William J. Whelan

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.

A novel glycosyl-amino acid linkage: Rabbit-muscle glycogen is covalently linked to a protein via tyrosine

A recent review summarizes our identification in rabbit-muscle glycogen of a protein that resists all attempts at removal by means that should displace non-covalently bound protein [Kennedy et al. (1985) In Membranes and Muscle (Berman, M.C., Gevers, W. and Opie, L.H. eds.) pp. 65-84, ICSU Press/IRL Press, Oxford]. Here we confirm that the glycogen is covalently bonded to the protein and report that the attachment is via a novel glycosidic linkage involving the hydroxyl group of tyrosine.

A glycogen-debranching enzyme from Cytophaga

Potato R-enzyme [1 ] and bacterial pullulanase [2, 3] hydrolyse the a-1,6-linkages in pullulan and a-limit dextrins and also cleave the a-1,6-branch linkages in amylopectin [4]. The availability of highly purified preparations of pullulanase from Aerobacter aerogenes [5] has made this enzyme invaluable in the analysis of the fine structure of amylopectin [6]. Pullulanase, however, is of limited use in the analysis of glycogen structure because, although it is able to hydrolyse some branch linkages in degraded glycogen, it has little or no action on the undegraded macromolecule [7]. Yeast isoamylase [8] hydrolyses a limited proportion of the interchain linkages of amylopectin and glycogen but, unlike pullulanase, does not act on the 1,6-linkages of pullulan. Recently, an extracellular isoamylase from a new strain o f Pseudomonas was reported to hydrolyse almost all the branch linkages of amylopectin and glycogen [9, 10]. The importance of this type of enzyme for the analysis of glycogen structure prompts us to report the discovery of an isoamylase in a species of Cytophaga. Studies of the partially purified enzyme indicate that its specificity of action is similar to that of the Pseudomonas enzyme and that it has the ability to hydrolyse the branch linkages of amylopectin and glycogen with the complete dismemberment of the branched macromolecules.

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.

Incomplete conversion of glycogen and starch by crystalline amyloglucosidase and its importance in the determination of amylaceous polymers

Amyloglucosidase [EC 3.2.1.3.] hydrolysesboth the 1 -~ 4and 1 ~ 6-bonds of starch and glycogen, and is reportedly capable of causing a quantitative conversion of these polymers into glucose. It has become an agent of choice for the specific and quantitative determination of amylaceous polymers. Thus it was reported from our Laboratory that Aspergillus niger amyloglucosidase could be used for this purpose [1 ]. Recently we detected an o~-amylase-like impurity in our preparation of A. niger enzyme and turned to the use of a crystalline preparation from Rhizopus niveus. The unexpected finding was then made that the amylase-free enzyme is unable in many instances to bring about a complete conversion of the starch components and glycogens into glucose. Adulteration of the glucamylase with a-amylase restores the conversion to a quantitative level.

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.

The mechanism of Q-enzyme action and its influence on the structure of amylopectin

Abstract Q-Enzyme is responsible for the synthesis of the 1,6-branch linkages in amylopectin. Its action on a model amylodextrin containing a single branch linkage has been studied. It is concluded that the enzymic process whereby the branch linkages of amylopectin are synthesized is a random action of the branching enzyme on a complex—possibly a double helix—formed between two 1,4-α-glucan chains. This action pattern predicts a novel arrangement of the units chains in amylopectin.

Multiple branching in glycogen and amylopectin

Abstract The β-amylase limit dextrins of glycogen and amylopectin are completely debranched by joint action of isoamylase and pullulanase. Action of isoamylase alone results in incomplete debranching as a consequence of the inability of this enzyme to hydrolyze those A-chains that are two glucose units in length (half the total number of A-chains). From the reducing powers released by isoamylase acting (a) alone and (b) in conjunction with pullulanase, the relative numbers of A- (unsubstituted) and B- (substituted) chains in the β-dextrins, and therefore in the native polysaccharides themselves, can be calculated. Examination of a series of glycogens and amylopectins in this way showed that the ratio of A-chains: B-chains is markedly higher in amylopectins (1.5–2.6:1) than in glycogens (0.6–1.2:1). Glycogen typically contains A-chains and B-chains in approximately equal numbers; amylopectin typically contains approximately twice as many A-chains as B-chains. These polysaccharides therefore differ in degree of multiple branching as well as in average chain length. A consequence of these findings is that amylopectin cannot be formed in vivo by debranching of a glycogen precursor, as proposed by Erlander, since it is impossible to increase the A:B chain ratio by action of a debranching enzyme.