Joseph Lomako

A new look at the biogenesis of glycogen.

The discovery of glycogenin as a self‐ghicosylating protein that primes glycogen synthesis has significantly increased our understanding of the structure and metabolism of this storage polysaccharide The amount of glycogenin will influence how much glycogen the cell can store. Therefore, the production of active glycogenin primer in the cell has the potential to be the overall rate‐limiting process in glycogen formation, capable of overriding the better understood hormonally controlled mechanisms of protein phosphorylation/dephosphorylation that regulate the activities of glycogen synthase and Phosphorylase. There are indications that a similar covalent modification control is also being exerted on glycogenin. Glycogenin has the ability to glucosylate molecules other than itself and to hydrolyze UDPglu‐cose. These are independent of self‐glucosylation, so that glycogenin, even when it has completed its priming role and become part of the glycogen molecule, retains its catalytic potential. Another new component of glycogen metabolism has been discovered that may have even greater influence on total glycogen stores than does glycogenin. This is proglycogen, a low molecular mass (~400 kDa) form of glycogen that serves as a stable intermediate on the pathways to and from depot glycogen (macroglycogen, mass 107 Da, in muscle). It is suggested that glycogen oscillates, according to glucose supply and energy demand, between the macroglycogen and proglycogen, but not usually the glycogenin, forms. The proportion of proglycogen to macroglycogen varies widely between liver, skeletal muscle, and heart, from 3 to 15% to 50% by weight, respectively. On a molar basis, proglycogen is greatly in excess over macroglycogen in muscle and heart, meaning that if the proglycogen in these tissues could be converted into macroglycogen, they could store much more total glycogen. Discovering the factors that regulate the balance between glycogenin, proglycogen, and macroglycogen may have important implications for the understanding and management of noninsulin‐dependent diabetes and for exercise physiology.—Alonso M. D., Lomako, J., Lomako, W. M., Whelan, W. J. FASEB J. 9, 1126‐1137(1995)

A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis.

In this paper we elucidate part of the mechanism of the early stages of the biosynthesis of glycogen. This macromolecule is constructed by covalent apposition of glucose units to a protein, glycogenin, which remains covalently attached to the mature glycogen molecule. We have now isolated, in a 3500‐fold purification, a protein from rabbit muscle that has the same Mr as glycogenin, is immunologically similar, and proves to be a self‐glucosylating protein (SGP). When incubated with UDP‐[14C]glucose, an average of one molecular proportion of glucose is incorporated into the protein, which we conclude is the same as glycogenin isolated from native glycogen. The native SGP appears to exist as a high‐molecular‐weight species that contains many identical subunits. Because the glucose that is self‐incorporated can be released almost completely from the acceptor by glycogenolytic enzymes, the indication is that it was added to a preformed chain or chains of 1,4‐linked α‐glucose residues. This implies that SGP already carries an existing maltosaccharide chain or chains to which the glucose is added, rather than glucose being added directly to protein. The putative role of SGP in glycogen synthesis is confirmed by the fact that glucosylated SGP acts as a primer for glycogen synthase and branching enzyme to form high‐molecular‐weight material. SGP itself is completely free from glycogen synthase. The quantity of SGP in muscle is calculated to be about one‐half the amount of glycogenin bound in glycogen.— Lomako, J.; Lomako, W. M.; Whelan, W. J. A self‐glucosylating protein is the primer for rabbit muscle‐glycogen biosynthesis. FASEB J. 2: 3097‐3103; 1988.

Glycogen synthesis in the astrocyte: from glycogenin to proglycogen to glycogen.

The astrocyte of the newborn rat brain has proven to be a versatile system in which to study glycogen biogenesis. We have taken advantage of the rapid stimulation of glycogen synthesis that occurs when glucose is fed to astrocytes, and the marked limitation on this synthesis that occurs in astrocytes previously exposed to ammonium ions. These observations have been related to our earlier reports of the initiation of glycogen synthesis on a protein primer, glycogenin, and the discovery of a low‐molecular‐weight form of glycogen, proglycogen. The following conclusions have been drawn: 1) In the ammonia‐treated astrocytes starved of glucose, free glycogenin is present. 2) When these astrocytes are fed with glucose, proglycogen is synthesized from the glycogenin primer by a glycogen‐synthase‐like UDPglucose transglucosylase activity (proglycogen synthase) distinct from the well‐recognized glycogen synthase, and synthesis stops at this point. 3) Proglycogen is the precursor of macromolecular glycogen, which is synthesized from proglycogen by glycogen synthase when glucose is fed to untreated astrocytes, accounting for the much greater accumulation of total glycogen. 4) The stimulus to proglycogen and macroglycogen synthesis that occurs on feeding glucose to untreated or ammonia‐treated astrocytes is the result of the activation of proglycogen synthase, not of glycogen synthase. 5) Therefore, in the synthesis of macromolecular glycogen from glycogenin via proglycogen, the step between glycogenin and proglycogen is rate‐limiting. 6) The discovery of additional potential control points in glycogen synthesis, now emerging, may assist the identification of so‐far‐unexplained aberrations of glycogen metabolism.—Lomako, J., Lomako, W. M., Whelan, W. J., Dombro, R. S., Neary, J. T., and Norenberg, M. D. Glycogen synthesis in the astrocyte: from glycogen to proglycogen to glycogen. FASEB J. 7: 1386‐1393; 1993.

