Molecular Pathogenesis of a New Glycogenosis Caused by a Glycogenin-1 Mutation

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Biochimica et Biophysica Acta 1822 (2012) 493–499

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Biochimica et Biophysica Acta

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Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation

Johanna Nilsson a,⁎, Adnan Halim b, Ali-Reza Moslemi a, Anders Pedersen c, Jonas Nilsson b, Göran Larson b, Anders Oldfors a

a Department of Pathology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden b Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden c Swedish NMR Centre, Gothenburg, Sweden

article info abstract

Article history: Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short ol- Received 27 June 2011 igosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glu- Received in revised form 7 November 2011 cosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with Accepted 28 November 2011 glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Available online 9 December 2011 Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83

Keywords: for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino Glycogen acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosy- Glycogenin lated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled Glycogen storage disease UDP-14C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We dem- Cell-free expression onstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage Mass spectrometry at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1. © 2011 Elsevier B.V. All rights reserved.

1. Introduction substrate (Fig. 1A). First, a glucose-O-tyrosine linkage is formed with the hydroxyl group of tyrosine 195 [10–14] and second, several Glycogen is composed of highly branched glucose polymers and α1,4 glucosidic linkages are formed to produce an oligosaccharide serves as an important energy supply within cells. The main glycogen containing approximately 8–13 glucose residues [13,15,16]. The depots are found in the liver and in the skeletal muscle. Liver glyco- priming oligosaccharide chain is required for elongation by glycogen gen releases glucose to the blood stream to maintain blood glucose synthase (EC 2.4.1.11) and the introduction of α1,6 branching points levels and skeletal muscle glycogen serves as rapidly accessible ener- by branching enzyme (EC 2.4.1.18) to produce glycogen [17]. Each gy during muscle contraction. The high energy consumption in skele- glycogen granule in muscle cells contains approximately 30,000 glu- tal muscle makes it susceptible to metabolic disorders, which cose residues with numerous non-reducing ends for rapid access to constitute a heterogenous group of diseases with impaired energy glucose [18]. production due to enzymatic deﬁciencies in the metabolism of fatty We recently described the ﬁrst patient with a disease associated acids or glycogen. A subgroup of the metabolic disorders are the gly- with glycogenin-1 mutations [19]. The patient was admitted with cogenoses characterized by accumulation or depletion of glycogen sudden cardiac arrest at age 27 and skeletal muscle revealed pro- due to defects in the glycogen degradation or synthesis pathways found glycogen depletion (Fig. 1B). Sequencing of the glycogenin-1 [1]. Glycogen synthesis is initiated by the autoglucosylation of the gene, GYG1, showed a nonsense mutation in one allele and a missense protein glycogenin [2,3]. Two different genes code for the two iso- mutation, Thr83Met, in the other. Only the allele with the missense forms of glycogenin: glycogenin-1 is a 38 kDa protein expressed in mutation was expressed and resulted in inactivation of the autoglu- muscle and heart [4–6] and glycogenin-2 is a 66 kDa protein and cosylation of glycogenin-1 necessary for the priming of glycogen syn- the predominant isoform in liver [7–9]. thesis in muscle. In normal controls virtually all glycogenin-1 Glycogenin-1 is a glycosyltransferase (EC 2.4.1.186) catalyzing molecules were autoglucosylated whereas a heterozygous carrier of two autoglucosylation reactions using UDP-glucose as donor the Thr83Met mutation demonstrated a small proportion of unglucosylated glycogenin-1 in skeletal muscle [19]. In this study we have investigated the importance of the Thr83 in ⁎ Corresponding author. Tel.: +46 313428548; fax: +46 313422886. human glycogenin-1 for autoglucosylation and the formation of a E-mail address: [email protected] (J. Nilsson). glucose-O-tyrosine linkage at tyrosine 195. We demonstrate that

