Glyoxalase I polymorphism rs2736654 causing the Ala111Glu substitution modulates enzyme activity--implications for autism

Abstract

Autism is a pervasive, heterogeneous, neurodevelopmental disability characterized by impairments in verbal communications, reciprocal social interactions, and restricted repetitive stereotyped behaviors. Evidence suggests the involvement of multiple genetic factors in the etiology of autism, and extensive genome-wide association studies have revealed several candidate genes that bear single nucleotide polymorphisms (SNPs) in non-coding and coding regions. We have shown that a non-conservative, non-synonymous SNP in the glyoxalase I gene, GLOI, may be an autism susceptibility factor. The GLOI rs2736654 SNP is a C→A change that causes an Ala111Glu change in the Glo1 enzyme. To identify the significance of the SNP, we have conducted functional assays for Glo1. We now present evidence that the presence of the A-allele at rs2736654 results in reduced enzyme activity. Glo1 activity is decreased in lymphoblastoid cells that are homozygous for the A allele. The Glu-isoform of Glo1 in these cells is hyperphosphorylated. Direct HPLC measurements of the glyoxalase I substrate, methylglyoxal (MG), show an increase in MG in these cells. Western blot analysis revealed elevated levels of the receptor for advanced glycation end products (RAGEs). We also show that MG is toxic to the developing neuronal cells. We suggest that accumulation of MG results in the formation of AGEs, which induce expression of the RAGE that during crucial neuronal development may be a factor in the pathology of autism.

Figure 1

Western blot analysis and ³²P labeling of different isoforms of Glo1 in lymphoblastoid cells in culture. Equal amounts of proteins (50 μg) were resolved on a 10–20% gradient SDS-PAGE and transferred onto a nitrocellulose membrane. The nitrocellulose membrane was probed with anti-Glo1 (rabbit polyclonal) antibody (I) or by autoradiography on an X-ray film (II). The cell lines used and the corresponding genotype are indicated.

Figure 2

Western blot analysis for the RAGE in lymphoblastoid cells expressing different isoforms of the Glo1. Equal amounts of proteins (50 μg) from particulate (a) and soluble (b) fractions were resolved on a 10–20% gradient SDS-PAGE followed by transfer onto a nitrocellulose membrane. The nitrocellulose membrane was probed with an anti-RAGE (rabbit polyclonal) antibody, and the blot was also probed with actin as a loading control.

Figure 3

Effect of MG on developing neurons. NHNP cells were grown on lamin-coated cover slips in the presence of BDNF. MG was added at the indicated concentrations and cells were allowed to differentiate for two weeks, following which cells were fixed and stained with primary antibodies for βIII tubulin (A), nestin (B), and DAPI (C). Panel (D) is a phase-contrast image.