PRiMA

106

128

To explore the scope and significance of alternate promoter usage and its putative inter-relationship to alternative splicing, we searched expression sequence tags for the 5 region of acetylcholinesterase (ACHE) genes. Three and five novel first exons were identified in human and mouse ACHE genes, respectively. Reverse transcription-PCR and in situ hybridization validated most of the predicted transcripts, and sequence analyses of the corresponding genomic DNA regions suggest evolutionarily conserved promoters for each of the novel exons identified. Distinct tissue specificity and stress-related expression patterns of these exons predict combinatorial complexity with known 3 alternative AChE mRNA transcripts. Unexpectedly one of the 5 exons encodes an extended N terminus in-frame with the known AChE sequence, extending the increased complexity to the protein level. The resultant membrane variant(s), designated N-AChE, is developmentally regulated in human brain neurons and blood mononuclear cells. Alternative promoter usage combined with alternative splicing may thus lead to stress-dependent combinatorial complexity of AChE mRNA transcripts and their protein products.Alternative splicing and alternate promoter usage both expand the complexity of gene products. While the massive contribution of alternative splicing to such expansion is widely recognized (1), less is known about the scope and significance of alternate promoter usage. Moreover the directionality of transcription processes raises the yet unresolved possibility that these two phenomena are inter-related, namely that the choice of the first exon determines downstream splice choices (2). The recent accumulation of genomic and gene expression data bases together with the development of sophisticated bioinformatic tools makes these questions amenable for experimental analysis as multiple gene products display both alternate promoter usage and alternative splicing variations. By alignment of expression sequence tags (ESTs) 1 against genomic sequences, for example, it is possible to explore the different alternatively spliced products of a single gene (3, 4). However, EST data bases are biased toward the 3Ј end of mRNAs and occasionally contain genomic contaminations that may cause misinterpretation of the genomic information (5). To correctly evaluate the inter-relationship between alternate promoter usage and alternative splicing, it is therefore necessary to characterize the identified variants using traditional molecular biology tools at the RNA level and, if applicable, at the protein level as well.The acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE) provides an adequate example for such a study. AChE pre-mRNA is subject to stimulus-induced 3Ј alternative splicing (6), and previous evidence has suggested that it is also subject to alternate promoter usage (7,8). We analyzed the genomic regions flanking the human and mouse ACHE genes and found an unexpected, evolutionarily conserved diversity of alternate exons at their 5Ј end. The newly id...

Section: Discussionmentioning

confidence: 99%

Combinatorial Complexity of 5′ Alternative Acetylcholinesterase Transcripts and Protein Products

Meshorer

Toiber

Zurel

et al. 2004

106

128

“…Because CutA was discovered in connection with membrane-bound AChE T tetramers, which are associated with the transmembrane protein PRiMA, we analyzed the possible effect of CutA on the formation of tetramers induced by PRiMA or by another AChE-associated protein, Qs, which also interacts with the t peptides through a proline-rich motif (5,19,26,30). In co-transfected COS cells, CutA did not appear to affect the recruitment of AChE T subunits into tetramers by these proteins.…”

Section: In Mammals Cuta Produces Several Variants Differing Inmentioning

confidence: 99%

“…In these hetero-oligomers, four catalytic subunits, corresponding to the AChE T variant that possesses a C-terminal t peptide (2), are associated with a hydrophobic 20-kDa protein (3,4). This protein has now been cloned and named PRiMA (proline-rich membrane anchor) (5). However, before the characterization of PRiMA, another protein was identified independently by different groups as a component of AChE preparations purified from mammalian brain (6,7).…”

mentioning

confidence: 99%

Protein CutA Undergoes an Unusual Transfer into the Secretory Pathway and Affects the Folding, Oligomerization, and Secretion of Acetylcholinesterase

Liang

Nunes-Tavares

Xie

et al. 2009

Self Cite

The mammalian protein CutA was first discovered in a search for the membrane anchor of mammalian brain acetylcholinesterase (AChE). It was co-purified with AChE, but it is distinct from the real transmembrane anchor protein, PRiMA. CutA is a ubiquitous trimeric protein, homologous to the bacterial CutA1 protein that belongs to an operon involved in resistance to divalent ions ("copper tolerance A"). The function of this protein in plants and animals is unknown, and several hypotheses concerning its subcellular localization have been proposed. We analyzed the expression and the subcellular localization of mouse CutA variants, starting at three in-frame ATG codons, in transfected COS cells. We show that CutA produces 20-kDa (H) and 15-kDa (L) components. The H component is transferred into the secretory pathway and secreted, without cleavage of a signal peptide, whereas the L component is mostly cytosolic. We show that expression of the longer CutA variant reduces the level of AChE, that this effect depends on the AChE C-terminal peptides, and probably results from misfolding. Surprisingly, CutA increased the secretion of a mutant possessing a KDEL motif at its C terminus; it also increased the formation of AChE homotetramers. We found no evidence for a direct interaction between CutA and AChE. The longer CutA variant seems to affect the processing and trafficking of secretory proteins, whereas the shorter one may have a distinct function in the cytoplasm.

“…Heteromeric complexes containing ColQ are attached to the basal lamina at neuromuscular junctions, whereas complexes containing PRiMA are anchored in cell membranes and represent the major AChE species in the brain (3).…”

mentioning

confidence: 99%

“…Although AChE T and other AChE variants can form dimers through an association zone located in the catalytic domain (the "four-helix bundle," formed by two helices from each subunit) and an intercatenary disulfide bond through a C-terminal cysteine (5), only AChE T subunits form tetramers and associate with anchoring proteins through an assembly of four C-terminal t peptides, also called tryptophan amphiphilic tetramerization domains. Aromatic residues play a major role in the association of four t peptides with proline-rich motifs that exist in the N-terminal regions of ColQ and PRiMA (3,6). These motifs are sufficient for the association with t peptides, and have been called proline-rich attachment domains (or PRADs).…”

mentioning

confidence: 99%

Acetylcholinesterase Associates Differently with Its Anchoring Proteins ColQ and PRiMA

Noureddine

Carvalho

Schmitt

et al. 2008

Self Cite

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE T subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE T subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE T subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE T subunits with the four upstream cysteines of the associated protein.Mammalian cholinergic tissues mostly express the T ("tailed") variant of acetylcholinesterase (AChE T ), 2 in which the C-terminal 40-residue t peptide allows the formation of AChE T tetramers, associated with the collagen ColQ and the transmembrane protein PRiMA (1, 2). Heteromeric complexes containing ColQ are attached to the basal lamina at neuromuscular junctions, whereas complexes containing PRiMA are anchored in cell membranes and represent the major AChE species in the brain (3).We have previously shown that the t peptide forms an amphiphilic ␣-helix, with a sector containing seven aromatic residues that are strictly conserved in all vertebrate cholinesterases (AChE, as well as butyryl cholinesterase (BChE)) (4). Although AChE T and other AChE variants can form dimers through an association zone located in the catalytic domain (the "four-helix bundle," formed by two helices from each subunit) and an intercatenary disulfide bond through a C-terminal cysteine (5), only AChE T subunits form tetramers and associate with anchoring proteins through an assembly of four C-terminal t peptides, also called tryptophan amphiphilic tetramerization domains. Aromatic residues play a major role in the association of four t peptides with proline-rich motifs that exist in the N-terminal regions of ColQ and PRiMA (3, 6). These motifs are sufficient for the association with t peptides, an...