Biology subjects

Wongtrakul-Kish, K.

Publications and source records attributed to Wongtrakul-Kish, K..

3 recordsLinked to original sources

bioRxiv2026

De-N-glycosylation of in vivo and in vitro adipogenic stem cell products unmasks differential expression of CD36 glycoprotein in human adipogenesis

Adipogenesis is the process of adipose-derived stem cells (ADSCs) responding to extracellular signals from the stem cell niche to differentiate into adipocytes (fat cells) and may be studied in vitro using a cocktail of chemicals that promote adipogenic differentiation to produce differentiated ADSCs (dADSCs). The global membrane N- and O-glycosylation changes of this process have been previously analysed and compared to native adipocytes as a benchmark for a true adipocyte profile, and revealed that bisecting GlcNAc type N-glycans are characteristic of adipogenesis. As stem cell differentiation has been widely reported to result in cellular protein changes, the same cells (ADSCs, dADSCs and mature adipocytes) were characterised for their membrane proteome here using label-free quantitative shotgun proteomics analysis. The membrane proteome displayed more differences in protein numbers between the cell types compared to the previously reported N-glycome which had shown high identical glycomes between stem cells and in vitro dADSCs, suggesting that the proteome is more dynamic during in vitro adipogenesis. Following the global shotgun proteomics analysis, a more targeted approach of carrying out proteomic analysis of de-N-glycosylated peptides of gel-separated proteins unearthed new glycoproteins not detected in the shotgun proteomic analysis. This approach identified the adipogenic marker, CD36, to be under-represented in the shotgun proteome analysis, but as the dominant (glyco)protein in the adipocyte membrane proteome that was also up-regulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). A comparison of CD36 sequence coverage in the global shotgun analysis with the de-N-glycosylated CD36 revealed a 41% increase when N-glycans were removed prior to trypsin digestion, explaining its observed increased abundance and highlights the crucial need for de-N-glycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of previously reported glycomics data and the proteomics and transcriptomics analyses in this work extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/722121v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@189a786org.highwire.dtl.DTLVardef@5563b8org.highwire.dtl.DTLVardef@5cb5borg.highwire.dtl.DTLVardef@69e11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

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

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 naive 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 glycans. In overlay assays, these sugars were differentially recognized by the C-type lectins mannose receptor and DC-SIGN. 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.

biochemistry↗

bioRxiv2020

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

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 hosts 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 Man5GlcNAc2 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.

biochemistry↗