bims-supasi 2023-07-23 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2023–07–23
thirteen papers selected by
Jonathan Wolf Mueller, University of Birmingham

Mapping the human chondroitin sulfate glycoproteome reveals an unexpected correlation between glycan sulfation and attachment site characteristics.
Switching mechanism from AR to EGFR signaling via 3-O-sulfated heparan sulfate in castration-resistant prostate cancer.
Effect of glycosaminoglycans with different degrees of sulfation on chondrogenesis.
The effect of the sulfation patterns of dermatan and chondroitin sulfate from vertebrates and ascidians on their neuritogenic and neuroprotective properties.
Identification of potential non-invasive biomarkers in diastrophic dysplasia.
Discovery of raffinose sulfate as a potential SARS CoV-2 inhibitor via blocking its binding with angiotensin converting enzyme 2.
Molecular implications of glycosaminoglycans in diabetes pharmacotherapy.
Pleiotrophin Signals Through ALK Receptor to Enhance the Growth of Neurons in the Presence of Inhibitory Chondroitin Sulfate Proteoglycans.
N-acetylglucosamine-6-O-sulfation on intestinal mucins prevents obesity and intestinal inflammation by regulating gut microbiota.
Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation.
Affinity-selected heparan sulfate collagen device promotes periodontal regeneration in an intrabony defect model in Macaca fascicularis.
Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis.
The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment.

Mol Cell Proteomics. 2023 Jul 13. pii: S1535-9476(23)00128-7. [Epub ahead of print] 100617

Mapping the human chondroitin sulfate glycoproteome reveals an unexpected correlation between glycan sulfation and attachment site characteristics.

Fredrik Noborn, Jonas Nilsson, Carina Sihlbom, Mahnaz Nikpour, Lena Kjellén, Göran Larson.

  Chondroitin sulfate proteoglycans (CSPGs) control key events in human health and disease and are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. Detailed information on the biological effects of site-specific CS structures is scarce as the polysaccharides are typically released from their core proteins prior to analysis. Here we present a novel glycoproteomic approach for site-specific sequencing of CS modifications from human urine. Software-assisted- and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that acidity of the attachment site motifs increased the levels of CS sulfation and statistical analysis confirmed this relationship. However, not only the acidity but also sequence and characteristics of specific amino acids in proximity of the serine glycosylation site correlated with the degree of sulfation. These results demonstrate attachment site-specific characteristics of CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.

Keywords:  chondroitin sulfate; glycopeptides; glycoproteomics; glycosaminoglycans; proteoglycans

DOI:  https://doi.org/10.1016/j.mcpro.2023.100617
Sci Rep. 2023 07 18. 13(1): 11618

Switching mechanism from AR to EGFR signaling via 3-O-sulfated heparan sulfate in castration-resistant prostate cancer.

Hayato Ota, Hirokazu Sato, Shuji Mizumoto, Ken Wakai, Kei Yoneda, Kazuo Yamamoto, Hayao Nakanishi, Jun-Ichiro Ikeda, Shinichi Sakamoto, Tomohiko Ichikawa, Shuhei Yamada, Satoru Takahashi, Yuzuru Ikehara, Shoko Nishihara.

Androgen deprivation therapy is given to suppress prostate cancer growth; however, some cells continue to grow hormone-independently as castration-resistant prostate cancer (CRPC). Sulfated glycosaminoglycans promote ligand binding to receptors as co-receptors, but their role in CRPC remains unknown. Using the human prostate cancer cell line C4-2, which can proliferate in hormone-dependent and hormone-independent conditions, we found that epidermal growth factor (EGF)-activated EGFR-ERK1/2 signaling via 3-O-sulfated heparan sulfate (HS) produced by HS 3-O-sulfotransferase 1 (HS3ST1) is activated in C4-2 cells under hormone depletion. Knockdown of HS3ST1 in C4-2 cells suppressed hormone-independent growth, and inhibited both EGF binding to the cell surface and activation of EGFR-ERK1/2 signaling. Gefitinib, an EGFR inhibitor, significantly suppressed C4-2 cell proliferation and growth of a xenografted C4-2 tumor in castrated mouse. Collectively, our study has revealed a mechanism by which cancer cells switch to hormone-independent growth and identified the key regulator as 3-O-sulfated HS.

