Patients with congenital myasthenia associated with end-plate acetylcholinesterase deficiency show normal sequence, mRNA splicing, and assembly of catalytic subunits.

Abstract

A congenital myasthenic condition has been described in several patients characterized by a deficiency in end-plate acetylcholinesterase (AChE). The characteristic form of AChE in the end-plate basal lamina has the catalytic subunits disulfide linked to a collagen-like tail unit. Southern analysis of the gene encoding the catalytic subunits revealed no differences between patient and control DNA. Genomic DNA clones covering exon 4 and the alternatively spliced exons 5 and 6 were analyzed by nuclease protection and sequencing. Although allelic differences were detected between controls, we found no differences in exonic and intronic areas that might yield distinctive splicing patterns in patients and controls. The ACHE gene was cloned from genomic libraries from a patient and a control. Transfection of the cloned genes revealed identical species of mRNA and expressed AChE. Cotransfection of the genes expressing the catalytic subunits with a cDNA from Torpedo encoding the tail unit yielded asymmetric species that require assembly of catalytic subunits and tail unit. Thus the catalytic subunits of AChE expressed in the congenital myasthenic syndrome appear identical in sequence, arise from similar splicing patterns, and assemble normally with a tail unit to form a heteromeric species. (J. Clin. Invest. 1995. 95:333-340.)

Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme

“…In these patients, the kinetic properties of residual AChE in muscle as well as the activity and kinetic properties of erythrocyte AChE were normal. Mutation analysis of ACHE T in two EAD patients not included in this report revealed no abnormality, and SV40-transformed monkey kidney fibroblast (COS) cells cotransfected with ACHE T cloned from these two patients together with Torpedo COLQ readily expressed asymmetric AChE (18).…”

Quaternary Associations of Acetylcholinesterase

“…Using antibodies directed against Q C , we showed that this COOHterminal region could be removed by collagenase without disrupting the assembly of catalytic tetramers. This experiment suggested that AChE T subunits were linked to the Q N domain (8). We further showed that an isolated Q N domain was sufficient to bind one AChE T tetramer, by constructing a chimeric protein in which Q N is fused to the COOH-terminal glycolipid (GPI) addition signal of the H subunit of Torpedo AChE (H C ).…”

“…We previously cloned the Q subunit from collagen-tailed AChE of Torpedo electric organs (5) and showed that it is able to associate with AChE T subunits of Torpedo, rat, or human AChE, forming collagen-tailed molecules in transfected COS cells (5)(6)(7)(8). The primary sequence deduced from the cDNA encoding Torpedo Q subunits comprises a signal peptide, an NH 2 -terminal domain (Q N ) which contains a pair of adjacent cysteines, a central collagen domain flanked by two pairs of cysteine residues, and a COOH-terminal domain (Q C ).…”

“…We further showed that an isolated Q N domain was sufficient to bind one AChE T tetramer, by constructing a chimeric protein in which Q N is fused to the COOH-terminal glycolipid (GPI) addition signal of the H subunit of Torpedo AChE (H C ). Coexpression of this Q N /H C protein with catalytic T subunits of Torpedo, rat, or human AChE produces GPI-anchored AChE tetramers (8,9).…”

C-terminal and Heparin-binding Domains of Collagenic Tail Subunit Are Both Essential for Anchoring Acetylcholinesterase at the Synapse

“…The causative molecular defects reside in COLQ (21)(22)(23)25) and not in ACHE T (26). Expression studies in COS cells of genetically engineered ColQ mutants along with wild-type AChE T reveal four categories of COLQ mutations (23) as follows: 1) N-terminal mutations that prevent association of AChE T with ColQ; 2) collagen domain truncation mutations that prevent the formation of A 12 ; 3) a CTD missense mutation that prevents triple helical association of ColQ; and 4) CTD mutations that do not abolish A 12 formation but likely affect insertion of ColQ into the synaptic basal lamina.…”