Positioning of a Polymorphic Quantitative Trait Nucleotide in theNcf1Gene Controlling Oxidative Burst Response and Arthritis Severity in Rats

Abstract

The Ncf1 gene, encoding the P47(PHOX) protein that regulates production of reactive oxygen species (ROS) by the phagocyte NADPH oxidase (NOX2) complex, is associated with autoimmunity and arthritis severity in rats. We have now identified that the single-nucleotide polymorphism (SNP) resulting in an M153T amino acid substitution mediates arthritis resistance and thus explains the molecular polymorphism underlying the earlier identified Ncf1 gene effect. We identified the SNP in position 153 to regulate ROS production using COS(PHOX) cells transfected with mutated Ncf1. To determine the role of this SNP for control of arthritis, we used the Wistar strain, identified to carry only the postulated arthritis resistant SNP in position 153. When this Ncf1 allele was backcrossed to the arthritis susceptible DA strain, both granulocyte ROS production and arthritis resistance were restored. Position 153 is located in the hinge region between the PX and SH3 domains of P47(PHOX). Mutational analysis of this position revealed a need for an -OH group in the side chain but we found no evidence for phosphorylation. The polymorphism did not affect assembly of the P47(PHOX)/P67(PHOX) complex in the cytosol or membrane localization, but is likely to operate downstream of assembly, affecting activity of the membrane NOX2 complex.

Identification of candidate risk gene variations by whole-genome sequence analysis of four rat strains commonly used in inflammation research

“…Five SNVs are in potentially conserved regions, but only one of them, the nsSNV, is in Ncf1 . Hence, the genome sequences support the previous findings that the inflammation regulatory effect from this congenic is mediated by the Ncf1 nsSNV [61]. …”

Identification of a region in p47phox/NCF1 crucial for phagocytic NADPH oxidase (NOX2) activation

“…The PX domain (aa 3-121) of p47phox is responsible for the binding to phosphoinositides, such as phosphatidylinositol (3,4)-bisphosphate, and phosphatidic acid [24 -26], which is important for the membrane localization of the cytosolic NOX2 complex components [20,25,27]. Mutations in the linker between the PX and SH3A domains affect NOX2 activation [18,28] and decouple phosphatidylinositol binding from NOX2 activation [29]. It is not likely that changes in the region found in this study (aa 228 -235) would prohibit the PX-coordinated membrane association of p47phox.…”

“…The level of oxidative burst ex vivo was measured with a protocol described earlier [17], applying the modifications described recently [18]. After the removal of erythrocytes, the bone marrow cells were stained with APC-labeled Gr-1 antibody (BD Biosciences, San Jose, CA, USA), incubated with DHR-123, and stimulated with PMA.…”

Rheumatoid arthritis: identifying and characterising polymorphisms using rat models

“…The arthritis-protective congenic rat with an E3-derived Ncf1 on a susceptible DA background showed an increased oxidative-burst response (Olofsson et al, 2003c). Further studies using a Wistar congenic strain that differs from DA at only position 153 in Ncf1 conclusively showed that this position regulates ROS and mediates arthritis resistance in rats (Hultqvist et al, 2011). Mutational analysis of this SNP (substitution of methionine to threonine at position 153) demonstrated that this polymorphism did not affect the cytosolic assembly or the localisation of the NOX2 complex, but operates downstream of NOX2 assembly, thereby affecting the superoxide production of the NOX2 complex (Hultqvist et al, 2011).…”

“…Further studies using a Wistar congenic strain that differs from DA at only position 153 in Ncf1 conclusively showed that this position regulates ROS and mediates arthritis resistance in rats (Hultqvist et al, 2011). Mutational analysis of this SNP (substitution of methionine to threonine at position 153) demonstrated that this polymorphism did not affect the cytosolic assembly or the localisation of the NOX2 complex, but operates downstream of NOX2 assembly, thereby affecting the superoxide production of the NOX2 complex (Hultqvist et al, 2011). This discovery in rats was strengthened by the finding that a spontaneous mutation (Box 1) in the mouse Ncf1 gene, which reduces Ncf1 expression and produces an undetectable ROS response, gives rise to enhanced arthritis (Hultqvist et al, 2004).…”