DOI: https://doi.org/10.1038/s41598-023-38746-x
Hua Xi Kou Qiang Yi Xue Za Zhi. 2023 Aug 01. pii: 1000-1182(2023)04-0395-10. [Epub ahead of print]41(4): 395-404

Effect of glycosaminoglycans with different degrees of sulfation on chondrogenesis.

Wen Zheng, Ming-Xiang Cai, Huizhen Peng, Minyi Liu, Xiangning Liu.

   OBJECTIVES: This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro.
METHODS: ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively.
RESULTS: Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression.
CONCLUSIONS: This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.

Keywords:  cartilage repair; chondrogenesis; degree of sulfation; sulfated glycosaminoglycan

DOI:  https://doi.org/10.7518/hxkq.2023.2023055
Int J Biol Macromol. 2023 Jul 14. pii: S0141-8130(23)02725-3. [Epub ahead of print]247 125830

The effect of the sulfation patterns of dermatan and chondroitin sulfate from vertebrates and ascidians on their neuritogenic and neuroprotective properties.

Taiane Barreto Medeiros, Paloma Cosendey, Diovana Ramos Gerin, Graziele Fonseca de Sousa, Taynan Motta Portal, Cintia Monteiro-de-Barros.

  Neurodegeneration is caused by the progressive loss of the structure and function of neurons, leading to cell death, and it is the main cause of many neurodegenerative diseases. Many molecules, such as glycosaminoglycans (GAGs), have been studied for their potential to prevent or treat these diseases. They are widespread in nature and perform an important role in neuritogenesis and neuroprotection. Here we investigated the neuritogenic and neuroprotective role of Phallusia nigra dermatan sulfate (PnD2,6S) and compared it with two distinct structures of chondroitin sulfate (C6S) and dermatan sulfate (D4S). For this study, a neuro 2A murine neuroblastoma cell line was used, and a chemical lesion was induced by the pesticide rotenone (ROT). We observed that PnD2,6S + ROT had a better neuritogenic effect than either C6S + ROT or D4S + ROT at a lower concentration (0.05 μg/mL). When evaluating the mitochondrial membrane potential, PnD2,6S showed a neuroprotective effect at a concentration of 0.4 μg/mL. These data indicate different mechanisms underlying this neuronal potential, in which the sulfation pattern is important for neuritogenic activity, while for neuroprotection all DS/CS structures had similar effects. This finding leads to a better understanding the chemical structures of PnD2,6S, C6S, and D4S and their therapeutic potential.

Keywords:  Glycosaminoglycan; Neuro 2A; Rotenone

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.125830
Bone. 2023 Jul 14. pii: S8756-3282(23)00171-0. [Epub ahead of print] 116838

Identification of potential non-invasive biomarkers in diastrophic dysplasia.

Chiara Paganini, Ricki S Carroll, Chiara Gramegna Tota, Andrea J Schelhaas, Alessandra Leone, Angela L Duker, David A O'Connell, Ryan F Coghlan, Brian Johnstone, Carlos R Ferreira, Sabrina Peressini, Riccardo Albertini, Antonella Forlino, Luisa Bonafè, Ana Belinda Campos Fernandes Xavier, Andrea Superti-Furga, Andreas Zankl, Antonio Rossi, Michael B Bober.

  Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by pathogenic variants in the SLC26A2 gene encoding for a cell membrane sulfate/chloride antiporter crucial for sulfate uptake and glycosaminoglycan (GAG) sulfation. Research on a DTD animal model has suggested possible pharmacological treatment approaches. In view of future clinical trials, the identification of non-invasive biomarkers is crucial to assess the efficacy of treatments. Urinary GAG composition has been analyzed in several metabolic disorders including mucopolysaccharidoses. Moreover, the N-terminal fragment of collagen X, known as collagen X marker (CXM), is considered a real-time marker of endochondral ossification and growth velocity and was studied in individuals with achondroplasia and osteogenesis imperfecta. In this work, urinary GAG sulfation and blood CXM levels were investigated as potential biomarkers for individuals affected by DTD. Chondroitin sulfate disaccharide analysis was performed on GAGs isolated from urine by HPLC after GAG digestion with chondroitinase ABC and ACII, while CXM was assessed in dried blood spots. Results from DTD patients were compared with an age-matched control population. Undersulfation of urinary GAGs was observed in DTD patients with some relationship to the clinical severity and underlying SLC26A2 variants. Lower than normal CXM levels were observed in most patients, even if the marker did not show a clear pattern in our small patient cohort because CXM values are highly dependent on age, gender and growth velocity. In summary, both non-invasive biomarkers are promising assays targeting various aspects of the disorder including overall metabolism of sulfated GAGs and endochondral ossification.

