In addition to gene mutations, chemical modification of the enzyme Laforin induced by nitrosative stress has been found to be a likely cause of Lafora disease as well, researchers at Okayama University report.
Lafora disease is a fatal illness characterized by the abnormal generation of glycogens within neurons and other cells, for which no cure exists. Classified as a neurodegenerative disorder, it is caused by gene mutations that affect the production of two enzymes in the human body, Laforin and Malin.
The third potential cause of Lafora disease the study found, is that when Laforin undergoes a particular reaction known as S-nitrosylation, the enzyme stops functioning properly.
The researchers reasoned that since S-nitrosylation has been linked to the development of other neurodegenerative diseases like Alzheimer’s and Parkinson’s, the reaction may also play a role in the pathogenesis of Laforin disease.
They first confirmed that S-nitrosylation does take place by letting Laforin react with nitric oxide (NO). The authors then investigated whether the S-nitrosylation of Laforin affected its three biochemical functions.
One of Laforin’s roles is to remove the phosphates that get incorporated, due to enzymatic errors, in glycogens. This is a three-step process: Laforin binds to the glycogen, removes the phosphate, and then binds to Malin.
Satoh and colleagues checked whether S-nitrosylated Laforin still binds to glycogens and to Malin; their results suggest that modified Laforin has the same binding capacities as unmodified Laforin. Laforin’s ability to remove phosphate groups—its phosphatase function—was, however, found to be inhibited after
The authors then looked at what happened when treating cells expressing non-mutated Laforin with an NO donor.
A comparison with untreated cells revealed that 40% more Lafora bodies were formed when Laforin was treated with NO. Lafora bodies are the inclusion bodies characteristic of Lafora disease, consisting of accumulated glycogen with excessive phosphate content and precipitating inside cells.
Known causes of the generation of Lafora bodies are mutations of genes that encode two enzymes, Laforin and Malin. The latter is a so-called E3 ubiquitin ligase: a protein that assists in transferring a ubiquitin molecule (a very common regulatory protein) from an E2 enzyme (an enzyme capable of carrying ubiquitin) to a substrate peptide.
Laforin is a so-called glucan phosphatase: its function is to liberate phosphate groups from glucans (clusters consisting of a large number of glucose molecules).
In the context of Lafora disease, the relevant glucans are glycogens (multi-branched strands of glucose molecules organized into a globular structure); Lafora bodies largely consist of phospho-glycogens. The presence of phosphates in glycogen makes it insoluble—hence the precipitation of Lafora bodies.
Additional research is needed for precisely determining Laforin’s S-nitrosylation sites, and a further next step will be repeating the experiments in animal cells or neurons.
Rikako Toyota et al.
S-nitrosylation of laforin inhibits its phosphatase activity and is implicated in Lafora disease
Matters (2016). DOI: 10.19185/matters.201606000014
Image: (a) untreated or (b) NO donor treated wild-type Laforin-expressing cells are shown. Green, cells stained for Laforin and LB; blue, nuclei. Credit: Okayama University
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