Use of Genomic DNA as an Indirect Reference for Identifying Gender-Associated Transcripts in Morphologically Identical, but Chromosomally Distinct, Schistosoma mansoni Cercariae

BACKGROUND: The use of DNA microarray technology to study global Schistosoma gene expression has led to the rapid identification of novel biological processes, pathways or associations. Implementation of standardized DNA microarray protocols across laboratories would assist maximal interpretation of generated datasets and extend productive application of this technology. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing a new Schistosoma mansoni oligonucleotide DNA microarray composed of 37,632 elements, we show that schistosome genomic DNA (gDNA) hybridizes with less variation compared to complex mixed pools of S. mansoni cDNA material (R = 0.993 for gDNA compared to R = 0.956 for cDNA during ‘self versus self’ hybridizations). Furthermore, these effects are species-specific, with S. japonicum or Mus musculus gDNA failing to bind significantly to S. mansoni oligonucleotide DNA microarrays (e.g R = 0.350 when S. mansoni gDNA is co-hybridized with S. japonicum gDNA). Increased median fluorescent intensities (209.9) were also observed for DNA microarray elements hybridized with S. mansoni gDNA compared to complex mixed pools of S. mansoni cDNA (112.2). Exploiting these valuable characteristics, S. mansoni gDNA was used in two-channel DNA microarray hybridization experiments as a common reference for indirect identification of gender-associated transcripts in cercariae, a schistosome life-stage in which there is no overt sexual dimorphism. This led to the identification of 2,648 gender-associated transcripts. When compared to the 780 gender-associated transcripts identified by hybridization experiments utilizing a two-channel direct method (co-hybridization of male and female cercariae cDNA), indirect methods using gDNA were far superior in identifying greater quantities of differentially expressed transcripts. Interestingly, both methods identified a concordant subset of 188 male-associated and 156 female-associated cercarial transcripts, respectively. Gene ontology classification of these differentially expressed transcripts revealed a greater diversity of categories in male cercariae. Quantitative real-time PCR analysis confirmed the DNA microarray results and supported the reliability of this platform for identifying gender-associated transcripts. CONCLUSIONS/SIGNIFICANCE: Schistosome gDNA displays characteristics highly suitable for the comparison of two-channel DNA microarray results obtained from experiments conducted independently across laboratories. The schistosome transcripts identified here demonstrate, for the first time, that gender-associated patterns of expression are already well established in the morphologically identical, but chromosomally distinct, cercariae stage

Cardiomyopathy as presenting sign of glycogenin-1 deficiency-report of three cases and review of the literature.

We describe a new type of cardiomyopathy caused by a mutation in the glycogenin-1 gene (GYG1). Three unrelated male patients aged 34 to 52 years with cardiomyopathy and abnormal glycogen storage on endomyocardial biopsy were homozygous for the missense mutation p.Asp102His in GYG1. The mutated glycogenin-1 protein was expressed in cardiac tissue but had lost its ability to autoglucosylate as demonstrated by an in vitro assay and western blot analysis. It was therefore unable to form the primer for normal glycogen synthesis. Two of the patients showed similar patterns of heart dilatation, reduced ejection fraction and extensive late gadolinium enhancement on cardiac magnetic resonance imaging. These two patients were severely affected, necessitating cardiac transplantation. The cardiomyocyte storage material was characterized by large inclusions of periodic acid and Schiff positive material that was partly resistant to alpha-amylase treatment consistent with polyglucosan. The storage material had, unlike normal glycogen, a partly fibrillar structure by electron microscopy. None of the patients showed signs or symptoms of muscle weakness but a skeletal muscle biopsy in one case revealed muscle fibres with abnormal glycogen storage. Glycogenin-1 deficiency is known as a rare cause of skeletal muscle glycogen storage disease, usually without cardiomyopathy. We demonstrate that it may also be the cause of severe cardiomyopathy and cardiac failure without skeletal muscle weakness. GYG1 should be included in cardiomyopathy gene panels

The substrate specificity of isoamylase and the preparation of apo-glycogenin

A new facet of the specificity of the glycogen-debranching enzyme, isoamylase, namely, the hydrolysis of a carbohydrate-amino acid linkage, is described. This bond joins the terminal, reducing-end d-glucose unit of glycogen to the hydroxyl group of tyrosine in glycogenin, the primer protein for glycogen biogenesis. The specificity was further defined by demonstrating that 4-nitrophenyl α-maltotrioside and higher homologs also act as substrates. The splitting of the glycogen-glycogenin bond by isoamylase indicates the α-anomeric configuration of the terminal d-glucose unit. It also provides a means of preparing apoglycogenin. Pullulanase, a somewhat similar starch- and glycogen-debranching enzyme, does not split these new isoamylase substrates, permitting the 4-nitrophenyl saccharides to be used in distinguishing between isoamylase and pullulanase

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

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

Glycogenin: the primer for mammalian and yeast glycogen synthesis

Glycogen synthesis, whether in mammalian tissue, yeast, or Agrobacterium tumefaciens or other bacteria, is initiated by autoglucosylation of a protein. Initiation in muscle, by a self-glucosylating protein, glycogenin-1, is the most thoroughly studied system, as is described here. These relatively recent findings have prompted a rekindling of interest in the intermediates lying between the primer and mature mammalian glycogen

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