-H₂O (2.9, 2.8)	Tyr 1 123 (2.8) Tyr 1 273 (2.5) Gly 1 285 (3.1) Arg 1 282 (3.8)	Tyr 1 117 (2.8) Tyr 1 267 (2.7) Gly 1 279 (3.2)	Tyr 1 267 (2.6) Gly 1 279 (2.9)
Gal⁴	Glu 1 203 (2.8) Phe 1 252 (2.7) His 1 253 (2.7) Ser 1 264 (2.8)	Glu 1 203 (2.7) Phe 1 252 (2.6) His 1 253 (2.8) Gln 1 254-H₂O (2.6, 2.5) Ser 1 264 (2.7)	Glu 1 203 (2.6) Phe 1 252 (2.5) His 1 253 (2.9) Leu 1 254-H₂O (2.9, 3.1) Ser 1 264 (2.7)	Glu 1 199 (2.7) Phe 1 248 (2.5) His 1 249 (3.1) Ser 1 260 (2.7) Leu 1 250-H₂O (2.9, 3.0)	Glu 1 209 (2.4) Phe 1 258 (2.8) His 1 259 (2.7) Ser 1 270 (2.5)	Glu 1 203 (2.6) Phe 1 252 (2.8) His 1 253 (3.1) Ser 1 264 (2.9)	Glu 1 203 (2.7) Phe 1 252 (2.7) His 1 253 (3.0) Ser 1 264 (2.7)
GalNAc³	Glu 1 203 (2.5)	Glu 1 203 (2.6) Arg 1 269-H₂O (2.9, 3.1)	Glu 1 203 (2.7)	Glu 1 199 (2.5)	Glu 1 209 (2.6)		Glu 1 203 (2.5)

Subtype	B1	B2	B3	B4	B5	B6	B7	B8
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/B isoforms. For H_C alignment, residues 862−1 291 of the BoNT/B1 sequence were used. The sequence numbers of BoNT/B1 to /B8 in NCBI were APH17270.1^[67], RUT52409.1^[68], WP_061310563.1^[69], CAA50482.1^[70], BDB03763.1^[71], BAO05199.1^[72], WP_129265872.1^[69], and AFN61309.1^[73].
B1	−	91.78	93.15	89.04	94.98	93.82	90.41	92.24
B2	91.78	−	95.78	89.97	91.56	96.56	90.41	93.68
B3	95.97	98.22	−	89.27	92.92	96.34	92.92	93.14
B4	92.80	92.78	92.95	−	88.13	88.79	89.27	87.44
B5	96.20	95.27	95.74	92.41	−	92.91	90.41	91.78
B6	96.20	98.22	98.22	92.49	95.51	−	92.65	93.19
B7	94.81	95.82	95.74	92.72	94.42	91.99	−	90.64
B8	95.51	95.74	95.90	92.18	94.74	92.69	90.64	−

Subtype	B1	B2	B3	B4	B5	B6	B7	B8
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/B isoforms. For H_C alignment, residues 862−1 291 of the BoNT/B1 sequence were used. The sequence numbers of BoNT/B1 to /B8 in NCBI were APH17270.1^[67], RUT52409.1^[68], WP_061310563.1^[69], CAA50482.1^[70], BDB03763.1^[71], BAO05199.1^[72], WP_129265872.1^[69], and AFN61309.1^[73].
B1	−	91.78	93.15	89.04	94.98	93.82	90.41	92.24
B2	91.78	−	95.78	89.97	91.56	96.56	90.41	93.68
B3	95.97	98.22	−	89.27	92.92	96.34	92.92	93.14
B4	92.80	92.78	92.95	−	88.13	88.79	89.27	87.44
B5	96.20	95.27	95.74	92.41	−	92.91	90.41	91.78
B6	96.20	98.22	98.22	92.49	95.51	−	92.65	93.19
B7	94.81	95.82	95.74	92.72	94.42	91.99	−	90.64
B8	95.51	95.74	95.90	92.18	94.74	92.69	90.64	−

Subtype	E1	E2	E3	E4	E5	E6	E7	E8	E9	E10	E11	E12
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/E isoforms. For H_C alignment, residues 848−1 252 of the BoNT/E1 sequence were used. The sequence numbers of BoNT/E1 to/E12 in NCBI were E1: ADF42615.1^[92], E4: APF24160.1^[93], E10: AII82313.1^[93], and E11: AII82281.1^[94]. The sequence numbers in Uniprot were E2: A2I2S6^[95], E3: A2I2S5^[95], E5: Q9K395^[96], E6: A8Y878^[97], E7: G8I2N7^[95], E8: G8I2N8^[95], E9: K7S9Y2^[95], and E12: W8FNB6^[98].
E1	−	97.35	100	97.78	92.82	97.28	100	97.28	83.17	95.31	93.58	87.65
E2	99.12	−	97.53	97.28	91.11	96.79	97.53	99.75	84.16	96.54	95.31	88.40
E3	98.24	97.36	−	97.78	92.59	97.28	100	99.75	83.17	95.31	93.58	87.65
E4	97.36	97.12	95.69	−	90.62	99.51	97.78	97.53	82.67	94.81	93.09	86.67
E5	96.88	96.41	95.21	95.05	−	90.84	92.82	91.09	83.91	88.86	89.60	90.84
E6	97.04	96.81	95.93	96.96	94.89	−	97.28	97.04	82.67	93.83	93.09	86.88
E7	97.92	97.12	97.36	96.25	94.81	96.41	−	97.28	83.17	94.57	93.58	87.87
E8	96.25	97.04	95.69	96.17	94.09	96.81	98.32	−	83.91	96.05	95.56	88.37
E9	89.13	89.37	88.73	90.01	89.52	88.25	89.21	89.45	−	83.66	85.15	88.37
E10	95.61	95.93	95.13	95.13	93.16	95.85	96.88	97.84	89.21	−	97.04	87.38
E11	93.37	93.85	92.57	92.73	91.93	93.13	93.45	94.41	89.05	95.29	−	87.62
E12	92.97	93.13	92.57	92.57	93.16	91.13	92.49	92.01	91.52	91.77	91.05	−

Subtype	E1	E2	E3	E4	E5	E6	E7	E8	E9	E10	E11	E12
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/E isoforms. For H_C alignment, residues 848−1 252 of the BoNT/E1 sequence were used. The sequence numbers of BoNT/E1 to/E12 in NCBI were E1: ADF42615.1^[92], E4: APF24160.1^[93], E10: AII82313.1^[93], and E11: AII82281.1^[94]. The sequence numbers in Uniprot were E2: A2I2S6^[95], E3: A2I2S5^[95], E5: Q9K395^[96], E6: A8Y878^[97], E7: G8I2N7^[95], E8: G8I2N8^[95], E9: K7S9Y2^[95], and E12: W8FNB6^[98].
E1	−	97.35	100	97.78	92.82	97.28	100	97.28	83.17	95.31	93.58	87.65
E2	99.12	−	97.53	97.28	91.11	96.79	97.53	99.75	84.16	96.54	95.31	88.40
E3	98.24	97.36	−	97.78	92.59	97.28	100	99.75	83.17	95.31	93.58	87.65
E4	97.36	97.12	95.69	−	90.62	99.51	97.78	97.53	82.67	94.81	93.09	86.67
E5	96.88	96.41	95.21	95.05	−	90.84	92.82	91.09	83.91	88.86	89.60	90.84
E6	97.04	96.81	95.93	96.96	94.89	−	97.28	97.04	82.67	93.83	93.09	86.88
E7	97.92	97.12	97.36	96.25	94.81	96.41	−	97.28	83.17	94.57	93.58	87.87
E8	96.25	97.04	95.69	96.17	94.09	96.81	98.32	−	83.91	96.05	95.56	88.37
E9	89.13	89.37	88.73	90.01	89.52	88.25	89.21	89.45	−	83.66	85.15	88.37
E10	95.61	95.93	95.13	95.13	93.16	95.85	96.88	97.84	89.21	−	97.04	87.38
E11	93.37	93.85	92.57	92.73	91.93	93.13	93.45	94.41	89.05	95.29	−	87.62
E12	92.97	93.13	92.57	92.57	93.16	91.13	92.49	92.01	91.52	91.77	91.05	−

