Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

Abstract

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

From cyanobacteria to plants: conservation of PII functions during plastid evolution

“…These proteins form a widespread superfamily of trimeric proteins with potential regulatory roles and occur in almost all known organisms (Kinch and Grishin 2002;Arnesano et al 2003;Saikatendu et al 2006). While in most cases their function is unknown, in some cases they are known to be involved in diverse functions such as copper tolerance (Arnesano et al 2003) or anchoring acetylcholinesterase in mammalian neurons (Perrier et al 2000). Whether these proteins are evolutionary related to PII proteins or not remains to be clarified.…”

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

“…In addition, there is no obvious correlation between the expression of CutA and that of membrane-bound AChE, because CutA is expressed in brain and also in all other mammalian tissues. However, stable transfection with an antisense construct designed to block expression of CutA was found to suppress the membrane anchoring of AChE in a murine neuroblastoma cell line, N18TG2 (7). Unless some other modification occurred during the derivation of this cell line, this suggested that CutA might play a role in the assembly of AChE T subunits with PRiMA.…”

“…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). This protein was called CutA because of its homology with a bacterial protein (Cu 2ϩ tolerance A), derived from an operon involved in resistance to copper and other divalent metal ions (8).…”

“…The presence of both CutA and PRiMA in AChE preparations was established by sequencing tryptic peptides and detection in Western blots by different antibodies; in contrast with PRiMA, CutA was not disulfide-linked to AChE subunits, as shown by electrophoresis in SDS-polyacrylamide gels under nonreducing conditions (7).…”

CutA Divalent Cation Tolerance Homolog (Escherichia coli) (CUTA) Regulates β-Cleavage of β-Amyloid Precursor Protein (APP) through Interacting with β-Site APP Cleaving Protein 1 (BACE1)

“…The mammalian CutA divalent cation tolerance homolog (Escherichia coli), CUTA, was discovered in a search for the membrane anchor of acetylcholinesterase (AChE), an enzyme that degrades acetylcholine and an important target for inhibition during AD treatment (16). Although it was later determined that CUTA does not directly interact with AChE and that the real membrane anchor protein of AChE is PRiMA (17), mouse CUTA was recently found to affect the processing and trafficking of AChE (18).…”