Cloning, expression and characterization of aβ1,4-GalNAc Transferase from Schistosoma mansoni. Pubblico
Leon, Kristoffer Edgar (2015)
Abstract
Schistosomiasis is an insidious parasitic helminth infection that affects millions of people worldwide. Infection is marked by a number of clinical manifestations, ranging from dermatitis to chronic inflammation of the liver and spleen. Schistosoma mansoni is one of the helminth species responsible for schistosomiasis, and has been previously studied for its interactions with the human immune system and its carbohydrate (glycan) biochemistry. The entire genome has recently been published online and partially annotated. Computational tools were used to select a gene coding for a β1,4-N-acetylgalactosaminyltransferase (smβ1,4-GalNAcT), a class of enzymes responsible for synthesizing LacDiNac (LDN). LDN plays an important role in the immune response to S. mansoni and is a parent glycan that is further modified by the addition of fucose(s) to generate other antigenic carbohydrates. The gene was cloned into a mammalian expression vector, expressed in a mammalian cell line grown in suspension, purified and then used in activity assays to prove the computationally determined gene was a smβ1,4-GalNAcT. Activity assays were also used to determine metal cofactor necessity, and the specificity for the nucleotide sugar donor UDP-GalNAc. The enzyme itself is an important member of S. mansoni glycosylation pathways and immunology, and could potentially represent a target for pharmaceutical development. Lastly, the smβ1,4-GalNAcT provides a link between the glycome and genome. Glycomics is becoming more important, especially in understanding host-pathogen interactions, and knowing the enzymes necessary for glycan synthesis is important in understanding the complex and fascinating glycobiology of S. mansoni.
Table of Contents
Introduction...1
Schistosomiasis Epidemiology...1
Schistosoma mansoni life cycle...2
Schistosoma mansoni clinical manifestation...3
Immune Response to schistosomiasis...4
Carbohydrate Immunomodulation...5
Schistosoma mansoni and LDN...5
Figure 1: LDN and LDN Derived glycans...6
The glycogenome and glycosyltransferases...6
Figure 2: Glycosyltransferase Mechanism Scheme...7
Materials and Methods...8
Selection of a putativeβ 1,4-GalNacT...8
Cloning ofsmβ1,4-GalNacT...9
Expression of smβ1,4-GalNacT in HEK-Freestyle Cells...10
Purification of smβ1,4-GalNacT-GFP...10
Western Blots and Silver Stains...11
Enzyme Assays...11
Table 1: Endpoint Enzyme Assays...12
Table 2: Timepoint Enzyme Assays...12
HPLC Purification...13
MALDI-TOF Mass Spectrometry Analysis of Glycans...13
Results...14
Selection of a smβ1,4-GalNacT-GFP...14
Table 3: Gene IDs of the GalNAcTs...14
Figure 3: Amino acid alignments of putative GalNacTs...16
Figure 4: Phylogenetic trees of putative GalNacTs...16
Figure 5: Expression levels of putative β1,4-GalNacTs...16
Synthesis and cloning of smβ1,4-GalNacT-GFP...16
Figure 6A: Vector map of the smGalNAcT-pGEn2 vector...17
Figure 6B: Restriction digest analysis of smGalNAcT plasmids...18
Figure 7A,B: Flow cytometry of transfected Cells...19
Figure 7C,D,E: GFP expression by flouresence microscopy...19
Figure 8: Western blots of smGalNacT transfected cells...21
Activity Assays of smβ1,4-GalNacT-GFP...21
Figure 9: HPLC profiles of triose timepoint assays...22
Figure 10: HPLC profiles of chitobiose timepoint assays...22
Figure 11: MALDI-TOF Mass data of LDN...23
Discussion...24
Conclusion...28
References...32
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Cloning, expression and characterization of aβ1,4-GalNAc Transferase from Schistosoma mansoni. () | 2018-08-28 12:52:34 -0400 |
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