Recombinant mucin biotechnology and engineering

Abstract

Mucins represent a largely untapped class of polymeric building block for biomaterials, therapeutics, and other biotechnology. Because the mucin polymer backbone is genetically encoded, sequence-specific mucins with defined physical and biochemical properties can be fabricated using recombinant technologies. The pendent O-glycans of mucins are increasingly implicated in immunomodulation, suppression of pathogen virulence, and other biochemical activities. Recent advances in engineered cell production systems are enabling the scalable synthesis of recombinant mucins with precisely tuned glycan side chains, offering exciting possibilities to tune the biological functionality of mucin-based products. New metabolic and chemoenzymatic strategies enable further tuning and functionalization of mucin O-glycans, opening new possibilities to expand the chemical diversity and functionality of mucin building blocks. In this review, we discuss these advances, and the opportunities for engineered mucins in biomedical applications ranging from in vitro models to therapeutics.

Keywords: Biomaterial; Biopolymer; Drug delivery; Glycan; Lubrication; Mucin; Recombinant.

Fig. 1.. Mucin backbone and glycosylation.

(A) Mucins can be divided into secreted and membrane-associated family members. All mucins contain a serine, threonine, and proline rich region, often composed of variable number of tandem repeats (TRs), that are sites of O-glycosylation. In membrane-associated mucins, the heavily glycosylated domain is linked to a single pass transmembrane anchor and short cytoplasmic tail. Secreted mucins can be divided into gel-forming and non-gel forming subtypes. Gel-forming mucins contain N- and C-terminal cysteine-rich domains (orange, round) and contribute to the viscoelastic properties of mucus. (B) Mucins are defined by their densely grafted O-glycan structures on serine and threonine residues. O-glycosylation in mammals is initiated by transfer of N-acetylgalactosamine (GalNAc; shown) to the side chain of threonine or serine. Abbreviations: Gal, galactose; GlcNac, N-acetylglucosamine; GalNAc, N-glycolyl neuraminic acid; Ser, serine; Thr, threonine.

Fig. 2.. Applications of recombinant mucins.

Biomaterials: mucin biopolymers are under active investigation as building blocks for biocompatible materials and hydrogels. Therapeutics: mucin glycans have bioactivities that are being explored for immune modulation, attenuation of microbial virulence, and other applications. Drug delivery: mucins can be chemically functionalized to serve as carriers for drugs and other therapeutic agents. Lubrication: mucin-based lubricants like lubricin (shown) can hydrate, protect, and lubricate materials ranging from cartilage to contact lenses. Non-fouling coatings: mucin surface coatings can resist protein deposition and microbial interactions. Anti-adhesive coatings: mucins have potential as non-immunogenic alternatives to polyethylene glycol (PEG) and other synthetic polymers for surface coatings on liposomes and nanoparticles.

Fig. 3.. Human O-glycosylation pathway map.

Graphic depiction of O-glycosylation pathways with mucin-related glycosyltransferase genes. The basic O-GalNAc structure (Tn-antigen) is generated by a family of ppGalNAcT enzymes on serine or threonine residues primarily in the Golgi apparatus. The Tn-antigen can be extended to form one of the primary core structures (core 1–4) or capped with sialic acid to form the sialyl-Tn antigen. The core structures can subsequently be elongated or branched and capped through sialyltransferases, fucosyltransferases, sulfotransferases, and other enzymes. Main core structures and examples of their extended structures are shown in bold. Glycan symbols are drawn according to the Symbol Nomenclature for Glycans (SNFG) format.

Fig. 4.. Validated probes for evaluation of glycosylation.

All indicated probes have been validated for specificity on printed or cell-based glycan arrays in the indicated references. Additional binding motifs are indicated, if known.

Fig. 5.. Approaches for engineering and functionalization of mucin O-glycans.

(A) Aldehydes are selectively introduced into sialic acids by periodate oxidation or into terminal galactose and N-acetylgalactosamine with galactose oxidase. The aldehydes can subsequently be coupled with aminooxy containing chemistries using aniline as a catalyst. (B) Glycan structures can be labelled, extended, and modified in vitro through chemoenzymatic approaches. (C) The cellular glycome and the glycosylation patterns on individual mucins can be tuned through individual or combinatorial knockout and knockin of glycosyltransferases (D) Metabolic glycoengineering exploits exogenous unnatural sugars, such as Ac4ManNAz, to functionalize glycan structures for subsequent bio-conjugation with click chemistry.