Using FACE, we depict and practically demonstrate the isolation and visualization of glycans liberated during the digestion of oligosaccharides by glycoside hydrolases (GHs). This is exemplified through two cases: (i) the digestion of chitobiose by the streptococcal -hexosaminidase GH20C and (ii) the digestion of glycogen by the GH13 member SpuA.
Fourier transform mid-infrared spectroscopy (FTIR) is a robust method for compositional characterization of plant cell walls. Each absorption peak in the infrared spectrum of a sample corresponds to a vibrational frequency between the bonds of the atoms, thus creating a distinct material fingerprint. This paper introduces a technique centered around the utilization of FTIR spectroscopy in conjunction with principal component analysis (PCA) for characterizing the chemical makeup of the plant cell wall. In a cost-effective and non-destructive manner, the described FTIR approach allows for high-throughput identification of the essential compositional distinctions within a vast collection of samples.
The protective roles of gel-forming mucins, highly O-glycosylated polymeric glycoproteins, are crucial for shielding tissues from environmental insult. PF-3644022 To decipher their biochemical properties, these samples must undergo an extraction and enrichment procedure starting from the biological samples. A method for obtaining and partially refining human and murine mucins from intestinal scrapings and/or fecal material is presented. Traditional gel electrophoresis methods are insufficient for separating mucins, given their substantial molecular weights, thereby hindering effective analysis of these glycoproteins. The creation of composite sodium dodecyl sulfate urea agarose-polyacrylamide (SDS-UAgPAGE) gels is described, enabling accurate band confirmation and resolution of extracted mucins.
Situated on white blood cells, a family of receptors called Siglecs are known for their immunomodulatory functions. By binding to cell surface sialic acid-containing glycans, Siglecs modify the closeness of their interaction with other receptors that they control. Immune response modulation is directly influenced by the proximity-based signaling motifs located on the cytosolic domain of Siglecs. As Siglecs play pivotal roles in maintaining immune homeostasis, a more profound understanding of their glycan ligands is vital for a clearer comprehension of their significance in health and disease. For exploring Siglec ligands on cellular surfaces, soluble forms of recombinant Siglecs are often employed in conjunction with flow cytometry. A key benefit of flow cytometry is the ability to quickly determine the relative levels of Siglec ligands among different cellular constituents. Detailed instructions are given on how to perform the most accurate and sensitive detection of Siglec ligands on cells through the use of flow cytometry, following a sequential process.
Intact tissues are routinely assessed for antigen localization using the immunocytochemistry technique. The intricate structure of plant cell walls, a matrix of highly decorated polysaccharides, underscores the vast array of CBM families, each uniquely recognizing their substrates. Large proteins, such as antibodies, may encounter difficulties in reaching their cell wall epitopes, potentially due to steric hindrance. In view of their smaller size, CBMs are a compelling substitute for probes. This chapter describes how CBM probes are used to examine the intricate polysaccharide topochemistry in the cell wall and to quantify the enzymatic degradation.
Plant cell wall hydrolysis is substantially influenced by the interplay of proteins like enzymes and CBMs, thereby shaping their specific roles and operational effectiveness. Highlighting the importance of various parameters associated with protein affinity and polymer type and organization in assemblies, bioinspired assemblies coupled with FRAP diffusion and interaction measurements represent a crucial alternative to simple ligand interaction characterizations.
The last two decades have witnessed the emergence of surface plasmon resonance (SPR) analysis as a key tool for scrutinizing protein-carbohydrate interactions, offering various commercial instruments for researchers. Measurable nM to mM binding affinities are possible; however, the associated risks necessitate cautious experimental planning. pediatric infection We present a comprehensive overview of the SPR analysis process, covering all steps from immobilization to data interpretation, and offering key considerations for practitioners seeking reproducible results.
Isothermal titration calorimetry provides a means of determining the thermodynamic parameters for the interaction between proteins and mono- or oligosaccharides dissolved in solution. The determination of stoichiometry and affinity in protein-carbohydrate interactions, coupled with the evaluation of enthalpic and entropic contributions, can be reliably achieved using a robust method, which doesn't require labeled proteins or substrates. A method for measuring binding energetics involving multiple injections is described in this section, specifically for the interaction between an oligosaccharide and a carbohydrate-binding protein.