Subtype	F1	F2	F3	F4	F5	F6	F7	F8	F9
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/F isoforms. For H_C alignment, residues 866−12 778 of the BoNT/F1 sequence were used. The sequence numbers of BoNT/F1−BoNT/F9 in NCBI were F1: WP_011987710.1^[69], F2: UYX45882.1^[100], F3: APQ97862.1^[100], F4: APQ71923.1^[101], F5: APR02750.1^[101], F6: ADU57954.1^[102], F7: AQA28590.1^[103], F8: KEJ01913.1^[104], and F9: AQA28591.1^[105].
F1	−	82.97	84.63	89.29	83.94	82.51	79.56	98.05	84.71
F2	83.71	−	96.37	81.51	92.03	93.89	72.02	83.70	90.34
F3	84.25	97.19	−	82.44	93.46	93.64	73.17	84.39	92.49
F4	92.33	83.71	84.09	−	82.00	81.03	76.40	88.56	83.45
F5	70.31	74.37	74.35	69.84	−	90.22	73.48	84.18	92.03
F6	88.05	90.02	90.04	87.42	74.11	−	72.17	82.27	88.75
F7	74.43	69.53	69.91	72.77	64.45	70.84	−	79.32	73.48
F8 F9	96.24 84.27	83.71 89.92	84.17 81.63	93.19 84.03	69.84 73.75	87.81 87.37	73.01 69.85	− 84.18	84.67 −

Subtype	F1	F2	F3	F4	F5	F6	F7	F8	F9
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/F isoforms. For H_C alignment, residues 866−12 778 of the BoNT/F1 sequence were used. The sequence numbers of BoNT/F1−BoNT/F9 in NCBI were F1: WP_011987710.1^[69], F2: UYX45882.1^[100], F3: APQ97862.1^[100], F4: APQ71923.1^[101], F5: APR02750.1^[101], F6: ADU57954.1^[102], F7: AQA28590.1^[103], F8: KEJ01913.1^[104], and F9: AQA28591.1^[105].
F1	−	82.97	84.63	89.29	83.94	82.51	79.56	98.05	84.71
F2	83.71	−	96.37	81.51	92.03	93.89	72.02	83.70	90.34
F3	84.25	97.19	−	82.44	93.46	93.64	73.17	84.39	92.49
F4	92.33	83.71	84.09	−	82.00	81.03	76.40	88.56	83.45
F5	70.31	74.37	74.35	69.84	−	90.22	73.48	84.18	92.03
F6	88.05	90.02	90.04	87.42	74.11	−	72.17	82.27	88.75
F7	74.43	69.53	69.91	72.77	64.45	70.84	−	79.32	73.48
F8 F9	96.24 84.27	83.71 89.92	84.17 81.63	93.19 84.03	69.84 73.75	87.81 87.37	73.01 69.85	− 84.18	84.67 −

不同血清型肉毒毒素受体结合域研究进展

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尹凡铭 ¹^,² , 朱晨思 ² , 李涛 ² , 王慧 ¹^,²^,^*

微生物学报 | 综述 2024,64(7): 2172-2193

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微生物学报 | 综述 2024, 64(7): 2172-2193

不同血清型肉毒毒素受体结合域研究进展

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尹凡铭¹^,², 朱晨思², 李涛², 王慧¹^,²^,^*

作者信息

1 牡丹江医学院公共卫生学院, 黑龙江牡丹江 157011

2 军事科学院军事医学研究院微生物流行病研究所病原微生物生物安全全国重点实验室, 北京 100071

Research progress in receptor-binding domains of different serotypes of botulinum neurotoxins

Fanming YIN¹^,², Chensi ZHU², Tao LI², Hui WANG¹^,²^,^*

Affiliations

1 School of Public Health, Mudanjiang Medical University, Mudanjiang 157011, Heilongjiang, China

2 State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China

出版时间: 2024-07-04 doi: 10.13343/j.cnki.wsxb.20230603

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摘要

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肉毒毒素(botulinum neurotoxin, BoNT)是人类已知毒性最强的蛋白质之一，可以引起肌肉松弛麻痹，严重时可导致死亡。肉毒毒素共分为7种血清型(BoNT/A−BoNT/G)，根据氨基酸序列差异可进一步分为40多种亚型。肉毒毒素分子结构由3个基本结构域组成：重链羧基端细胞受体结合域、氨基端的易位域和轻链催化域。在运动神经元表面，受体结合域首先与聚唾液酸神经节苷脂结合，随后与突触囊泡蛋白2或突触囊泡结合蛋白结合形成双受体复合物。每种血清型的受体结合域都必须与其相应受体结合才能发挥作用。肉毒毒素的结构功能及其对宿主的作用一直都是研究热点。近年来，因受体结合域可以促进肉毒毒素与运动神经元膜特异性结合，而成为新的研究方向。本综述将概述不同血清型肉毒毒素与受体结合过程中受体结合域结构变化和结合位点差异。通过分析不同血清型及亚型的序列以及受体结合域结构特征，可以更好地了解细胞受体结合域的序列差异和功能，并为肉毒毒素的治疗策略提供新思路。

关键词

肉毒毒素 / 受体结合域 / 神经节苷脂 / 突触囊泡蛋白2 / 突触囊泡结合蛋白

Abstract

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Botulinum neurotoxins (BoNTs), a group of the most toxic proteins, can cause muscle paralysis and even lead to death in severe cases. BoNTs can be classified into 7 serotypes (BoNT/A−BoNT/G) and further classified into more than 40 subtypes according to the differences in amino acid sequences. BoNTs consist of three basic domains: the C-terminal receptor-binding domain of the heavy chain, the N-terminal translocation domain, and the light-chain catalytic domain. On the surface of motor neurons, the receptor-binding domain binds first to polysialoganglioside and subsequently to synaptic vesicle protein 2 or synaptotagmin to form a two-receptor complex. The functioning of each serotype relies on the binding of the receptor-binding domain to the corresponding receptor. BoNTs have always been a research hotspot in terms of the structure, function, and effect on the host. The role of the receptor-binding domain in promoting the specific binding of BoNTs to motor neurons has become a new research direction. This review summarizes the structural changes of the receptor-binding domains and the differences in binding sites during the binding of different serotypes of BoNTs to receptors. By analyzing the sequences and structural characteristics of the receptor-binding domains of different serotypes and subtypes, we can fully understand the sequence differences and functions of the receptor-binding domain and give insights into the treatment of BoNTs.

Key words

botulinum neurotoxin / receptor-binding domain / ganglioside / synaptic vesicle protein 2 / synaptotagmin

引用本文

尹凡铭, 朱晨思, 李涛, 王慧. 不同血清型肉毒毒素受体结合域研究进展. 微生物学报, 2024 , 64 (7) : 2172 -2193 . DOI: 10.13343/j.cnki.wsxb.20230603

Fanming YIN, Chensi ZHU, Tao LI, Hui WANG. Research progress in receptor-binding domains of different serotypes of botulinum neurotoxins[J]. Acta Microbiologica Sinica, 2024 , 64 (7) : 2172 -2193 . DOI: 10.13343/j.cnki.wsxb.20230603

