A cataract‐causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain

Abstract

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231–294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

A novel Cx50 (GJA8) p.H277Y mutation associated with autosomal dominant congenital cataract identified with targeted next-generation sequencing

“…Studies of certain connexin mutants linked to congenital cataracts have implicated hemichannels with aberrant voltage-dependent gating or modulation by divalent cations in disease pathogenesis [25]. Further deletion analysis has shown that the fourth transmembrane domain (M4) and a membrane proximal region (codons 231-294) of the cytoplasmic domain are needed for transport from the endoplasmic reticulum and localization to the plasma membrane [30]. However, unlike previously characterized Cx50 mutants that exhibited impaired intracellular transport and subsequent insertion into the plasma membrane and/or lack of function, the Cx50 mutant p.G46V with functional hemichannels and gap junction channels causes cell death even when expressed at minute levels.…”

“…Since there are many different derangements in the cellular/ biochemical behavior and function of the Cx50 mutants, further functional impact of the mutant protein on cell growth [16,42] or hemichannels voltage-dependent gating [30,43], will improve our understanding of the underlying pathogenesis of cataract formation and illuminate the role of the connexin family in the lens. Also, since this is a dominant disease, it would be really interesting to assess whether the mutant protein dimerize with the wild-type form would impede the proper localization or function of the wild-type [44], which would help to design possible genetic therapies in the future.…”

Evaluating gap junction variants for a role in pediatric cataract: an overview of the genetic landscape and clinical classification of variants in the GJA3 and GJA8 genes

“…The second was an autosomal recessive c.607dup p.(Thr203Asnfs*47) change reported in two affected children in an Indian family, with heterozygous parents and relatives all presenting as unaffected [129]. This variant resulted in the loss of the second extracellular domain through to the C-terminal protein tail, with both this variant and a truncated p.Thr203Ter protein shown to result in disrupted localization to the plasma membrane and retention in the endoplasmic reticulum [130].…”