Protein-carbohydrate interactions can be scrutinized by employing solution-state nuclear magnetic resonance (NMR) spectroscopy techniques. Within this chapter, two-dimensional 1H-15N heteronuclear single quantum coherence (HSQC) techniques are presented enabling the swift and effective screening of a panel of carbohydrate-binding partners, enabling the measurement of the dissociation constant (Kd), and allowing for mapping of the carbohydrate-binding site onto the protein's structural layout. This study details the titration of CpCBM32, a carbohydrate-binding module from Clostridium perfringens, family 32, with N-acetylgalactosamine (GalNAc). The investigation encompasses calculating the apparent dissociation constant and mapping the binding site of GalNAc onto the three-dimensional structure of CpCBM32. This strategy can be implemented in various CBM- and protein-ligand systems.
Microscale thermophoresis (MST) is an emerging technology, displaying high sensitivity, for the investigation of a wide assortment of biomolecular interactions. For a comprehensive selection of molecules, affinity constants can be obtained quickly, utilizing microliter-scale reactions within minutes. Employing the MST algorithm, we assess and quantify the interactions between proteins and carbohydrates in this study. Titration of a CBM3a occurs with insoluble cellulose nanocrystals, and a separate titration of a CBM4 is performed with soluble xylohexaose.
The interaction of proteins with sizable soluble ligands has been a long-standing subject of study utilizing affinity electrophoresis. This technique has proven exceptionally valuable in investigating the binding of polysaccharides by proteins, notably carbohydrate-binding modules (CBMs). Employing this method, recent years have also witnessed investigations into carbohydrate-binding sites of proteins, frequently present on enzyme surfaces. The following protocol illustrates how to identify binding interactions between the catalytic domains of enzymes and various carbohydrate ligands.
Proteins known as expansins, devoid of enzymatic activity, are essential for the relaxation of plant cell walls in plants. Bacterial expansin's biomechanical activity is measured via two custom protocols, which are detailed below. The initial investigation employs expansin to weaken the filter paper, a key element of the procedure. Creep (long-term, irreversible extension) is the focus of the second assay, applied to plant cell wall samples.
Cellulosomes, meticulously refined through evolution, are multi-enzymatic nanomachines that expertly break down plant biomass. Cellulosomal component integration is orchestrated by precisely arranged protein-protein interactions, linking the enzyme-associated dockerin modules to the numerous cohesin modules present on the scaffoldin. To effectively degrade plant cell wall polysaccharides, designer cellulosome technology was developed to provide insights into the roles of the catalytic (enzymatic) and structural (scaffoldin) cellulosomal components. Genomics and proteomics advancements have led to the discovery of intricately structured cellulosome complexes, consequently boosting the sophistication of designer-cellulosome technology. Consequently, our capacity to elevate the catalytic potential of artificial cellulolytic structures has been advanced by these higher-order designer cellulosomes. Procedures for the generation and application of such complex cellulosomal arrangements are documented in this chapter.
Lytic polysaccharide monooxygenases participate in the oxidative cleavage of glycosidic bonds present in a variety of polysaccharides. medical autonomy Cellulose or chitin activity is a common characteristic of the LMPOs examined so far, making the analysis of these activities the principal subject of this review. Significantly, the count of LPMOs engaged with different polysaccharides is on the rise. Cellulose, after processing by LPMOs, can undergo oxidation at either the C1 position, the C4 position, or both. These alterations, though resulting in only slight structural changes, nonetheless render both chromatographic separation and mass spectrometry-based product identification difficult tasks. The modifications in physicochemical characteristics stemming from oxidation must be considered when selecting analytical procedures. The oxidation of carbon one leads to a sugar that loses its reducing capacity, gaining instead acidic characteristics; oxidation at carbon four, in contrast, yields products that are highly susceptible to degradation at both extremely acidic and extremely alkaline conditions. These products display a keto-gemdiol equilibrium, which favors the gemdiol form significantly in aqueous solutions. Native products arise from the partial deterioration of C4-oxidized byproducts, which might explain claims of glycoside hydrolase activity in studies of LPMOs. Importantly, apparent glycoside hydrolase activity might be explained by the presence of trace levels of contaminating glycoside hydrolases, as these typically have significantly higher catalytic rates than LPMOs. In order to compensate for the low catalytic turnover rates of LPMOs, sensitive product detection methods are indispensable, consequently limiting the range of analytical procedures.