Glycosaminoglycan Chains Research Articles

Endothelin‐1 dependent expression of GAG genes involves NOX and p38 mediated Smad linker region phosphorylation

Endothelin‐1 (ET‐1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET‐1‐mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho‐Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST‐1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real‐time polymerase chain reaction (qRT‐PCR). ET‐1 treatment of human VSMCs resulted in an increase in phospho‐Smad2L level. The TGF‐β receptor antagonist, SB431542 and the mixed ETA and ETB receptor antagonist bosentan, inhibited ET‐1‐mediated phospho‐Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET‐1‐mediated phospho‐Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post‐ET‐1 treatment were increased compared to untreated controls (p < 0.01). The ET‐mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N‐acetyl‐L‐cysteine (NAC). ET‐1‐mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation‐dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation.

Biologically Derived Neoproteoglycans for Profiling Protein–Glycosaminoglycan Interactions

Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme-substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.

Chemical editing of proteoglycan architecture.

Proteoglycans are heterogeneous macromolecular glycoconjugates that orchestrate many important cellular processes. While much attention has focused on the poly-sulfated glycosaminoglycan chains that decorate proteoglycans, other important elements of their architecture, such as core proteins and membrane localization, have garnered less emphasis. Hence, comprehensive structure-function relationships that consider the replete proteoglycan architecture as glycoconjugates are limited. Here we present an extensive approach to study proteoglycan structure and biology by fabricating defined semisynthetic modular proteoglycans that can be tailored for cell surface display. The expression of proteoglycan core proteins with unnatural amino acids permits bioorthogonal click chemistry with functionalized glycosaminoglycans for methodical dissection of the parameters required for optimal binding and function of various proteoglycan-binding proteins. We demonstrate that these sophisticated materials can recapitulate the functions of native proteoglycan ectodomains in mouse embryonic stem cell differentiation and cancer cell spreading while permitting the analysis of the contributing architectural elements toward function.

Isolation and structural characterization of bioactive glycosaminoglycans from the green-lipped mussel Perna canaliculus

Chondroitin sulfate (CS) and heparan sulfate (HS) are sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide units composed of hexosamine and hexuronic acid. GAG chains exhibit diverse bioactivities in a structure-specific manner. Marine invertebrates are a rich source of highly sulfated and rare structures of GAG chains. Here, we isolated GAGs from the green-lipped mussel Perna canaliculus, an aquaculture species that is produced on a large scale. We separated GAGs based on the degree of negative charges and analyzed their disaccharide compositions. CS and HS both exhibited characteristic compositions of differently sulfated disaccharides. CS chains showed a higher degree of sulfation than HS chains and contained a high percentage of the E unit disaccharide GlcA-GalNAc(4,6-O-disulfate). Furthermore, CS chains rich in the E unit stimulated the neurite outgrowth of primary cultured neurons. The present results indicate the potential of P. canaliculus GAGs as biomaterials to study the structure-function relationships of GAGs.

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation.

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC‐chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D‐pellet culture led to significant increases in the expression of ECM genes for type‐II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific –10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N‐acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co‐IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B‐SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC‐chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators.

Emerging role of syndecans in maintaining homeostasis of colon epithelium during inflammation.

The syndecans are a family of transmembrane proteoglycans that are widespread in mammalian tissues. Located at the cell surface membrane, they contribute to modulating the composition of the extracellular matrix via glycosaminoglycan chains (GAGs) attached to their extracellular domains. Syndecans can interact with a variety of extracellular ligands through their core proteins and GAGs, and may also transmit signals through their transmembrane domain to regulate intracellular functions. These properties enable syndecan to modulate glycocalyx formation, epithelial cell-to-cell connections for cell barrier formation, and epithelial cell-lamina propria interactions in the colon epithelium, all of which are crucial for the homeostasis of this tissue. Inflammation induces structural alterations of the colon epithelium, and accumulating evidence suggests that syndecan expression might play important regulatory functions during inflammation. This review summarizes the possible roles of syndecans in maintaining tissue homeostasis in the colon epithelium, especially under inflammation.

ChGn-2 Plays a Cardioprotective Role in Heart Failure Caused by Acute Pressure Overload.