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肉毒毒素由肉毒梭状芽孢杆菌在厌氧条件下产生^[1]。根据细菌之间的表型差异，肉毒梭状芽孢杆菌分类群可分为6组(I−VI)^[2] (图1)。I组肉毒梭菌菌株在10−37 ℃之间生长，产生高度耐热的孢子。II组菌株是中等耐热性孢子，具有非蛋白水解的特性，在低温3.5 ℃下生长。III组在37−40 ℃较高温度下生长，主要为引起动物肉毒中毒的C型和D型。产生G型的阿根廷梭菌是第IV组^[3]。此外，其他几种梭状芽孢杆菌菌株虽然不典型，但也具有神经毒性，如巴氏梭菌菌株产生F型肉毒毒素(F7亚型)，一些丁酸梭菌菌株产生E型(E4亚型和E5亚型)，属于Ⅵ组。肉毒毒素根据中和抗体不同可以分为不同血清型，目前已鉴定出7种血清型(BoNT/A−BoNT/G)，以及一种不能被任何已知肉毒毒素抗体中和的新型血清型(BoNT/X)^[4]。通过基因组测序技术发现嵌合型肉毒素BoNT/FA (HA)、BoNT/CD和BoNT/DC^[5-8]，以及来自米饭魏斯氏菌的BoNT/Wo^[9-11]、来自肠球菌的BoNT/En^[12-13]和来自双发酵副梭菌的肉毒毒素样蛋白等^[14]。不同血清型之间毒性具有很大差异，BoNT/A、BoNT/B、BoNT/E和BoNT/F与人类肉毒中毒相关^[15]。由于氨基酸序列的差异，BoNT内的每种血清型进一步分为亚型(如BoNT/A1−BoNT/A8、BoNT/B1−BoNT/B8、BoNT/E1−BoNT/E12、BoNT/F1−BoNT/F9)^[16] (图1)。如图1所示，使用MEGA 11软件对A−X型序列进行分析，整体来看G型与B型序列相似性更高，H型与A型序列相似度较高，E型与F型序列相似性较高，而每种血清型内不同亚型一致性更高。

BoNT最初是作为单个可溶性多肽链生产的，约150 kDa^[17]。这种蛋白对神经组织无毒性，但当它被蛋白酶或靶细胞本身产生的蛋白酶切割时，则具有毒性。前体蛋白被切割产生2条多肽链，它们通过一个二硫键连接。其中一个多肽链为大小50 kDa的轻链(light chain, LC)，具有锌内肽酶活性，因此被称为催化结构域^[18]。目前，轻链是肉毒中毒抑制剂开发的重要方向，研究者根据肉毒毒素的结构设计药物或通过高通量筛选不同的小分子文库发现多种针对肉毒中毒的潜在抑制剂^[19-21]，并以先导化合物为基础不断改进及合成类似的化合物以进一步提高药效。

肉毒毒素另一个多肽链为100 kDa的重链(heavy chain, HC)，由2个结构域组成：N端易位域(translocation domain, H_N)和C端受体结合域(cell-binding domain, H_C)^[22]。受体结合域与神经元表面受体结合，使BoNT进入神经元并发挥毒性作用。BoNT进入神经元过程如图2所示，第一步，BoNT的受体结合域与神经元膜上的“第一受体”聚唾液酸神经节苷脂结合，主要包括GT1b、GD1a、GD1b和GM1b。不同血清型肉毒毒素结合不同神经节苷脂，如A型可以与GD1A、GT1b或GM1b结合，BoNT/B、BoNT/E和BoNT/F均可与GD1a或GT1b结合，BoNT/C与GT1b或GM1b结合，BoNT/G只与GT1b结合，目前研究认为BoNT/D不依赖于与神经节苷脂结合发挥作用^[23]。第二步，细胞外K⁺浓度升高导致细胞膜去极化，使Ca²⁺通道开放，Ca²⁺流入细胞内与“第二受体” SV2或Syt结合并发信号。其中BoNT/A、BoNT/D、BoNT/E和BoNT/F与SV2结合，BoNT/B和BoNT/G与Syt结合。BoNT与聚唾液酸神经节苷脂以及SV2或Syt结合，共同组成“双受体模型”。BoNT进入神经元后，经过进一步作用发挥毒性，引起机体神经肌肉麻痹。

1 肉毒毒素受体

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1.1 神经节苷脂

聚唾液酸神经节苷脂由具有许多唾液酸残基的亲水复合多糖组成，能够与疏水性神经酰胺尾结合，但不同神经节苷脂之间单糖单元的数量、组成和位置有所不同(图3)。这些神经节苷脂可以通过不同形式嵌入细胞膜中，并且在细胞表面展示各种糖部分。神经元膜上最常见的神经节苷脂包括GT1b、GD1a、GD1b和GM1b^[24]。受体结构域的羧基末端包含肽基序H...SxWY...G，它构成神经节苷脂结合位点(ganglioside binding site, GBS)的核心。神经节苷脂和神经节苷脂结合位点之间的相互作用，可以根据H_C与寡糖结合后的晶体结构确定。

所有血清型毒素(D型除外)均利用神经节苷脂作为辅助受体^[25-26]。其中GT1b是对肉毒毒素具有最高亲和力的神经节苷脂，其碳水化合物部分由7个单糖组成(图3)。

1.2 突触囊泡蛋白2

突触囊泡蛋白2 (synaptic vesicle protein 2, SV2)是硫酸角质素蛋白聚糖^[27]，是神经元和内分泌细胞中存在的蛋白质家族^[28-29]。SV2具有糖基化位点^[30]以及可被钙离子抑制的突触蛋白结合位点，BoNT/A^[31]、BoNT/D^[32]、BoNT/E^[33]和BoNT/F^[34]利用SV2管腔结构域进入细胞。SV2家族有3个成员：SV2A、SV2B和SV2C，它们结构相似但表达位置不同。SV2A是主要的异构体，普遍存在于所有大脑区域，SV2B主要存在于皮质和海马体中^[35]，SV2C亚型在大脑区域(如纹状体、黑质、脑桥和延髓)中水平较高。SV2与其他蛋白的相互作用可以对囊泡胞吐作用产生影响。

1.3 突触囊泡结合蛋白

Nishiki等在20世纪90年代首次发现了同源突触囊泡结合蛋白synaptotagmin I和II (Syt-I和Syt-II)可以作为BoNT/B的结合受体^[36-37]。后来研究证实，Syt-I和Syt-II是介导BoNT/B结合和进入细胞的功能性蛋白受体^[38]。随后BoNT/G和BoNT/DC也被报道利用Syt-I和Syt-II作为功能受体^[39-40]。Syt由含有糖基化位点的小腔内N端片段、单个跨膜结构域、参与SV融合所需的细胞内钙离子、磷脂和可溶性N-乙酰亚胺敏感因子连接的蛋白质受体(soluble N-ethylmaleimide-sensitive factor attachment protein receptors, SNARE)复合物结构域组成。Syt-I和Syt-II在突触囊泡中表达，在神经递质释放中，作为触发胞吐作用的钙离子传感器发挥重要作用，随后在活性区依赖网格蛋白的内吞作用进入^[41]。胞吐作用后，在细胞表面Syt的N端结构域更容易与肉毒毒素结合^[42]。

2 肉毒毒素与受体结合

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2.1 A型肉毒毒素

在BoNT/A血清型中，目前有8个亚型(A1−A8)，其氨基酸水平差异在3%−16%。由于BoNT/A1可用于治疗痉挛、肌张力障碍和眉间纹等多种疾病^[43-44]，是目前表征最充分的亚型。

如表1所示，不论是从全长还是H_C序列比对来看，其中A1与A5相似性最高，达到97.15%以及93.90%，A1与A3相似性最低。

2.1.1 A型肉毒毒素与神经节苷脂结合

通过神经节苷脂与H_C结合后的一些变化，分析亚型(H_C/A1−H_C/A6)中有6个结合残基是保守的(表2)^[52]。根据每个H_C/A亚型对神经节苷脂的相对亲和力，可以从界面上存在的氢键数量推断出来H_C/A1和H_C/A2对GD1a的亲和力最高(A亚型与GD1a亲和力排序为A1/A2 > A3/A4 > A6)。

