Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Bioreversible Anionic Cloaking Enables Intracellular Protein Delivery with Ionizable Lipid Nanoparticles.

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic "cloak" to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic ß-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation.

N-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N-linked glycans is the difficulty of installing a single N-acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (N-GlcNAcylation). Here, we develop an in vitro method for N-GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N-GlcNAc and successfully determine that PglB with an N311V mutation (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy.

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O -glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O -glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O -glycosylation in the first larval stage and identify O -glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans , we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Cell-Free Systems for the Production of Glycoproteins.

Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction.

Proteomic profiling of membrane vesicles from Mycobacterium avium subsp. paratuberculosis: Navigating towards an insilico design of a multi-epitope vaccine targeting membrane vesicle proteins.

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SIGNIFICANCE: Johne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease.

SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders.

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding "guide" peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous ß-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation.

Glycovaccinology: The design and engineering of carbohydrate-based vaccine components.

Vaccines remain one of the most important pillars in preventative medicine, providing protection against a wide array of diseases by inducing humoral and/or cellular immunity. Of the many possible candidate antigens for subunit vaccine development, carbohydrates are particularly appealing because of their ubiquitous presence on the surface of all living cells, viruses, and parasites as well as their known interactions with both innate and adaptive immune cells. Indeed, several licensed vaccines leverage bacterial cell-surface carbohydrates as antigens for inducing antigen-specific plasma cells secreting protective antibodies and the development of memory T and B cells. Carbohydrates have also garnered attention in other aspects of vaccine development, for example, as adjuvants that enhance the immune response by either activating innate immune responses or targeting specific immune cells. Additionally, carbohydrates can function as immunomodulators that dampen undesired humoral immune responses to entire protein antigens or specific, conserved regions on antigenic proteins. In this review, we highlight how the interplay between carbohydrates and the adaptive and innate arms of the immune response is guiding the development of glycans as vaccine components that act as antigens, adjuvants, and immunomodulators. We also discuss how advances in the field of synthetic glycobiology are enabling the design, engineering, and production of this new generation of carbohydrate-containing vaccine formulations with the potential to prevent infectious diseases, malignancies, and complex immune disorders.

Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates.

The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Profiling Germinal Center-like B Cell Responses to Conjugate Vaccines Using Synthetic Immune Organoids.

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice.

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

A Low-Cost, Thermostable, Cell-Free Protein Synthesis Platform for On-Demand Production of Conjugate Vaccines.

Cell-free protein synthesis systems that can be lyophilized for long-term, non-refrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability and a low-cost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ∼$0.50 per dose after storage at room temperature (∼22 °C) or 37 °C for up to 4 weeks, and ∼$1.00 per dose after storage at 50 °C for up to 4 weeks, with costs based on raw materials purchased at the laboratory scale. We show that these conjugate vaccines generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our low-cost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.

Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts.

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N-glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Twin-arginine translocase component TatB performs folding quality control via a chaperone-like activity.

The twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) system that synchronizes the proofreading of substrate protein folding with lipid bilayer transport. However, the molecular details of this QC mechanism remain poorly understood. Here, we hypothesized that the conformational state of Tat substrates is directly sensed by the TatB component of the bacterial Tat translocase. In support of this hypothesis, several TatB variants were observed to form functional translocases in vivo that had compromised QC activity as evidenced by the uncharacteristic export of several misfolded protein substrates. These variants each possessed cytoplasmic membrane-extrinsic domains that were either truncated or mutated in the vicinity of a conserved, highly flexible α-helical domain. In vitro folding experiments revealed that the TatB membrane-extrinsic domain behaved like a general molecular chaperone, transiently binding to highly structured, partially unfolded intermediates of a model protein, citrate synthase, in a manner that prevented its irreversible aggregation and stabilized the active species. Collectively, these results suggest that the Tat translocase may use chaperone-like client recognition to monitor the conformational status of its substrates.

Two-Tiered Selection and Screening Strategy to Increase Functional Enzyme Production in E. coli.

Development of recombinant enzymes as industrial biocatalysts or metabolic pathway elements requires soluble expression of active protein. Here we present a two-step strategy, combining a directed evolution selection with an enzyme activity screen, to increase the soluble production of enzymes in the cytoplasm of E. coli. The directed evolution component relies on the innate quality control of the twin-arginine translocation pathway coupled with antibiotic selection to isolate point mutations that promote intracellular solubility. A secondary screen is applied to ensure the solubility enhancement has not compromised enzyme activity. This strategy has been successfully applied to increase the soluble production of a fungal endocellulase by 30-fold in E. coli without change in enzyme specific activity through two rounds of directed evolution.