Biochemistry

Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-α1,3-GlcA-β1,3]_n that is uniquely synthesized on α-dystroglycan (α-DG) and is essential for neuromuscular function and brain development. It binds several extracellular matrix proteins that contain laminin-globular domains and is a receptor for Old World arenaviruses such as Lassa Fever virus. Monoclonal antibodies such as IIH6 are commonly used to detect matriglycan on α-DG. However, endogenous expression levels are not sufficient to detect and analyze matriglycan by mass spectrometry approaches. Thus, there is a growing need to independently confirm the presence of matriglycan on α-DG and possibly other proteins. We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: β-Glucuronidase from Thermotoga maritima and α-xylosidase from Sulfolobus solfataricus. This allowed us to identify and categorize matriglycan on α-DG by studying post-digestion changes in the molecular weight of α-DG using SDS-PAGE followed by western blotting with anti-matriglycan antibodies, anti-core α-DG antibodies, and/or laminin binding assay. In some tissues, matriglycan is capped by a sulfate group, which renders it resistant to digestion by these dual exoglycosidases. Thus, this method can be used to determine the capping status of matriglycan. To date, matriglycan has only been identified on vertebrate α-DG. We anticipate that this method will facilitate the discovery of matriglycan on α-DG in other species and possibly on other proteins.

Key features

• Analysis of endogenous matriglycan on dystroglycan from any animal tissue.

• Matriglycan is digested using thermophilic enzymes, which require optimum thermophilic conditions.

• Western blotting is used to assay the success and extent of digestion.

• Freshly purified enzymes work best to digest matriglycan.

Graphical overview

α-Dystroglycan (α-DG) from muscle is shown here modified by a phosphorylated core M3 glycan, which extends further and terminates in a repeating disaccharide of xylose (Xyl) and glucuronic acid (GlcA) called matriglycan. β-glucuronidase (Bgus) and α-xylosidase (Xyls) hydrolyze the β-1,3-linked GlcA and α-1,3 linked-Xyl, starting from the terminal residues.

Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography–mass spectrometry (GC–MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC–MS.

Key features

• Comparative analysis of monosaccharide content in Arabidopsis-derived pectin between different genotypes or different treatments.

• Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue.

• Allows quick analyses of neutral and acidic monosaccharides simultaneously.

Graphical overview

Lipopolysaccharides (LPS) (or lipooligosaccharides [LOS], which lack the O-antigen side chains characteristic of LPS), and outer membrane proteins (OMP) are major cell-surface molecules in the outer membrane (OM) of gram-negative bacteria. The LPS is responsible for causing endotoxic shock in infected hosts and, in conjunction with some OMPs, provides protection to the bacterium against host innate immune defenses and attachment to host cells. Electrophoretic analysis can provide valuable information regarding the size, number, and variability of LPS/LOS and OMP components between bacterial strains and mutants, which aids in understanding the basic biology and virulence factors of a particular species. Furthermore, highly purified extracts are normally not required if only electrophoretic analysis is to be done, and various methods have been established for such procedures. Here, we review ameliorated procedures for fast and convenient extraction of LPS/LOS and protein-enriched outer membranes (PEOM) for optimal electrophoretic resolution. Specifically, we will describe the phenol-based micro-method for LPS/LOS extraction, a differential extraction procedure with sodium lauryl sarcosinate for PEOM, and gel preparation for electrophoretic analysis of LPS/LOS samples in detail.

Graphic abstract:

Workflow for the preparation and analysis of LPS/LOS and PEOM.

Colloidal chitin (CC) is a common substrate used in research work involving chitin-active enzymes (chitinases). Cell free supernatant (CFS) is prepared from fermented broth. Preparation of CC and CFS usually involve large amounts of liquid, which must be separated from the solids. This necessitates the use of a large volume centrifugation facility, which may not be accessible to everyone. Filtration is a viable alternative to centrifugation, and several filter elements are described in the literature. Each of those elements has its own set of disadvantages like non-availability, high cost, fragility, and non-reusability. Here we describe the use of lab coat clothing material (LCCM) for the preparation of CC and CFS. For filtration purposes, the LCCM was found to be functional, rugged, reusable, and cost-effective. Also described here is a new method for the estimation of laminarinase using a laminarin infused agarose gel plate. An easily available optical fabric brightener (OFB) was used as a stain for the agarose plate. The laminarin infused agarose plate assay is simple, inexpensive, and was found to be impervious to high amounts of ammonium sulfate (AS) in enzyme precipitates.

Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis.

Graphical abstract:

Overview of chromatographic methods for assessing the enzymatic degradation of chitin

(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely uncharacterized due to the absence of informative activity assays. Previously, a radioactive assay and reducing end modification have been used to characterize GS activity. The radioactive assay determines only the total amount of glucan formed through glucose incorporation and does not report the length of the polymers produced. The glucan length has been characterized by reducing end modification, but this method is unsuitable for mechanistic studies due to the very high detection limit of millimolar amounts and the labor intensiveness of the technique. Consequently, fundamental aspects of GS catalysis, such as the polymer length specificity, remain ambiguous. We have developed a size exclusion chromatography (SEC)-based method that allows detailed functional and mechanistic characterization of GS. The approach harnesses the pH-dependent solubility of (1,3)-β-d-glucan, where (1,3)-β-d-glucan forms water-soluble random coils under basic pH conditions, and can be analyzed by SEC using pulsed amperometric detection (PAD) and radioactivity counting (RC). This approach allows quantitative characterization of the total amount and length of glucan produced by GS with minimal workup and a d-glucose (Glc) detection limit of ~100 pmol. Consequently, this approach was successfully used for the kinetic characterization of GS, providing the first detailed mechanistic insight into GS catalysis. Due to its sensitivity, the assay is applicable to the characterization of GS from any fungi and can be adapted to study other polysaccharide synthases.

We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on permethylated di- and multiply sulfated glycans will survive in nanoESI conditions to allow detection of multiply charged intact molecular ions, and more comprehensive MS/MS can be performed in an automated fashion at higher sensitivity, compared with MALDI-MS/MS. Parallel higher energy collision dissociation (HCD) and ion trap collision induced dissociation (CID)-based MS², coupled with product-dependent MS³ in data dependent acquisition mode proved to be highly productive when applied to resolve and identify the isomeric sulfated glycan structures. In-house glycomic data mining software, GlyPick, was developed and used to automate the downstream process of identification and relative quantification of target sulfated glycotopes based on summed intensity of their diagnostic MS² ions extracted from thousands of HCD-MS² and/or CID-MS² data.

Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous for selective detection in the negative ion mode if the sialic acids can first be neutralized. This is most conveniently achieved by a concerted sample preparation workflow in which permethylation is followed by solid phase fractionation to isolate the sulfated glycans prior to MS analysis. Importantly, we demonstrated that conventional NaOH/DMSO slurry permethylation method can retain the sulfates. Instead of extracting permethylated glycans into chloroform for sample clean-up, reverse phase C18 cartridge coupled with self-packed amine-tip or mixed mode weak anion exchange cartridge can be utilized to obtain in good yield the non-sulfated, mono-sulfated, and multiply sulfated permethylated glycans in separate fractions for sulfoglycomic analysis.

Pseudomonas syringae is a model plant pathogen that infects more than 50 plant species worldwide, thus leading to significant yield loss. Pseudomonas biofilm always adheres to the surfaces of medical devices or host cells, thereby contributing to infection. Biofilm formation can be visualized on numerous matrixes, including coverslips, silicone tubes, polypropylene and polystyrene. Confocal laser scanning microscopy can be used to visualize and analyze biofilm structure. In this study, we modified and applied the current method of P. aeruginosa biofilm measurement to P. syringae, and developed a convenient protocol to visualize P. syringae biofilm formation using a borosilicate glass tube as the matrix coupled with crystal violet staining.

Cyanobacteria, which have the extraordinary ability to grow using sunlight and carbon dioxide, are emerging as a green host to produce value-added products. Exploitation of this highly promising host to make products may depend on the ability to modulate the glucose metabolic pathway; it is the key metabolic pathway that generates intermediates that feed many industrially important pathways. Thus, before cyanobacteria can be considered as a leading source to produce value-added products, we must understand the interaction between glucose metabolism and other important cellular activities such as photosynthesis and chlorophyll metabolism. Here we describe reproducible and reliable methods for measuring extracellular glucose and glycogen levels from cyanobacteria.