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Although the discovery of glycogen in the liver, attributed to Claude Bernard, happened more than 160 years ago, the mechanism involved in the initiation of glucose polymerization remained unknown. The discovery of glycogenin at the core of glycogen's structure and the initiation of its glucopolymerization is among one of the most exciting and relatively recent findings in Biochemistry. This review focuses on the initial steps leading to the seminal discoveries of proteoglycogen and glycogenin at the beginning of the 1980s, which paved the way for subsequent foundational breakthroughs that propelled forward this new research field. We also explore the current, as well as potential, impact this research field is having on human health and disease from the perspective of glycogen storage diseases. Important new questions arising from recent studies, their links to basic mechanisms involved in the de novo glycogen biogenesis, and the pervading presence of glycogenin across the evolutionary scale, fueled by high throughput -omics technologies, are also addressed., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Triosephosphate isomerase (TPI), the glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), has been frequently identified as a target of S-nitrosylation by proteomic studies. However, the effect of S-nitrosylation on its activity has only been explored in plants and algae. Here, we describe the in vitro S-nitrosylation of human TPI (hTPI), and the effect of the modification on its enzymatic parameters. NO-incorporation into the enzyme cysteine residues occurred by a time-dependent S-transnitrosylation from both, S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO), with CySNO being the more efficient NO-donor. Both X-ray crystal structure and mass spectrometry analyses showed that only Cys217 was S-nitrosylated. hTPI S-nitrosylation produced a 30% inhibition of the Vmax of the DHAP conversion to G3P, without affecting the Km for DHAP. This is the first study describing features of human TPI S-nitrosylation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The X-ray structure of rabbit glycogenin containing the T82M (T83M according to previous authors amino acid numbering) mutation causing glycogenosis showed the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP-glucose hydrolyzing activities were abolished even though affinity and interactions with UDP-glucose and positioning of Tyr194 acceptor were conserved. Substitution of Thr82 for serine but not for valine restored the maximum extent of autoglucosylation as well as transglucosylation and UDP-glucose hydrolysis rate. Results provided evidence sustaining the essential role of the lost single hydrogen bond for UDP-glucose activation leading to glycogenin-bound glycogen primer synthesis., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Initiation of glucose polymerization by glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization.

Glycogenin initiates the biosynthesis of proteoglycogen, the mammalian glycogenin-bound glycogen, by intramolecular autoglucosylation. The incubation of glycogenin with UDP-glucose results in formation of a tyrosine-bound maltosaccharide, reaching maximum polymerization degree of 13 glucose units at cessation of the reaction. No exhaustion of the substrate donor occurred at the autoglucosylation end and the full autoglucosylated enzyme continued catalytically active for transglucosylation of the alternative substrate dodecyl-maltose. Even the autoglucosylation cessation once glycogenin acquired a mature maltosaccharide moiety, proteoglycogen and glycogenin species ranging rM 47-200kDa, derived from proteoglycogen, showed to be autoglucosylable. The results describe for the first time the ability of polysaccharide-bound glycogenin for intramolecular autoglucosylation, providing evidence for cessation of the glucose polymerization initiated into the tyrosine residue, by inaccessibility of the acquired maltosaccharide moiety to further autoglucosylation.

The ability of monomeric glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant glycogenin. We determined that monomer glycogenin exists in solution at concentration below 0.60-0.85 microM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step.

Glycogen is found in mammals and yeast bound to glycogenin forming proteoglycogen. The branched polysaccharide is joined to the protein through the C-chain, a maltosaccharide considered to be 13 glucose units long and double branched as the other branched glycogen B-chains. We described before the isolation of c-glycogenin, the debranched C-chain bound to glycogenin, from muscle proteoglycogen. In this work, the size of the C-chain is analyzed for the first time. The maltosaccharide moiety of c-glycogenin was auto[14C]glucosylated by a short incubation with UDP-[14C]glucose, and the labeled maltosaccharide was released by heating in 2 M NaOH containing 0.1 M NaBH4 and analyzed by high-performance thin layer chromatography (HPTLC). The results indicate that the C-chain is about half the size of the B-chains, not long enough to be double branched.

