Day 1 :
Keynote Forum
Liang Lin
Northwestern University, IL 60208, USA
Keynote: SITE-SPECIFIC GLYCOSYLATION OF PROTEINS VIA N-GLYCOSYLTRANSFERACES WITH SUBSTRATE ORTHOGONALITY
Biography:
Liang has completed his PhD from Peking University in Chemical Biology at 2015, and now is a postdoc at Department of Biomedical Engineering, Northwestern University from then. Liang’s main work at Northwestern University is using high-throughput MALDI technique (SAMDI) combining cell-free protein synthesis to fast and systematic characterize glycosyltransferases in glycoprotein engineering.
Abstract:
Glycosylation is the most complex post-translational modification and modulates the functions of many proteins. Glycoproteins isolated from living cells are generally a heterogeneous mixture of glycoforms, making it difficult to understand how specific glycan modifications effect protein function. Two-step chemoenzymatic glycosylation approach introduces glycoforms by first truncating the heterogeneous glycoforms into a monosaccharide primer and then using glycosyltransferases or glycosidases to elaborate the desired glycoform. However, this method lacks control in directing the initial glycosylation and therefore is typically not suited to preparing proteins having multiple, site-specifically defined oligosaccharide modifications. Here we introduce orthogonal N-glycosyltransferases (NGTs) to selectively direct the initial monosaccharide modification to a specific sequence in the protein. To identify the orthogonal NGTs, we developed an approach for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), which combines cell-free protein synthesis and high throughput screening with self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. We screened 41 putative NGTs and one reported NGT mutant and found four NGTs having orthogonal peptide substrate specificities. To further refine the specificities of NGTs, we prepared and characterized hundreds of NGT mutants and found sets of engineered mutants that have even greater orthogonality for peptide substrates. We then engineered the model protein colicin E7 immunity proteins with the four orthogonal sequences and sequentially treated the protein with the four corresponding NGTs to give a tetraglycosylated protein with 62% purity. Importantly, the hundreds of NGTs, forming an NGT library, that we characterized now provide the ability to modify most canonical N-glycosylation sequences, N-X-S/T (X is not P), and some non-canonical sequences, N-X-Z (Z is not S/T). Therefore, we can glycosylate approved or potential therapeutic proteins and vaccines (even those not containing canonical glycosylation sequences) using suitable NGTs from the NGT library, with site selectivity, to obtain desired properties such as prolonged serum life time and increased immunogenicity.
- Glycobiology and Glycochemistry, Proteoglycan and Sialic acid, Protein engineering and Glycoimmunology
Chair
Liang
Northwestern University, IL 60208, USA
Session Introduction
Muhammad. I. K.Afridi
University North Cyprus.Institute of Health Science
Title: Role of Adiponectin in Menopausal Women
Biography:
Muhammad. I. K.Afridi, University North Cyprus.Institute of Health Science
Abstract:
Adipogenesis refers to the differentiation of pre-adipocytes into mature fat cells, i.e. the development of adipose tissue, which varies according to sex and age. Adipocytes differentiate from stellate or fusiform precursor cells of mesenchymal origin.Adiponectin has been postulated to act an important role in the modulation of glucose and lipd metabolism in insulin-sensitive tissue in both humans and animals. The transition from pre to post menopause is associated with the emergency of many features of metabolic state. The intraabdominal body fat increases, low density lipoprotein and triglyceride levels increase while high density lipoprotein decreases. As the results to date are conflicting. In our study we aimed to study the changes in adiponectin and anthropometric parameters after menopause.For this purpose the ELISA methods was used in the study to evaulate the values of adiponectin. A total of 70 female in menopause and 90 control subjects were included in this study.The results showed that adiponectin , BMI and blood pressure increased with menopause and in order to investigate the effect of menopause on these parameters, further work must be carried out in the near future.
Erin Smith
University of Otago, Wellington, New Zealand.
Title: Increased maternal fructose intake alters milk composition, offspring plasma free fatty acids and hepatic lipid deposition
Biography:
Erin is currently in her second year of her PhD at the University of Otago School of Medicine in Wellington, New Zealand. Her study focus is to investigate the effects of excess maternal fructose on pregnancy, lactation and offspring development and adult health status.
