On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure

Abstract

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200mg/kg linamarin, or 200mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease.

Figure 1

(A) Highlighted sites of carbamoylation (lysine residues 103 and 549) on serum albumin in animals treated with vehicle. (B) Highlighted sites of carbamoylation (lysine residues 103 and 438) on serum albumin in linamarin-treated animals. Peptide 435-452 (underlined) was also found carbamoylated in animals treated with NaOCN but not vehicle. (C) Serum albumin sequence with 23 highlighted lysine (K) carbamoylated residues and underlined peptide 435-452 (also found in linamarin-treated animals), in animals treated with NaOCN.

Figure 2

(A) Representative 2D-DIGE images displaying differential expression of lumbosacral proteins from animals treated with vehicle (saline) while maintained on normal AAA diet versus 75% SAA-deficient diet. Green spot (e.g. spot # 2): higher protein abundance in saline/AAA-treated samples; red spot (e.g. spot # 44): higher abundance of respective protein in saline/SAA-treated animals; yellow spot (e.g. spot # 19): no differential change of respective protein abundance in saline-treated animals fed normal AAA versus 75% SAA-deficient diet. Protein names of spots are given in Table 1. Differential migration of proteins e.g. spots 11-14 identified as dynamin 1 indicates that posttranslational modifications occur in our model. (B) Graph depicting relative abundance of a selected protein, i.e., N-ethylmaleimide sensitive fusion protein (isoform CRA_a, NSF) in samples from animals treated with either one of the 6 treatment conditions (1= linamarin, 2= NaOCN, 3= vehicle; A= normal AAA diet and B= 75% SAA-deficient diet). Differential protein abundance of NSF in the pair comparison 1A versus 1B, or 2A versus 2B, or 3A versus 3B demonstrates the impact of diet on protein expression for each treatment condition. (C) Decyder output showing differential abundance of NSF in 3A (left, vehicle/AAA) versus 3B (right, vehicle/SAA) protein samples.