H_C/A1 (PDB: 3BTA)与H_C/A1: GD1a (PDB: 5TPB)或H_C/A1: GT1b (PDB: 2VU9) [结合与未结合的分子相比，均方根差(root mean square deviation, RMSD)分别为0.5 Å和0.3 Å (对于Cα原子)]^[51]。GD1a和GT1b仅相差1个单糖(图3)，两者均对毒素具有较高亲和力。H_C/A2与H_C/A1相比，整体结构在结合GD1a后GBS附近的环(残基1 269−1 277)具有构象变化(图4)。在H_C/A2中环定位的变化随着Phe 1 278向GBS旋转，这一特征在H_C/A3: GD1a、H_C/A4: GD1a和H_C/A5: GM1b结构中也可以观察到^[3,53]。

H_C/A3: GD1a (PDB: 6THY)和H_C/A3 (PDB: 6F0O)的构象非常相似，RMSD为1.0 Å^[53]。H_C/A3在与GD1a结合后，除了与神经节苷脂结合后残基的相对位置发生变化外，氢键也有明显的差异，尤其是与Sia⁵结合的氢键。Tyr 1 263移动到Sia⁵附近取代了1个水分子，并与Phe 1 248进一步相互作用，Asn 126与Phe 1 245形成氢键，His 1 249接近GBS与Gal4和几个疏水残基(Phe 1 113、Val 1 198、Glu 1 199、Tyr 1 250、Trp 1 262)形成2个氢键^[54] (图4)。

H_C/A4 (PDB: 6F0P)在与GD1a结合后，Arg 1 282和Tyr 1 123与Sia⁵形成氢键相互作用^[53] (表2)。此外，H_C/A4与神经节苷脂无水介导的相互作用，而H_C/A1、H_C/A2和H_C/A3都有水介导的相互作用。

H_C/A5: GM1b (PDB: 7QPPU)结构与H_C/A5 (PDB: 6TWP)结构非常相似，在与神经节苷脂结合后，H_C/A5: GM1b结构的N端发生了显著的构象变化。Arg 893和Tyr 894的侧链在与GM1b结合后向蛋白质结构主体旋转，导致结构更加紧凑(图4)。另一个研究发现，H_C/A5与BoNT/A1: GT1b (PDB: 2VU9)的结构相比，含有神经节苷脂相互作用的残基1 260−1 280环采用了不同的排列方式^[55]。认为是因为S 1 275和R 1 276参与相互作，使环的构象发生改变。

H_C/A6具有两种晶体形式，晶体I (PDB: 6TWO)和晶体II (PDB: 8ALP)之间的晶体结构存在差异(图4)。H_C/A6 (晶体形式I)与神经节苷脂结合后残基Phe 1 278向Sia⁵旋转时1 269– 1 277环变宽(图4)。这与H_C/A2、H_C/A3和H_C/A5中发生的现象一致。H_C/A6 (晶体形式II)中其铰链旋转约16.8°，这是在BoNT/A亚型结构中观察到的子结构域最大的铰链运动^[56]。H_C/A6与H_C/A5一样，蛋白质整体折叠与其他肉毒毒素H_C结构高度相似。除了残基1 117，它是一个Phe而不是一个Tyr以外，神经节苷脂结合位点都与H_C/A5相同(图4)。虽然缺少羟基会导致GT1b的末端唾液酸失去氢键，但侧链仍然可以继续与碳环相互作用^[57]。

研究证明，神经节苷脂和不同亚型H_C之间相互作用具有差异，这种差异来自水介导的相互作用以及氢键产生的相互作用^[57]。之前报道H_C/A3与H_C/A1相比，H_C/A3与GD1a的结合亲和力低于H_C/A1，研究者认为H_C/A3与GD1a的氢键相互作用减少是毒性降低的一个原因^[3]。而BoNT/A2比BoNT/A1对神经节苷脂具有更高的亲和力，这与BoNT/A2可以更快进入神经元细胞现象一致^[59-60]。H_C/A4结构显示出与GD1a氢键相互作用的减少，研究证明其在小鼠中的活性与BoNT/A1相比降低很多^[61]，并且BoNT/A4进入细胞的效率也较低^[62-63]，这些可以证明细胞结合域与肉毒毒素毒性以及进入神经元细胞的速度等方面息息相关。

2.1.2 A型肉毒毒素与SV2受体结合

与BoNT/A受体结合域结合的SV2分为2部分结合位点。第一部分是蛋白结合位点，在H_C/A亚型中有一些残基并不保守，如H_C/A1和H_C/A2中的残基Thr 1 142、Thr 1 144和Thr 1 146^[64] (图5)，它们容易与SV2C的β片段堆积产生相互作用。然而这种相互作用只能产生很小结构变化。另一方面，所有H_C/A亚型都有2个高度保守的残基，如H_C/A1、H_C/A2、H_C/A5和H_C/A6亚型中的Thr 1 145和Thr 1 146，HC/A3中的Thr 1 141和Thr 1 142，以及H_C/A4中的Thr 1 151和Thr 1 152 (图5)，这些保守残基对于SV2C与受体结合域结合至关重要^[65]。

H_C/A2结构域与非糖基化SV2C-LD4复合物结合的晶体结构表明，尽管不同A亚型间存在一些残基差异，但结合模式是相似的。如结构高度相似的H_C/A5和H_C/A6也可能以相同的方式结合SV2C。这主要是因为β-链之间的主干-主干相互作用的性质，导致大多数A亚型与SV2C间的相互作用一致。然而，一个值得注意的变异点是上述H_C/A3中的Thr 1 152和H_C/A6的Thr 1 156，它们可以与SV2C的H563形成亲电相互作用(图5)。在H_C/A1中的等效位置有一个精氨酸(图5)，可以形成阳离子-π堆叠相互作用，在H_C/A2中的等效位置有一个谷氨酸，可以与SV2C组氨酸形成盐桥。

第二部分聚糖结合位点，对于BoNT/A与SV2的高亲和力结合至关重要，因为只有糖基化的SV2才能增强受体结合域与SV2结合。BoNT/A1与人的糖基化SV2C (H_C/A1: gSV2C)结合的晶体结构显示，Asn-559上的N-连接聚糖与gSV2C产生大量相互作用。参与H_C/A1聚糖结合的2个关键残基是Phe 953和His 1 064 (图6)，它们与Asn 559聚糖的2个GlcNAc分子形成π堆积相互作用。另外12个残基与聚糖形成氢键网络通过水介导的相互作用。

研究者检测SV2C糖结合位点发现，残基Gln106会阻碍糖的结合，该残基与BoNT/A1中的His 1 064相对应，因此不会使受体结合域与SV2C结合的亲和力降低^[64]。然而，与BoNT/A1相比，Gln106残基在BoNT/A2、BoNT/A3、BoNT/A5、BoNT/A6和BoNT/A8中，与位于1 060位的精氨酸残基相对应，可能阻碍糖的结合，而导致与SV2C亲和力降低^[53] (图6)。这几种亚型对SV2C的亲和力可能低于BoNT/A1，然而在体内外实验中发现，BoNT/A2和BoNT/A6均比BoNT/A1能够更快地进入神经元细胞^[60]。

2.2 B型肉毒毒素

目前，BoNT/B血清型有8种亚型(B1−B8)，它们在氨基酸水平上的差异介于1.5%−7.0% (表3)^[3]。如表3所示，从全长序列比对来看，其中B1−B8相似性较高，达到90%以上；从受体结合域序列比对来看，B1与B6相似性最高，与B4相似性最低。虽然BoNT/B的晶体结构与BoNT/A类似^[11]，但BoNT/B靶向神经元细胞膜上Syt-I或Syt-II蛋白受体^[39]。

2.2.1 B型肉毒毒素与神经节苷脂结合

据报道，BoNT/B通过与神经细胞膜上的Syt-II和神经节苷脂GD1a或GT1b结合而产生毒性。BoNT/B的毒性与受体结合有关，但与单独的GT1b或GD1a无关^[74]。GD1a的4个亚单位与BoNT/B的GBS结合，其中Sia⁵与N 1 273、N 1 105、G 1 277和Y 1 263形成氢键，Gal4与I 1 240、H 1 241、S 1 260和E 1 190形成氢键(图7A)^[75]。GalNAc³和Sia⁶分别与E 1 190和W 1 262形成一个氢键(图7A)。W 1 262进一步与Gal4形成关键的芳族堆叠相互作用^[76]。

