PhD research assistant State University of New York-Buffalo, United States
Introduction: : Glycosylation is an essential post-translational modification in mammals that mediates molecular recognition and diverse fundamental biological processes. Glycans are assembled by the concerted action of an enzyme family called glycosyltransferases, primarily in the endoplasmic reticulum (ER) and Golgi. In humans, this family consists of a set of 200+ members that form a range of complex extracellular carbohydrate structures, and simpler modifications inside cells. Due to their unique substrate specificity that distinguishes glycan stereoisomers, engineering these enzymes could result in new avenues for developing biocatalysts. Understanding the molecular features regulating glycosyltransferase activity is also important during the diagnosis of congenital disorders of glycosylation (CDG), as some mutations may have only minor effects on enzyme activity whereas other point mutations could result in clinical phenotype. High-throughput methods are needed to study structure-function relationships of mammalian glycosyltransferases due to their essential role in assembling the glycocalyx. However, such methods are currently unavailable. We address this shortcoming in this abstract.
Materials and
Methods: : The presentation will describe the development of a novel biomolecular engineering technology to quantify mammalian glycosyltransferase activity following surface display on human cells. Here, to mimic the natural presentation of glycosyltransferases that express with an N-terminal transmembrane domain and C-terminal catalytic domain within the ER/Golgi lumen, we incorporate the transmembrane and cytoplasmic domains of two type-II transmembrane proteins, CD94/NKG2 and CD26/DPP4 (dipeptidyl peptidase-4) at the N-terminus of Fc-fusion glycosyltransferases. This enables glycosyltransferase presentation both in the ER/Golgi lumen and on the cell surface. Click chemistry with azido-derivatized nucleotide sugars are then used to quantify enzyme activity using flow cytometry. This paper demonstrates the use of this technology for screening sialyltransferase mutations en mass, assaying sialyltransferases from various organisms reported in the CAZy (Carbohydrate-Active EnZymes) database, and also characterizing diverse sub-types of human sialyltransferases.
Results, Conclusions, and Discussions:: Results &
Discussion: The paper tested the hypothesis that attachment of Type II transmembrane and cytoplasmic domains of CD26 and CD94 can result in surface display of active, mammalian glycosyltransferase. To this end HEK293T knockout cells lacking human ST3Gal1, were used as a platform. In one aspect using lectins to quantify cell surface enzyme activity, we confirmed that such surface display was possible. As the signal change due to lectins was small, we additionally developed a click chemistry-based strategy coupled with flow cytometry to quantify enzyme activity. Here, Neisseria meningitidis CMP-sialic acid synthase (NmCSS), CTP and 9-azido-Neu5Ac (Neu5Ac,9Az) were used in a one pot-reaction to generate CMP-Neu5Ac,9Az in situ. The presence of surface displayed active sialyltransferase then enabled the formation of azido derivatized cell-surface sialoglycans that could be quantified using either fluorescent DBCO (DBCO-AF488) or DBCO-biotin followed by addition of fluorescent anti-biotin antibodies (Figure). This platform was then used to screening of 1680 different pig ST3Gal1 mutants to yield α(2,3)sialyltransferases with improved enzymatic properties. These are so called “super enzymes”. Using endogenous cell-surface substrates, the method was also extended to other human sialyltransferases, including ST6Gal1, ST3Gal4, and ST6GalNAc2. Additionally, the approach was used to screen putative sialyltransferases from diverse organisms not characterized in the CAZy (Carbohydrate-Active EnZymes) database. This yield new insights into the natural evolution of sialyltransferases across the phylogenetic tree.
Conclusions: Overall, the paper describes a facile, robust, high-throughput, low-cost strategy to study glycosyltransferase structure-function relationships using mammalian surface display and click-chemistry. Using this approach, high-throughput analysis of glycosyltransferase structure-function relationships is possible. We demonstrate that this can be applied across diverse CAZymes. While the current study focusses on sialyltransferases, the technology should also be readily scalable to other enzyme families. This approach may enable large-scale mutagenesis studies to generate vast amounts of data needed for modern machine learning applications, exploration of glycosyltransferase activity across organisms, and provide new opportunities for glycoengineering and translational sciences.
