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ALG9-CDG is a CDG-I defect within the group of Congenital Disorders of Glycosylation (CDG). We here describe the clinical symptoms of two new and unrelated ALG9-CDG patients, both carrying the novel homozygous missense variant c.1460 T > C (p.L487P) in the ALG9 gene which led to global developmental delay, psychomotor disability, facial dysmorphisms, brain and heart defects, hearing loss, hypotonia, as well as feeding problems. New clinical symptoms comprised West syndrome with hypsarrhythmia. Quantitative RT-PCR analysis revealed a significantly enhanced ALG9 mRNA transcript level, whereas the protein amount in fibroblasts was significantly reduced. This could be ascribed to a stronger degradation of the mutated ALG9 protein in patient fibroblasts. Lipid-linked oligosaccharide analysis showed an ALG9-CDG characteristic accumulation of Man 6 GlcNAc 2 -PP-dolichol and Man 8 GlcNAc 2 -PP-dolichol in patient cells. The clinical findings of our patients and of all previously published ALG9-CDG patients are brought together to further expand the knowledge about this rare N-glycosylation disorder. SYNOPSIS: Homozygosity for p.L487P in ALG9 causes protein degradation and leads to West syndrome. [ABSTRACT FROM AUTHOR]

Yeast α-mannan (YM) is a densely branched N-linked glycan that decorates the surface of yeast cell walls. Owing to the high degree of branching, cleavage of the backbone of YM appears to rely on the coupled action of side-chain-cleaving enzymes. Upon examining the genome sequences of bovine-adapted Bacteroides thetaiotaomicron strains, isolated for their ability to degrade YM, we have identified a tandem pair of genes inserted into an orphan pathway predicted to be involved in YM metabolism. Here, we investigated the activity of one of these enzymes, a predicted endo-mannanase from glycoside hydrolase (GH) family 76 (BtGH76-MD40). Purified recombinant BtGH76-MD40 displayed activity on structurally distinct YMs from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Linkage analysis of released oligosaccharide products from S. cerevisiae and S. pombe mannan determined BtGH76-MD40 targets a specific linkage that is conserved in structurally diverse YM substrates. In addition, using two differential derivatization methods, we have shown that there is an absolute requirement for undecorated d -mannopyranose in the −1 subsite. Determination of the BtGH76-MD40 X-ray crystal structure and structural superimposition and molecular docking of a branched alpha-mannopentatose substrate supported these findings. In contrast, BtGH76-MD40 can accommodate extended side chains in the +1 and −2 subsites, highlighting that a single alpha-1,6-mannosyl residue is a prerequisite for activity, and cleavage occurs at the reducing end of the undecorated monosaccharide. Collectively these results demonstrate how acquisition of new enzymes within extant pathways contributes to the functional abilities of saccharolytic bacteria persisting in complex digestive ecosystems. Image 1 • Yeast mannan (YM) is a highly branched polysaccharide with species-dependent structural diversity. • An enzyme has been found within the bovine rumen that cleaves the backbone of YM. • Cleavage requires the presence of an unbranched 6-linked mannose. • Glycosidic linkage analysis and crystallography elucidate the nature of interactions. [ABSTRACT FROM AUTHOR]

N -glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1 . The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available. [ABSTRACT FROM AUTHOR]

Although lotus root powder has high nutritional value and good therapeutic effects, there is still few reports on quality analysis of lotus root powder. In this study, we have established a novel method to identify the source and level of adulteration in lotus root powder, based on self-developed derivatization reagents and narrow range mass loss filtration technology. By using this approach, 80 species of oligosaccharides have been identified in lotus root powder, and 18 of them are characteristic and common oligosaccharides for lotus root powder from different places, which might be helpful to distinguish between oligosaccharides and their common adulterants. More importantly, in combination with multivariate statistical methods, the composition proportion of adulterants can also be effectively determined (from 5% to 95%). Furthermore, it is worthy to be mentioned that it is a pioneer report of using narrow range quality loss filtering technology towards successful detection and identification of food ingredients, which achieves rapid detection of adulterated components and their proportions in lotus root powder. In short, the approach of using mass defect filter is effective in sugar spectra analysis, which might be promising for simple, sensitive, and accurate food quality control. • A rapid detection method for adulterated components and their proportions in lotus root powder has been successfully established. • For the first time, oligosaccharide analysis of lotus root powder can be realized. • For the first time, narrow range mass loss filtration technology is applied to the detection and identification of food ingredients. [ABSTRACT FROM AUTHOR]

The influence of the preparation method and the effect of in vitro digestion on the fructo-oligosaccharides (FOS) and malto-oligosaccharides (MALTOS) of sweet potato ( Ipomoea batatas Lam), mandioquinha ( Arracacia xanthorrhiza ), white yam ( Dioscorea alata L.), cassava ( Manihot esculenta Crantz), and taro ( Colocasia esculenta ) were evaluated. Tubers/roots were cooked in two ways: whole/unpeeled and peeled/in cubes. In vitro digestion was performed on whole/unpeeled cooked vegetables. An oligosaccharide analysis was carried out by HPAE-PAD. The largest amounts of oligosaccharides were found in the whole/unpeeled cooked tubers/roots. Sweet potato showed the highest content of FOS (29.08 ± 1.60 mg/100 g) and the highest concentrations of MALTOS were observed in white yam (113.70 ± 3.41 mg/100 g). In vitro digestion promoted an increase in maltotriose in all tubers/roots. Sweet potato FOS were not changed by digestion. However, in mandioquinha , white yam, and cassava, FOS levels were higher after digestion. The increase in MALTOS after in vitro digestion may be associated with both the hydrolysis of oligosaccharides with a higher degree of polymerization and starch. FOS contents increased after digestion possibly by hydrolysis of inulin or release from the food matrix. Sweet potato, cassava, and white yams can contribute to an increased intake of prebiotics. [ABSTRACT FROM AUTHOR]

Abstract: Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. [Copyright &y& Elsevier]

Abstract: Glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction of heparin currently used as an anticoagulant and antithrombotic drug, are arousing increasing interest in anticancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs heparins has not been studied yet in detail. In this work, ion pair reversed-phase high-performance liquid chromatography (IPRP–HPLC) coupled with electrospray ionization mass spectrometry (ESI–MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs heparins usually prepared from porcine mucosal heparin. The method was also found to be very effective in analyzing gs derivatives obtained from heparins of different animal and tissue origins. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs heparins contain mainly tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues was shown to be a marker of the gs-modified AT binding site within heparin chains. [Copyright &y& Elsevier]

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides. [Copyright &y& Elsevier]