Jean Massoulié

Molecular and cellular biology of cholinesterases

Abbreviations

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non‐amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N‐glycans of the ‘complex’ and ‘hybrid’ types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non‐denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

Cholinesterases: Tissue and Cellular Distribution of Molecular Forms and Their Physiological Regulation

In Chapter 8 a we have described the molecular structure and the interactions of the multiple molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) in vertebrates. We show there that the two enzymes present globular forms (G1 G2, G4) which exist as non-hydrophobic as well as amphiphilic molecules, and asymmetric, collagen-tailed molecules (A4, A8, A12). These molecular forms may be further subdivided according to the binding of lectins or according to their electrophoretic migrations in nondenaturing electrophoresis.

Amphiphilic, glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase

Summary1.In a recent study, we distinguished two classes of amphiphilic AChE dimers inTorpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent.2.In the present report, we analyze the amphiphilic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles.3.We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers ofTorpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationships with other cholinesterase molecular forms.

The catecholamine transporter of adrenal medulla chromaffin granules.

The hormone concentration in the secretory granules of endocrine cells is very large. In adrenal medulla, the concentration of adrenaline and noradrenaline in chromaffin granules has been estimated to be 0.5 M, indicating a concentration gradient between the organelle matrix and the cytoplasm of 5 orders of magnitude. Such a gradient results from a well-described ATP-dependent transport, involving two different steps (for a review, see refs. 1-4). In the first step, an ATP-dependent H+ pump of the V-type generates an H+-electrochemical gradient, the matrix becoming acidic and positively charged with respect to the cytoplasm. In the second step, a transporter, the vesicular monoamine transporter, uses the H+-electrochemical gradient to drive monoamine uptake by catalyzing an electrogenic H+/monoamine antiport. The vesicular monoamine transporter has a very low substrate specificity, and it will transport all monoamines (TABLE l), those present in adrenal medulla, such as adrenaline, noradrenaline, or dopamine, as well as others, such as serotonin or histamine. We have shown that it also transports other unrelated components, such as meta-iodobenzylguanidine (MIBGY or the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+).6 Advantage has been taken of these transports: MIBG, accumulated in the secretory pool, has allowed imaging of pheochromocytoma or neuroblastoma by scintigraphy; and MPP+ transport has allowed expression cloning of the vesicular transporter. Several drugs inhibit monoamine uptake by interacting directly with the vesicular transporter. These inhibitors have been developed as ligands of the monoamine

Biosynthesis of the Molecular Forms of Acetylcholinesterase

Vertebrates possess a variety of forms of acetylcholinesterase (AChE), as schematically illustrated in Figure 1. These forms present the same catalytic activity, but differ in their quaternary structure and in their interactions with membranes or with the extra-cellular matrix (basal lamina) (for reviews, see Massoulie and Bon, 1982; Massoulie and Toutant, 1988; Massoulie et al., in press). The catalytic subunits are generated from a single gene, they undergo various post-translational modifications and in some cases associate with structural subunits. Alternative splicing, together with the use of several transcription origins and polyadenylation signals, generate multiple mRNAs (Sikorav et al., 1987, 1988; Schumacher et al., 1988; Maulet et al., 1990). The coding sequence, however, seems to be modified only by alternative splicing of its 3 region. In Torpedo, the major part of this coding sequence, common to all mRNAs, is included in a large exon I (1678 nucleotides, from the initiation codon of T. marmorata AChE) and a small exon H (167 nucleotides) (Maulet et al., 1990). Exon II may be spliced to either exon IIIH or exon IIIT, generating the two kinds of catalytic subunits, H and T. These subunits constitute, respectively, the two main molecular forms occurring in the electric organ: the glycolipid (GPI)-anchored dimers and the collagen-tailed asymmetric forms. We do not designate the alternative exons and the corresponding catalytic subunits by the same letters as the globular (G) and asymmetric (A) molecular forms, because the distinction based on the general quaternary structure of the molecules does not coincide with the nature of their catalytic subunits: while collagen-tailed forms A forms contain only T subunits, different types of globular G forms may include either H or T subunits. In addition, a third type of catalytic subunit may be generated by an mRNA structure in which exon II is continued by the adjacent genomic sequence.

Unusual transfer of CutA into the secretory pathway, evidenced by fusion proteins with acetylcholinesterase

The mouse CutA protein exists as long and short components of 20 and 15u2003kDa, produced by the use of different in‐frame ATGs initiation codons, and by proteolytic cleavage. We recently showed that, surprisingly, the longer, uncleaved component resides mostly in the secretory pathway and is secreted, whereas the shorter component resides mostly in the cytoplasm. To confirm these subcellular localizations, we constructed fusion proteins in which the catalytic domain of rat acetylcholinesterase was placed downstream of the CutA variants. The acquisition of an active conformation and N‐glycosylation of the fusion proteins proved their transfer into the secretory pathway. We show that the CutA‐AChE fusion proteins produced and secreted active, N‐glycosylated molecules, while an AChE mutant lacking its secretory signal peptide did not produce any significant activity. Thus, an N‐terminal CutA domain actually drives AChE into the endoplasmic reticulum and allows its secretion. This was observed with full length CutA, starting at Met1, and at a much lower level with the shorter mutants starting at Met24 and Met44, although the latter is not predicted to possess any signal peptide. These experiments illustrate the value of using AChE as a reporter and reveals an unusual protein trafficking and secretory process.

Acetylcholinesterase in Elapid Snakes

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission, but is also present in non-cholinergic contexts where its function is not clearly known (1). This is the case in pulmonary and intestinal epitheliums, salivary glands, or blood cells surface. AChE is a very abundant protein in the venom of some Elapids snakes (2–3).