The lectin from the common edible mushroom Agaricus bisporus (ABL) belongs to the group of proteins that have the property of binding the Thomsen-Friedenreich antigen (T-antigen) selectively and with high affinity, but does not show any sequence similarity to the other proteins that share this property. The ABL sequence is instead similar to those of members of the saline-soluble fungal lectins, a protein family with pesticidal properties. The presence of different isoforms has been reported. It has been found that in order to be able to grow diffraction-quality crystals of the lectin, it is essential to separate the isoforms, which was performed by preparative isoelectric focusing. Using standard procedures, it was possible to crystallize the most basic of the forms by either vapour diffusion or equilibrium dialysis, but attempts to grow crystals of the other more acidic forms were unsuccessful. The ABL crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 93.06, b = 98.16, c = 76.38 A, and diffract to a resolution of 2.2 A on a conventional source at room temperature. It is expected that the solution of this structure will yield further valuable information on the differences in the T-antigen-binding folds and will perhaps help to clarify the details of the ligand binding to the protein.

Proteoglycogen glycogenin is linked to the glucose residue of the C-chain reducing end of glycogen. We describe for the first time the release by isoamylase and isolation of C-chain-bound glycogenin (C-glycogenin) from proteoglycogen. The treatment of proteoglycogen with alpha-amylase releases monoglucosylated and diglucosylated glycogenin (a-glycogenin) which is able to autoglucosylate. It had been described that isoamylase splits the glucose-glycogenin linkage of fully autoglucosylated glycogenin previously digested with trypsin, releasing the maltosaccharide moiety. It was also described that carbohydrate-free apo-glycogenin shows higher mobility in SDS-PAGE and twice the autoglucosylation capacity of partly glucosylated glycogenin. On the contrary, we found that the C-glycogenin released from proteoglycogen by isoamylolysis shows lower mobility in SDS-PAGE and about half the autoglucosylation acceptor capacity of the partly glucosylated a-glycogenin. This behavior is consistent with the release of maltosaccharide-bound glycogenin instead of apo-glycogenin. No label was split from auto-[14C]glucosylated C-glycogenin or fully auto-[14C]glucosylated a-glycogenin subjected to isoamylolysis without previous trypsinolysis, thus proving no hydrolysis of the maltosaccharide-tyrosine linkage. The ability of C-glycogenin for autoglucosylation would indicate that the size of the C-chain is lower than the average length of the other glycogen chains.

This study describes for the first time the amphiphilicity of the protein moiety of proteoglycogen. Glycogenin but not proteoglycogen associates to phospholipid vesicles and forms by itself stable Gibbs and Langmuir monolayers at the air-buffer interface. The adsorption free energy (-6.7 kcal/mol) and the glycogenin collapse pressure (47 mN/m) are indicative of its high surface activity which can thermodynamically drive and retain the protein at the membrane interface to a maximum equilibrium adsorption surface pressure of 21 mN/m. The marked surface activity of glycogenin is further enhanced by its thermodynamically favorable penetration into zwitterionic and anionic phospholipids with a high cut-off surface pressure point above 30 mN/m. The strong association to phospholipid vesicles and the marked surface activity of glycogenin correspond to a high amphiphilic character which supports its spontaneous association to membrane interfaces, in which the de novo biosynthesis of glycogen was proposed to initiate.

Glycogen-free but not glycogen-bound glycogenin transglucosylates dodecyl-beta-maltoside. Furthermore, its sugar nucleotide-binding site can be photoaffinity labeled using [beta-(32)P]5-azido-UDP-glucose. Disruption with DMSO of the hydrogen bonds that stabilize the alpha-helical structure of glycogen restored the photoaffinity labeling of the glycogen-bound enzyme but not its transglucosylation activity. The larger size polysaccharide that linked to glycogenin allowed transglucosylation corresponding to that of PG-200, a proteoglycogen species of M(r) 200 kDa. PG-200 showed lower activity and increased activation energy than glycogen-free glycogenin. Heat denaturation of glycogen-free and glycogen-bound glycogenin occurred at 51 and 64 degrees C, respectively. Active glycogenin was recovered after the glycogen-bound form was heated at 60-70 degrees C and immediately cooled. Treatment at 60 degrees C of the glycogen-free enzyme resulted in inactivation. This is the first report describing the inactivation and thermal stabilization of an enzyme by linked polysaccharide., (Copyright 2001 Academic Press.)

Glycogen synthase of bovine retina was found associated with the acid-insoluble and acid-soluble proteoglycogen fractions. The synthase associated with the acid-insoluble proteoglycogen precursor showed an 8-fold lower Km for UDP-glucose than the synthase associated with the acid-soluble fraction, and was inhibited by detergent. A short digestion with pronase resulted in conversion of the acid insoluble fraction into acid-soluble. The results lead us to postulate that the acid-insolubility of the proteoglycogen fraction and the association with retina membrane proposed before, is caused by glycogen synthase strongly associated to its polysaccharide moiety. The enlargement of the polysaccharide moiety during proteoglycogen biosynthesis, from glycogenin linked to a few 11 to 12 glucose units to the acid-insoluble proteoglycogen precursor (Mr 470,000) would be carried out, together with the branching enzyme, by the glycogen synthase showing a low Km for UDP-glucose. The glycogen synthase with the highest Km for UDP-glucose would participate in conversion of the precursor into mature acid-soluble proteoglycogen.