BackgroundCardiac extracellular matrix is critically involved in cardiac homeostasis, and accumulation of chondroitin sulfate glycosaminoglycans (CS‐GAGs) was previously shown to exacerbate heart failure by augmenting inflammation and fibrosis at the chronic phase. However, the mechanism by which CS‐GAGs affect cardiac functions remains unclear, especially at the acute phase.Methods and ResultsWe explored a role of CS‐GAG in heart failure using mice with target deletion of ChGn‐2 (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) that elongates CS chains of glycosaminoglycans. Heart failure was induced by transverse aortic constriction in mice. The role of CS‐GAG derived from cardiac fibroblasts in cardiomyocyte death was analyzed. Cardiac fibroblasts were subjected to cyclic mechanical stretch that mimics increased workload in the heart. Significant CS‐GAGs accumulation was detected in the heart of wild‐type mice after transverse aortic constriction, which was substantially reduced in ChGn‐2‐/‐ mice. Loss of ChGn‐2 deteriorated the cardiac dysfunction caused by pressure overload, accompanied by augmented cardiac hypertrophy and increased cardiomyocyte apoptosis. Cyclic mechanical stretch increased ChGn‐2 expression and enhanced glycosaminoglycan production in cardiac fibroblasts. Conditioned medium derived from the stretched cardiac fibroblasts showed cardioprotective effects, which was abolished by CS‐GAGs degradation. We found that CS‐GAGs elicits cardioprotective effects via dual pathway; direct pathway through interaction with CD44, and indirect pathway through binding to and activating insulin‐like growth factor‐1.ConclusionsOur data revealed the cardioprotective effects of CS‐GAGs; therefore, CS‐GAGs may play biphasic role in the development of heart failure; cardioprotective role at acute phase despite its possible unfavorable role in the advanced phase.

Hedgehog pathway modulation by glypican 3-conjugated heparan sulfate.

Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson-Golabi-Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.

Proteoglycans and proteoglycan mimetics for tissue engineering.

Proteoglycans play a crucial role in proper tissue morphology and function throughout the body that is defined by a combination of their core protein and the attached glycosaminoglycan chains. Although they serve a myriad of roles, the functions of extracellular proteoglycans can be generally sorted into four categories: modulation of tissue mechanical properties, regulation and protection of the extracellular matrix, sequestering of proteins, and regulation of cell signaling. The loss of proteoglycans can result in significant tissue dysfunction, ranging from poor mechanical properties to uncontrolled inflammation. Because of the key roles they play in proper tissue function and due to their complex synthesis, the past two decades have seen significant research into the development of proteoglycan mimetic molecules to recapitulate the function of proteoglycans for therapeutic and tissue engineering applications. These strategies have ranged from semisynthetic graft copolymers to recombinant proteoglycan domains synthesized by genetically engineered cells. In this review, we highlight some of the important functions of extracellular proteoglycans, as well as the strategies developed to recapitulate these functions.

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity.

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their invivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity invitro and invivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

Bacteria associated with acne use glycosaminoglycans as cell adhesion receptors and promote changes in the expression of the genes involved in their biosynthesis

BackgroundCell surface glycosaminoglycans (GAGs) participate in many physiological and pathological processes, including infections and inflammatory response. Acne is a common chronic inflammatory skin disorder that affects the pilosebaceous unit and has a multifactorial etiology, including bacterial colonization of the hair follicle. This study aimed to investigate the participation of GAG in the adhesion of Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis to keratinocytes and fibroblasts of the skin by competition experiments and cell surface removal using specific liases. The alteration in the transcription of the genes responsible for the synthesis of GAG induced by the adhesion of these bacteria was also analyzed by qRT-PCR.ResultsGAGs are involved in bacterial adherence to skin cells, especially fibroblasts, where chondroitin sulfate displayed the higher effect. Bacterial adherence produced different alterations in the transcription of the genes responsible for GAG structures. P. acnes induced mostly changes in keratinocytes, while S. epidermidis was the main cause of alterations in fibroblasts. These variations in gene expression affected all the stages in the biosynthesis of the main species of GAGs, heparan and chondroitin sulphate.ConclusionsGAGs species are involved in the adhesion of acne-related bacteria to skin cells in a differential manner depending on each microorganism and cellular type, although other receptors seem to exist. Bacterial adherence led to variations on gene expression in skin cells affecting GAG chains structure what, consequently, should alter their interactions with different ligands, affecting the development of acne disease.

Internalization and trafficking of CSPG-bound recombinant VAR2CSA lectins in cancer cells

Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.

