Juan A. Curtino

Enzymatic synthesis of glucosylsphingosine by rat brain microsomes

Labeled glucosylceramide was formed when rat brain microsomes were incubated with14C-UDP-glucose. When sphingosine was added to the incubation mixture, labeled glucosylsphingosine was synthesized, and the formation of glucosylceramide increased.

Enzymic synthesis of cerebroside from glycosylsphingosine and stearoyl-CoA by an embryonic chicken brain preparation

Summary Glucosylceramide and galactosylceramide were enzymically synthesized by incubating an embryonic chicken brain microsomal fraction with stearoyl-CoA and, respectivelly, glucosylsphingosine or galactosylsphingosine. The formation of free sphingosine by enzymic splitting of glycosylsphingosine prior to the synthesis of cerebroside, was not required.

The amphiphilic character of glycogenin.

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.

Two Glycogen Synthase Activities Associated with Proteoglycogen in Retina

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.

Mechanisms of monomeric and dimeric glycogenin autoglucosylation.

Background: Glycogenin autoglucosylation, required to prime glycogen glucopolymerization, can be produced by the monomeric and dimeric forms of the enzyme. Results: Glycogenin intramonomer glucosylation produced full autoglucopolymerization, and intrasubunit glucosylation was necessary to complete dimer autoglucosylation. Conclusion: Glycogenin dimerization is not required for full autoglucosylation. Significance: De novo glycogen biosynthesis can be sustained by monomeric glycogenin. 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.

The intramolecular autoglucosylation of monomeric glycogenin.

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.

Evidence for glycogenin autoglucosylation cessation by inaccessibility of the acquired maltosaccharide.

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.

Cellular and subcellular localization of glycogenin in chicken retina

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.

Crystallization and preliminary X-ray study of the common edible mushroom (Agaricus bisporus) lectin.

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.

C-chain-bound glycogenin is released from proteoglycogen by isoamylase and is able to autoglucosylate

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.