High sensitivity analysis of nanogram quantities of glycosaminoglycans using ToF-SIMS

Glycosaminoglycans (GAGs) are important biopolymers that differ in the sequence of saccharide units and in post polymerization alterations at various positions, making these complex molecules challenging to analyse. Here we describe an approach that enables small quantities (< 200 ng) of over 400 different GAGs to be analysed within a short time frame (3-4 hours). Time of flight secondary ion mass spectrometry (ToF-SIMS) together with multivariate analysis is used to analyse the entire set of GAG samples. Resultant spectra are derived from the whole molecules and do not require pre-digestion. All 6 possible GAG types are successfully discriminated, both alone and in the presence of fibronectin. We also distinguish between pharmaceutical grade heparin, derived from different animal species and from different suppliers, to a sensitivity as low as 0.001 wt%. This approach is likely to be highly beneficial in the quality control of GAGs produced for therapeutic applications and for characterising GAGs within biomaterials or from in vitro cell culture

Bi-allelic pathogenic variants in HS2ST1 cause a syndrome characterized by developmental delay and corpus callosum, skeletal and renal abnormalities

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulphated linear polysaccharides. Heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report biallelic pathogenic variants in the HS2ST1 gene in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2-O-sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2-O-sulfated domains but had an increase in N- and 6-O-sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals showed an increased length and concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a novel recognizable syndrome and emphasize a role for 2-O-sulfated heparan sulfate in human neuronal, skeletal and renal development

Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCα signaling and cross-talk with TGFβ

Aims: Vascular calcification is a major cause of morbidity and mortality. Fibroblast growth factor-2 (FGF-2) plays an instructive role in osteogenesis and bone development, but its role in vascular calcification was unknown. Therefore, we investigated the involvement of FGF-2 in vascular calcification and determined the mechanism by which it regulates this process. Methods and Results: We demonstrate that FGF-2 expression is increased in vascular smooth muscle cells (VSMCs) induced to deposit a mineralized matrix by incubation with β-glycerophosphate. FGF-2 is also localized to sites of calcification within human atherosclerotic plaques. The expression of syndecan-4, a heparan sulfate proteoglycan which regulates FGF-2 signaling, is also increased in mineralizing VSMCs and co-localizes with FGF-2 in human calcified atherosclerotic plaques. Exogenous FGF-2 inhibits VSMC mineralization, and this inhibition is reduced when syndecan-4 expression is knocked-down using siRNA. Biochemical inhibition of FGFR signaling using a pan FGFR inhibitor (BGJ398) or knocking-down syndecan-4 expression in VSMCs using siRNA increases VSMC mineralization. These increases are prevented by inhibiting transforming growth factor-β (TGFβ) signaling with SB431542, suggesting cross-talk between FGF-2 and TGFβ signaling is crucial for the regulation of VSMC mineralization. Syndecan-4 can also regulate FGF-2 signaling directly via protein kinase Cα (PKCα) activation. Biochemical inhibition of PKCα activity using Gö6976, or siRNA-mediated suppression of PKCα expression increases VSMC mineralization; this increase is also prevented with SB431542. Finally, the ability of FGF-2 to inhibit VSMC mineralization is reduced when PKCα expression is knocked-down. Conclusion: This is the first demonstration that syndecan-4 promotes FGF-2 signaling, and in turn, suppresses VSMC mineralization by down-regulating TGFβ signaling. Our discoveries that FGF-2 and syndecan-4 expression is increased in mineralizing VSMCs and that PKCα regulates FGF-2 and TGFβ signaling in VSMCs suggests that the syndecan-4/FGF-2/TGFβ signaling axis could represent a new therapeutic target for vascular calcification

Glycoscience finally comes of age

To take its place alongside genomics and proteomics, glycoscience needs recognition from scientist

Comparative quantification of the surfaceome of human multipotent mesenchymal progenitor cells

SummaryMesenchymal progenitor cells have great therapeutic potential, yet incomplete characterization of their cell-surface interface limits their clinical exploitation. We have employed subcellular fractionation with quantitative discovery proteomics to define the cell-surface interface proteome of human bone marrow mesenchymal stromal/stem cells (MSCs) and human umbilical cord perivascular cells (HUCPVCs). We compared cell-surface-enriched fractions from MSCs and HUCPVCs (three donors each) with adult mesenchymal fibroblasts using eight-channel isobaric-tagging mass spectrometry, yielding relative quantification on >6,000 proteins with high confidence. This approach identified 186 upregulated mesenchymal progenitor biomarkers. Validation of 10 of these markers, including ROR2, EPHA2, and PLXNA2, confirmed upregulated expression in mesenchymal progenitor populations and distinct roles in progenitor cell proliferation, migration, and differentiation. Our approach has delivered a cell-surface proteome repository that now enables improved selection and characterization of human mesenchymal progenitor populations