In silico and in vitro mapping of specificity patterns of glycosaminoglycans towards cysteine cathepsins B, L, K, S and V

Human cysteine cathepsins are lysosomal proteases, which are involved in different biological processes. Their enzymatic activity can be regulated by glycosaminoglycans (GAGs): long linear periodic negatively charged polysaccharides, which dimeric building blocks consist of uronic acid and hexosamine monosaccharide units. In this study, molecular docking simulations of chondroitin 4-sulfate, chondroitin 6-sulfate, heparin, heparan sulfate, dermatan sulfate and hyaluronic acid of various chain lengths were performed with cathepsins B, L, K, S and V and followed by molecular dynamics-based refinement and binding free energy analysis. We concluded that electrostatics might be a driving force for cathepsin-GAG interactions; indeed as in most of characterised systems, the increase of GAG chain length consequently leads to a more pronounced effect on the strength of cathepsin-GAG interactions. Results also suggest that binding of GAGs at different regions on cathepsins surface affect differently their enzymatic activity and could is dependent on cathepsin and GAG type. Present data contribute to systematic description of cathepsin-GAG interactions, which is helpful in understanding the subtle molecular mechanisms of protease regulation behind their biological functions.

Mutations in the heparan sulfate backbone elongating enzymes EXT1 and EXT2 have no major effect on endothelial glycocalyx and the glomerular filtration barrier

In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.

Experimental and numerical analysis of electroactive characteristics of scleral tissue

The sclera provides mechanical support to retina and protects internal contents of the eye against external injuries. The scleral extracellular matrix is mainly composed of collagen fibers and proteoglycans (PGs). At physiological pH, collagen molecules are neutral but PGs contain negatively charged glycosaminoglycan chains. Thus, the sclera can be considered as a polyelectrolyte hydrogel and is expected to exhibit mechanical response when subjected to electrical stimulations. In this study, we mounted scleral strips, dissected from the posterior part of porcine eyes, at the center of a custom-designed container between two electrodes. The container was filled with NaCl solution and the bending deformation of scleral strips as a function of the applied electric voltage was measured experimentally. It was found that scleral strips reached to an average bending angle of 3°, 10° and 23° when subjected to 5V, 10V, and 15V, respectively. We also created a chemo-electro-mechanical finite element model for simulating the experimental measurements by solving coupled Poisson-Nernst-Plank and equilibrium mechanical field equations. The scleral fixed charge density and modulus of elasticity were found by fitting the experimental data. The ion concentration distribution inside the domain was found numerically and was used to explain the underlying mechanisms for the scleral electroactive response. The numerical simulations were also used to investigate the effects of various parameters such as the electric voltage and fixed charge density on the scleral deformation under an electric field. Statement of significanceThis manuscript investigates the electroactive response of scleral tissue. It demonstrates that the sclera deforms mechanically when subjected to electrical stimulations. A chemo-electro-mechanical model is also presented in order to numerically capture the electromechanical response of the sclera. This numerical model is used to explain the experimental observations by finding the ion distribution inside the tissue under an electric field. This work is significant because it shows that the sclera is an electroactive polyanionic hydrogel and it provides new information about the underlying mechanisms governing its mechanical and electrical properties.

Endothelin-1 mediated glycosaminoglycan synthesizing gene expression involves NOX-dependent transactivation of the transforming growth factor-β receptor.

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor(TGF) β receptor (TGFBR1) stimulates glycosaminoglycan (GAG) elongation on proteoglycans. GPCR agonists thrombin and lysophosphatidic acid (LPA) via respective receptors transactivate the TGFBR1 via Rho/ROCK dependent pathways however mechanistic insight for ET-1 transactivation of the TGFBR1 remains unknown. NADPH oxidase (NOX) generates reactive oxygen species (ROS) and is a signalling entity implicated in the pathogenesis of many diseases including atherosclerosis. If implicated in this pathway, NOX/ROS would be a potential therapeutic target. In this study, we investigated the involvement of NOX in ET-1/ET receptor-mediated transactivation of TGFBR1 to stimulate mRNA expression of GAG chain synthesizing enzymes chondroitin 4-O-sulfotransferase 1 (C4ST-1) and chondroitin sulfate synthase 1 (ChSy-1). The invitro model used vascular smooth muscle cells that were treated with pharmacological antagonists in the presence and absence of ET-1 or TGF-β. Proteins and phosphoproteins isolated from treated cells were quantified by western blotting and quantitative real-time PCR was used to assess mRNA expression of GAG synthesizing enzymes. In the presence of diphenyliodonium (DPI) (NOX inhibitor), ET-1 stimulated phospho-Smad2C levels were inhibited. ET-1 mediated mRNA expression of GAG synthesizing enzymes C4ST-1 and ChSy-1 was also blocked by TGBFR1 antagonists, SB431542, broad spectrum ET receptor antagonist bosentan, DPI and ROS scavenger N-acetyl-L-cysteine. This work shows that NOX and ROS play an important role in ET-1mediated transactivation of the TGFBR1 and downstream gene targets associated with GAG chainelongation. As ROS is involved in GPCR to protein tyrosine kinase receptor transactivation, the NOX/ROS axis presents as the first common biochemical target in all GPCR to kinase receptor transactivation signalling.