Keywords:  Biomarker; Collagen X; Diastrophic dysplasia; Glycosaminoglycan; Sulfation; Urine

DOI:  https://doi.org/10.1016/j.bone.2023.116838
Int J Biol Macromol. 2023 Jul 18. pii: S0141-8130(23)02713-7. [Epub ahead of print] 125818

Discovery of raffinose sulfate as a potential SARS CoV-2 inhibitor via blocking its binding with angiotensin converting enzyme 2.

Rami Adel Pashameah, Raya Soltane, Ahmed M Sayed.

  The present study aimed to characterize the possible binding sites on the SARS CoV-2 RBD-ACE2 complex and to highlight sulfated oligosaccharides as potential anti-SARS CoV-2 via inducing RBD-ACE2 complex destabilization and dissociation. By combining pharmacophore-based and structural-based virtual screening approaches we were able to discover raffinose sulfate (RS) as a potential antiviral sulfated oligosaccharide against two SARS CoV-2 variants (i.e., wild type and Omicron) (IC50 = 4.45 ± 0.28 μM and 4.65 ± 0.32 μM, respectively). Upon MD simulation, RS was able to establish stable binding at the RBD-ACE2 interface inducing a rapid dissociation. Accordingly, and by using bio-layer interferometry (BLI) assays, RS was able to significantly weaken the affinity between RBD (of both variants) and ACE2. Additionally, we found that RS has a poor cellular permeability indicating that its interaction with the RBD-ACE2 complex may be the main mechanism by which it mediates its antiviral activity against SARS CoV-2. Despite its proposed interaction with the RBD-ACE2 complex, RS did not show any inhibitory activity against ACE2 catalytic activity. In light of these findings, the RS scaffold can be further developed into a novel anti-SARS CoV-2 drug with improved activity and tolerability in comparison with other sulfated polysaccharides e.g., heparin and heparan.

Keywords:  Bio-layer interferometry; Molecular dynamics simulation; Pharmacophore; RBD-ACE2; SARS CoV-2; Sulfated oligosaccharides; Virtual screening

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.125818
Int J Biol Macromol. 2023 Jul 17. pii: S0141-8130(23)02716-2. [Epub ahead of print] 125821

Molecular implications of glycosaminoglycans in diabetes pharmacotherapy.

Tanya Waseem, Madiha Ahmed, Tausif Ahmed Rajput, Mustafeez Mujtaba Babar.

  Diabetes mellitus causes a wide range of metabolic derangements with multiple organ damage. The microvascular and macrovascular complications of diabetes result partly from the damage to the glycosaminoglycans (GAG) in the basement membrane. GAGs are negatively charged polysaccharides with repeating disaccharide units. They play a significant role in cellular proliferation and signal transduction. Destruction of extracellular matrix results in diseases in various organs including myocardial fibrosis, retinal damage and nephropathy. To substitute the natural GAGs pharmacotherapeutically, they have been synthesized by using basic disaccharide units. Among the four classes of GAGs, heparin is the most widely studied. Recent studies have revealed multiple significant GAG-protein interactions suggesting their use for the management of diabetic complications. Moreover, they can act as biomarkers for assessing the disease progression. A number of GAG-based therapeutic agents are being evaluated for managing diabetic complications. The current review provides an outline of the role of GAGs in diabetes while covering their interaction with different molecular players that can serve as targets for the diagnosis, management and prevention of diabetes and its complications. The medicinal chemistry and clinical pharmacotherapeutics aspects have are covered to aid in the establishment of GAG-based therapies as a possible avenue for diabetes.

Keywords:  Advanced glycation end products; Diabetes; Glycosaminoglycans; Heparan sulfate; Interactomics; Proteoglycans

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.125821
Neurosci Insights. 2023 ;18 26331055231186993

Pleiotrophin Signals Through ALK Receptor to Enhance the Growth of Neurons in the Presence of Inhibitory Chondroitin Sulfate Proteoglycans.