Abstract:
Globally, dietary fructose is a major public health concern. Fructose can contribute to insulin resistance, hepatic de novo lipogenesis, hypertriglyceridemia and obesity. Since diet during pregnancy can influence fetal growth and later-life disease predisposition. Little is known regarding the effects of fructose during pregnancy and the influence on offspring development and predisposition to later-life disease. Female guinea pigs were randomly allocated to control (CD) or fructose (FD) (10% in drinking water) groups. Following 60 days of fructose intake, guinea pigs were mated. Pregnant dams continued ad libitum access to fructose water throughout pregnancy. Oral Glucose Tolerance Tests (OGTT) were performed prior to fructose feeding at 12 weeks of age, 60 days after fructose feeding and at mid-gestation day 35. Following birth, all litters were standardized to 4 pups/litter. Offspring groups were weighed daily and underwent blood collection on day 0, 7, and 14. On day 21 offspring underwent an OGTT and post-mortem tissues were also collected. Maternal response to glucose was significantly increased in the FD group (P>0.05). Significant increases in milk pentadecanoic(P>0.01), vaccenic (P>0.01), cis-vaccenic (P>0.01) and palmitoleic acids (P>0.08) were observed. At day 7 offspring plasma glucose was significantly increased in FD males and females. Likewise, free fatty acids were increased in offspring plasma, Total saturates (P=0.01), C18:0 (P=0.02), C16:0 (P=0.002). Metabolomics and proteomics are currently being analysed.
Elrefaei EH
Tanta University, Egypt
Title: Effects of berberine on high-fat/high-sucrose induced non-alcoholic steatohepatitis (NASH) in experimental rats
Biography:
Biography
Eman Elrefaei has completed her bachelor degree in medicine and general surgery at age of 25 years from Tanta university, school of medicine Egypt. Then got her MD in medical biochemistry and molecular biology at age of 28 years.
Abstract:
Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the world, characterized by hepatic steatosis, inflammation, hepatocyte injury with or without fibrogenesis, which might lead to cirrhosis. Berberine (BBR) is a natural isoquinoline alkaloid with very impressive health benefits. The aim of this study is to evaluate the protective effect of BBR in experimental NASH induced by high fat/high-sucrose diet in male albino rats. Methods: 60 male albino rats divided randomly into four equal groups: group I (normal control group), group II (BBR treated control group), group III (NASH group) and group IV (BBR treated NASH group). Levels of PGC-1α in hepatic nuclear extract were measured by ELISA, while activity of cytosolic glycerol 3 phosphate dehydrogenase (GPDH1) in liver tissue homogenate, liver enzymes, lipid profile and plasma FRAP were measured spectrophotometrically. Results: There was a statistically significant decrease of hepatic PGC-1α, plasma FRAP, serum HDL-C along with significant increase in the activity of GPDH1, liver enzymes as well as hyperlipidemia in NASH group compared to both normal control and BBR treated control groups. These pathological disturbances were significantly ameliorated by BBR supplementation. In conclusion, the present study provided unequivocal evidence that disturbed hepatic PGC-1α and altered redox status acted as major contributing factors for the pathogenesis of high-fat/high-sucrose induced NASH in rats. It also shed some light on the potential therapeutic value of BBR in NASH; partly accredited to its hypolipidemic and antioxidant effects, in addition to upregulating the levels of PGC-1α in hepatic nuclear extracts.
Sarita Aryal1
National Tsinghua University, Hsinchu 300, Taiwan
Title: Structural basis of investigation of cyclophilin 1 and cyclophilin 2 with Myb 3 transcription factor in Trichomonas veginalis
Biography:
Sarita Aryal1, National Tsinghua University, Hsinchu 300, Taiwan
Abstract:
Trichomoniasis, caused by Trichomonas vaginalis is the most common non-viral sexually transmitted disease in humans and is correlated with elevated susceptibility to HIV and HPV transmission. During pregnancy overload of iron can express hydrogenosomal enzymes and help in cytoadherence of T. vaginalis for its growth. Previous studies suggested that ap65-1 gene is involving in parasitic cytoadherence and its transcription is mediated by the coordinated action of Myb1, Myb2, and Myb3 transcription factors. Cyclophilin 1 (TvCyP1) and Cyclophilin 2 (TvCyP2), are peptidyl-prolyl isomerase present in the human parasite Trichomonas vaginalis, interacts with Myb transcription factors. In this study, we determined the unique structure of TvCyP2 as a monomer where its extra N-terminus loop goes in to the catalytic pocket of its own monomer fitting well into the active site and auxiliary pocket, respectively. Surprisingly N-terminus loop is mimicking the substrate as cyclosporine or Myb1 peptide which showed to have auto inhibition mechanism for TvCyP2. Using NMR and other techniques, we are now trying to find the role of this extra N-terminus extension on enzyme catalytic activity and auto inhibition. For the protein-protein interactions, our NMR data further showed that TvCyP1 and TvCyP2 both have interaction with minimum TvCyP1-binding sequence of Myb3 (Myb3 50–87). This result is further confirmed using ITC which also showed this myb3 peptide does bind with TvCyP1 and TvCyP2.Together with the finding we are further focusing on X-Ray studies for the complex structures TvCyP1 and TvCyP2 with Myb3 peptide. Together, the structure of TvCyP2 and detailed structural insights on TvCyP1-Myb3 interaction could pave the way for newer drugs to treat drug resistant strains.