Identification of rare protein disulfide isomerase gene variants in amyotrophic lateral sclerosis patients

Abstract

Disruption of endoplasmic reticulum (ER) proteostasis is a salient feature of amyotrophic lateral sclerosis (ALS). Upregulation of ER foldases of the protein disulfide isomerase (PDI) family has been reported in ALS mouse models and spinal cord tissue and body fluids derived from sporadic ALS cases. Although in vitro studies suggest a neuroprotective role of PDIs in ALS, the possible contribution of genetic mutations of these ER foldases in the disease process remains unknown. Interestingly, intronic variants of the PDIA1 gene were recently reported as a risk factor for ALS. Here, we initially screened for mutations in two major PDI genes (PDIA1/P4HB and PDIA3/ERp57) in a US cohort of 96 familial and 96 sporadic ALS patients using direct DNA sequencing. Then, 463 familial and 445 sporadic ALS patients from two independent cohorts were also screened for mutations in these two genes using whole exome sequencing. A total of nine PDIA1 missense variants and seven PDIA3 missense variants were identified in 16 ALS patients. We have identified several novel and rare single nucleotide polymorphisms (SNPs) in both genes that are enriched in ALS cases compared with a large group of control subjects showing a frequency of around 1% in ALS cases. The possible biological and structural impact of these ALS-linked PDI variants is also discussed.

Keywords: Amyotrophic lateral sclerosis; ER stress; ERp57; PDIA1; Protein disulfide isomerase.

Fig. 1

Location and sequence conservation of missense variants of PDIA1 and ERp57 identified in US ALS cases by direct sequencing. (A) PDI primary structure: catalytic a and a′ domains containing the active site motif CXXC sequence (black), non-catalytic domains b and b′ containing ligand binding sites (gray), and x-linker region (white). Alignment of PDIA1 (B) and ERp57 (C) sequences from indicated species. Amino acid conservation across species of the missense mutations of PDIA1 and ERp57 identified in ALS patients.

Fig. 2

Structural analysis of amino acid substitutions caused by ALS-linked mutations in PDIA1 and ERp57. (A) Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg³⁰⁰ located to the b′ domain of PDIA1 with Trp³⁹⁶ located to the a′ domain adjacent to the active site motif CGHC (designatedas AS in yellow) is shown in comparison to the mutated version of PDIA1^R300H highlighting the same residues. A potential stabilization of the interaction between the b′ and a′ domains is shown that maybe caused by the interaction of the aromatic rings of mutated His³⁰⁰ with Trp³⁹⁶. (B) Structural overlay of abb′ domains of wild-type Asp²⁹² (residue in red on white backbone) to mutant Asn²⁹² PDIA1 (residue in gray on orange backbone). A shift in the backbone structure was predicted using the SWISSMODEL server. The local surface surrounding the mutated residue is shown indicating the loss of a negative charge. (C) Structural overlay of the ERp57 abb′a′ domains highlighting wild-type Asp²¹⁷ (residue in red on white backbone) and mutant Asn²¹⁷ (residue in gray on orange backbone) at the hinge between b and b′ domains of ERp57. A shift in the backbone structure was predicted using the SWISSMODEL server. The local surface surrounding the mutated residue is shown indicating the loss of a negative charge, which may impact the binding to CNX, since positively charged residues (e.g. Lys²¹⁴) important for the interaction of ERp57 with CNX are in the vicinity of the mutated site (Pollock et al., 2004; Kozlov et al., 2006). (D) The surface of the abb′a′ domains of wild-type ERp57 and mutant ERp57^Q481K is shown. The mutation to Lys⁴⁸¹ leads to an additional positive charge in the a′ domain of ERp57. This site is predicted to be located in proximity to highly positively charged C-terminal region with the amino acid sequence KPKKKKK for which the structure is not available. The removal or mutation of this region completely abolishes the binding of ERp57 with the negatively charged P-domain of CNX (Pollock et al., 2004). We hypothesize that the additional positive charge acquired by the mutation of Gln⁴⁸¹ to Lys⁴⁸¹ may lead to an alteration in the interaction between CNX and ERp57 with potential functional consequences for the folding of client proteins of the CNX/CRT cycle.