Fig. 1. A: Schematic illustration of glycogen synthesis. Glycogenin-1 initially autoglucosylates at tyrosine 195 and the oligosaccharide is then elongated by glycogen synthase and branching enzyme into a glycogen granule. The electron micrograph shows glycogen granules (arrow) within skeletal muscle. B: Cross sections of skeletal muscle from the patient with the glycogenin-1 Thr83Met mutation and a control stained with Periodic Acid Schiff (PAS) reagent to detect glycogen. The muscle fibers of the patient demonstrate glycogen depletion. glycogenin-1 with the Thr83Met substitution is unable by itself to (Calbiochem) or 0.01 μM UDP-14C-glucose (PerkinElmer) in 30 °C form the glucose-O-tyrosine linkage at tyrosine 195. However, for 1 h [20]. The recombinant glycogenin-1 was denatured using glycogenin-1 with the Thr83Met mutation is glucosylated by a cata- Laemmli sample buffer with 5% β-mercaptoethanol and run on a lytically active glycogenin-1 protein. 10% Bis–Tris gel (Invitrogen) for 1 h with 200 V followed by electro- blotting to Invitrolon™ PVDF filters at 20 V for 10 min. The incorpora- 2. Material and methods tion of UDP-14C-glucose was detected on X-ray film and glycogenin-1 was detected with Anti-human glycogenin antibody clone M07 2.1. Vector construction (Abnova, Taipei City, Taiwan) 1:500 and Western Breeze™ (Invitrogen). The coding region of GYG1 was amplified from cDNA prepared from patient or control skeletal muscle using the primers 5′-ATG 2.3. Purification, gel filtration and alpha-amylase treatment of ACA AGA TCA GGC CTT T-3′ and 5′-CTA CTG GAG GTA AGT GTC-3′ glycogenin-1 and inserted downstream of the 6xHis tag in the pEXP5-NT/TOPO vector (Invitrogen, Carlsbad, CA). The cloned sequence was identical Autoglucosylated glycogenin-1 was purified with the 6xHis tag to GenBank accession number NM_001184720 with the exception using Ni-NTA Magnetic Agarose beads (Qiagen) according to the of c.248C>T causing the amino acid change Thr83Met in the patient manufacturer's instruction. cDNA [19]. The wild type vector prepared from the control muscle Glycogenin-1 eluates from Ni-NTA affinity purification were run was used as a template to make additional constructs with amino on a Superdex 200 5/150 analytical gel filtration column connected acid alteration at position 83 or 195 of glycogenin-1. The amino acid to an ÄKTA purifier (GE Healthcare). The buffer was 25 mM sodium substitutions were induced by using QuikChange®II Site-Directed phosphate pH 7.4, 150 mM sodium chloride, 0.05% Tween 20. Flow Mutagenesis kit (Stratagene, La Jolla, CA) according to manufacturer's rate was 0.15 ml/min and 100 μl fractions were collected. protocol and the primers listed in Supplementary Table 1. All con- Glycogenin-1 chromatograms were compared with a column stan- structs were confirmed by sequencing. dard curve of ferritin (440 kDa), aldolase (158 kDa), ovalbumin (43 kDa) and RNase A (13.7 kDa). Peak fractions were analyzed 2.2. Cell-free expression and in vitro glucosylation with Western blot using the Anti-human glycogenin antibody clone M07. Recombinant glycogenin-1 was expressed from the pEXP5-NT/ The glycogenin-1 was treated with 10 μg/ml human alpha- TOPO vectors (above) using EasyExpress Protein Synthesis Kit (Qia- amylase (Sigma-Aldrich, Schnelldorf, Germany) in a total volume of gen, Hilden, Germany) according to the manufacturer's protocol. 200 μl PBS pH 6.5 for 1 h at 37 °C while still attached to the beads. Glycogenin-1 was allowed to autoglucosylate in 50 mM Hepes pH The alpha-amylase was then removed by rinsing. Glycogenin-1 was

7.5, 5 mM MnCl2, 0.5 mM DTT in the presence of 0.1 M UDP-glucose released from the beads by boiling in Laemmli sample buffer prior J. Nilsson et al. / Biochimica et Biophysica Acta 1822 (2012) 493–499 495 to the SDS-PAGE. The gel was stained with Coomassie stain and carbamidomethyl modiﬁcation of cysteine and variable oxidation of glycogenin-1 was excised for in-gel trypsinization. methionine.

2.4. In-gel tryptic digestion and mass spectrometry 3. Results

In-gel trypsinization was performed as described by Shevchenko 3.1. Glycogenin-1 Thr83Met is incapable of autoglucosylation [21]. The excised gel fragments were destained in 12.5 mM