Glycogenin, the autoglucosyltransferase that initiates the de novo biosynthesis of glycogen, photoaffinity labeled with [beta32P]5-azido-UDP-glucose. The photoinsertion of the azidouridine derivative showed activating ultraviolet light dependency, saturation effects, and inhibition by UDP-glucose, thus demonstrating the specificity of the interaction. In the absence of Mn2+, the requirement for the catalytic activity of glycogenin, the photolabeling decreased by 70%. Competitive binding experiments indicated that the pyrophosphate or a phosphate was the moiety of UDP-glucose implicated in the strongest interaction at the binding site. Proteolytic digestion of photolabeled glycogenin resulted in the identification of two labeled fragments, 89-143 and 168-233, that carried the uridine binding sites. This is the first report of the region of glycogenin that harbors the UDP-glucose-binding domain.

Glycogenin, the autoglucosyltransferase that primes the biosynthesis of proteoglycogen, is found in the polysaccharide linked proteoglycogen form in mammals and chicken. Glycogenin was released from proteoglycogen and its activity was measured, together with that of glycogen synthase as well as glycogen content, in muscle, liver, and brain during chicken development. The specific activity of glycogenin, expressed per protein, increased with development only in muscle and was higher than the specific activities measured in liver and brain at any time. Concomitant with the rise in activity, an enhanced expression of the protein was observed with Western blot. The specific activity of glycogen synthase increased with development in muscle and liver, while glycogen accumulation was noticeable only in liver. The results indicate that the molar concentration of proteoglycogen is higher in muscle than in liver. The high glycogen content of liver may indicate that the size of the polysaccharide moiety of proteoglycogen is larger in liver than in muscle. This is the first report of developmental modulation of de novo biosynthesis of glycogen at the level of the primer that initiates glucose polymerization.

Proteoglycogen is the end product in the process of glycogen biogenesis. We have purified rabbit muscle proteoglycogen and studied the glucosyltransferase reactions catalyzed by its protein moiety, glycogenin, free or bound to the polysaccharide. The purification strategy involved dissolution of proteoglycogen and cosedimenting membrane vesicles in a Triton X-114/Triton X-45 mixture followed by partition in the aqueous phase, potassium iodide precipitation of accompanying proteins, and washing by high-speed centrifugation. Glycogenin or a proteoglycogen species of an average molecular mass of 200 kDa was isolated by ion-exchange chromatography after the purified proteoglycogen had been subjected to long or short amylolytic digestion, respectively. Besides autoglucosylation from UDP-glucose, glycogenin was capable of autogalactosylation from UDP-galactose. The autoglucosylation reaction was not inhibited by the simultaneous glucosylation of the exogenous acceptors N-(maltosyl-alpha-1-4-(1-deoxiglucitol))-peptide or n-dodecyl-beta-D-maltoside. The polysaccharide-bound glycogenin species of 200 kDa showed to be active for the glucosylation of exogenous acceptor and represented the isolated proteoglycogen of higher size having glucosyl transferase activity. This is the first description of the isolation of native proteoglycogen and a proteoglycogen species having glucosyltransferase activity.

Neurospora crassa proteoglycogen was purified and its protein moiety, M-glycogenin, was released by amylolytic treatment. The released protein was capable of autoglucosylation from UDP-glucose forming glucosyl-alpha 1,4-glucosyl linkage. The kinetics of autoglucosylation suggested an intramolecular mechanism of reaction. M-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently. Both auto- and transglucosylation reactions were dependent on Mn2+. Thus, M-glycogenin, which has also been described as the constituent of Escherichia coli proteoglycogen (A. Goldraij and J. A. Curtino. 1993, Biochem. Mol. Biol. Int. 30, 453-458), is a glucosyltransferase that bears similar catalytic properties with mammalian glycogenin. This is the first report on the enzymatic character of the protein constituent of proteoglycogen in primitive organisms, which suggest that the mechanism for the de novo biosynthesis of glycogen was conserved over a very long period of evolution.