虽然GD1a和GT1b中存在的Sia⁵和Sia⁶均与GBS相互作用，但GT1b结构与GD1a相比多一个Sia⁷ (图3)，研究者认为其可以加强BoNT/B与受体的结合^[76]。

在Syt-II和GD1a共同结合过程中，BoNT/B1结合域的晶体结构与Sia⁵片段表现出强烈的相互作用^[76]。虽然Syt-II和GD1a之间无直接联系，但有证据表明，两者均可以影响彼此的结合，这可能与两者结合位点的空间排列有关^[77]。

2.2.2 B型肉毒毒素与Syt结合

BoNT/B可以与Syt-I或Syt-II形成一个短螺旋，与BoNT/B结合区域内的疏水凹槽结合^[78]进入细胞。Chai等对BoNT/B1与Syt之间的结合机制研究发现^[79]，Syt-I与BoNT/B的亲和力较低。

BoNT/B2与BoNT/B1具有相似的受体结合机制，但研究发现B2比B1的毒性低，认为与Syt亲和力有关。BoNT/B1和BoNT/B2之间有4个残基差异，为疏水结合机制的关键残基。由于这4个残基与Syt的保守残基邻近，因此被认为与Syt亲和力变化相关^[80]。在BoNT/B1中，S 1 199通过芳香族与F47的相互作用增加与Syt的亲和力；在BoNT/B2中，这种疏水残基相互作用被S 1 117和N 1 197与Syt-I之间的静电作用所取代(图7B)，可以导致B2和Syt之间的疏水相互作用丧失，从而使亲和力降低^[76]。

另一个研究发现，BoNT/B与Syt-II的亲和力取决于实验是在人还是大鼠的Syt-II上进行的。与大鼠和小鼠相比，BoNT/B与人Syt-II的结合亲和力降低^[81-82]。这种显著的差异是由于Phe54氨基酸的差异而产生^[81]。研究者对Syt-II的管腔结构域^[83]进行研究发现，重组人Syt-II-LD在小鼠体内对BoNT/B中毒具有保护作用。证明重组人Syt-II的管腔结构域与神经节苷脂联合可以抑制BoNT/B对小鼠的毒性^[84]。正是因为Syt受体可以与BoNT/B特异性结合的特点，使Syt成为肉毒毒素抑制剂研究新的方向，后来研究者发现了一种可以中和Syt-II受体结合位点的抗体SC12^[85]，它可以有效地阻止BoNT/B与其受体的结合。

2.3 C型肉毒毒素

BoNT/C主要与动物肉毒中毒有关^[86-87]。BoNT/C血清型没有亚型，截至目前仅鉴定出2种不同的蛋白序列，它们具有99.9%的一致性。

一项早期研究发现，BoNT/C可以与GD1b和GT1b结合，实验中发现，在神经节苷脂缺陷小鼠中BoNT/C毒性会降低，证实了BoNT/C利用神经节苷脂进入细胞^[88]。BoNT/C的受体结构域包含一个SxWY不保守基序和一个额外的唾液酸结合位点^[89]，这个结合位点在BoNT/C中称为Sia-1。Sia-1被发现可以结合含Sia⁵以及Sia⁷的神经节苷脂(GD1b > GT1b)^[90]。后来还发现BoNT/C可能含有2个神经节苷结合位点，经研究证实了唾液酸结合位点与任一神经节苷脂结合位点被破坏，都会阻止H_C进入神经元细胞^[88]。

2.4 D型肉毒毒素

与BoNT/C一样，BoNT/D没有亚型，有多个相似性超过96%的一级序列。

神经节苷脂类是BoNT/D进入细胞所必需的。研究发现，BoNT/D中毒不依赖于神经节苷脂与受体的结合^[23]，其毒性取决于神经节苷脂的表达，β-发夹环是神经节苷脂和神经元结合所必需的，被称为神经节苷脂结合环，β-发夹环内的Asp 1 233有利于毒性的表达。另外，BoNT/D进入神经元依赖于多种碳水化合物的相互作用^[91]。

BoNT/D可以识别SV2的3种亚型。缺乏SV2的细胞与BoNT/D结合后不会产生毒性，但可以通过表达3种SV2亚型(SV2A、SV2B和SV2C)中的任何一种来恢复这种毒性。N-链糖基化位点的突变也对BoNT/D进入神经元没有影响，这表明BoNT/D中的SV2受体结合域可能与其他SV2相互作用的肉毒毒素不同^[89]。

2.5 E型肉毒毒素

目前已知有12种BoNT/E亚型(E1−E12)，其氨基酸特征差异高达12% (表4)。如表4所示，从全长序列比对来看，与E1比较，E9序列差异较大，与E2最相似为99.12%。从受体结合域序列比对来看，E1与E3和E7序列完全一致，而与E9序列差异仍然最大，为83.17%。

2.5.1 E型肉毒毒素与神经节苷脂结合

BoNT/E识别GD1a和GT1b的亲和力高于GM1。H_C/E与GD1a结合后，可以观察到明显的结构变化。特别是在C端β-三叶草形亚结构域可以观察到环1 228−1 237发生了构象变化^[18] (图8)。侧链残基R 1 230在这个环中发挥重要作用，其产生的静电作用可以推动主链和环远离神经节苷脂。这种现象可以通过与侧链相邻的L 1 092和F 1 214残基的疏水作用进一步稳定(图8A)。Gal4首先与S 1 222形成氢键，而后与SxWY基序相互作用，其次与残基Y 1 225相互作用，最后通过与W 1 224的吲哚环堆积而保持结合稳定(图8A)。GalNAc3也与K 1 215和K 1 171侧链形成强氢键来增强稳定性(图8A)。这些残基与神经节苷脂相互作用使H_C/E与GD1a结合更加稳定。

2.5.2 E型肉毒毒素与SV2结合

BoNT/E只能与SV2A或SV2B结合^[18]。研究表明，BoNT/A的受体结合域可识别SV2管腔结构域四边形β螺旋折叠的C端、β链的开放边缘以及N-聚糖，它们一起形成H/CA的复合结合位点。然而，BoNT/E的受体结合域与SV2结合位点有8个氨基酸缺失(图8B)，表明BoNT/E利用一种独特的机制来识别SV2A或SV2B^[97]。

2.6 F型肉毒毒素

目前BoNT/F血清型有9种亚型(F1−F9)，它们的序列同一性差异高达30% (表5)。如表5所示，从全长序列比对来看，与F1相比，F1亚型与F4亚型序列相似性最高，为92.33%；与F5相似性最低，为70.31%。从受体结合域序列比较，F1与F8序列相似性最高，为98.05%。

2.6.1 F型肉毒毒素与神经节苷脂结合

BoNT/F1需要与GT1b或GD1a中含有α2, 3-链唾液酸的神经节苷脂结合^[78]。GT1b和GD1a对BoNT/F1表现出较高的亲和力。BoNT/F1的H_C结构域表面含有大量的阳离子，由于GT1b神经节苷脂带负电荷的唾液酸，因此H_C可以与GT1b神经节苷脂表面的阴离子相互作用。在GBS区域，BoNT/F1主要通过其芳香族残基H 1 241和W 1 250与神经节苷脂相互作用。在GBS区域之外，研究者发现氨基酸N 1 121、N 1 119、R 1 179、R 1 202、K 1 199、S 1 190、K 1 193和Y 1 183也通过氢键与神经节苷脂相互作用^[106]。

2.6.2 F型肉毒毒素与SV结合

据报道，BoNT/F1的蛋白受体是否是糖基化的SV2仍有待最终确定。因为模拟预测BoNT/F1与SV2A的蛋白质部分之间不存在结合，因为在BoNT/F1序列的1 135位处存在脯氨酸，其占据与BoNT/A结构中残基T 1 146相同的位置^[106]。然而，T 1 146是一个通过与SV2形成氢键来介导受体相互作用关键残基^[93]。