NH4HCO3 with 50% acetonitrile and in 25 mM NH4HCO3 with 50% To investigate the effect of the glycogenin-1 Thr83Met mutation in methanol. The samples were incubated in 10 mM DTT in 56 °C for vitro we expressed apo-glycogenin-1 in an E. coli based cell-free pro- 1 h and in 55 mM iodoacetamide for 45 min at room temperature. tein expression system and studied the autoglucosylation after addi- The gel pieces were rinsed as described above, dried in a vacuum cen- tion of UDP-glucose. Kinetics of wild type glycogenin-1 have trifuge and incubated with digestion buffer (50 mM NH4HCO3, 0.1% demonstrated that the autoglucosylation of wild type glycogenin-1 acetonitrile, 10 ng/μl trypsin, proteomics grade, Sigma-Aldrich) at is nearly complete within 20 min [23]. 37 °C overnight. Peptides were extracted in 50% acetonitrile/1% tri- The recombinant unglucosylated glycogenin-1 was detected as a fluoracetic acid and the supernatant was evaporated to dryness in a 40 kDa band including the 6xHis tag on Western blot using a mono- vacuum centrifuge. Prior to MS analysis, the peptides were reconsti- clonal antibody to human glycogenin-1 (Fig. 2A, lane 2). After the ad- tuted in 0.2% formic acid. dition of UDP-glucose to wild type glycogenin-1 a mobility shift of approximately 1 kDa was observed consistent with autoglucosylation 2.5. Nano-LC/MS/MS analysis of 8–13 glucose residues (Fig. 2A, lane 1). About half of the glycogenin-1 molecules demonstrated this shift in molecular weight The samples were injected using a HTC-PAL autosampler (CTC An- indicating that the recombinant wild type glycogenin-1 molecules alytics AG, Zwingen, Switzerland) connected to an Agilent 1100 bina- were not optimally autoglucosylated in this system. To further con- ry pump (Agilent Technologies, Palo Alto, CA). The peptides were firm that the gel-shift was a result of autoglucosylation the samples trapped on a precolumn (45×0.075 mm i.d.) and separated on re- were treated with alpha-amylase that specifically cleaves the α1,4 versed phase column, 200×0.050 mm. Both columns were packed glucosidic linkages between the glucose residues of the oligosaccha- in-house with Reprosil-Pur 3 mm C18-AQ particles. The flow through ride chain. This treatment resulted in a reduction of the molecular the analytical column was reduced by a split to approximately 100 nl/ weight similar to that of wild type glycogenin-1 before the addition min. A linear gradient of 10–50% acetonitrile in 0.2% formic acid dur- of UDP-glucose (Fig. 2A, lane 3). ing 40 min was used for separation of the peptides. The nanoflow LS/ Glycogenin-1 with the threonine to methionine substitution in MS/MS was performed on a hybrid linear ion trap-Fourier transform position 83 showed no size shift after 60 min in the presence of ion cyclotron resonance (FTICR) mass spectrometer equipped with a UDP-glucose and the molecular weight was unchanged by the 7 T ICR magnet (LTQ-FT, Thermo Fisher Scientific, Bremen, Germany). alpha-amylase treatment indicating that it was not at all autoglucosy- The spectrometer was operated in data-dependent mode, automati- lated (Fig. 2A, lanes 4–6). cally switching to MS/MS mode. MS-spectra were acquired in the FTICR, while MS/MS-spectra were acquired in the linear ion trap. For each scan of FTICR, the five most intense, doubly or triply charged, ions were sequentially fragmented in the linear ion trap by collision induced dissociation (CID).

2.6. MS data analysis

For peptide identification, all tandem mass spectra were searched by an in-house version of MASCOT (Version 2.2.0; Matrix Science, London). LTQ-FT .raw files were converted to .dta files by the Xcalibur software utility extract_msn (Thermo Fisher Scientific). The search parameters were set to: peptide tolerance, 10 ppm; MS/MS accuracy, 0.6 Da; enzyme, trypsin; one missed cleavage allowed, taxonomy; human, database; SwissProt 101005 (20,259 sequences), fixed carbamidomethyl modification of cysteine; variable oxidation of methionine and variable substitution of tyrosine by phenylalanine. The .dta file searches were repeated according to the parameters above, with the exception of using semitryptic enzyme specificity to account for non-tryptic N- or C-terminal end of the peptides. For glycopeptide identification, LTQ-FT .raw files were evaluated manually and searched for precursor ions that were 1–8 hexose residues (162.0528 Da–1296.4224 Da) heavier than co-eluting (±5 min) non-glucosylated peptides that included tyrosine 195. The MS2 spectra of such precursor ions were surveyed for neutral losses corresponding to hexose residues and individually converted to .mzXML fi format using the readW application. mzXML le searches were per- Fig. 2. Wild type and Thr83Met glycogenin-1 proteins expressed in a cell-free expres- formed with the mMass application [22] with precursor ion masses sion system, autoglucosylated in vitro in the presence of UDP-glucose and visualized manually defined by subtracting the monoisotopic mass of hexose with western blot (A) and autoradiography after incorporation of UDP-14C-glucose residues from the FTICR-MS1 mass measured precursor ion. Search (B). Wild type glycogenin-1 incorporated glucose in the presence of UDP-glucose (A and B, lane 1) that was cleaved with alpha-amylase (A and B, lane 3). Thr83Met parameters were set to: peptide tolerance, 10 ppm; MS/MS accuracy, glycogenin-1 did not incorporate glucose (A and B, lane 4) and was not affected by 0.6 Da, enzyme semitrypsin, one missed cleavage allowed, taxonomy; alpha-amylase (A and B, lane 6) indicating that Thr83Met glycogenin-1 was not cata- human, database; Swissprot 101005 (20,259 sequences), fixed lyzing the glucose-O-tyrosine 195 linkage. 496 J. Nilsson et al. / Biochimica et Biophysica Acta 1822 (2012) 493–499