We investigated the cellular and subcellular localization of glycogenin in chicken retina using a polyclonal antibody raised against chicken muscle glycogenin. The antiserum recognized both free and glycogen-bound glycogenin on dot blots. Immunocytochemistry revealed an uniform staining of all the retina layers except for the outer segments and ganglion cells layers that were very weakly stained and for the inner segments layer and a zone between the inner nuclear and inner plexiform layers that were heavily stained. Electron microscopy of neuronal cells showed immunoreactivity localized in the cytoplasm and nucleus. This is the first description of the cellular and subcellular localization of glycogenin. Our results suggest that the biosynthesis of glycogen could begin in both, the cytoplasm and nucleus of the neurone.

The proteoglycogen fraction of Neurospora crassa was purified and subjected to radioiodination with [125I]iodide. Amylolysis of the polysaccharide moiety led to the isolation of a labelled 31 kDa-protein. The NH2-terminal amino acid sequence of 10 residues of the 31 kDa-protein was determined. A 31 kDa-protein was also bound to glycogen in Escherichia coli. Proteoglycogen has not been heretofore found in any primitive unicellular organism.

The incorporation of [14C]glucose from UDP-[14C]glucose into proteoglycogen fractions of a retinal microsomal preparation was studied. From the rate of labelling of acid-insoluble and -soluble proteoglycogen at different sugar-donor concentrations, and from the conversion of the labelled acid-insoluble into an acid-soluble form measured by a 'chase' with unlabelled UDP-glucose, it was concluded that acid-insoluble 42 kDa protein (p42)-bound glycogen of weight-average Mr 4.7 x 10(5) and acid-soluble p42-bound glycogen of weight-average Mr 7.0 x 10(5) [Miozzo, Lacoste & Curtino (1989) Biochem. J. 260, 287-289] are related as precursor and product respectively. About one-third of the acid-insoluble proteoglycogen was excluded from a Sephacryl S-500 column and was associated with large membrane vesicles. Proteoglycogen was not dissociated from the membranes by treatment with saline solutions or with SDS at a low detergent-to-protein ratio. It was dissociated by treatment with detergents under conditions which were shown to solubilize integral membrane sialoglycoconjugates of retina. These results lead us to postulate that the biogenesis of retina glycogen starts on membrane-associated p42 to form acid-insoluble proteoglycogen, which is then dissociated from membranes and converted into acid-soluble proteoglycogen by the 'growth' of its polysaccharide moiety.

Bovine retina membrane proteins and glycoproteins were insoluble in chloroform/methanol (2:1, v/v) unless the membrane suspension was precipitated with trichloroacetic acid and the organic solvent mixture added to the precipitated membranes. The presence of millimolar amount of trichloroacetic acid in the organic solvent led to the total solubilization of membranes. The glycoproteins precipitated at the interphase after partition of the acidified chloroform/methanol solution with water and were resolubilized from the interphase with chloroform/methanol/water (1:1:0.3, by vol). The solubility properties of the membrane glycoproteins in the acidified organic solvent mixtures allow to remove the bulk of membrane lipids and to recover from the chloroform/methanol/water solution the glycoprotein of rod outer segment membranes, rhodopsin, as protonated N-retinylidene opsin in a water soluble form.

Tyrosine-glycogen obtained from retina proteoglycogen by exhaustive proteolytic digestion was radiolabelled with 125I. The 125I-labelled tyrosine-glycogen was degraded by amylolytic digestion to a very small radioactive product, which was identified as iodotyrosine by h.p.l.c. The amylolytic mixture used released glucose and maltose that were alpha-linked to the phenolic hydroxy group of p-nitrophenol. No free iodotyrosine was found before or after the intact [125I]iodotyrosine-glycogen was subjected to two cycles of the Edman degradation procedure. The linkage between protein and glycogen was alkali-stable. Therefore it is concluded that the protein-bound glycogen was O-glycosidically linked to the phenolic hydroxy group of tyrosine. The amino acid has not been heretofore found to be involved in the linkage of carbohydrates to proteins.

The trichloroacetic acid-insoluble 1,4-alpha-glucan fraction from bovine retina was purified and characterized. It is a proteoglycogen fraction containing a 42 kDa protein moiety similar in size to the protein moiety of the trichloroacetic acid-soluble proteoglycogen fraction. The apparent weight-average Mr of acid-insoluble and acid-soluble proteoglycogens are 4.7 x 10(5) and 7.0 x 10(5) respectively. The present results support suggestions from earlier studies indicating that acid-insoluble proteoglycogen is the precursor of the acid-soluble form.

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.