2.7 G型肉毒毒素

目前已知的BoNT/G蛋白序列只有2个，它们具有99.9%的一级序列同源性。

BoNT/G具有保守的SxWY基序，并优先结合GT1b^[107]。其亲和力顺序(GT1b > GD1a > GD1b)与BoNT/A、BoNT/B和BoNT/F相似^[108]。除了双受体相互作用外，BoNT/G还含有与BoNT/B类似的脂质结合环，该环可直接与细胞膜相互作用增强亲和力，去掉该环可显著降低神经毒性^[109-110]。

据报道，BoNT/G和BoNT/B的蛋白受体是Syt-I或Syt-II。BoNT/B和BoNT/G对Syt-I和Syt-II的管腔结构域的亲合力按照B-Syt-II > G-Syt-I > G-Syt-II > B-Syt-I的顺序递减。Syt-I和Syt-II之间有3个相互作用氨基酸不同，即A38与M 46、M 47与F 55、L 50与I 58。与H_C/B-Syt-II类似，野生型H_C/G的Y 1 186和L 1 191侧链与Syt-II的M 46、F 55和I 58相比，分别更好地与Syt-I的A 38、M 47和L 50结合，因此H_C/G对Syt-I的亲和力高于Syt-II^[108,111]。

2.8 HA (FA)型肉毒毒素

BoNT/HA是2014年在一例婴儿肉毒杆菌中毒中被发现的^[112-113]。由于其不可中和的抗原性，它被称为BoNT/H (BoNT/HA)，根据序列分析将该基因置于与其他血清型不同的谱系中^[113]。随后确定该分子为一种花叶毒素，由与BoNT/F5相似的LC、与BoNT/F1相似的H_N结构域和与BoNT/A1相似的受体结构域组成^[6]。已经证实糖基化的SV2C-LD4与BoNT/FA的受体结构域可以直接结合^[114]，但是该结合结构域的晶体结构与BoNT/A1有一些细微的差异^[115]。神经节苷脂结合位点能够保持与BoNT/A1相同的折叠，但尚未存在神经节苷脂结合结构，因此确切的相互作用仍不确定^[116]。BoNT/FA序列包含BoNT/A1的突变，研究者通过对未糖基化的SV2C的下拉试验确定BoNT/A1对SV2蛋白骨架的亲和力降低，而参与聚糖结合的等效残基保持不变^[114]。这些突变对SV2不同亚型的影响还有待进一步研究。

2.9 DC型肉毒毒素

BoNT/DC是一种自然存在的嵌合毒素，与BoNT/C和BoNT/D进化相关。它的LC和H_N几乎与BoNT/D相同，而它的H_C与C型的相似性约为77%^[117]。SxWY基序在H_C/DC中不保守。在所有BoNT中，BoNT/DC的独特之处是：在缺乏神经节苷的培养神经元中，BoNT/DC与受体的结合及进入细胞不受影响，H_C/DC与神经节苷脂晶体结构表明，H_C/DC仅识别唾液酸部分，不与碳水化合物主链相互作用。因此，BoNT/DC能够利用含唾液酸的分子作为受体进入细胞。

3 总结与展望

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BoNT除致病外也可用于美容^[116]和疾病治疗(如治疗帕金森病^[118]、膀胱炎^[119]和偏头痛等^[120])。近年来，受体复合物的晶体结构为点突变奠定了基础，也为肉毒毒素的治疗提供新的方向。有研究者通过在BoNT重链上进行点突变，发现这种突变可以提高毒素活性。例如Yin等对H_C/A和H_C/B进行了点突变，发现可以增强它们受体结合域与神经节苷脂和蛋白质受体的结合，从而提高毒素靶向进入神经元的能力^[121]。Burns等对BoNT/A的神经节苷脂结合位点进行突变，增强了其与神经节苷脂之间的相互作用，从而增强了BoNT/A的靶向效力^[122]。这种受体结合域的点突变，为肉毒毒素在治疗方面的研究提供更多可能。

目前，受体结合域研究存在局限性，大多仅限于1种或2种血清型，很多血清型受体结合域与受体结合机制尚不清楚。如上述中提到，BoNT/E的受体结合域与SV2结合位点有8个氨基酸缺失，但仍以未知作用机制与受体结合并发挥毒理作用。BoNT/D引起中毒不依赖于与神经节苷脂的结合^[23]，其毒性取决于神经节苷脂的表达。然而嵌合型BoNT/DC虽然与BoNT/D和BoNT/C在结构上有高度相似性，但其进入神经元可以不需要与神经节苷脂结合直接进入。此外，人和动物中受体结构不同，也可以导致一些血清型只会引起动物中毒，而不会引起人中毒。

另一方面，尽管BoNT亚型之间的序列同一性很高，但它们的中毒特性仍存在显著差异，例如它们的发作症状和作用持续时间。如前面所述BoNT/A2在神经元细胞中比BoNT/A1更有效并且可以更快地进入细胞，BoNT/A1的效力为BoNT/A4的1 000倍，A3尾静脉注射发现与A1症状不同等。目前大多数研究认为这些是由LC决定的，但根据目前对于重链的研究，认为这很可能是3种结构域共同作用的结果，而不单是某一个单独结构域作用的结果。

基金

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病原微生物生物安全全国重点实验室课题(SKLPBS2223)

参考文献

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文献

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参考文献引证文献

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GARCIA-RODRIGUEZ C, YAN SD, GEREN IN, KNOPP KA, DONG JB, SUN ZD, LOU JL, CONRAD F, WEN WH, FARR-JONES S, SMITH TJ, BROWN JL, SKERRY JC, SMITH LA, MARKS JD.A four-monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotypes C and D[J].Toxins,2021,13(9):641.

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文章信息

doi: 10.13343/j.cnki.wsxb.20230603

接收时间：2023-09-25
首发时间：2026-03-19
出版时间：2024-07-04

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收稿日期：2023-09-25
录用日期：2024-03-13

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State Key Laboratory of Pathogen and Biosecurity(SKLPBS2223)

病原微生物生物安全全国重点实验室课题(SKLPBS2223)

作者信息

1 牡丹江医学院公共卫生学院, 黑龙江牡丹江 157011

2 军事科学院军事医学研究院微生物流行病研究所病原微生物生物安全全国重点实验室, 北京 100071

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Subtype	A1	A2	A3	A4	A5	A6	A7	A8
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/A isoforms. For H_C alignment, residues 870−1 296 of the BoNT/A1 sequence were used. The sequence numbers of BoNT/A1−BoNTA8 in NCBI were CAL82360.1^[45], CAA51824.1^[46], ACA57525.1^[47], ACQ51417.1^[47], ACG50065.1^[48], ACW83608.1^[49], AFV13854.1^[50], and AJA05787.1^[51].
A1	−	87.29	86.82	91.55	93.90	90.61	91.78	87.79
A2	89.97	−	98.83	88.47	89.65	90.12	90.35	93.43
A3	84.65	93.13	−	88.24	88.94	89.65	89.88	92.72
A4	89.35	83.35	84.49	−	86.85	88.94	85.92	90.14
A5	97.15	90.35	85.11	87.50	−	95.43	92.72	89.91
A6	95.68	91.67	86.27	87.89	95.83	−	91.08	87.32
A7	93.75	89.74	84.88	86.81	94.37	92.98	−	89.67
A8	93.51	93.52	87.81	89.34	93.59	93.20	91.36	−

Subtype

Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/A isoforms. For H_C alignment, residues 870−1 296 of the BoNT/A1 sequence were used. The sequence numbers of BoNT/A1−BoNTA8 in NCBI were CAL82360.1^[45], CAA51824.1^[46], ACA57525.1^[47], ACQ51417.1^[47], ACG50065.1^[48], ACW83608.1^[49], AFV13854.1^[50], and AJA05787.1^[51].

