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

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

K Ohno et al. Proc Natl Acad Sci U S A. 1998.

Abstract

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHET) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHET attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHET gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHET or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHET. We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ, and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHET in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHET prevents association of ColQ with ACHET; mutations distal to the attachment domain generate a mutant approximately 10.5S species of AChE composed of one ACHET tetramer and a truncated ColQ strand. The approximately 10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Density gradient centrifugation of AChE extracted from muscle in control subject (A) and EAD patients 1, 4, 5, and 6 (B_–_E). Mutations in each case are indicated. A and G denote asymmetric and globular species. In each patient extract, the heteromeric A12 and A8 species are absent and the composite G4/A4 peak is replaced by a single mutant ≈10.5S peak. (M, mutant peak.)

Figure 2

Figure 2

cDNA sequence of the human COLQ gene (GenBank accession no. AF057036) and truncation mutations in the patients. Mutations are underlined. The 107del215 mutation results from skipping of exons 2 and 3. Vertical lines indicate exon boundaries. The left and right columns show nucleotide and codon numbers from the translational start site. The 3′ untranslated region is not shown.

Figure 3

Figure 3

Alignment of the amino acid sequences of human, rat (14), partial mouse (14), and Torpedo (13) ColQ and truncation mutations in the patients. Gaps are inserted for alignment. The amino acid sequence encoded by the alternatively transcribed exon 1A also is shown. Closed arrowheads point to the first amino acid deleted by the truncation mutations. Dashes show identical amino acids. Secretion signal peptides are italicized for all species. Collagen domains consisting of GXY triplets are underlined. A black bar indicates the PRAD. Hatched bars indicate heparan sulfate proteoglycan binding domains. Open arrowheads show essential cysteines. The right column indicates codon numbers.

Figure 4

Figure 4

Genomic structure of the human COLQ gene. Exons (boxes) and introns (horizontal lines) are drawn to scale. Splicing marks indicate seven alternative transcripts found in normal controls. Hatched areas show untranslated regions. Closed arrowheads indicate point mutations; black bar indicates skipping of exons 2 and 3.

Figure 5

Figure 5

Chromosomal localization of COLQ by FISH. In situ hybridization was performed by using a digoxigenin-labeled human COLQ cDNA probe and normal metaphase cells from stimulated blood culture. (A) The digitized image of the COLQ probe signal is localized on both chromatids of 4′,6-diamidino-2-phenylindole-enhanced G-banded chromosome 3 from reverse imaging by the Vysis

smartcapture

system (Vysis, Downers Grove, IL). (B) COLQ is mapped to 3p25

Figure 6

Figure 6

Restriction analysis (A, B, D, and F) and allele-specific PCR (ASP) (C and E) using genomic DNA from patients and their respective relatives. (A to F) Families of patients 1 to 6, respectively. Closed and open arrowheads point to mutant and wild-type fragments. Closed symbols and arrows indicate patients; half-shaded symbols represent asymptomatic carriers. The −46G/A polymorphism in A is linked to the 107del215 mutation.

Figure 7

Figure 7

Sedimentation profiles of AChE species extracted from COS cells transfected with wild-type _ACHE_T and COLQ (A), wild-type _ACHE_T (B), and with wild-type _ACHE_T plus COLQ mutants (C_–_H). Solid lines in all panels indicate sedimentation in the presence of 0.5% Triton X-100; interrupted lines in A and E show sedimentation in the presence of 1% Brij-97. (M, mutant peak.)

Comment in

Similar articles

Cited by

References

    1. Katz B, Miledi R. J Physiol (London) 1973;231:549–574. - PMC - PubMed
    1. Katz B, Thesleff S. J Physiol (London) 1957;138:63–80. - PMC - PubMed
    1. Maselli R A, Soliven B C. Muscle Nerve. 1991;14:1182–1188. - PubMed
    1. Salpeter M M, Kasprzak H, Feng H, Fertuck H. J Neurocytol. 1979;8:95–115. - PubMed
    1. Dettbarn W D. Fundam Appl Toxicol. 1984;4:S18–S26. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources