Chemically tunable mucin chimeras assembled on living cells

Abstract

Mucins are a family of secreted and transmembrane glycoproteins characterized by a massive domain of dense O-glycosylation on serine and threonine residues. Mucins are intimately involved in immunity and cancer, yet elucidation of the biological roles of their glycodomains has been complicated by their massive size, domain polymorphisms, and variable glycosylation patterns. Here we developed a synthetic route to a library of compositionally defined, high-molecular weight, dual end-functionalized mucin glycodomain constructs via N-carboxyanhydride polymerization. These glycopolypeptides are the first synthetic analogs to our knowledge to feature the native α-GalNAc linkage to serine with molecular weights similar to native mucins, solving a nearly 50-year synthetic challenge. Physical characterization of the mimics revealed insights into the structure and properties of mucins. The synthetic glycodomains were end-functionalized with an optical probe and a tetrazine moiety, which allowed site-specific bioorthogonal conjugation to an engineered membrane protein on live mammalian cells. This strategy in protein engineering will open avenues to explore the biological roles of cell surface mucins.

Equilibrium Modeling of the Mechanics and Structure of the Cancer Glycocalyx

“…Literature on experimental measurements of the properties of the glycocalyx is emerging. Constituents, such as HA and a variety of mucins, have been characterized through the measurement of molecular size (43)(44)(45) and persistence length (6,19,20,(46)(47)(48)(49). However, these glycocalyx constituents are highly variable because of dynamically changing glycosylation states and decoration with side chains.…”

“…However, these glycocalyx constituents are highly variable because of dynamically changing glycosylation states and decoration with side chains. The stiffness of bottlebrush mucins or proteindecorated HA chains likely depends strongly on the side chain density, size, composition, and charge (6,10). Although bottlebrush polymer theory provides relations between the polymer persistence length and the side chain properties (50), detailed experimental measurements of the decoration-dependent stiffness would be useful to develop a quantitative understanding of the glycocalyx and its functions on the cell membrane.…”

“…Mucins are defined by their densely clustered O-glycans along the central polypeptide backbone, giving rise to a molecular structure that resembles a comb or bottlebrush polymer. The bottlebrush structure is proposed to vary from flexible chains to relatively rigid rods upon increasing the glycosylation and the density of glycan side chains along the mucin backbone (6). Assembly of other biopolymer types on the cell surface has also been linked to cancer (1).…”

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins

“…Deglycosylation of rhMUC16 was performed according to the manufacturer's instructions (Deglycosylation Mix; Promega). Mucin mimetic copolymer consisting of 50% GalNAc-Ser and 50% Lys was synthesized as previously described (38). Both deglycosylated polymer and untreated polymer were subjected to StcE cleavage and gel staining as described above for recombinant protein substrates.…”

Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering

“…[62] The unnatural amino acid can carry variouso rthogonal chemical groups,s uch as ketone,a ldehyde, azide and norbornene, to functionalize ac ell surfacep rotein for subsequent modification. [63][64][65] This amber codon suppression methodr equires the expression of exogenous amino acid-tRNA synthetase and tRNA pair in the cells. Nevertheless, it unprecedentedly expands the chemical functionality of cells, which therefore enables us to manipulate cellular proteinsi nv ivo and in vitro with great liberty.M oreover,i ncorporation of two differentfunctionalities such as azide andt etrazine may afford more opportunities for cellular manipulation, such as tandem labeling on the cell surface.…”