Both wild type and Thr83Met glycogenin-1 from the cell-free ex- site-directed mutagenesis we changed threonine at position 83 to pression system were analyzed with size-exclusion chromatography. amino acids with different side chains and chemical properties Western blot of chromatography fractions showed detectable (Fig. 3A). The different glycogenin-1 variants were expressed in the glycogenin-1 as dimers or tetramers in both samples. However, the cell-free expression system. We then analyzed the ability of recombinant protein was also found as aggregates which might be glycogenin-1 with these different modifications to autoglucosylate an explanation for the incomplete autoglucosylation of wild type with the gel-shift assay. In addition to wild type glycogenin-1 with glycogenin-1 (Fig. 2A, lane 1). threonine at position 83, glycogenin-1 with serine at position 83 To study the glucose-O-tyrosine linkage at position 195 in mutant was also capable of autoglucosylation (Fig. 3B). This was not the and wild type glycogenin-1 the experiment was repeated with UDP- case for glycogenin-1 with methionine, alanine, cysteine, phenylala- 14C-glucose and analyzed by autoradiography (Fig. 2B). Wild type nine, tyrosine or valine substitutions at position 83 (Fig. 3B). Autoglu- 14 glycogenin-1 showed incorporation of glucose in the presence of cosylation experiments with UDP- C-glucose demonstrated glucose UDP-glucose (Fig. 2B, lane 1) and most of it was cleaved off by incorporation of glycogenin-1 with either threonine or serine in posi- alpha-amylase treatment (Fig. 2B, lane 3). Glycogenin-1 with the tion 83 but not with the other amino acids tested (Fig. 3C). These re- Thr83Met mutations showed no incorporation of glucose (Fig. 2B, sults demonstrate that threonine in position 83 was not lanes 4–6). These results indicate that the glycogenin-1 with the indispensable for catalytic function of glycogenin-1. A change of thre- Thr83Met mutation was not capable of catalyzing the glucose-O- onine to serine, which is similar to threonine with respect to size and tyrosine 195 linkage. To structurally characterize the glucosylation presence of a hydroxyl group, will thus retain the catalytic properties pattern mass spectrometry was applied, using nano-LC/MS/MS. of the protein. Glycogenin-1 constructs with the bulkier tyrosine, After adding UDP-glucose, the samples were treated with alpha- which also contains a hydroxyl group, as well as alanine, cysteine amylase in order to simplify the mass spectrometric analysis of gluco- and valine that have a similar size as threonine but lack a hydroxyl sylated peptides. Several semitryptic and tryptic peptides of different group, showed no autoglucosylation. length containing tyrosine 195 were detected and identified by the MASCOT algorithm from both wild type and Thr83Met glycogenin-1 3.3. Thr83Met glycogenin-1 is glucosylated by a catalytically active (Table 1). Most peptides derived from wild type glycogenin-1 sam- glycogenin-1 ples were modified with hexose (glucose) in the range between two and eight glucose molecules. For several of these glucosylated pep- Gel filtration analysis has shown that 95% of the glycogenin-1 mol- tides, MS/MS experiments also revealed the glucose moieties to be se- ecules exist as dimers [20] and it has been demonstrated that the cat- lectively attached to Tyr195 (Suppl Fig. 1E–G). Interestingly, not a alytically inactive Lys85Gln rabbit glycogenin-1 could be glucosylated single peptide containing only one glucose was detected suggesting by a catalytically active glycogenin-1 within a heterodimer [26]. Re- that alpha-amylase is unable to cleave α1,4 glucosidic linkages closer placement of the glucose-binding tyrosine 195 with phenylalanine than two glucose residues from the peptide backbone. None of the (Tyr195Phe) produces a glycogenin-1 that is catalytically active but identified peptides derived from Thr83Met glycogenin-1 that includ- incapable of binding glucose [27,28]. ed Tyr195 showed any glucose modifications confirming that To study whether the catalytically inactive human glycogenin-1 Thr83Met glycogenin-1 was unable to form the glucose-O-tyrosine with the Thr83Met mutation could be glucosylated we co-expressed linkage at position 195. Tyrosines at position 187 and 197 were recombinant Thr83Met and Tyr195Phe glycogenin-1 and studied never found to be glucosylated. autoglucosylation after the addition of UDP-14C-glucose (Fig. 4A). No incorporation of glucose in the Tyr195Phe glycogenin-1 sam- 3.2. Threonine at position 83 is not specific for catalytically active ple was detected (Fig. 4A, lane 3) but when Thr83Met and Tyr195Phe glycogenin-1 glycogenin-1 were co-expressed the Thr83Met glycogenin-1 was glucosylated (Fig. 4A, lane 2) indicating a glucosylation at Tyr195 of Threonine in position 83 of glycogenin-1 is a phylogenetically con- glycogenin-1 with the Thr83Met mutation, catalyzed by glycogenin- served amino acid within a potential UDP-binding fold [24,25].By 1 with the Tyr195Phe mutation.

