Analysis of Mannose 6-Phosphate Uncovering Enzyme Mutations Associated with Persistent Stuttering

Wang-Sik Lee,Changsoo Kang,Dennis Drayna,Stuart Kornfeld

doi:10.1074/jbc.m111.295899

Abstract

GlcNAc-1-phosphodiester-N-acetylglucosaminidase ("uncovering enzyme" (UCE); EC 3.1.4.45) is a Golgi enzyme that mediates the second step in the synthesis of the mannose 6-phosphate lysosomal targeting signal on acid hydrolases. Recently, three mutations (two missense and one deletion/frameshift) in the NAGPA gene that encodes UCE have been identified in individuals with persistent stuttering. We now demonstrate that each mutation leads to lower cellular UCE activity. The p.R328C mutation impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The p.H84Q mutation also impairs folding and, in addition, decreases the specific activity of the enzyme that folds sufficiently to traffic to the Golgi. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system. These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype.

Highlights

Three mutations in the NAGPA gene encoding uncovering enzyme (UCE) have been found in some individuals with persistent stuttering
Fibroblasts of Individuals with Persistent Stuttering Have Decreased UCE Activity—Skin fibroblasts from two individuals with persistent stuttering and mutations in the NAGPA gene, one with a p.R328C mutation and the other with a p.F513SfsX113 deletion resulting in a frameshift, along with WT fibroblasts were assayed for UCE activity
The portion of the mutant UCE molecules that successfully trafficked to the trans-Golgi network (TGN) appeared to have a normal catalytic function

Summary

Background

Three mutations in the NAGPA gene encoding uncovering enzyme (UCE) have been found in some individuals with persistent stuttering. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype. Kang et al [3] reported 10 different mutations (nine missense) in the GNPTAB, GNPTG, and NAGPA genes in 25 of 393 alleles of unrelated Pakistani and North American stutterers, whereas only 1 of 372 alleles of control individuals had a mutation in any of these genes These genes encode the two enzymes that generate the Man-6-P recognition marker on lysosomal acid hydrolases. We found that all three mutations diminish total cellular UCE activity, but each impacts the enzyme in a different manner

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION