Within the body, energy is stored as glycogen in muscle and liver tissues as well as triglycerides in fatty adipose deposits. Glycogen itself is a polysaccharide, composed of branched glucose chains built up around glycogenin-1 and -2, glycosyltransferase units that self-initiate glycogen synthesis. The two enzymes are 70% homologous even though they are the products of two different genes. They frequently co-exist in tissues as homodimers but have also been found as interactive heterodimers. In the presence of substrate, one unit catalyzes the addition of glucose moieties to a specific tyrosine residue on the other; this continues until a chain of a certain length is achieved. Once this happens, glycogen synthase takes over, building larger branched chain molecules from the initial 13 units.
Nilsson et al. (2014) used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to characterize self-glucosylation by glycogenin and to investigate whether the two forms interact with each other to regulate this process.1 The researchers used a cell-free experimental system to examine glucosylation in the absence of conflicting metabolic enzymes involved in glycogen synthesis.
First, the researchers created glycogenin-1 and -2 constructs and mutant non-binding forms, cloning them into an expression vector. Using a cell-free system, they synthesized the proteins before subjecting them to in vitro glucosylation studies. Expression products were purified, then separated, using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) before identification by Western blotting.
Using the in vitro system, Nilsson et al. found that, although glycogenin-1 could initiate its own glucosylation, glycogenin-2 was able to add glucose units only when co-expressed with glycogenin-1. The team confirmed their results using mutated, inactive or non-binding synthetic protein forms and also in a human embryonic kidney cell culture system. The researchers then confirmed this finding with autoradiography of glucosylation study products, using labeled UDP-14C-glucose as the substrate in the cell-free system.
The scientists also examined in vivo glucosylation of the two enzymes, selecting target tissues with high levels of analyte mRNA for analysis. Using cryostat sections of human tissue, they found that glycogenin-1 and -2 are present in their glucosylated forms in the liver. Glucosylated glycogenin-2 was found most abundantly in adipose tissue, alongside its unglucosylated form.
The researchers followed the in vitro and in vivo studies by investigating self-glucosylation of glycogenin-1 and -2 using LC-MS/MS in an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Using a previously validated glycoproteomics method,2 they examined tryptic glycopeptide fragments following alpha-amylase treatment of purified, cell-free glucosylated products. Alpha-amylase treatment reduced the glucose chains to simplify the MS analysis, by reducing the m/z values.
When expressed alone, the majority of tryptic peptides found by LC-MS/MS following in-gel digestion for glycogenin-2 were unglucosylated. Following manual data sorting, however, the more sensitive glycoproteomics method did show the presence of glucosylated glycogenin-2 tryptic peptides, suggesting that glucosylation was occurring. The researchers comment that this finding might be due to the relative over-abundance of glucose substrate saturating the LC-MS/MS method, compared with lower concentrations in the cell-free experimental protocol. Furthermore, when Nilsson and co-workers used electron transfer dissociation as the fragmentation method, they identified Tyr-228 as the glycogenin-2 glucosylation site. Additional analysis of the data revealed that glucosylated glycogenin-1 tryptic fragments contained four to eight glucose units, whereas a maximum of four were seen on glycogenin-2. Nevertheless, LC-MS/MS confirmed that glycogenin-2 glucosylation was more apparent when co-expressed with glycogenin-1.
Nilsson et al. propose that their cell-free expression system allows accurate and close examination of glycogenin-1 and -2 glucosylation, free from interference by other enzymes involved in glycogen metabolism. By examining glucosylation in a patient with severe glycogen depletion and showing that glycogenin-2 did not compensate for a glycogenin-1 mutation, the researchers have already expanded the scope of this technique into clinical studies.
1. Nilsson, J., et al. (2014) “LC–MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences,” Biochimica et Biophysica Acta, 1844 (pp. 398–405).
2. Nilsson, J., et al. (2012) “Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation,” Biochimica et Biophysica Acta, 1822 (pp. 493–9).
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