Glycogen phosphorylase of shrimp (Litopenaeus vannamei): Structure, expression and anti-WSSV function.
Zhang Yong-Sheng,Li Fei-Xiang,Yao Cui-Luan
Fish & shellfish immunology
Glycogen phosphorylase (GP, EC 18.104.22.168) catalyze the rate-limiting step in glycogenolysis in animals, forming glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Therefore, GP plays a crucial role in carbohydrate metabolism. In the present study, the full-length cDNA sequence of GP (LvGP) was cloned from shrimp, Litopenaeus vannamei. The obtained 3242-bp LvGP cDNA sequence included a 5'-terminal untranslated region (UTR) of 48 bp, an open reading frame (ORF) of 2559 bp encoding a polypeptide of 852 amino acids (aa) and a 3'-UTR of 635 bp. The predicted LvGP protein sequence contained a typical phosphorylase domain (113-829 aa) and shared 72%-97% identities with GP from other species. Phylogenetic analysis revealed that LvGP showed the closest relationship with GP from Marsupenaeus japonicus. Tissue expression profiles showed that LvGP existed in most examined tissues, with the most predominant expression in the brain, followed by the muscles and stomach. LvGP transcripts in hepatopancreas and hemocytes were up regulated after the WSSV challenge. Furthermore, the role of LvGP in shrimp defending against WSSV infection was investigated by RNA interference (RNAi). Our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvGP RNAi (P < 0.01). The glycogen, glucose, and pyruvate content decreased in GP RNAi shrimp after WSSV injection, however, the lactate and ATP concentration enhanced. Moreover, lectin and anti-lipopolysaccharide factor2 (ALF2) were induced in LvGP silencing shrimp after WSSV infection, whereas the expression levels of crustin, ALF1 and ALF3 decreased. Our results suggested that the LvGP might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious gene expression.
Affinity Crystallography Reveals the Bioactive Compounds of Industrial Juicing Byproducts of Punica granatum for Glycogen Phosphorylase.
Stravodimos George A,Kantsadi Anastassia L,Apostolou Anna,Kyriakis Efthimios,Kafaski-Kanelli Vassiliki-Nafsika,Solovou Theodora,Gatzona Pagona,Liggri Panagiota G V,Theofanous Stavroula,Gorgogietas Vyron A,Kissa Apostolia,Psachoula Chariklia,Lemonakis Angelos,Chatzileontiadou Demetra S M,Psarra Anna-Maria G,Skamnaki Vassiliki T,Haroutounian Serkos A,Leonidas Demetres D
Current drug discovery technologies
BACKGROUND:Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. OBJECTIVE:The aim was to examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. METHOD:Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. RESULTS:All extracts exhibited significant in vitro inhibitory potency (IC50 values in the range of low μg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively.While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3Mthat acts synergistically with ellagic acid. CONCLUSION:Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements, our study provides a first positive step towards this process.
Candidate Gene Polymorphisms and their Association with Glycogen Content in the Pacific Oyster Crassostrea gigas.
She Zhicai,Li Li,Qi Haigang,Song Kai,Que Huayong,Zhang Guofan
BACKGROUND:The Pacific oyster Crassostrea gigas is an important cultivated shellfish that is rich in nutrients. It contains high levels of glycogen, which is of high nutritional value. To investigate the genetic basis of this high glycogen content and its variation, we conducted a candidate gene association analysis using a wild population, and confirmed our results using an independent population, via targeted gene resequencing and mRNA expression analysis. RESULTS:We validated 295 SNPs in the 90 candidate genes surveyed for association with glycogen content, 86 of were ultimately genotyped in all 144 experimental individuals from Jiaonan (JN). In addition, 732 SNPs were genotyped via targeted gene resequencing. Two SNPs (Cg_SNP_TY202 and Cg_SNP_3021) in Cg_GD1 (glycogen debranching enzyme) and one SNP (Cg_SNP_4) in Cg_GP1 (glycogen phosphorylase) were identified as being associated with glycogen content. The glycogen content of individuals with genotypes TT and TC in Cg_SNP_TY202 was higher than that of individuals with genotype CC. The transcript abundance of both glycogen-associated genes was differentially expressed in high glycogen content and low glycogen content individuals. CONCLUSIONS:This study identified three polymorphisms in two genes associated with oyster glycogen content, via candidate gene association analysis. The transcript abundance differences in Cg_GD1 and Cg_GP1 between low- and the high-glycogen content individuals suggests that it is possible that transcript regulation is mediated by variations of Cg_SNP_TY202, Cg_SNP_3021, and Cg_SNP_4. These findings will not only provide insights into the genetic basis of oyster quality, but also promote research into the molecular breeding of oysters.