Capillary Zone Electrophoresis-Electrospray Ionization Tandem Mass Spectrometry for Total Analysis of Chondroitin/Dermatan Sulfate Oligosaccharides.

Proteoglycans are heavily glycosylated proteins, covalently linked to one or more glycosaminoglycan (GAG) chains, abundantly expressed in the extracellular matrix (ECM). Among GAGs, chondroitin sulfate (CS) and dermatan sulfate (DS) play an essential role at the ECM level; however, the composition of the hybrid CS/DS as well as the distribution of the sulfate groups along the chain were also shown to influence biological activities in brain. The elevated structural diversity of CS/DS motifs, in which sulfation may occur at GalNAc and/or IdoA/GlcA in various combinations, requires the development of specific high performance analytical methods for reliable elucidation. Due to its sensitivity, reproducibility, and efficiency, capillary zone electrophoresis (CZE) for separation of CS/DS oligosaccharides coupled to electrospray ionization mass spectrometry (ESI-MS) for their structure determination contributed an essential progress to this field.In the present chapter, two powerful methods based on CZE for separation and ESI-MS for identification and structural analysis of CS/DS are presented. The first part is devoted to offline CZE-ESI-MS based on fraction collection, screening by negative ion mode nanoESI, and fragmentation analysis in tandem MS using collision-induced dissociation (CID) at low ion acceleration energies. In the second part of the chapter, a strategy for online CZE-ESI-MS in normal polarity and negative mode ESI followed by tandem MS in real-time data-dependent acquisition mode for CS/DS separation, screening, and fragmentation is described in detail. The latter method entails the in-laboratory manufacturing of a simple yet sturdy interface for the online CZE coupling to ESI-MS and the optimization of the coupled system for total analysis of regularly sulfated and irregularly, i.e., under- and oversulfated CS/DS domains.

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

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.

Decorin: a multifunctional proteoglycan involved in oocyte maturation and trophoblast migration.

Decorin (DCN) is a proteoglycan belonging to the small leucine-rich proteoglycan family. It is composed of a protein core containing leucine repeats with a glycosaminoglycan chain consisting of either chondroitin sulfate or dermatan sulfate. DCN is a structural component of connective tissues that can bind to type I collagen. It plays a role in the assembly of the extracellular matrix (ECM), and it is related to fibrillogenesis. It can interact with fibronectin, thrombospondin, complement component C1, transforming growth factor (TGF), and epidermal growth factor receptor. Normal DCN expression regulates a wide range of cellular processes, including proliferation, migration, apoptosis, and autophagy, through interactions with various molecules. However, its aberrant expression is associated with oocyte maturation, oocyte quality, and poor extravillous trophoblast invasion of the uterus, which underlies the occurrence of preeclampsia and intrauterine growth restriction. Spatiotemporal hormonal control of successful pregnancy should regulate the concentration and activity of specific proteins such as proteoglycan participating in the ECM remodeling of trophoblastic and uterine cells in fetal membranes and uterus. At the human feto-maternal interface, TGF-β and DCN play crucial roles in the regulation of trophoblast invasion of the uterus. This review summarizes the role of the proteoglycan DCN as an important and multifunctional molecule in the physiological regulation of oocyte maturation and trophoblast migration. This review also shows that recombinant DCN proteins might be useful for substantiating diverse functions in both animal and in vitro models of oogenesis and implantation.

Quality control and product differentiation of LMWHs marketed in China using 1H NMR spectroscopy and chemometric tools

Low molecular weight heparins (LMWHs) are heterogeneous mixtures of glycosaminoglycan chains composed of mixture of different lengths and substitution patterns. Structural characterization and quality control of LMWHs have always been challenging. The Chinese drug regulatory authorities have been committed to improve the supervision standards of LMWHs to better regulate the quality and safety of LMWHs in current Chinese market. In the present paper, 80 batches of three types LMWHs (dalteparin, enoxaparin and naldroparin) marketed in China from different manufacturers were studied by 1H NMR experiments and chemometric analysis. The method can be used not only to monitor impurities and contaminants, but also to check the batch-to-batch consistency of each manufacture. Moreover, for the biosimilar LMWHs from different manufactures, they can be differentiated and clustered according to their slightly different structural compositions originated from production process. By using this method, the quality and safety of LMWHs marketed in China were initially assessed.