570 Biowissenschaften; Biologie
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- (glycogen) starch phosphorylase (1)
- Chlamydomonas (1)
- Determination of crystallinity (1)
- Determination of hydration (1)
- GWD (1)
- Label-free imaging (1)
- Nonlinear optical microscopy (1)
- P-31 NMR (1)
- PWD (1)
- Structural determination (1)
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Lafora disease (LD, OMIM #254780) is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.
Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wildtype plants and mutants lacking either GWD or PWD using P-31 NMR. Phosphorylation at both C6- and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid-hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively.
Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii
(2006)
Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.
Second harmonic generation (SHG) is a nonlinear optical process that inherently generates signal in non-centrosymmetric materials, such as starch granules, and therefore can be used for label-free imaging. Both intensity and polarization of SHG are determined by material properties that are characterized by the nonlinear susceptibility tensor, ((2)). Examination of the tensor is performed for each focal volume of the image by measuring the outgoing polarization state of the SHG signal for a set of incoming laser beam polarizations. Mapping of nonlinear properties expressed as the susceptibility ratio reveals structural features including the organization of crystalline material within a single starch granule, and the distribution of structural properties in a population of granules. Isolated granules, as well as in situ starch, can be analyzed using polarimetric SHG microscopy. Due to the fast sample preparation and short imaging times, polarimetric SHG microscopy allows for a quick assessment of starch structure and permits rapid feedback for bioengineering applications. This article presents the basics of SHG theory and microscopy applications for starch-containing materials. Quantification of ultrastructural features within individual starch granules is described. New results obtained by polarization resolved SHG microscopy of starch granules are presented for various maize genotypes revealing heterogeneity within a single starch particle and between various granules.