Proglycogen: A low-molecular-weight form of muscle glycogen

We recently reported that muscle contains a trichloroacetic acid‐precipitable component having M r approx. 400 kDa that can be glucosylated by an endogenous enzyme acting on UDPglucose. This component contains within itself the autocatalytic, self glucosylating protein glycogenin, the primer for glycogen synthesis. We now report that this substance, to which we give the name proglycogen, is a glycogen‐like molecule constituting about 15% or total glycogen. It acts as a very efficient acceptor of glucose residues added from UDPglucose. Further, that the endogenous enzyme that adds the glucose to proglycogen is not the autocatalytic protein but a glycogen synthase‐like enzyme. Proglycogen may be an intermediate in the synthesis and degradation of macromolecular glycogen and may exist and be metabolized as a separate entity. Consideration should now be given to the revival of the concept that tissue contains two forms of glycogen. One is proglycogen. The other is the ‘classical’, macromolecular glycogen. Additionally, proglycogen and glycogen may be glucosylated by different forms of synthase.

β‐Glucosylarginine: a new glucose‐protein bond in a self‐glucosylating protein from sweet corn

In the search for a protein primer for starch synthesis, an autocatalytic self‐glucosylating protein has been isolated from sweet corn. Several tryptic peptides were obtained from the [14C]glucosylated protein and were sequenced, corresponding to over 40% of the estimated total sequence (molecular mass 42 kDa). There is no homology with the amino acid sequence of the autocatalytic glycogen primer, glycogenin, nor in respect of the nature of the union between the autocatalytically added glucose and the protein, which, in the case of the corn protein, now named amylogenin, is a novel glucose‐protein bond, a single β‐glucose residue joined to an arginine residue.

Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity

Glycogenin, the self‐glucosylating primer for glycogen synthesis is expressed in wild‐type E. coli as a recombinant protein in an already partly glucosylated form owing to the presence of its substrate UDP‐glucose. By using an E. coli mutant strain lacking in UDP‐glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate‐free glycogenin (apo‐glycogenin) in good yield. When provided with UDPxy‐lose, it autocatalytically adds 1 xylose residue. With UDP‐glucose, an average of 8 glucose residues are added. However release of the self‐synthesized maltosaccharide chains with isoamylase reveals them to be a mixture. Chains as long as 11 glucose residues (maltoundecaose) are present. The ability of recombinant apo‐glycogenin to self‐glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr‐194 of the protein.

Glycogen contains phosphodiester groups that can be introduced by UDPglucose:glycogen glucose 1-phosphotransferase

Rabbit‐muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C‐6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose:glycogen glucose 1 ‐phosphotransferase that positions glucose 1 ‐phosphate on C‐6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1‐phosphate.

Tyrosine-194 of glycogenin undergoes autocatalytic glucosylation but is not essential for catalytic function and activity

Glycogenin is the protein primer for glycogen synthesis. By autocatalytic transglucosylation from UDPglucose, it creates a malto‐octaose chain attached to its Tyr‐194. It has been uncertain whether the autocatalysis includes the addition of the first glucose residue to Tyr‐194. We now show this to be the case. However, we also demonstrate, contrary to a claim by others, that Tyr‐194 is not necessary for the catalytic function and activity of glycogenin.

The nature of the primer for glycogen synthesis in muscle

We and others have reported that glycogenin, the covalently bound protein found in muscle glycogen, also exists in muscle in a glycogen‐free form (M r, 38 000–39 000) that is autocatalytic, undergoes self‐glucosylation and acts as a primer for glycogen synthesis. We now report that this entity is not present in a fresh muscle extract. Instead it exists within a pro form of much higher molecular mass which breaks down spontaneously to the M r, 38 000–39 000 form. Such breakdown is accelerated by the addition of α‐amylase and is prevented by protease inhibitors. Multiple intermediates of the breakdown process have been detected, each capable of undergoing glucosylation.

Substrate specificity of the autocatalytic protein that primes glycogen synthesis

The autocatalytic protein that primes muscle‐glycogen synthesis, and which glucosylates itself from UDPglucose, is inhibited by maltose. Investigation of the reason for the inhibition led to the finding that the protein will glucosylate substrates other than itself. p‐Nitrophenyl αglucoside, αmaltoside, αmaltotrioside and αmaltotetraoside each inhibit self‐glucosylation of the protein by acting as alternative acceptor substrates. The αmaltoside is the best acceptor. The αmaltohexaoside did not act as an acceptor but was an effective inhibitor. These findings help to explain the self‐limiting nature of the autocatalytic extension of the maltosaccharide chain of the protein and suggest that protein self‐glucosylation may be an intennolecular event. They may also point to the mechanism by which the autocatalytic protein is initially glycosylated.