Sequence-Specific Mucins for Glycocalyx Engineering

Abstract

Few approaches exist for the stable and controllable synthesis of customized mucin glycoproteins for glycocalyx editing in eukaryotic cells. Taking advantage of custom gene synthesis and a biology-by-parts approach to cDNA construction, we build a library of swappable DNA bricks for mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized native mucin repeats and newly designed domains for synthetic mucins. We construct a library of over 50 mucins, each with unique chemical, structural, and optical properties and describe how additional permutations could readily be constructed. We apply the library to explore sequence-specific effects on glycosylation for engineering of mucins. We find that the extension of the immature α-GalNAc Tn-antigen to Core 1 and Core 2 glycan structures depends on the underlying peptide backbone sequence. Glycosylation could also be influenced through recycling motifs on the mucin cytoplasmic tail. We expect that the mucin parts inventory presented here can be broadly applied for glycocalyx research and mucin-based biotechnologies.

Keywords: custom gene synthesis; engineering; glycan; glycosylation; mucin; synthetic biology.

Figure 1.

Combinatorial genetic encoded library for sequence-specific mucins. (a) Schematic diagram of the combinatorial sequence-specific mucins. (b) Schematic shows the swappable biobricks and flanking restriction sites for complete mucin construction. (c) Workflow for the design and fabrication of cDNAs for the mucin tandem-repeat backbones. (d) Summary of codon-scrambled mucin backbones in the library.

Figure 2.

Construction and validation of sequence-specific mucin expression. (a) Components and features of codon-optimized Muc1 variants with GFP reporters. (b) Predicted molecular weight of the polypeptide backbone. (c) Biosynthesis of Tn antigen, Core 1, and Core 2 glycans, and specificity of relevant lectins for their detection. (d) Western blot analysis of Native Muc1 expression and glycosylation in wild-type and Core-1 β-3-T specific molecular chaperone (COSMC) knockout MCF10A cells. The MCF10A cells were stably transfected with native Muc1. The surface sialic acids were labeled with AFDye 568 through periodate labeling prior to lysate collection. The blot was stained in multiple colors with MUC1 TR (CD227 HPMV), Ab-FITC, and PNA-CF640 or biotinylated VVA (Secondary: NeutrAvidin-Dylight 650). (e) Western blot analysis of native and codon-scrambled Muc1 in extracts of transiently transfected HEK293T cells. (f) Immunofluorescence images of transiently transfected HEK293T cells expressing indicated constructs and probed with PNA lectin (left), anti-Muc1 antibody (center left), GFP (center right), and Hoescht nuclear stain (right) (scale bar 10 μm). (g) PNA lectin blot analysis (left) and intensity profiles (right) of mucins of varying sizes in extracts of transiently transfected HEK293T cells.

Figure 3.

Engineering the frequency of glycosylation sites in the Muc1 polymer backbone tunes O-glycan maturation. (a) Components and features of secreted Muc1 and engineered variants each with 21 tandem repeats. (b) Tandem repeat sequences of secreted mucin mutants and the molecular weight of the polypeptide backbones. Single, double, and triple glycosylation mutants (sMuc1S, sMuc1D, and sMuc1T) have one, two, or three, serine/threonine (S/T) to alanine substitutions per repeat, respectively. (c) Representative Western blot analysis of affinity-purified recombinant secreted mucins from FreeStyle 293-F cell culture media probed with anti-SUMOstar antibody and PNA, s-WGA, and VVA lectins (of three independent experiments). The lectin blot was costained in multiple colors with PNA-Alexa Fluor 568, s-WGA-FITC, and biotinylated VVA (Secondary: NeutrAvidin-Dylight 650). (d) Representative fluorescence intensity electrophoretograms of the blots in part c. (e) Ratiometric intensity analysis of PNA to VVA signal (upper) and s-WGA to VVA signal (lower) for the indicated mucins and their corresponding frequency of S/T glycosylation sites in the polymer backbone. Ratiometric fluorescence intensity was quantified along each lane and normalized to the signal from the secreted mucin with wild-type Muc1 tandem repeats (sMuc1); data presented as the mean and SEM from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (f) Left: MALDI-TOF mass spectra registered for samples of permethylated glycan alditols from secreted mucins with wild-type Muc1 tandem repeats (sMuc1) and triple mutant (sMuc1T) from HEK293T cell culture media. The ion signals were annotated with respect to the relative masses of molecular ions (m/z) detected as sodium adducts and by assignment of the respective core structure (red for Core 1 and black for Core 2). Right: Schematic presentation of O-linked glycans detected on the secreted mucins.

Figure 4.

Designer mucin domains reveal sequence-specific effects on glycosylation. (a) Components and features of designer mucins. (b) Predicted molecular weight of the mucin polypeptide backbones. (c) Representatie Western blot analysis (from three independent experiments) of indicated constructs in extracts of transiently transfected HEK293T cells probed with anti-GFP antibody or costained with PNA and VVA lectins. (d) Representative fluorescence intensity electrophoretograms of the Western blots in part c for indicated constructs from three independent experiments. Dashed lines indicate the peak of the glycoform visible in the PNA blot. Shaded boxes indicate the regions between the bands on the anti-GFP blot with the highest and second highest apparent molecular weights. (e) Ratiometric intensity analysis of PNA to VVA staining for the indicated mucins and their corresponding frequency of serine and threonine glycosylation sites in the polymer backbone. Fluorescence intensity was quantified along each lane of the dual-probed lectin blot, and the PNA/VVA ratio was normalized to that of the KEPAPTTP x20 mucin; data presented as the mean and SEM from three independent experiments. (f) The fold change in the PNA/VVA ratio with doubling the indicated mucin backbone size from 40 to 80 tandem repeats; data presented as the mean and SEM from three independent experiments. *p < 0.05

Figure 5.

Tuning mucin glycosylation through cytoplasmic tail engineering. (a) Components and features of cell-surface mucins with synthetic 21-amino-acid transmembrane anchors (TM21) and engineered cytoplasmic motifs; native CT refers to a native cytoplasmic tail adapted from Muc1. (b) Lectin blot analysis of the indicated mucin isoforms from transiently transfected HEK293T cells to detect sialylated O-glycans by periodate oxidation and Core-I structures by PNA; blots are representative of three independent experiments. (c) PNA-lectin blot analysis of the indicated mucin isoforms before and after sialidase treatment; blots are representative of three independent experiments. (d) Top: Representative MAA and PNA lectin blot analysis (from four independent experiments) of the indicated mucin isoforms immunoprecipitated from transiently transfected HEK293T cells. Bottom: Ratiometric intensity of sialic acid to Core 1 glycan signal (MAA/PNA); data presented as the mean and SEM from four independent experiments. *P < 0.05.