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Loss of heparan sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria.

ABSTRACT: Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier.

SUBMITTER: Chen S

PROVIDER: S-EPMC8092444 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor.

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GAG-ID: Heparan Sulfate (HS) and Heparin Glycosaminoglycan High-Throughput Identification Software.

Project description:Heparin and heparan sulfate are very large linear polysaccharides that undergo a complex variety of modifications and are known to play important roles in human development, cell-cell communication and disease. Sequencing of highly sulfated glycosaminoglycan oligosaccharides like heparin and heparan sulfate by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains challenging because of the presence of multiple isomeric sequences in a complex mixture of oligosaccharides, the difficulties in separation of these isomers, and the facile loss of sulfates in MS/MS. We have previously introduced a method for structural sequencing of heparin/heparan sulfate oligosaccharides involving chemical derivatizations that replace labile sulfates with stable acetyl groups. This chemical derivatization scheme allows the use of reversed phase LC for high-resolution separation and MS/MS for sequencing of isomeric heparan sulfate oligosaccharides. However, because of the large number of analytes present in complex mixtures of heparin/HS oligosaccharides, the resulting LC-MS/MS data sets are large and cannot be annotated with existing glycomics software because of the specifically designed chemical derivatization strategy. We have developed a tool, called GAG-ID, to automate the interpretation of derivatized heparin/heparan sulfate LC-MS/MS data based on a modified multivariate hypergeometric distribution to weight the annotation of more intense peaks. The software is tested on a LC-MS/MS data set collected from a mixture of 21 synthesized heparan sulfate tetrasaccharides. By testing the discrimination of scoring with this system, we show that stratifying peaks into different intensity classes benefits the discrimination of scoring, and GAG-ID is able to properly assign all 21 synthetic tetrasaccharides in a defined mixture from a single LC-MS/MS run.

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Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices.

Project description:Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase C?, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

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TFCP2 is a transcriptional regulator of heparan sulfate assembly and melanoma cell growth

Project description:Heparan sulfate (HS) is a long, linear polysaccharide that is ubiquitously expressed in all animal cells and plays a key role in many cellular processes, including cell signaling and development. Dysregulation of HS assembly has been implicated in pathophysiological conditions, such as tumorigenesis and rare genetic disorders. HS biosynthesis occurs in a non-template driven manner in the endoplasmic reticulum and Golgi through the activity a large group of biosynthetic enzymes. While much is known about its biosynthesis, little is understood about the regulation of HS assembly across diverse tissue types and disease states. To address this gap in knowledge, we recently performed genome-wide CRISPR/Cas9 screens to identify novel regulatory factors of HS biosynthesis. From these screens, we identified the alpha globin transcription factor, TFCP2, as a top hit. To investigate the role of TFCP2 in HS assembly, we targeted TFCP2 expression in human melanoma cells using the CRISPR/Cas9 system. TFCP2 knockout cells exhibited decreased fibroblast growth factor binding to cell surface HS, alterations in HS composition, and slowed cell growth compared to wildtype cells. Additionally, RNA sequencing revealed that TFCP2 regulates expression of multiple enzymes involved in HS assembly, including the secreted endosulfatase, SULF1. Pharmacological targeting of TFCP2 activity similarly reduced growth factor binding and increased SULF1 expression, and knockdown of SULF1 expression in TFCP2 mutant cells restored melanoma cell growth. Overall, these studies identify TFCP2 as a novel transcriptional regulator of HS and highlight HS-protein interactions as a possible target to slow melanoma growth.

2023-05-01 | GSE224599 | GEO

Heparan sulfate glycomimetics <i>via</i> iterative assembly of "clickable" disaccharides.

Project description:Heparan sulfate (HS) glycosaminoglycans are widely expressed on the mammalian cell surfaces and extracellular matrices and play important roles in a variety of cell functions. Studies on the structure-activity relationships of HS have long been hampered by the challenges in obtaining chemically defined HS structures with unique sulfation patterns. Here, we report a new approach to HS glycomimetics based on iterative assembly of clickable disaccharide building blocks that mimic the disaccharide repeating units of native HS. Variably sulfated clickable disaccharides were facilely assembled into a library of mass spec-sequenceable HS-mimetic oligomers with defined sulfation patterns by solution-phase iterative syntheses. Microarray and surface plasmon resonance (SPR) binding assays corroborated molecular dynamics (MD) simulations and confirmed that these HS-mimetic oligomers bind protein fibroblast growth factor 2 (FGF2) in a sulfation-dependent manner consistent with that of the native HS. This work established a general approach to HS glycomimetics that can potentially serve as alternatives to native HS in both fundamental research and disease models.

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Heparan sulfate in skin wounds

Project description:Standardized skin wounds were established surgically on mice and allowed to heal during a 15-day period. Expression of genes related to heparan sulfate biosynthesis was studied in wound bed and edges during the healing process. Total RNA was isolated from wound edge (regenerating skin) and wound bed at 2, 6 and 15 days post wounding, as well as from intact control skin. Three animals were used for each time point.

2010-05-22 | E-GEOD-7435 | biostudies-arrayexpress

Heparan sulfate in skin wounds

2008-04-01 | GSE7435 | GEO

Heparan Sulfate Glycosaminoglycan Is Predicted to Stabilize Inflammatory Infiltrate Formation and RANKL/OPG Ratio in Severe Periodontitis in Humans.

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| S-EPMC9598302 | biostudies-literature

TMEM165 a new player in proteoglycan synthesis: loss of TMEM165 impairs elongation of chondroitin- and heparan-sulfate glycosaminoglycan chains of proteoglycans and triggers early chondrocyte differentiation and hypertrophy.

Project description:TMEM165 deficiency leads to skeletal disorder characterized by major skeletal dysplasia and pronounced dwarfism. However, the molecular mechanisms involved have not been fully understood. Here, we uncover that TMEM165 deficiency impairs the synthesis of proteoglycans by producing a blockage in the elongation of chondroitin-and heparan-sulfate glycosaminoglycan chains leading to the synthesis of proteoglycans with shorter glycosaminoglycan chains. We demonstrated that the blockage in elongation of glycosaminoglycan chains is not due to defect in the Golgi elongating enzymes but rather to availability of the co-factor Mn2+. Supplementation of cell with Mn2+ rescue the elongation process, confirming a role of TMEM165 in Mn2+ Golgi homeostasis. Additionally, we showed that TMEM165 deficiency functionally impairs TGFβ and BMP signaling pathways in chondrocytes and in fibroblast cells of TMEM165 deficient patients. Finally, we found that loss of TMEM165 impairs chondrogenic differentiation by accelerating the timing of Ihh expression and promoting early chondrocyte maturation and hypertrophy. Collectively, our results indicate that TMEM165 plays an important role in proteoglycan synthesis and underline the critical role of glycosaminoglycan chains structure in the regulation of chondrogenesis. Our data also suggest that Mn2+ supplementation may be a promising therapeutic strategy in the treatment of TMEM165 deficient patients.

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