黑曼巴蛇（Dendroaspis polylepis）的毒蕈碱毒素

Muscarinic toxins from the black mamba Dendroaspis polylepis.

作者信息

Jolkkonen M, Van Giersbergen P L, Hellman U, Wernstedt C, Oras A, Satyapan N, Adem A, Karlsson E

机构信息

Department of Biochemistry, Biomedical Centre, Uppsala, Sweden.

出版信息

Eur J Biochem. 1995 Dec 1;234(2):579-85. doi: 10.1111/j.1432-1033.1995.579_b.x.

DOI:10.1111/j.1432-1033.1995.579_b.x

PMID:8536706

Abstract

Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins alpha, beta, and gamma (MT alpha, MT beta, and MT gamma). All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MT alpha and MT beta were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60-98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as alpha-neurotoxins, cardiotoxins, and fasciculins. In contrast to the alpha-neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors. The binding constants of MT alpha and MT beta were determined for human muscarinic receptors of subtypes m1-m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps. MT alpha and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31-33), which are Leu-Asn-His in MT alpha and Ile-Val-Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MT alpha has high affinity to all subtypes, with Ki (inhibition constant) values of 23 nM (m1; pKi = 7.64 +/- 0.10), 44 nM (m2; pKi = 7.36 +/- 0.06), 3 nM (m3; pKi = 8.46 +/- 0.14), 5 nM (m4; pKi = 8.32 +/- 0.07), and 8 nM (m5; pKi = 8.09 +/- 0.07). MT4 has high affinity only to m1 (Ki = 62 nM) and m4 (87 nM) receptors, and low (Ki > 1 microM) affinity to m2, m3, and m5. The region at positions 31-33 evidently plays an important role in the toxin-receptor interaction. MT beta has low affinity for m1 and m2 receptors (Ki > 1 microM) and intermediate affinity for m3 (140 nM; pKi = 6.85 +/- 0.03), m4 (120 nM; pKi = 6.90 +/- 0.06), and m5 (350 nM; pKi = 6.46 +/- 0.01). The low affinity of MT beta may reflect a tendency for spontaneous inactivation.

摘要

从黑曼巴蛇（Dendroaspis polylepis）毒液中分离出了三种作用于毒蕈碱受体的新毒素。它们被命名为毒蕈碱毒素α、β和γ（MTα、MTβ和MTγ）。所有这些毒素都有四个二硫键和65或66个氨基酸。已确定MTα和MTβ的序列。从绿曼巴蛇和黑曼巴蛇毒液中已分离出约12种毒蕈碱毒素，它们彼此之间的序列同一性为60 - 98%，并且与许多（约180种）其他蛇毒成分相似，如α-神经毒素、心脏毒素和束丝菌素。与α-神经毒素不同，毒蕈碱毒素不与烟碱型乙酰胆碱受体结合。测定了MTα和MTβ对稳定表达于中国仓鼠卵巢细胞中的m1 - m5亚型人毒蕈碱受体的结合常数。这些毒素的选择性不如早期从绿曼巴蛇（Dendroaspis angusticeps）发现的毒蕈碱毒素。MTα和来自D. angusticeps的毒蕈碱毒素MT4仅在三个氨基酸区域（第31 - 33位残基）存在差异，在MTα中为Leu - Asn - His，在MT4中为Ile - Val - Pro。这种差异导致亚型选择性发生明显变化。MTα对所有亚型都有高亲和力，其抑制常数（Ki）值分别为：m1为23 nM（pKi = 7.64 ± 0.10），m2为44 nM（pKi = 7.36 ± 0.06），m3为3 nM（pKi = 8.46 ± 0.14），m4为5 nM（pKi = 8.32 ± 0.07），m5为8 nM（pKi = 8.09 ± 0.07）。MT4仅对m1（Ki = 62 nM）和m4（87 nM）受体有高亲和力，对m2、m3和m5的亲和力较低（Ki > 1 μM）。第31 - 33位区域显然在毒素与受体的相互作用中起重要作用。MTβ对m1和m2受体的亲和力较低（Ki > 1 μM），对m3（140 nM；pKi = 6.85 ± 0.03）、m4（120 nM；pKi = 6.90 ± 0.06）和m5（350 nM；pKi = 6.46 ± 0.01）具有中等亲和力。MTβ的低亲和力可能反映了其自发失活的倾向。