Background

Characterization of polysaccharides is fundamental to our understanding of various biological processes, such as cell wall biosynthesis in bacteria, fungi, and plants, biofilm formation by microbes, and the formation of extracellular matrix in humans. Although the characterization of short and soluble oligosaccharides can be performed using a variety of methods including thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry, significant technical challenges remain with respect to the characterization of long, biologically relevant polymers. As a result, molecular details regarding the size and mechanism of biosynthesis of large polysaccharides remain mostly unknown.

Over the past two decades, many methods have been developed to study charged and water-soluble polysaccharides. For example, hyaluronan has been characterized using paper chromatography (Tlapak-Simmons et al., 2005) or electrophoresis (Krupa et al., 2007), and bacterial peptidoglycan has been characterized by electrophoresis (Barrett et al., 2007); however, few methods are available for the characterization of charge-neutral and water-insoluble polysaccharides. Among such polysaccharides, (1,3)-β-d-glucan is an essential structural component of the fungal cell wall (Munro, 2013), and its biosynthetic enzyme, (1,3)-β-d-glucan synthase (GS), is a proven target of FDA-approved antifungal antibiotics (Douglas, 2001). Thus, characterizing the mechanism of catalysis and inhibition of GS is imperative to understand fungal cell wall biosynthesis and the mechanism of action and resistance against GS-targeted antifungal drugs. However, despite GS activity having been known since the 1980s (Shematek et al., 1980), detailed mechanistic characterization has not been possible due to the absence of appropriate methods to quantitatively evaluate the amount and length of (1,3)-β-d-glucan. Consequently, many fundamental aspects of this enzyme, such as the product length specificity, remain ambiguous.

GS has been characterized using radioactive assays (Shematek et al., 1980) that quantitate the total amount of water-insoluble (1,3)-β-d-glucan by determining the amount of incorporated d-glucose (Glc). While this method determines the overall activity of GS, it does not reveal the length of the polymer products. The length of GS products has been characterized using reducing end modification (Shematek et al., 1980), where the reducing end of (1,3)-β-d-glucan is reduced to sorbitol and the polymer hydrolyzed to monosaccharides. Subsequently, the length is determined based on the ratio between Glc and sorbitol. This analysis suggests that the average length of (1,3)-β-d-glucan produced by GS in a crude membrane preparation is 60–80 mer (Shematek et al., 1980); however, this approach requires a large amount of (1,3)-β-d-glucan, usually in millimolar quantities. Moreover, the method can underestimate the length by cleavage via a peeling reaction or other mechanisms during the workup or purification (Chhetri et al., 2020); therefore, there currently exists no suitable method for the detailed mechanistic characterization of GS.

More recently, size exclusion chromatography (SEC) has been used to characterize the length of water-insoluble polysaccharides; however, the chromatography conditions and detection methods frequently limit its use in detailed mechanistic investigations. For example, fungal cell wall glucan and chitin were analyzed by SEC after carboxymethyl derivatization with monochloroacetic acid using radioactivity as the detection method (Cabib and Duran, 2005; Cabib, 2009). This chemical derivatization solubilizes otherwise water-insoluble glucan and chitin and allows SEC analysis using an aqueous solvent. However, since the derivatization is not quantitative, the absolute length of the polymers was not determined, and only the relative lengths of glucan- and chitin-containing isolated cell walls were determined (Cabib et al., 2012).

SEC has also been applied to characterize bacterial cellulose synthase. In this case, cellulose was solubilized in dimethylacetamide containing 8% LiCl (w/v) and analyzed by gel permeation chromatography coupled to multi-angle light scattering (GPC-MALS) (McManus et al., 2018). This approach avoids the pitfalls of chemical derivatization and is potentially applicable to many other polysaccharides. However, the length was determined only under the steady-state of enzyme catalysis, and elongation of the cellulose polymer was not detectable, likely due to the limited sensitivity of refractive index detection.

Here, we report the protocol used for the determination of the amount and length of (1,3)-β-d-glucan using SEC in aqueous sodium hydroxide with pulsed amperometric detection (PAD) and radioactivity counting (RC). This protocol overcomes the aforementioned limitations: PAD and RC allow the characterization of the length and distribution of glucan polymers at sensitivities appropriate for the mechanistic characterization of GS, and the use of aqueous sodium hydroxide as a solvent allows solubilization of the otherwise water-insoluble (1,3)-β-d-glucan. The detection limit of this approach (~100 pmol) is greater than four orders of magnitude lower than reducing end modification previously reported for (1,3)-β-d-glucan characterization. One important limitation of this assay is that it requires reasonably pure GS. So far, the method does not work with crude membrane fractions due to the presence of proteins that perturb the migration of glucan through the SEC column. Therefore, in this protocol, we describe both the preparation of partially purified GS using the product entrapment method and the SEC assay. The product entrapment yields GS with 20–30% purity on SDS-PAGE and a specific activity of ~1,000 nmol/min/mg. While our characterization suggests that the impurities in this preparation does not affect the apparent function of GS (either glucan length or kinetics) (Chhetri et al., 2020), it is critical to remove proteins from the glucan samples by washing with SDS. This protocol has been used to study the mechanism of GS catalysis and successfully detected (1,3)-β-d-glucan elongation between ~1,000 and ~8,000 mer for the first time in the over 40-year-long history of GS (Chhetri et al., 2020). The facile chain length determination was also coupled to blocked substrate analogs to unambiguously determine the direction of polymerization, one of the key but challenging mechanistic questions in polysaccharide biosynthesis (Chhetri et al., 2020). These applications demonstrate the significance of the SEC-based GS activity assays. Similar approaches could be adapted to study the activities of other polysaccharide synthases such as glycogen and hyaluronan synthases.