(I-321) Chemoenzymatic Synthesis of Immune-Regulating Mucin Mimics

Introduction:: The mucosal immune system is the first obstacle a pathogen must overcome to gain entry to a host. For humans, mucosal membranes cover 400m² to separate the self from the non-self. Mucus and the glycocalyx are comprised of secreted or tethered glycoproteins called mucins. These high molecular weight structures consist of a backbone of amino acids with O-linked glycans on serine and threonine residues.

Mucin composition is highly variable between regions of the body and between members of the same species. Although heterogeneous, mucins exhibit conserved glycosylation motifs in specific disease states. There is not currently a method to model native mucin structure and mucins are either ignored entirely or modelled with unnatural linkages and a lack of tunability to the structure.

Specific O-linked glycans are over and under expressed in specific pathologies. We are interested in studying the glycans fucose and sialic acid. An overexpression of fucose terminated glycan structures on mucins has been observed in chronic conditions of the upper airways, including asthma and cystic fibrosis. Sialic acids are found in excess on mucins in infection and cancer progression, and are important immune regulators by interacting with siglec receptors on immune cells.

This presents an opportunity to engineer mucosal environments to elucidate the tole of mucins in the human immune response. In this study, we developed a method for chemoenzymatic synthesis of mucin mimics with tunable structure that matches native mucin conformation. Overall, we are designing and engineering models to probe mucosal immunity.


Materials and Methods:: A combination of chemical and enzymatic approaches were utilized to generate glycosylated polypeptides. Tunable synthetic mucin backbones were generated via ring-opening n-carboxyanhydride (NCA) polymerization with a transition metal catalyst. Terminal glycan decoration was accomplished with recombinantly expressed, bacterial derived glycosylation enzymes in a one-pot multi-enzyme reaction. Enzymes were expressed in e. coli competent cells and purified via divalent metal ion affinity chromatography to an N-terminal polyhistidine tag. Glycosylation efficiency for was characterized with commercially available assay kits for each sialic acid or fucose.




Results, Conclusions, and Discussions:: We were able to show the percent glycosylation of a polymer substrate via glycan quantification assays to determine the efficiency of our glycosylation procedures. This was accomplished via synthesis of NCA polymerized native mucin structures in combination with one-pot multi-enzyme reactions to tune glycosylation patterns of interest.

Here we report a streamlined procedure to fine tune glycosylation patterns on synthetic mucins. Chemically synthesized mucin glycopolypeptides were efficiently synthesized via NCA polymerization in a tunable, living polymerization while terminal bioactive glycans were appended at specific linkages via enzymatic reactions in vitro. Variations in synthetic mucin composition were studied, including backbone glycosylation density, serine or threonine sites for glycosylation initiation, non-glycosylated amino acid composition, and mucin glycan core structure variation. Terminal glycosylation density was quantified with previously established colorimetric assay methods.


Our protocol to generate tunable synthetic mucins opens opportunity to study mucosal immunity in a controlled in vitro and vivo environment. These mucins can be modelled to replicate aberrant mucin presentation in disease states from inflammatory conditions and cancer to pathogen infection and beyond. The materials generated from the protocols in this study will allow precise modeling of mucosal immunity. The ability to immunoengineer mucosal tissues will allow more efficient diagnosis, treatment, and therapy of a plethora of disease states caused by both external pathogens and internal chronic conditions.






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