Dataset Information

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

ABSTRACT: The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.

SUBMITTER: Flowers SA

PROVIDER: S-EPMC7681006 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Decrease of core 2 <i>O-</i>glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

Flowers Sarah A SA Thomsson Kristina A KA Ali Liaqat L Huang Shan S Mthembu Yolanda Y Regmi Suresh C SC Holgersson Jan J Schmidt Tannin A TA Rolfson Ola O Björkman Lena I LI Sundqvist Martina M Karlsson-Bengtsson Anna A Jay Gregory D GD Eisler Thomas T Krawetz Roman R Karlsson Niclas G NG

The Journal of biological chemistry 20200914 47

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type <i>O-</i>linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cart ...[more]

PMID: 32928962

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Project description:Lubricin/proteoglycan-4 (PRG-4) is a mucinous glycoprotein that lubricates cartilage and maintains normal tissue function and cell homeostasis. Altered O-glycoproteforms of lubricin have been found in osteoarthritis (OA) synovial fluid (SF), which could ostensibly be used to diagnose early onset OA. However, SF is invasive to obtain and generally would not be surveyed from otherwise healthy individuals. Thus, a plasma-based OA screening tool focused on lubricin glycosylation could be a less invasive method to aid in early-stage OA diagnosis. In this report, we used glycomics and glycoproteomics to characterize glycoproteoforms of OA lubricin in SF and plasma. We obtained near-complete sequence coverage of lubricin's mucin domain and its glycosylation using matched SF and plasma from patients with OA (N = 5). From SF lubricin we observed a spectrum of O-glycans ranging from a single GalNAcα1-Ser/Thr monosaccharide up to branched pentasaccharides. In contrast, plasma based lubricin was predominantly decorated with sialylated Galβ1-3GalNAcα1-Ser/Thr (Sialyl T). To explain the glycosylation differences observed between SF and plasma lubricin, we present splice variant-specific peptides found within the non-glycosylated region, revealing that that the longest spliceoform of lubricin was present exclusively in SF, while additional shorter splice variants could only be detected in plasma. Based on our glycoproteomic data, we developed and validated a lectin assay for lubricin, and applied this on a larger cohort of matched SF/plasma (N = 19) to confirm the glycosylation differences between SF and plasma proteoforms. Next, we leveraged our assay to screen over 100 patient with OA samples (OA patients N = 108/controls N = 38) to probe plasma lubricin as an OA biomarker. Here, we detected a decrease in α2,6 linked sialic acid in patients with OA and further show that the extent of α2,6 and α2,3 sialylation on plasma-associated lubricin correlated with patient characteristics, especially Body Mass Index (BMI).

Project description:Lubricin or proteoglycan-4 (PRG-4) is a mucinous glycoprotein that lubricates the cartilage and maintains normal tissue function and cell homeostasis. Altered O- glycoproteforms of lubricin have been found in osteoarthritis (OA) synovial fluid (SF), which could ostensibly be used as a biomarker to diagnose early onset OA. However, SF is invasive to obtain and generally would not be surveyed on otherwise healthy individuals. Thus, a plasma-based OA screening tool focused on lubricin glycosylation could serve as an improved biomarker. In this report, we used glycomics and glycoproteomics to identify glycoproteoforms of OA lubricin in SF and plasma. We obtained near-complete sequence coverage of the mucin domain of lubricin and its glycosylation using matched SF/plasma from OA patients (N=5). In SF we observed a spectrum of O-glycans on lubricin ranging from the single GalNAc monosaccharide up to branched pentasaccharide and low in sialylation compared lubricin in plasma, decorated with sialylated Gal1-3GalNAc. We also detected splice variant-specific peptides from lubricin non-glycosylated region showing that the longest splice form of lubricin was exclusively present in SF, while in addition shorter splice variants were also present in plasma.Following elution, the mucin-enriched plasma or synovial fluid was subjected to reduction, alkylation, and proteolytic digestion. To begin, dithiothreitol (DTT, Sigma) was added to a concentration of 2 mM and reacted at 65 °C for 20 min followed by alkylation in 5 mM iodoacetamide (IAA, sigma) for 15 min in the dark at RT. Subsequently, mucinase SmE was added at an enzyme:substrate (E:S) ratio of 1:20 and allowed to react overnight at 37 °C. At this point, the samples were split into two aliquots, one containing 90% and the other containing the remaining 10%. The former represented the “mucinase-only” digest that did not undergo any further proteolysis. The latter was subjected to an additional digestion with trypsin at a 1:50 E:S ratio for 6 hours at 37 °C. All reactions were quenched by adding 1 µL of formic acid (Thermo Scientific) and diluted to a volume of 200 µL prior to desalting. Addition of formic acid also caused sodium deoxycholate to precipitate out of solution, and the resulting supernatant was transferred to a new tube before desalting. Desalting was performed using 10 mg Strata-X 33 µm polymeric reversed phase SPE columns (Phenomenex). Each column was activated using 500 µL acetonitrile (ACN) (Honeywell) followed by of 500 µL of 0.1% formic acid, 500 µL of 0.1% formic acid in 40% ACN, and equilibration with two additions of 500 µL of 0.1% formic acid. After equilibration, the samples were added to the column and rinsed twice with 200 µL of 0.1% formic acid. The columns were transferred to a 1.5 mL tube for elution by two additions of 150 µL of 0.1% formic acid in 40% ACN. The eluent was then dried using a vacuum concentrator (LabConco) prior to reconstitution in 10 µL of 0.1% formic acid.

Project description:The chronicity of osteoarthritis (OA), characterized by pain and inflammation in the joints, is linked to a glutamate receptor, N-methyl-D-aspartate (NMDA). The use of plant species such as Chenopodium ambrosioides L. (Amaranthaceae) as NMDA antagonists offers a promising perspective. This work aims to analyze the antinociceptive and anti-inflammatory responses of the crude hydroalcoholic extract (HCE) of C. ambrosioides leaves in an experimental OA model. Wistar rats were separated into six groups (n = 24): clean (C), negative control (CTL-), positive control (CTL+), HCE0.5, HCE5 and HCE50. The first group received no intervention. The other groups received an intra-articular injection of sodium monoiodoacetate (MIA) (8 mg/kg) on day 0. After six hours, they were orally treated with saline, Maxicam plus (meloxicam + chondroitin sulfate) and HCE at doses of 0.5 mg/kg, 5 mg/kg and 50 mg/kg, respectively. After three, seven and ten days, clinical evaluations were performed (knee diameter, mechanical allodynia, mechanical hyperalgesia and motor activity). On the tenth day, after euthanasia, synovial fluid and draining lymph node were collected for cellular quantification, and cartilage was collected for histopathological analysis. Finally, molecular docking was performed to evaluate the compatibility of ascaridole, a monoterpene found in HCE, with the NMDA receptor. After the third day, HCE reduced knee edema. HCE5 showed less cellular infiltrate in the cartilage and synovium and lower intensities of allodynia from the third day and of hyperalgesia from the seventh day up to the last treatment day. The HCE5 and HCE50 groups improved in forced walking. In relation to molecular docking, ascaridole showed NMDA receptor binding affinity. C. ambrosioides HCE was effective in the treatment of OA because it reduced synovial inflammation and behavioral changes due to pain. This effect may be related to the antagonistic effect of ascaridole on the NMDA receptor.

Dataset Information

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

Publications

Decrease of core 2 <i>O-</i>glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

Similar Datasets

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