Table 1 Peptides containing tyrosine 195 from wild type and Thr83Met glycogenin-1 detected by mass spectrometry.

Hexose modiﬁed tyrosin is underlined and Δppm larger than 10 is marked in red. *MASCOT ion scores are not available for all glucosylated peptides due to the inability of the algorithm to resolve complex post-translational modiﬁcations. J. Nilsson et al. / Biochimica et Biophysica Acta 1822 (2012) 493–499 497

Fig. 3. Threonine 83 of human glycogenin-1 was replaced using site-directed mutagenesis by amino acids with different side-chains (A). Mutated glycogenin-1 proteins were autoglucosylated in vitro.2μg of protein were separated on a SDS-PAGE and the autoglucosylation was assayed with gel-shift on western blot (B) and for incorporation of UDP-14C-glucose with autoradiography (C). Only threonine and serine in position 83 produced a catalytically active glycogenin-1 that incorporated glucose (B and C, lanes 1 and 3).

Nano-LC/MS/MS confirmed the presence of both glycogenin-1 mutation, Thr83Met, in the other. A heterozygous carrier of only the variants in the co-expression experiment and showed that at least Thr83Met mutation had no clinical signs or symptoms but a small two hexoses had been added to tyrosine 195 of Thr83Met proportion, less than 50%, of unglucosylated glycogenin-1 in muscle glycogenin-1 (Fig. 4B, Table 2). and cultured myoblasts [19]. These findings raised questions related to autoglucosylation of human glycogenin-1 and the importance 4. Discussion and function of threonine at position 83. Autoglucosylation of glycogenin-1 has previously been studied in rabbit skeletal muscle We recently described the first disease associated with a severe glycogenin-1, which is highly homologous to human glycogenin-1 defect in glycogenin-1, a protein necessary for priming of glycogen [5]. The autoglucosylation reaction includes binding and hydrolysing synthesis in muscle. The investigations demonstrated profound gly- UDP-glucose, the formation of a glucose-O-tyrosine linkage at posi- cogen depletion in muscle and mutations in the gene coding for tion 195 and the elongation of the oligosaccharide with several α1,4 glycogenin-1, GYG1. The patient was compound heterozygous for a glucosidic linkages. In this study we investigated autoglucosylation nontranslated nonsense mutation in one allele and a missense of the human glycogenin-1 and the effect of the Thr83Met

Fig. 4. A: The catalytically active Tyr195Phe glycogenin-1 and Thr83Met glycogenin-1 proteins were co-expressed and allowed to autoglucosylate in vitro. Incorporation of UDP-14C- glucose was detected with autoradiography. Tyr195Phe glycogenin-1 was unable to bind glucose (lane 3) but glucosylated the Thr83Met glycogenin-1 when co-expressed (lane 2). B: MS2 spectrum showing the fragmentation pattern of the glycopeptide IYNLSSISIY195SYLPAFK from Thr83Met glycogenin-1 co-expressed with Tyr195Phe glycogenin-1 detected with nano-LC/MS/MS. The peptide had two hexoses (glucoses) attached to tyrosine 195. 498 J. Nilsson et al. / Biochimica et Biophysica Acta 1822 (2012) 493–499

Table 2 MS detection of peptides containing tyrosine 195 or the mutated phenylalanine 195 from a co-expression of Thr83Met and Tyr195Phe glycogenin-1.

