Biology subjects

Jay, G. D.

Publications and source records attributed to Jay, G. D..

6 recordsLinked to original sources

bioRxiv2025

Lubricin's Mucin Domain Has Strong Polyproline Type-II Helical Character

Lubricin is a glycoprotein that is crucial for maintaining joint health by preventing joint wear by reducing joint friction in the boundary mode. Lubricin was recently observed to hinder the formation of uric acid crystals in the joint and prevent a form of gouty arthritis. However, despite lubricins great physiological importance, our current understanding of the molecular origins of lubricins beneficial properties is limited by a lack of detailed structural information regarding its central mucin domain: lubricins large size (227.5 kDa) and numerous glycosylations pose a substantial obstacle to conventional experimental methods for solving protein structures. In this work, we employ a combination of physics-based replica exchange molecular dynamics (REMD) simulations and circular dichroism (CD) experiments to shed light on the structure of lubricins central mucin domain. Using REMD, we model [KEPAPTTP]2, an amino acid repeat found throughout the mucin domain, and find that the mucin domain is likely to exhibit polyproline type II (PPII) helices, which are further stabilized by O-linked oligosaccharide chains. Motivated by these simulation results, we performed circular dichroism spectroscopy on fragments of the mucin domain that also show clear polyproline-II helical character, corroborating our computational findings. Altogether, this work provides strong evidence of a lubricin mucin domain with significant polyproline type II content. As polyproline helices are often also found in other glycoproteins with antifreeze properties, this work may also explain the atomistic underpinnings of their interfacial functions, including lubrication and competition with crystal formation. SIGNIFICANCELubricin is a mucinous glycoprotein containing a central heavily glycosylated domain that plays a crucial role in the lubrication of joints. However, little is known about the structure of this large central mucin domain, which makes the development of related therapeutics or biomedical devices challenging. In this work, we provide strong evidence that lubricins mucin domain possesses polyproline type II (PPII) character that is enhanced by glycosylation upon the basis of a combination of molecular dynamics simulations and circular dichroism experiments. Lubricins PPII character provides a molecular basis for its lubricating properties, which may provide insights into related antifreeze glycoproteins (AFGP) and the development of new biocompatible lubricants and ryo-preservatives.

biophysics↗

bioRxiv2022

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the NF{kappa}B pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects TNF-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells {+/-}TNF {+/-}rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNF-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleu-kins, and transcription factors. TNF treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified FAT10, which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNF and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NF{kappa}B activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

molecular biology↗

bioRxiv2021

The level of synovial human VEGFA, IL-8 and MIP-1α correlate with truncation of lubricin glycans in osteoarthritis

Osteoarthrithis (OA) is an endemic disease due to the increase of the worlds elderly population. Previously thought to be a consequence of an imbalance between cartilage degradation and biosynthesis, it is now recognized as a disease also involving inflammation, hence influencing the level of inflammatory cytokines, growth factors and chemokines. Lubricin is a mucin type molecule where its OA induced glycosylation truncation propels a deteriorating lubrication of the articular cartilage. The objective of this study was to explore the OA driven truncation of O-linked glycosylation of synovial lubricin and its cross talk with systemic and local (synovial fluid, SF) inflammation. We compared the systemic level of cytokines/chemokine in OA patients and controls plasma with their local level in SF using a 44 plex screen. The level of 27 cytokines and chemokines was consistently measured in both plasma and SF. The data showed that the levels of cytokines and chemokines in OA plasma display limited correlation to their counterpart in SF. The level of synovial IL-8 and MIP-1 and VEGFA in OA patients, but not their plasma level, where the only cytokines that displayed a significant correlation to the observed lubricin O-linked glycosylation truncation. These cytokines were also shown to be upregulated exposing fibroblast like synoviocytes from healthy and OA patients to recombinant lubricin with truncated glycans mainly consisting of Tn-antigens, while lubricin with sialylated and non-sialylated T anigens did not have any effect. The data suggest that truncated glycans of lubricin, as found in OA, promotes the synovial cytokine production and exerebate the local synovial inflammation.

pathology↗

bioRxiv2020

Proteoglycan 4 (PRG4) expression and functionin dry eye associated inflammation

PurposeDry eye disease (DED) affects hundreds of millions worldwide. Proteoglycan 4 (PRG4) has been shown to improve signs and symptoms of DED in humans. The objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine how exogenous recombinant human PRG4 (rhPRG4) modulates cytokine and chemokine secretion in response to TNF and IL-1{beta}, explore rhPRG4 as a potential substrate and/or inhibitor of MMP-9, and to understand how experimental dry (EDE) in mice affects PRG4 expression. MethodsPRG4 secretion was quantified by Western blotting and PRG4 expression by immunocytochemistry. Cytokine/chemokine release was measured by ELISA, and MMP-9 inhibition was quantified using an MMP-9 inhibitor kit. EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and Western blotting in lacrimal gland lysate. ResultshTCEpi cells synthesize and secrete PRG4 in vitro, which is inhibited by TNF and IL-1{beta}. TNF and IL-1{beta} significantly increased secretion of cytokine IL-6 and chemokines IL-8, IP-10, RANTES, and ENA-78, and several of these chemokines were downregulated after cotreatment with rhPRG4. Fluorescently-labelled rhPRG4 was internalized by hTCEpi cells. rhPRG4 was not digested by MMP-9 and inhibited in vitro activity of exogenous MMP-9 both in solution and in the presence of human tears. Finally, EDE decreased corneal and lacrimal gland expression of PRG4. ConclusionsThese results demonstrate rhPRG4s anti-inflammatory properties in the corneal epithelium and its contribution to ocular surface homeostasis, furthering our understanding of PRG4s immunomodulatory properties in the context of DED inflammation.

biochemistry↗

bioRxiv2019

Core-2 O-glycans are required for Galectin-3 interaction with the osteoarthritis related protein lubricin

Synovial fluid lubricin (proteoglycan 4) is a mucin-type O-linked glycosylated (60% of the mass) biological lubricant involved in osteoarthritis (OA) development. Lubricin has been reported to be cross-linked by synovial galectin-3 on the lubricating articular surface. Here, we confirm that binding to galectin-3 depended on core-2 O-linked glycans, where surface plasmon resonance of a recombinant lubricin (rhPRG4) devoid of core-2 structures lacked binding capacity to recombinant galectin-3. Both galectin-3 levels and interactions with synovial lubricin were found to be decreased in late-stage OA patients coinciding with an increase of truncated and less sialylated core 1 O-glycans. These data suggest a defect cross-linking of surface active molecules in OA and provides novel insights into OA molecular pathology.

biochemistry↗

bioRxiv2019

Cathepsin G Degrades Synovial Fluid Lubricin: Relevance For Osteoarthritis Pathogenesis

Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff in gels, and with proteomics. Full length lubricin ([~]300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain ([~]250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in different domains of lubricin. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the lubrication of OA joints.

pathology↗