Glycogen and its breakdown products, maltose and malto-oligosaccharides, are important carbon sources for vaginal bacteria including Gardnerella species. MusEFGKI transport systems for maltose and malto-oligosaccharides have been identified in all Gardnerella species; however, unlike in other species, the Gardnerella swidsinskii operon encodes two substrate-binding proteins (SBPs) (MusE1345, MusE1346, ~60% amino acid identity). Two SBPs could allow binding of additional ligands, providing a competitive advantage to G. swidsinskii relative to other species with only one SBP. Our objectives were to determine if both genes are expressed in G. swidsinskii and compare the specificity and affinity of G. swidsinskii MusE SBPs for glycogen breakdown products. Gene expression analysis showed the presence of a polycistronic transcript spanning both SBP encoding genes; however, musE1346 transcripts were more abundant, likely due to the presence of an additional promoter identified in the intergenic region. No difference in the relative expression of either gene was observed in isolates grown in media supplemented with glycogen or maltotriose. Predicted structures of both SBPs were highly similar and characteristic of previously characterized maltose-binding proteins. Both proteins had a high affinity for maltose, maltotriose and maltotetraose (K d 10-6 to 10-7 M) and much lower affinities to maltopentaose and maltohexaose (K d 10-3 to 10-4 M). Our results demonstrate that the affinities of G. swidsinskii MusE SBPs for maltose and malto-oligosaccharides are similar under the same experimental conditions.

Glycans are some of the most difficult biomolecules to analyze owing to their branching tendencies as well as their regiochemical and stereochemical diversity. Yet, the correlation between various pathological states and glycan quantity or structural alterations has demonstrated the importance and urgency for the development of a more robust glycan analytical technique. Furthermore, the manufacturing and regulation of biopharmaceuticals demands a feasible and improved analytical approach toward the characterization and quantitation of glycosylations. Unfortunately, multiple commercially available glycan tags lack, in combination, liquid chromatography detection sensitivity, chemical stability and, most importantly, optimal glycan characterization capabilities. Therefore, a novel fluorescent tag coupled with a free radical approach for glycan characterization was designed and developed to help address this gap in glycan analysis. The analytical capabilities of this novel tag were assessed via hydrophilic liquid chromatography-fluorescence quantitation and ESI/MS free radical-mediated characterization by using linear glycan standards, branched isobaric glycans lacto-N-difucohexaose I and lacto-N-difucohexaose II, and N-glycans released from ribonuclease B.

The human vaginal microbiota is frequently dominated by lactobacilli and transition to a more diverse community of anaerobic microbes is associated with health risks. Glycogen released by lysed epithelial cells is believed to be an important nutrient source in the vagina. However, the mechanism by which vaginal bacteria metabolize glycogen is unclear, with evidence implicating both bacterial and human enzymes. Here we biochemically characterize six glycogen-degrading enzymes (GDEs), all of which are pullanases (PulA homologues), from vaginal bacteria that support the growth of amylase-deficient Lactobacillus crispatus on glycogen. We reveal variations in their pH tolerance, substrate preferences, breakdown products and susceptibility to inhibition. Analysis of vaginal microbiome datasets shows that these enzymes are expressed in all community state types. Finally, we confirm the presence and activity of bacterial and human GDEs in cervicovaginal fluid. This work establishes that bacterial GDEs can participate in the breakdown of glycogen, providing insight into metabolism that may shape the vaginal microbiota.
© 2023. The Author(s).

Urine and plasma biomarker testing for lysosomal storage disorders by liquid chromatography mass spectrometry (LC-MS) currently requires multiple analytical methods to detect the abnormal accumulation of oligosaccharides, mucopolysaccharides, and glycolipids. To improve clinical testing efficiency, we developed a single LC-MS method to simultaneously identify disorders of oligosaccharide, mucopolysaccharide, and glycolipid metabolism with minimal sample preparation.
We created a single chromatographic method for separating free glycans and glycolipids in their native form, using an amide column and high pH conditions. We used this glycomic profiling method both in untargeted analyses of patient and control urines using LC ion-mobility high-resolution MS (biomarker discovery), and targeted analyses of urine, serum, and dried blood spot samples by LC-MS/MS (clinical validation).
Untargeted glycomic profiling revealed twenty biomarkers that could identify and subtype mucopolysaccharidoses. We incorporated these with known oligosaccharide and glycolipid biomarkers into a rapid test that identifies at least 27 lysosomal storage disorders, including oligosaccharidoses, mucopolysaccharidoses, sphingolipidoses, glycogen storage disorders, and congenital disorders of glycosylation and de-glycosylation. In a validation set containing 115 urine samples from patients with lysosomal storage disorders, all were unambiguously distinguished from normal controls, with correct disease subtyping for 88% (101/115) of cases. Glucosylsphingosine was reliably elevated in dried blood spots from Gaucher disease patients with baseline resolution from galactosylsphingosine.
Glycomic profiling by liquid chromatography mass spectrometry identifies a range of lysosomal storage disorders. This test can be used in clinical evaluations to rapidly focus a diagnosis, as well as to clarify or support additional gene sequencing and enzyme studies.
Copyright © 2021 Elsevier Inc. All rights reserved.

Following clinical indications, the laboratory diagnosis of the inherited metabolic myopathy, Pompe disease (PD), typically begins with demonstrating a reduction in acid alpha-glucosidase (GAA), the enzyme required for lysosomal glycogen degradation. Although simple in concept, a major challenge is defining reference intervals, as even carriers can have reduced GAA, and pseudodeficiencies complicate interpretation. Here, we developed a mass spectrometric assay for quantification of a urinary glycogen metabolite (tetrasaccharide) and reported on its utility as a confirmatory test for PD in a diagnostic laboratory. Using two age-related reference intervals, eight returned tetrasaccharide concentrations above the calculated reference interval but did not have PD, highlighting non-specificity. However, retrospective analysis revealed elevated tetrasaccharide in seven infantile-onset (IOPD) cases and sixteen late-onset (LOPD) cases, and normal concentrations in one heterozygote. Prospective tetrasaccharide analysis in nine individuals with reduced GAA confirmed IOPD in one, LOPD in six and identified two heterozygotes. Using this metabolite as a biomarker of therapeutic response was not overly informative; although most patients showed an initial drop following therapy initiation, tetrasaccharide concentrations fluctuated considerably and remained above reference intervals in all patients. While useful as a confirmation of PD, its utility as a biomarker for monitoring treatment warrants further investigation.