Khadra Mohamed
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E-mail: khadra.mohamed[at]ru.nl Phone: +31(0)24 3610559 HG: lab 03.314 |
Functional characterisation of the human sialyltransferase isoenzyme family
Glycans cover the surfaces of all living cells and are displayed on lipids (glycolipids) and proteins (glycoproteins) and central to most cell functions and interactions. Glycans are complex and highly diverse biomolecules consisting of different monosaccharides that are assembled into a staggering array of small, long, linear, or branched structures – the glycome. Unlike DNA, RNA, and proteins, glycan assembly follows non-template-driven biosynthetic steps performed by ca. 200 glycosyltransferase (GTfs) enzymes. Furthermore, metabolic proteins that biosynthesize, modify, and degrade the monosaccharides building blocks influence glycan structures produced by cells. Hence, glycans are considered the most complex and diverse biomolecules found in nature that can encode a vast amount of biological information that we are just beginning to understand. Over the past decades, the field was mainly focused on the development of analytical and synthetic approaches to identify and produce the diverse glycan structures. Their cellular biosynthesis and biological interactions and functions and role in pathologies are more recently entering the focus of glycoscience. Altered glycosylation is now associated with number of diseases such as cancer, pathogen infections, and neurodegeneration.
One highly versatile glycan building block are the sialic acids (Sia), 9-carbon carboxylated sugars prominently found on the terminal position of glycans. This prominent location positions sialic acids at the nexus of numerous molecular interactions for example with the immune system and sialic acids are often the molecular target for pathogens to enter host cells. More than 50 chemically distinct sialic acid family members were identified so far. This is why sialic acid-capped sialoglycans form a subclass within the glycome – the sialome. In humans, the most common sialic acid derivative is N-acetylneuraminic acid (Neu5Ac) that carries an acetyl moiety at the C5 position. Sialic acids are synthesized inside the cell from N-acetylmannosamine (MaNAc) and activated through the addition of cytidine monophosphate (CMP) inside the cell. CMP-sialic acids are transported into the Golgi apparatus by a specific transporter SLC35A1 and incorporated into glycans by sialyltransferases (STfs). In humans, there are 20 STfs isoenzymes that can be grouped based on the type of acceptor sugar (galactose (Gal), N-acetylgalactosamine (GalNAc) or Sia) and glycosidic linkage they produce between the C2 position of the Sia and the C3, C6, or C8 position (α2-3/6/8) respectively, of the penultimate sugar. The four major groups are ST3Gal1-6, ST6Gal1/2, ST6GalNAc1-6, and ST8Sia1-6. The individual contribution of the 20 STf isoenzymes to sialylation, particularly their glycan and protein substrate context, is poorly understood and their tissue and cell type specific expression and regulation is not resolved.
With the advent of CRISPR-based precise genome editing, genetic engineering of GTfs expression has led to the generation of the cell-based glycan array, libraries of stably engineered human embryonic kidney (HEK293) cells with lack/expression of sets of GTfs each displaying specific glycan features of the glycome. A recently generated sialic acid library with combinatorial knockouts and knock-ins of STfs enables expression of sets or individual STfs isoenzymes and results in the display of specific Sia linkages on specific glycans. This revealed the fine binding specifies of 14 sialic acid-binding immune receptors, the Siglecs, that recognize specific products formed by individual or multiples STfs. This genetic glycoengineering strategy now enables dissection of the specific functions of the STfs isoenzymes. However, despite this current advance, the molecular mechanisms regulating STfs expression, localisation, and STf-STf interactions are scarcely known. Furthermore, differences in the abundances of certain sialoform or their discrepancy in e.g., inflammatory diseases can be addressed more precisely if we obtain a better knowledge of the expression patterns of the STfs, how changes in sialome arise, and more importantly, the specific activities of each STf isoenzyme. Thus, in this project, we aim to decipher the specific functions of individual STfs isoenzymes to elucidate their biological roles which could warrant further investigation into their roles in diseases such as inflammation where glycosylation changes are described.