−

87.29

86.82

91.55

93.90

90.61

91.78

87.79

89.97

−

98.83

88.47

89.65

90.12

90.35

93.43

84.65

93.13

−

88.24

88.94

89.65

89.88

92.72

89.35

83.35

84.49

−

86.85

88.94

85.92

90.14

97.15

90.35

85.11

87.50

−

95.43

92.72

89.91

95.68

91.67

86.27

87.89

95.83

−

91.08

87.32

93.75

89.74

84.88

86.81

94.37

92.98

−

89.67

93.51

93.52

87.81

89.34

93.59

93.20

91.36

−

Monosaccharide	H_C/A1: GD1a	H_C/A1: GT1b	H_C/A2: GD1a	H_C/A3: GD1a	H_C/A4: GD1a	H_C/A5: GM1b	H_C/A6: GD1a
Hydrogen bond distances for ganglioside binding in the structure H_C/A1: GD1a, H_C/A1: GT1b, H_C/A2: GD1a, H_C/A3: GD1a, H_C/A4: GD1a, H_C/A5: GM1b, and H_C/A6: GD1a. Water-mediated interactions are indicated in italics as “-H₂O molecules (n1, n2)”, where n1 is the distance between amino acid residues and water and n2 is the distance between water and monosaccharides.
Sia⁶	Trp 1 266 (3.5)	Trp 1 266 (3.1) Gln 1 270-H₂O (2.6, 2.5) Arg 1 276 (3.1)	Trp 1 266 (3.5)	Unmodelled	Unmodelled	Unmodelled	Unmodelled
Sia⁵	Tyr 1 117 (2.9) Tyr 1 267-H₂O (2.5, 3.5) Gly 1 279-H₂O (2.6, 2.8) Arg 1 276-H₂O (2.8, 2.8)	Tyr 1 117 (2.8, 3.0) Ser 1 275 (3.2) Arg 1 276-H₂O (3.1, 2.7) Gly 1 279-H₂O (2.7, 2.7)	Tyr 1 267-H₂O (2.5) Leu 1 250-H₂O (3.1, 2.8)	Tyr 1 263 (2.7) Gly 1 275 (2.9) Leu 1 250-H₂O (2.9, 2.8)	Tyr 1 123 (2.8) Tyr 1 273 (2.5) Gly 1 285 (3.1) Arg 1 282 (3.8)	Tyr 1 117 (2.8) Tyr 1 267 (2.7) Gly 1 279 (3.2)	Tyr 1 267 (2.6) Gly 1 279 (2.9)
Gal⁴	Glu 1 203 (2.8) Phe 1 252 (2.7) His 1 253 (2.7) Ser 1 264 (2.8)	Glu 1 203 (2.7) Phe 1 252 (2.6) His 1 253 (2.8) Gln 1 254-H₂O (2.6, 2.5) Ser 1 264 (2.7)	Glu 1 203 (2.6) Phe 1 252 (2.5) His 1 253 (2.9) Leu 1 254-H₂O (2.9, 3.1) Ser 1 264 (2.7)	Glu 1 199 (2.7) Phe 1 248 (2.5) His 1 249 (3.1) Ser 1 260 (2.7) Leu 1 250-H₂O (2.9, 3.0)	Glu 1 209 (2.4) Phe 1 258 (2.8) His 1 259 (2.7) Ser 1 270 (2.5)	Glu 1 203 (2.6) Phe 1 252 (2.8) His 1 253 (3.1) Ser 1 264 (2.9)	Glu 1 203 (2.7) Phe 1 252 (2.7) His 1 253 (3.0) Ser 1 264 (2.7)
GalNAc³	Glu 1 203 (2.5)	Glu 1 203 (2.6) Arg 1 269-H₂O (2.9, 3.1)	Glu 1 203 (2.7)	Glu 1 199 (2.5)	Glu 1 209 (2.6)		Glu 1 203 (2.5)

Monosaccharide

H_C/A1: GD1a

H_C/A1: GT1b

H_C/A2: GD1a

H_C/A3: GD1a

H_C/A4: GD1a

H_C/A5: GM1b

H_C/A6: GD1a

Hydrogen bond distances for ganglioside binding in the structure H_C/A1: GD1a, H_C/A1: GT1b, H_C/A2: GD1a, H_C/A3: GD1a, H_C/A4: GD1a, H_C/A5: GM1b, and H_C/A6: GD1a. Water-mediated interactions are indicated in italics as “-H₂O molecules (n1, n2)”, where n1 is the distance between amino acid residues and water and n2 is the distance between water and monosaccharides.

Sia⁶

Trp 1 266 (3.5)

Trp 1 266 (3.1)
Gln 1 270-H₂O
(2.6, 2.5)
Arg 1 276 (3.1)

Trp 1 266 (3.5)

Unmodelled

Sia⁵

Tyr 1 117 (2.9)
Tyr 1 267-H₂O (2.5, 3.5)
Gly 1 279-H₂O
(2.6, 2.8)
Arg 1 276-H₂O
(2.8, 2.8)

Tyr 1 117
(2.8, 3.0)
Ser 1 275 (3.2)
Arg 1 276-H₂O
(3.1, 2.7)
Gly 1 279-H₂O (2.7, 2.7)

Tyr 1 267-H₂O (2.5)
Leu 1 250-H₂O (3.1, 2.8)

Tyr 1 263 (2.7)
Gly 1 275 (2.9)
Leu 1 250-H₂O (2.9, 2.8)

Tyr 1 123 (2.8)
Tyr 1 273 (2.5)
Gly 1 285 (3.1)
Arg 1 282 (3.8)

Tyr 1 117 (2.8)
Tyr 1 267 (2.7)
Gly 1 279 (3.2)

Tyr 1 267 (2.6)
Gly 1 279 (2.9)

Gal⁴

Glu 1 203 (2.8)
Phe 1 252 (2.7)
His 1 253 (2.7)
Ser 1 264 (2.8)

Glu 1 203 (2.7)
Phe 1 252 (2.6)
His 1 253 (2.8)
Gln 1 254-H₂O (2.6, 2.5)
Ser 1 264 (2.7)

Glu 1 203 (2.6)
Phe 1 252 (2.5)
His 1 253 (2.9)
Leu 1 254-H₂O (2.9, 3.1)
Ser 1 264 (2.7)

Glu 1 199 (2.7)
Phe 1 248 (2.5)
His 1 249 (3.1)
Ser 1 260 (2.7)
Leu 1 250-H₂O (2.9, 3.0)

Glu 1 209 (2.4)
Phe 1 258 (2.8)
His 1 259 (2.7)
Ser 1 270 (2.5)

Glu 1 203 (2.6)
Phe 1 252 (2.8)
His 1 253 (3.1)
Ser 1 264 (2.9)

Glu 1 203 (2.7)
Phe 1 252 (2.7)
His 1 253 (3.0)
Ser 1 264 (2.7)

GalNAc³

Glu 1 203 (2.5)

Glu 1 203 (2.6)
Arg 1 269-H₂O (2.9, 3.1)

Glu 1 203 (2.7)

Glu 1 199 (2.5)

Glu 1 209 (2.6)

Glu 1 203 (2.5)

Subtype	B1	B2	B3	B4	B5	B6	B7	B8
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/B isoforms. For H_C alignment, residues 862−1 291 of the BoNT/B1 sequence were used. The sequence numbers of BoNT/B1 to /B8 in NCBI were APH17270.1^[67], RUT52409.1^[68], WP_061310563.1^[69], CAA50482.1^[70], BDB03763.1^[71], BAO05199.1^[72], WP_129265872.1^[69], and AFN61309.1^[73].
B1	−	91.78	93.15	89.04	94.98	93.82	90.41	92.24
B2	91.78	−	95.78	89.97	91.56	96.56	90.41	93.68
B3	95.97	98.22	−	89.27	92.92	96.34	92.92	93.14
B4	92.80	92.78	92.95	−	88.13	88.79	89.27	87.44
B5	96.20	95.27	95.74	92.41	−	92.91	90.41	91.78
B6	96.20	98.22	98.22	92.49	95.51	−	92.65	93.19
B7	94.81	95.82	95.74	92.72	94.42	91.99	−	90.64
B8	95.51	95.74	95.90	92.18	94.74	92.69	90.64	−

Subtype

Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/B isoforms. For H_C alignment, residues 862−1 291 of the BoNT/B1 sequence were used. The sequence numbers of BoNT/B1 to /B8 in NCBI were APH17270.1^[67], RUT52409.1^[68], WP_061310563.1^[69], CAA50482.1^[70], BDB03763.1^[71], BAO05199.1^[72], WP_129265872.1^[69], and AFN61309.1^[73].