Somnath J Gupta, Matthew A Churchward, Kathryn G Todd, Ian R Winship.

  Chondroitin sulfate proteoglycans (CSPGs), one of the major extracellular matrix components of the glial scar that surrounds central nervous system (CNS) injuries, are known to inhibit the regeneration of neurons. This study investigated whether pleiotrophin (PTN), a growth factor upregulated during early CNS development, can overcome the inhibition mediated by CSPGs and promote the neurite outgrowth of neurons in vitro. The data showed that a CSPG matrix inhibited the outgrowth of neurites in primary cortical neuron cultures compared to a control matrix. PTN elicited a dose-dependent increase in the neurite outgrowth even in the presence of the growth inhibitory CSPG matrix, with optimal growth at 15 ng mL-1 of PTN (114.8% of neuronal outgrowth relative to laminin control). The growth-promoting effect of PTN was blocked by inhibition of the receptor anaplastic lymphoma kinase (ALK) by alectinib in a dose-dependent manner. Neurite outgrowth in the presence of this CSPG matrix was induced by activation of the protein kinase B (AKT) pathway, a key downstream mediator of ALK activation. This study identified PTN as a dose-dependent regulator of neurite outgrowth in primary cortical neurons cultured in the presence of a CSPG matrix and identified ALK activation as a key driver of PTN-induced growth.

Keywords:  ALK; CSPGs; PTN; Plasticity; neuronal growth; neurons

DOI:  https://doi.org/10.1177/26331055231186993
JCI Insight. 2023 Jul 18. pii: e165944. [Epub ahead of print]

N-acetylglucosamine-6-O-sulfation on intestinal mucins prevents obesity and intestinal inflammation by regulating gut microbiota.

Hirohito Abo, Aoi Muraki, Akihito Harusato, Tetsuya Imura, Maki Suzuki, Kohta Takahashi, Timothy L Denning, Hiroto Kawashima.

  Intestinal mucins play an essential role in the defense against bacterial invasion and the maintenance of gut microbiota, which is instrumental in the regulation of host immune systems; hence, its dysregulation is a hallmark of metabolic disease and intestinal inflammation. However, the mechanism by which intestinal mucins control the gut microbiota as well as disease phenotypes remains nebulous. Herein, we report that N-acetylglucosamine (GlcNAc)-6-O-sulfation of O-glycans on intestinal mucins performs a protective role against obesity and intestinal inflammation. Chst4-/- mice, lacking GlcNAc-6-O-sulfation of the mucin O-glycans, showed significant weight gain and increased susceptibility to dextran sodium sulfate-induced colitis as well as colitis-associated cancer accompanied by significantly reduced immunoglobulin A (IgA) production caused by impaired T follicular helper cell-mediated IgA response. Interestingly, the protective effects of GlcNAc-6-O-sulfation against obesity and intestinal inflammation depend on the gut microbiota, evidenced by the modulation of the gut microbiota by co-housing or microbiota transplantation reversing disease phenotypes and IgA production. Collectively, our findings provide novel insight into the significance of host glycosylation, more specifically GlcNAc-6-O-sulfation on intestinal mucins, in protecting against obesity and intestinal inflammation via regulation of the gut microbiota.

Keywords:  Gastroenterology; Glycobiology; Inflammation; Mouse models; Obesity

DOI:  https://doi.org/10.1172/jci.insight.165944
Cell Chem Biol. 2023 Jul 13. pii: S2451-9456(23)00191-5. [Epub ahead of print]

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation.

Christoffer K Goth, Akul Y Mehta, Alyssa M McQuillan, Kelly J Baker, Melinda S Hanes, Simon S Park, Kathrin Stavenhagen, Gertrud M Hjortø, Jamie Heimburg-Molinaro, Elliot L Chaikof, Mette M Rosenkilde, Richard D Cummings.

  Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1 N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

Keywords:  CCL19; CCL21; CCL5; O-glycan; P-selectin; PSGL-1; chemokine; glycosulfopeptides; tyrosine sulfation

DOI:  https://doi.org/10.1016/j.chembiol.2023.06.013
Sci Rep. 2023 Jul 21. 13(1): 11774

Affinity-selected heparan sulfate collagen device promotes periodontal regeneration in an intrabony defect model in Macaca fascicularis.

