Fredrik Noborn

The Ehlers–Danlos syndromes (EDS) are a group of heritable connective tissue disorders, characterized by varying degrees of joint hypermobility, skin hyperextensibility and extracellular matrix defects. Spondylodysplastic type of EDS (spEDS) is a rare type of this group that can be caused by pathogenic variants in the B3GALT6 gene, coding for a galactosyltransferase essential for the formation of the canonical tetrasaccharide linkage region (GlcA–Gal–Gal–Xyl–O) in heparan sulfate and chondroitin sulfate proteoglycans. We previously showed that three siblings with spEDS, carrying biallelic pathogenic variants in B 3GALT6 , showed elevated levels of a non‐canonical trisaccharide linkage structure (GlcA–Gal–Xyl–O) on urinary bikunin, suggesting that this linkage structure can serve as a biomarker for the disease. To further validate the potential use of this, or similar, structures as biomarkers for spEDS‐ B3GALT6 we analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of two siblings from another family with this condition, also carrying biallelic but different pathogenic variants in the B3GALT6 gene. LC‐MS/MS analyses showed that the two siblings had a similar relative increase in the trisaccharide linkage structure of bikunin as compared to controls, corroborating our previous findings. Notably, this structure was exclusively found on the bikunin proteoglycan, whereas other proteoglycans displayed the canonical linkage region. Interestingly, the data also revealed a novel truncated sialylated disaccharide (Neu5Ac–Gal–Xyl–O) that was only found on a subset of the identified proteoglycans. The sialylated disaccharide was also identified in the datasets from our glycoproteomic study of the previously reported spEDS‐family. Together, these variants of the glycosaminoglycan linkage region may serve as disease biomarkers for spEDS‐ B3GALT6 . The findings additionally imply that the glycosite characteristics of specific core proteins influence the biosynthesis of the linkage region.

In recent years, several rational designed therapies have been developed for treatment of mucopolysaccharidoses (MPS), a group of inherited metabolic disorders in which glycosaminoglycans (GAGs) are accumulated in various tissues and organs. Thus, improved disease-specific biomarkers for diagnosis and monitoring treatment efficacy are of paramount importance. Specific non-reducing end GAG structures (GAG-NREs) have become promising biomarkers for MPS, as the compositions of the GAG-NREs depend on the nature of the lysosomal enzyme deficiency, thereby creating a specific pattern for each subgroup. However, there is yet no straightforward clinical laboratory platform which can assay all MPS-related GAG-NREs in one single analysis. Here, we developed and applied a GAG domain mapping approach for analyses of urine samples of ten MPS patients with various MPS diagnoses and corresponding aged-matched controls. We describe a nano-LC–MS/MS method of GAG-NRE profiling, utilizing 2-aminobenzamide reductive amination labeling to improve the sensitivity and the chromatographic resolution. Diagnostic urinary GAG-NREs were identified for MPS types IH/IS, II, IIIc, IVa and VI, corroborating GAG-NRE as biomarkers for these known enzyme deficiencies. Furthermore, a significant reduction of diagnostic urinary GAG-NREs in MPS IH (n = 2) and MPS VI (n = 1) patients under treatment was demonstrated. We argue that this straightforward glycomic workflow, designed for the clinical analysis of MPS-related GAG-NREs in one single analysis, will be of value for expanding the use of GAG-NREs as biomarkers for MPS diagnosis and treatment monitoring.

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human β3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

The increased risk of Alzheimer’s disease (AD) associated with specific apolipoprotein E (ApoE) isoforms appears to relate to altered amyloid-β (Aβ) homeostasis. Clearance of Aβ from the brain is reduced in the presence of the AD-associated ApoE4 isoform, which may contribute to the accumulation of Aβ deposits in the parenchyma and vasculature. The low-density lipoprotein receptor-related protein 1 (LRP1) and heparan sulfate proteoglycans (HSPGs), both established ApoE receptors, are involved in Aβ uptake, with LRP1 additionally implicated in Aβ transcytosis across the blood–brain barrier. In this study, we detected the co-distribution of heparan sulfate (HS), ApoE and LRP1 in Aβ(1-40)-positive brain microvessels from individuals with Down’s syndrome diagnosed with AD. In addition, ApoE was pulled-down from AD cerebrospinal fluid with anti-Aβ antibodies. Using Chinese hamster ovary cells deficient in HS or LRP1, we found that ApoE increases cell association of Aβ in a HSPG- and LRP1-dependent manner; and further, ApoE processing is altered in the absence of cellular HS. These interactions may facilitate Aβ clearance from the brain, but if overwhelmed could contribute to Aβ accumulation and the pathogenesis of AD.