Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.

Acetylation in Ionic Liquids Dramatically Increases Yield in the Glycosyl Composition and Linkage Analysis of Insoluble and Acidic Polysaccharides.

Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo ß-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal ß-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

A single amino acid change led to structural and functional differentiation of PvHd1 to control flowering in switchgrass.

Switchgrass, a forage and bioenergy crop, occurs as two main ecotypes with different but overlapping ranges of adaptation. The two ecotypes differ in a range of characteristics, including flowering time. Flowering time determines the duration of vegetative development and therefore biomass accumulation, a key trait in bioenergy crops. No causal variants for flowering time differences between switchgrass ecotypes have, as yet, been identified. In this study, we mapped a robust flowering time quantitative trait locus (QTL) on chromosome 4K in a biparental F2 population and characterized the flowering-associated transcription factor gene PvHd1, an ortholog of CONSTANS in Arabidopsis and Heading date 1 in rice, as the underlying causal gene. Protein modeling predicted that a serine to glycine substitution at position 35 (p.S35G) in B-Box domain 1 greatly altered the global structure of the PvHd1 protein. The predicted variation in protein compactness was supported in vitro by a 4 °C shift in denaturation temperature. Overexpressing the PvHd1-p.35S allele in a late-flowering CONSTANS-null Arabidopsis mutant rescued earlier flowering, whereas PvHd1-p.35G had a reduced ability to promote flowering, demonstrating that the structural variation led to functional divergence. Our findings provide us with a tool to manipulate the timing of floral transition in switchgrass cultivars and, potentially, expand their cultivation range.

Glycosyltransferase family 47 (GT47) proteins in plants and animals.

Glycosyltransferases (GTs) are carbohydrate-active enzymes that are encoded by the genomes of organisms spanning all domains of life. GTs catalyze glycosidic bond formation, transferring a sugar monomer from an activated donor to an acceptor substrate, often another saccharide. GTs from family 47 (GT47, PF03016) are involved in the synthesis of complex glycoproteins in mammals and insects and play a major role in the synthesis of almost every class of polysaccharide in plants, with the exception of cellulose, callose, and mixed linkage ß-1,3/1,4-glucan. GT47 enzymes adopt a GT-B fold and catalyze the formation of glycosidic bonds through an inverting mechanism. Unlike animal genomes, which encode few GT47 enzymes, plant genomes contain 30 or more diverse GT47 coding sequences. Our current knowledge of the GT47 family across plant species brings us an interesting view, showcasing how members exhibit a great diversity in both donor and acceptor substrate specificity, even for members that are classified in the same phylogenetic clade. Thus, we discuss how plant GT47 family members represent a great case to study the relationship between substrate specificity, protein structure, and protein evolution. Most of the plant GT47 enzymes that are identified to date are involved in biosynthesis of plant cell wall polysaccharides, including xyloglucan, xylan, mannan, and pectins. This indicates unique and crucial roles of plant GT47 enzymes in cell wall formation. The aim of this review is to summarize findings about GT47 enzymes and highlight new challenges and approaches on the horizon to study this family.

An update on xylan structure, biosynthesis, and potential commercial applications.

•Xylan is an abundant carbohydrate component of plant cell walls that is vital for proper cell wall structure and vascular tissue development.•Xylan structure is known to vary between different tissues and species.•The role of xylan in the plant cell wall is to interact with cellulose, lignin, and hemicelluloses.•Xylan synthesis is directed by several types of Golgi-localized enzymes.•Xylan is being explored as an eco-friendly resource for diverse commercial applications.

Structural and biochemical insight into a modular ß-1,4-galactan synthase in plants.

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1) transfers galactose and arabinose to either extend or cap the ß-1,4-galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBM95, and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a 'handshake' orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.

Insights into pectin O-acetylation in the plant cell wall: structure, synthesis, and modification.

O-Acetyl esterification is an important structural and functional feature of pectins present in the cell walls of all land plants. The amount and positions of pectin acetyl substituents varies across plant tissues and stages of development. Plant growth and response to biotic and abiotic stress are known to be significantly influenced by pectin O-acetylation. Gel formation is a key characteristic of pectins, and many studies have shown that gel formation is dependent upon the degree of acetylation. Previous studies have indicated that members of the TRICHOME BIREFRINGENCE-LIKE (TBL) family may play a role in the O-acetylation of pectin, however, biochemical evidence for acceptor specific pectin acetyltransferase activity remains to be confirmed and the exact mechanism(s) for catalysis must be determined. Pectin acetylesterases (PAEs) affect pectin acetylation as they hydrolyze acetylester bonds and have a role in the amount and distribution of O-acetylation. Several mutant studies suggest the critical role of pectin O-acetylation; however, additional research is required to fully understand this. This review aims to discuss the importance, role, and putative mechanism of pectin O-acetylation.

Chemo-Enzymatic Synthesis of Long-Chain Oligosaccharides for Studying Xylan-Modifying Enzymes.

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies.

Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure-function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga Klebsormidium flaccidum is a powerful biocatalytic tool for the bottom-up synthesis of pure ß-1,4 xylan polymers that self-assemble into microparticles in vitro. Using uridine diphosphate-xylose (UDP-xylose) and defined saccharide primers as substrates, we demonstrate that the shape, composition, and properties of the self-assembling xylan microparticles could be readily controlled via the fine structure of the xylan oligosaccharide primer used to initiate polymer elongation. Furthermore, we highlight two approaches for bottom-up and surface functionalization of xylan microparticles with chemical probes and explore the susceptibility of xylan microparticles to enzymatic hydrolysis. Together, these results provide a useful platform for structural and functional studies of xylans to investigate cell wall biosynthesis and polymer-polymer interactions and suggest possible routes to new biobased materials with favorable properties for biomedical and renewable applications.

Accelerating Biological Insight for Understudied Genes.

The rapid expansion of genome sequence data is increasing the discovery of protein-coding genes across all domains of life. Annotating these genes with reliable functional information is necessary to understand evolution, to define the full biochemical space accessed by nature, and to identify target genes for biotechnology improvements. The majority of proteins are annotated based on sequence conservation with no specific biological, biochemical, genetic, or cellular function identified. Recent technical advances throughout the biological sciences enable experimental research on these understudied protein-coding genes in a broader collection of species. However, scientists have incentives and biases to continue focusing on well documented genes within their preferred model organism. This perspective suggests a research model that seeks to break historic silos of research bias by enabling interdisciplinary teams to accelerate biological functional annotation. We propose an initiative to develop coordinated projects of collaborating evolutionary biologists, cell biologists, geneticists, and biochemists that will focus on subsets of target genes in multiple model organisms. Concurrent analysis in multiple organisms takes advantage of evolutionary divergence and selection, which causes individual species to be better suited as experimental models for specific genes. Most importantly, multisystem approaches would encourage transdisciplinary critical thinking and hypothesis testing that is inherently slow in current biological research.

Rational enzyme design for controlled functionalization of acetylated xylan for cell-free polymer biosynthesis.

Xylan O-acetyltransferase 1 (XOAT1) is involved in O-acetylating the backbone of hemicellulose xylan. Recent structural analysis of XOAT1 showed two unequal lobes forming a cleft that is predicted to accommodate and position xylan acceptors into proximity with the catalytic triad. Here, we used docking and molecular dynamics simulations to investigate the optimal orientation of xylan in the binding cleft of XOAT1 and identify putative key residues (Gln445 and Arg444 on Minor lobe & Asn312, Met311 and Asp403 on Major lobe) involved in substrate interactions. Site-directed mutagenesis coupled with biochemical analyses revealed the major lobe of XOAT1 is important for xylan binding. Mutation of single key residues yielded XOAT1 variants with various enzymatic efficiencies that are applicable to one-pot synthesis of xylan polymers with different degrees of O-acetylation. Taken together, our results demonstrate the effectiveness of computational modeling in guiding enzyme engineering aimed at modulating xylan and redesigning plant cell walls.

Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II.

BACKGROUND: In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature's most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. RESULTS: We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. CONCLUSIONS: The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

AtFUT4 and AtFUT6 Are Arabinofuranose-Specific Fucosyltransferases.

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), AtFUT4, and AtFUT6, are members of the plant-specific GT family 37 (GT37). AtFUT4 and AtFUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. AtFUT4 and AtFUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of AtFUT4 and AtFUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.

Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1.

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/ß/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases.

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized "on chip" by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.

Analytical Techniques for Determining the Role of Domain of Unknown Function 579 Proteins in the Synthesis of O-Methylated Plant Polysaccharides.

Matrix polysaccharides are a diverse group of structurally complex carbohydrates and account for a large portion of the biomass consumed as food or used to produce fuels and materials. Glucuronoxylan and arabinogalactan protein are matrix glycans that have sidechains decorated with 4-O-methyl glucuronosyl residues. Methylation is a key determinant of the physical properties of these wall glycopolymers and consequently affects both their biological function and ability to interact with other wall polymers. Indeed, there is increasing interest in determining the distribution and abundance of methyl-etherified polysaccharides in different plant species, tissues, and developmental stages. There is also a need to understand the mechanisms involved in their biosynthesis. Members of the Domain of Unknown Function (DUF) 579 family have been demonstrated to have a role in the biosynthesis of methyl-etherified glycans. Here we describe methods for the analysis of the 4-O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. We also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants. Our approach facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

The most abundant cyst wall proteins of Acanthamoeba castellanii are lectins that bind cellulose and localize to distinct structures in developing and mature cyst walls.

BACKGROUND: Acanthamoeba castellanii, which causes keratitis and blindness in under-resourced countries, is an emerging pathogen worldwide, because of its association with contact lens use. The wall makes cysts resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. METHODOLOGY/PRINCIPAL FINDINGS: Transmission electron microscopy and structured illumination microscopy (SIM) showed purified cyst walls of A. castellanii retained an outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect them. Mass spectrometry showed candidate cyst wall proteins were dominated by three families of lectins (named here Jonah, Luke, and Leo), which bound well to cellulose and less well to chitin. An abundant Jonah lectin, which has one choice-of-anchor A (CAA) domain, was made early during encystation and localized to the ectocyst layer of cyst walls. An abundant Luke lectin, which has two carbohydrate-binding modules (CBM49), outlined small, flat ostioles in a single-layered primordial wall and localized to the endocyst layer and ostioles of mature walls. An abundant Leo lectin, which has two unique domains with eight Cys residues each (8-Cys), localized to the endocyst layer and ostioles. The Jonah lectin and glycopolymers, to which it binds, were accessible in the ectocyst layer. In contrast, Luke and Leo lectins and the glycopolymers, to which they bind, were mostly inaccessible in the endocyst layer and ostioles. CONCLUSIONS/SIGNIFICANCE: The most abundant A. castellanii cyst wall proteins are three sets of lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Cyst wall formation is a tightly choreographed event, in which lectins and glycopolymers combine to form a mature wall with a protected endocyst layer. Because of its accessibility in the ectocyst layer, an abundant Jonah lectin is an excellent diagnostic target.