Identification of a Novel Type of Alternatively Spliced Exon from the Acetylcholinesterase Gene of Bungarus fasciatus

Abstract

The venom of the snake Bungarus fasciatus contains a hydrophilic, monomeric species of acetylcholinesterase (AChE), characterized by a C-terminal region that does not resemble the alternative T-or H-peptides. Here, we show that the snake contains a single gene for AChE, possessing a novel alternative exon (S) that encodes the C-terminal region of the venom enzyme, located downstream of the T exon. Alternative splicing generates S mRNA in the venom gland and S and T mRNAs in muscle and liver. We found no evidence for the presence of an H exon between the last common "catalytic" exon and the T exon, where H exons are located in Torpedo and in mammals. Moreover, COS cells that were transfected with AChE expression vectors containing the T exon with or without the preceding genomic region produced exclusively AChE T subunits. In the snake tissues, we could not detect any glycophosphatidylinositol-anchored AChE form that would have derived from H subunits. In the liver, the cholinesterase activity comprises both AChE and butyrylcholinesterase components; butyrylcholinesterase corresponds essentially to nonamphiphilic tetramers and AChE to nonamphiphilic monomers (G 1 na ). In muscle, AChE is largely predominant: it consists of globular forms (G 1 a and G 4 a ) and trace amounts of asymmetric forms (A 8 and A 12 ), which derive from AChE T subunits. Thus, the Bungarus AChE gene possesses alternatively spliced T and S exons but no H exon; the absence of an H exon may be a common feature of AChE genes in reptiles and birds.

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

“…2B). Asp 5 is conserved in all members of the ShKT domain family but is absent in the ICR domains of CRISPs (Fig. 2C).…”

“…2C). In ShK, the carboxylate of this aspartate (Asp 5 in ShK) forms a salt bridge with the ⑀-ammonium group of Lys 30 , and this salt bridge is necessary for proper folding of the peptide (18, 51, 52). Lys 32 and Arg 32 at the equivalent position in MMP23 TxD s from sea anemone, hydra, rat, mouse, and puffer fish ( Fig.…”

“…It has been suggested that toxins evolved from endogenous genes that function in normal cellular pathways (3,4). Indeed, venomous creatures possess toxins with homology to several proteins, including acetylcholinesterases (5), phospholipases (6,7), nerve growth factor (8), endothelins (9), Lynx-1 (10,11), Kunitz-type serine protease inhibitors (12), and the ion channel regulatory (ICR) 5 domains of cysteinerich secretory proteins (CRISPs) (3,13,14). Mammalian proteins containing toxin-like domains (TxDs) that block K ϩ channels have not been characterized previously.…”

Zebrafish Acetylcholinesterase Is Encoded by a Single Gene Localized on Linkage Group 7

“…In nematodes T exon is present in ace-1, whereas H exons are present in the three other ache genes (9). The absence of H exon appears to be frequent since it is lacking in Electrophorus (21), chick (46), or snake genes (47). In zebrafish AChE cDNA and gene, we identified only one kind of 3Ј exon encoding peptide T. The T peptide is characterized by the succession of regularly spaced aromatic residues (see Fig.…”

Testing the Toxicofera: Comparative transcriptomics casts doubt on the single, early evolution of the reptile venom system

“…(Mackessy, 2002), experiments with these secretions shows that several hours are needed to 229 achieve complete neuromuscular blockage. It should also be noted that the most frequently 230 cited sources for the generation of a toxic version of ache in banded krait via alternative 231 splicing include statements that ache "does not appear to contribute to the toxicity of the 232 venom" (Cousin et al, 1998), is "not toxic to mice, even at very high doses" (Cousin et al, 233 1996a) and is "neither toxic by itself nor acting in a synergistic manner with the toxic 234 components of venom" (Cousin et al, 1996b for snake venom cystatins as essential housekeeping or regulatory proteins, rather than 275 specific prey-targeted toxins…" Indeed, it is unclear why cystatins should be considered to be 276 conserved venom toxins, since even the original discovery of cystatin in the venom of the 277 puff adder (Bitis arietans) states that there is "…no evidence that it is connected to the 278 toxicity of the venom" (Ritonja et al, 1987). However, our analysis of these and related sequences suggests that they are likely part of a Kallikrein genes is currently unclear and the majority of these sequences can be found in our 331 serine protease tree (see later section and Supplementary figure S19).…”