−

91.78

93.15

89.04

94.98

93.82

90.41

92.24

91.78

−

95.78

89.97

91.56

96.56

90.41

93.68

95.97

98.22

−

89.27

92.92

96.34

92.92

93.14

92.80

92.78

92.95

−

88.13

88.79

89.27

87.44

96.20

95.27

95.74

92.41

−

92.91

90.41

91.78

96.20

98.22

92.49

95.51

−

92.65

93.19

94.81

95.82

95.74

92.72

94.42

91.99

−

90.64

95.51

95.74

95.90

92.18

94.74

92.69

90.64

−

Subtype	E1	E2	E3	E4	E5	E6	E7	E8	E9	E10	E11	E12
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/E isoforms. For H_C alignment, residues 848−1 252 of the BoNT/E1 sequence were used. The sequence numbers of BoNT/E1 to/E12 in NCBI were E1: ADF42615.1^[92], E4: APF24160.1^[93], E10: AII82313.1^[93], and E11: AII82281.1^[94]. The sequence numbers in Uniprot were E2: A2I2S6^[95], E3: A2I2S5^[95], E5: Q9K395^[96], E6: A8Y878^[97], E7: G8I2N7^[95], E8: G8I2N8^[95], E9: K7S9Y2^[95], and E12: W8FNB6^[98].
E1	−	97.35	100	97.78	92.82	97.28	100	97.28	83.17	95.31	93.58	87.65
E2	99.12	−	97.53	97.28	91.11	96.79	97.53	99.75	84.16	96.54	95.31	88.40
E3	98.24	97.36	−	97.78	92.59	97.28	100	99.75	83.17	95.31	93.58	87.65
E4	97.36	97.12	95.69	−	90.62	99.51	97.78	97.53	82.67	94.81	93.09	86.67
E5	96.88	96.41	95.21	95.05	−	90.84	92.82	91.09	83.91	88.86	89.60	90.84
E6	97.04	96.81	95.93	96.96	94.89	−	97.28	97.04	82.67	93.83	93.09	86.88
E7	97.92	97.12	97.36	96.25	94.81	96.41	−	97.28	83.17	94.57	93.58	87.87
E8	96.25	97.04	95.69	96.17	94.09	96.81	98.32	−	83.91	96.05	95.56	88.37
E9	89.13	89.37	88.73	90.01	89.52	88.25	89.21	89.45	−	83.66	85.15	88.37
E10	95.61	95.93	95.13	95.13	93.16	95.85	96.88	97.84	89.21	−	97.04	87.38
E11	93.37	93.85	92.57	92.73	91.93	93.13	93.45	94.41	89.05	95.29	−	87.62
E12	92.97	93.13	92.57	92.57	93.16	91.13	92.49	92.01	91.52	91.77	91.05	−

Subtype

E10

E11

E12

Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/E isoforms. For H_C alignment, residues 848−1 252 of the BoNT/E1 sequence were used. The sequence numbers of BoNT/E1 to/E12 in NCBI were E1: ADF42615.1^[92], E4: APF24160.1^[93], E10: AII82313.1^[93], and E11: AII82281.1^[94]. The sequence numbers in Uniprot were E2: A2I2S6^[95], E3: A2I2S5^[95], E5: Q9K395^[96], E6: A8Y878^[97], E7: G8I2N7^[95], E8: G8I2N8^[95], E9: K7S9Y2^[95], and E12: W8FNB6^[98].

−

97.35

100

97.78

92.82

97.28

100

97.28

83.17

95.31

93.58

87.65

99.12

−

97.53

97.28

91.11

96.79

97.53

99.75

84.16

96.54

95.31

88.40

98.24

97.36

−

97.78

92.59

97.28

100

99.75

83.17

95.31

93.58

87.65

97.36

97.12

95.69

−

90.62

99.51

97.78

97.53

82.67

94.81

93.09

86.67

96.88

96.41

95.21

95.05

−

90.84

92.82

91.09

83.91

88.86

89.60

90.84

97.04

96.81

95.93

96.96

94.89

−

97.28

97.04

82.67

93.83

93.09

86.88

97.92

97.12

97.36

96.25

94.81

96.41

−

97.28

83.17

94.57

93.58

87.87

96.25

97.04

95.69

96.17

94.09

96.81

98.32

−

83.91

96.05

95.56

88.37

89.13

89.37

88.73

90.01

89.52

88.25

89.21

89.45

−

83.66

85.15

88.37

E10

95.61

95.93

95.13

93.16

95.85

96.88

97.84

89.21

−

97.04

87.38

E11

93.37

93.85

92.57

92.73

91.93

93.13

93.45

94.41

89.05

95.29

−

87.62

E12

92.97

93.13

92.57

93.16

91.13

92.49

92.01

91.52

91.77

91.05

−

Subtype	F1	F2	F3	F4	F5	F6	F7	F8	F9
Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/F isoforms. For H_C alignment, residues 866−12 778 of the BoNT/F1 sequence were used. The sequence numbers of BoNT/F1−BoNT/F9 in NCBI were F1: WP_011987710.1^[69], F2: UYX45882.1^[100], F3: APQ97862.1^[100], F4: APQ71923.1^[101], F5: APR02750.1^[101], F6: ADU57954.1^[102], F7: AQA28590.1^[103], F8: KEJ01913.1^[104], and F9: AQA28591.1^[105].
F1	−	82.97	84.63	89.29	83.94	82.51	79.56	98.05	84.71
F2	83.71	−	96.37	81.51	92.03	93.89	72.02	83.70	90.34
F3	84.25	97.19	−	82.44	93.46	93.64	73.17	84.39	92.49
F4	92.33	83.71	84.09	−	82.00	81.03	76.40	88.56	83.45
F5	70.31	74.37	74.35	69.84	−	90.22	73.48	84.18	92.03
F6	88.05	90.02	90.04	87.42	74.11	−	72.17	82.27	88.75
F7	74.43	69.53	69.91	72.77	64.45	70.84	−	79.32	73.48
F8 F9	96.24 84.27	83.71 89.92	84.17 81.63	93.19 84.03	69.84 73.75	87.81 87.37	73.01 69.85	− 84.18	84.67 −

Subtype

Table shows the percentage of full-length and H_C domain sequence alignments for BoNT/F isoforms. For H_C alignment, residues 866−12 778 of the BoNT/F1 sequence were used. The sequence numbers of BoNT/F1−BoNT/F9 in NCBI were F1: WP_011987710.1^[69], F2: UYX45882.1^[100], F3: APQ97862.1^[100], F4: APQ71923.1^[101], F5: APR02750.1^[101], F6: ADU57954.1^[102], F7: AQA28590.1^[103], F8: KEJ01913.1^[104], and F9: AQA28591.1^[105].

−

82.97

84.63

89.29

83.94

82.51

79.56

98.05

84.71

83.71

−

96.37

81.51

92.03

93.89

72.02

83.70

90.34

84.25

97.19

−

82.44

93.46

93.64

73.17

84.39

92.49

92.33

83.71

84.09

−

82.00

81.03

76.40

88.56

83.45

70.31

74.37

74.35

69.84

−

90.22

73.48

84.18

92.03

88.05

90.02

90.04

87.42

74.11

−

72.17

82.27

88.75

74.43

69.53

69.91

72.77

64.45

70.84

−

79.32

73.48

F8
F9

96.24
84.27

83.71
89.92

84.17
81.63

93.19
84.03

69.84
73.75

87.81
87.37

73.01
69.85

−
84.18

84.67
−