Non-native phenylalanine is marked in red and hexose modiﬁed tyrosin is underlined.

substitution. Since glycogenin-1 most likely autoglucosylates in any when co-expressed with Tyr195Phe glycogenin-1 that is catalytically cell-type containing UDP-glucose we expressed glycogenin-1 in a active but cannot be autoglucosylated itself since the essential hy- cell-free system free from UDP-glucose and studied the autoglucosy- droxyl of the aromatic residue is missing at position 195 [12].By lation after addition of UDP-glucose in vitro. nano-LC/MS/MS we demonstrated two glucose residues attached to By means of autoradiography, gel-shift assay and mass spectrom- Tyr195 of the Thr83Met glycogenin-1 when it was co-expressed etry we demonstrated that glycogenin-1 carrying the Thr83Met sub- with Tyr195Phe glycogenin-1. One possible model is that the stitution is unable to catalyze the first steps in the autoglucosylation tyrosine-O-linkage as well as at least one α1,4 glucosidic linkage at- reaction to form the glucose-O-tyrosine linkages at position 195. tached to Thr83Met glycogenin-1 were catalyzed by the Tyr195Phe Threonine 83 is located within the conserved domain II of within a heterodimer. glycogenin-1 [25] and mutagenesis of the lysine at position 85 has That intra-dimeric glucosylation can take place also in vivo was ev- shown that it is necessary for the catalytic function of rabbit glyco- ident from the findings in a healthy heterozygous carrier of the genin [26]. Crystallization of rabbit glycogenin has demonstrated a Thr83Met mutation [19]. In this case a small proportion of the β-α-β Rossman-like fold, common to the nucleotide binding domains glycogenin-1 was unglucosylated compatible with the presence of of other glycosyltransferases, in the N-terminal part of the protein in approximately 25% nonglucosylated Thr83Met glycogenin-1in homo- close vicinity to threonine 83 [24], suggesting that threonine 83 may dimers and the others being either Thr83 glycogenin-1 homodimers also be important for the binding or hydrolyzing of UDP-glucose. or Thr83Met/Thr83 heterodimers. This explains why the heterozy- To investigate how specific threonine at position 83 is for the gous carriers so far identified have no signs or symptoms related to autoglucosylation of human glycogenin-1 we applied site-directed defective skeletal muscle energy metabolism. mutagenesis to study the effects of various amino acid substitutions In conclusion we demonstrate that glycogenin-1 with the disease at this position. Threonine 83 was exchanged to amino acids of differ- causing amino acid substitution Thr83Met is unable to catalyze the ent size and with different side chains. Our results indicate that the glucose-O-tyrosine 195 linkage, the first step in the autoglucosylation hydroxyl group on threonine is important for the function. Serine is of glycogenin-1. However, Thr83Met glycogenin-1 is glucosylated by like threonine a small amino acid with a hydroxyl group and a change the enzymatically active Tyr195Phe glycogenin-1 molecule. of threonine 83 to serine 83 did not inhibit autoglucosylation. Alanine Supplementary materials related to this article can be found on- and valine are both small amino acids and essentially isosteric with line at doi:10.1016/j.bbadis.2011.11.017. threonine but in contrast to serine and threonine they have no hydroxyl groups. Changing threonine 83 to alanine 83 or valine 83 abol- Acknowledgements ished autoglucosylation. The presence of a hydroxyl group indicates that the side chain of Thr83 may be involved in a hydrogen bond. MS data were obtained from the Proteomics Core Facility at Sahl- However, the crystal structure of rabbit glycogenin (PDB 1LL2 [24]) grenska Academy University of Gothenburg. The study was supported shows that the geometry is incorrect for a proper hydrogen bond to by grants from the Swedish Research Council (07122 to AO, 8266 to b form despite being in close proximity ( 3 Å) to the backbone amide GL) and West Swedish Muscle Foundation. proton of the conserved, catalytically important Asp163. It is still possible that a stabilizing hydrogen bond is important transiently during References the catalytic cycle. Thr83 is contiguous in sequence to Val82 which forms one side of [1] S. DiMauro, C. Garone, A. Naini, Metabolic myopathies, Curr. Rheumatol. Rep. 12 the uridine binding pocket [24]. 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