Screening for abnormal glycosylation in a cohort of adult liver disease patients

Congenital Disorders of Glycosylation (CDG) are a rapidly expanding group of rare genetic defects in glycosylation. In a novel CDG subgroup of Vacuolar-ATPase assembly defects various degrees of hepatic injury have been described, including end stage liver disease. However, the CDG diagnostic workflow can be complex as liver disease per se may be associated with abnormal glycosylation. Therefore, we collected serum samples of patients with a wide range of liver pathology to study the performance and yield of two CDG screening methods. Our aim was to identify glycosylation patterns that could help to differentiate between primary and secondary glycosylation defects in liver disease. To this end, we analyzed serum samples of 1042 adult liver disease patients. This cohort consisted of 567 liver transplant candidates and 475 chronic liver disease patients. Our workflow consisted of screening for abnormal glycosylation by transferrin isoelectric focusing (tIEF), followed by in-depth analysis of the abnormal samples with quadruple time-of-flight mass spectrometry (QTOF-MS). Screening with tIEF resulted in identification of 247 (26%) abnormal samples. QTOF-MS analysis of 110 of those did not reveal glycosylation abnormalities comparable with those seen in V-ATPase assembly factor deficiencies. However, two patients presented with isolated sialylation deficiency. Fucosylation was significantly increased in liver transplant candidates compared to healthy controls and patients with chronic liver disease. In conclusion, a significant percentage of patients with liver disease presented with abnormal CDG screening results, however, not indicative for a V-ATPase assembly factor defect. Advanced glycoanalytical techniques assist in the dissection of secondary and primary glycosylation defects. This article is protected by copyright. All rights reserved

Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5

Mutations in genes critical for proper intra-Golgi transport can cause human syndromes due to defects in glycosylation of proteins. Here, the authors identify a human variant of Syntaxin-5 that causes fatal multisystem disease and mislocalization of glycosyltransferases due to altered Golgi transport

Glycoproteomics in Cerebrospinal Fluid Reveals Brain-Specific Glycosylation Changes

The glycosylation of proteins plays an important role in neurological development and disease. Glycoproteomic studies on cerebrospinal fluid (CSF) are a valuable tool to gain insight into brain glycosylation and its changes in disease. However, it is important to consider that most proteins in CSFs originate from the blood and enter the CSF across the blood–CSF barrier, thus not reflecting the glycosylation status of the brain. Here, we apply a glycoproteomics method to human CSF, focusing on differences between brain- and blood-derived proteins. To facilitate the analysis of the glycan site occupancy, we refrain from glycopeptide enrichment. In healthy individuals, we describe the presence of heterogeneous brain-type N-glycans on prostaglandin H2-D isomerase alongside the dominant plasma-type N-glycans for proteins such as transferrin or haptoglobin, showing the tissue specificity of protein glycosylation. We apply our methodology to patients diagnosed with various genetic glycosylation disorders who have neurological impairments. In patients with severe glycosylation alterations, we observe that heavily truncated glycans and a complete loss of glycans are more pronounced in brain-derived proteins. We speculate that a similar effect can be observed in other neurological diseases where a focus on brain-derived proteins in the CSF could be similarly beneficial to gain insight into disease-related changes

NANS-mediated synthesis of sialic acid is required for brain and skeletal development.

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition