Publications by authors named "Katherine Wongtrakul-Kish"

8 Publications

  • Page 1 of 1

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

PLoS Negl Trop Dis 2021 Feb 2;15(2):e0009071. Epub 2021 Feb 2.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.
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http://dx.doi.org/10.1371/journal.pntd.0009071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880456PMC
February 2021

Bisecting GlcNAc Protein -Glycosylation Is Characteristic of Human Adipogenesis.

J Proteome Res 2021 Feb 31;20(2):1313-1327. Epub 2020 Dec 31.

Biomolecular Discovery Research Centre, Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Human adipose tissue contains a major source of adipose-derived stem cells (ADSCs) that have the ability to differentiate into various cell types: , ADSCs can differentiate into mesenchymal lineages including adipocytes, while , ADSCs become mature adipocytes. Protein glycosylation has been shown to change in stem cell differentiation, and while ADSCs have been acknowledged for their therapeutic potential, little is known about protein glycosylation during human ADSC adipogenic differentiation. In the present study, the global membrane protein glycosylation of native adipocytes was compared to ADSCs from the same individuals as a model of adipogenesis. For adipogenesis, ADSCs were adipogenically differentiated in cell culture using an optimized, large-scale differentiation procedure. The membrane glycome of the differentiated ADSCs (dADSCs) was compared with mature adipocytes and the progenitor ADSCs. A total of 137 glycan structures were characterized across the three cell types using PGC-LC coupled with negative-ion electrospray ionization mass spectrometry (ESI-MS)/MS. Significantly higher levels of bisecting GlcNAc-type -glycans were detected in mature adipocytes (32.1% of total glycans) and in dADSC progeny (1.9% of total glycans) compared to ADSCs. This was further correlated by the mRNA expression of the MGAT3 gene responsible for the enzymatic synthesis of this structural type. The bisecting GlcNAc structures were found on the majority of human native adipocyte membrane proteins, suggesting an important role in human adipocyte biology. Core fucosylation was also significantly increased during adipogenesis but did not correlate with an increase in Fut8 gene transcript. Unexpectedly, low abundance structures carrying rare β-linked Gal-Gal termini were also detected. Overall, the -glycan profiles of the differentiated progeny did not reflect native adipocytes, and the results show that bisecting GlcNAc structures are a characteristic feature of human adipocyte membrane protein -glycosylation. Raw MS files are available on GlycoPOST (ID: GPST000153 https://glycopost.glycosmos.org/).
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http://dx.doi.org/10.1021/acs.jproteome.0c00702DOI Listing
February 2021

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis.

Sci Rep 2020 07 31;10(1):12903. Epub 2020 Jul 31.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with ManGlcNAc being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.
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http://dx.doi.org/10.1038/s41598-020-69753-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395719PMC
July 2020

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

Proteomics 2019 11 16;19(21-22):e1900010. Epub 2019 Oct 16.

Department of Molecular Sciences and Biomolecular Discovery and Design Research Centre (BDDRC), Macquarie University, Sydney, 2109, Australia.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man GlcNAc Fuc ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man GlcNAc Fuc , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-β-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.
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http://dx.doi.org/10.1002/pmic.201900010DOI Listing
November 2019

Combining Glucose Units, /, and Collision Cross Section Values: Multiattribute Data for Increased Accuracy in Automated Glycosphingolipid Glycan Identifications and Its Application in Triple Negative Breast Cancer.

Anal Chem 2019 07 27;91(14):9078-9085. Epub 2019 Jun 27.

Analytics Group, Bioprocessing Technology Institute , Agency for Science, Technology and Research (A*STAR) , Singapore 138668.

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labeled glycans using hydrophilic interaction high-performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterization of closely related or isomeric structures through the generation of glycan collision cross section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labeled GSL glycans using HILIC-IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass, and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.
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http://dx.doi.org/10.1021/acs.analchem.9b01476DOI Listing
July 2019

GlycanAnalyzer: software for automated interpretation of N-glycan profiles after exoglycosidase digestions.

Bioinformatics 2019 02;35(4):688-690

Bioprocessing Technology Institute Agency for Science, Technology and Research (A*STAR), Singapore.

Summary: Many eukaryotic proteins are modified by N-glycans. Liquid chromatography (ultra-performance -UPLC and high-performance-HPLC) coupled with mass spectrometry (MS) is conventionally used to characterize N-glycan structures. Software can automatically assign glycan structures by matching their observed retention times and masses with standardized values in reference databases. However, more precise confirmation of N-glycan structures can be derived using exoglycosidases, enzymes that remove specific monosaccharides from glycans. Exoglycosidase removal of monosaccharides results in signature peak shifts, in both UPLC and MS1, yielding an effective way to verify N-glycan structure with high detail (down to the position and isomeric linkage of each monosaccharide). Because manual interpretation of exoglycosidase data is complex and time consuming, we developed GlycanAnalyzer, a web application that pattern matches N-glycan peak shifts following exoglycosidase digestion and automates structure assignments. GlycanAnalyzer significantly improves assignment accuracy over other auto-assignment methods on tests with a monoclonal antibody and four glycan standards (100% versus 82% for the next best software). By automating data interpretation, GlycanAnalyzer enables the easier use of exoglycosidases to precisely define N-glycan structure.

Availability And Implementation: http://glycananalyzer.neb.com. Datasets available online.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378934PMC
February 2019

Characterization of N- and O-linked glycosylation changes in milk of the tammar wallaby (Macropus eugenii) over lactation.

Glycoconj J 2013 Jul 10;30(5):523-36. Epub 2012 Oct 10.

Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Building E8C, Room 307, North Ryde, NSW 2109, Australia.

As one of several biologically active compounds in milk, glycoproteins have been indicated to be involved in the protection of newborns from bacterial infection. As much of the physical and immune development of the tammar wallaby (Macropus eugenii) young occurs during the early phases of lactation and not in utero, the tammar is a model species for the characterization of potential developmental support agents provided by maternal milk.In the present study, the N- and O-linked glycans from tammar wallaby milk glycoproteins from six individuals at different lactation time points were subjected to glycomics analyses using porous graphitized carbon liquid chromatography electrospray ionization mass spectrometry. Structural characterization identified a diverse range of glycan structures on wallaby milk glycoproteins including sialylated, sulphated, core fucosylated and O-fucosylated structures. 30 % of N-linked structures contained a core (α1-6) fucose. Several of these structures may play roles in development, and exhibit statistically significant temporal changes over the lactation period. The N-glycome was found to contain structures with NeuGc residues, while in contrast the O-glycome did not. O-fucosylated structures were identified in the early stages of lactation indicating a potential role in the early stages of development of the pouch young. Overall the results suggest that wallaby milk contains structures known to have developmental and immunological significance in human milk and reproduction in other animals, highlighting the importance of glycoproteins in milk.
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http://dx.doi.org/10.1007/s10719-012-9452-8DOI Listing
July 2013