Xiaoman Luo, Chau Sang Lau, Bach Quang Le, Tuan Chun Tan, Jian Hui Too, Raymond Alexander Alfred Smith, Na Yu, Simon M Cool.

It is challenging to regenerate periodontal tissues fully. We have previously reported a heparan sulfate variant with enhanced affinity for bone morphogenetic protein-2, termed HS3, that enhanced periodontal tissue regeneration in a rodent model. Here we seek to transition this work closer to the clinic and investigate the efficacy of the combination HS3 collagen device in a non-human primate (NHP) periodontitis model. Wire-induced periodontitis was generated in ten Macaca fascicularis, and defects were treated with Emdogain or collagen (CollaPlug) loaded with (1) distilled water, (2) HS low (36 µg of HS3), or (3) HS high (180 µg of HS3) for 3 months. At the endpoint, microscopic assessment showed significantly less epithelial down-growth, greater alveolar bone filling, and enhanced cementum and periodontal ligament regeneration following treatment with the HS-collagen combination devices. When evaluated using a periodontal regeneration assessment score (PRAS) on a scale of 0-16, collagen scored 6.78 (± 2.64), Emdogain scored 10.50 (± 1.73) and HS low scored 10.40 (± 1.82). Notably, treatment with HS high scored 12.27 (± 2.20), while healthy control scored 14.80 (± 1.15). This study highlights the efficacy of an HS-collagen device for periodontal regeneration in a clinically relevant NHP periodontitis model and warrants its application in clinical trials.

DOI: https://doi.org/10.1038/s41598-023-38818-y
Front Mol Biosci. 2023 ;10 1223972

Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis.

Baranca Buijsers, Marissa Maciej-Hulme, Maaike Jacobs, Marinka Bakker-van Bebber, Mark de Graaf, Rustem Salmenov, Naomi Parr, Ton J Rabelink, Tom Nijenhuis, Johan van der Vlag.

  Background: The glomerular endothelial glycocalyx is degraded during inflammation. The glycocalyx plays a pivotal role in endothelial function and is involved in many processes including binding of chemokines and cytokines, leukocyte trafficking, and preventing proteinuria. HS-based therapeutics are a promising novel class of anti-inflammatory drugs to restore a compromised endothelial glycocalyx under inflammatory conditions. Recently, we demonstrated that treatment with HS extracted from unstimulated glomerular endothelial glycocalyx (unstimulated HSglx) reduced albuminuria during anti-GBM induced glomerulonephritis. Since endothelial HS domains are distinct in unstimulated versus inflammatory conditions, we hypothesized that 1) unstimulated HSglx, 2) LPS-stimulated HSglx, 3) the HS-mimetic fucoidan and 4) the glycosaminoglycan preparation sulodexide, which is a mixture of low molecular weight heparin and dermatan sulfate, might have different beneficial effects in experimental glomerulonephritis. Methods: The effect of unstimulated HSglx, LPS HSglx, Laminaria japonica fucoidan, or sulodexide on experimental glomerulonephritis was tested in LPS-induced glomerulonephritis in mice. Analyses included urinary albumin creatinine measurement, cytokine expression in plasma and renal cortex, and renal influx of immune cells determined by flow cytometry and immunofluorescence staining. Furthermore, the observed in vivo effects were evaluated in cultured glomerular endothelial cells and peripheral blood mononuclear cells by measuring cytokine and ICAM-1 expression levels. The ability of the compounds to inhibit heparanase activity was assessed in a heparanase activity assay. Results: Treatment of mice with LPS HSglx or sulodexide near-significantly attenuated LPS-induced proteinuria. All treatments reduced plasma MCP-1 levels, whereas only fucoidan reduced IL-6 and IL-10 plasma levels. Moreover, all treatments reversed cortical ICAM-1 mRNA expression and both fucoidan and sulodexide reversed cortical IL-6 and nephrin mRNA expression. Sulodexide decreased renal influx of CD45+ immune cells whereas renal influx of macrophages and granulocytes remained unaltered for all treatments. Although all compounds inhibited HPSE activity, fucoidan and sulodexide were the most potent inhibitors. Notably, fucoidan and sulodexide decreased LPS-induced mRNA expression of ICAM-1 and IL-6 by cultured glomerular endothelial cells. Conclusion: Our data show a potentially protective effect of glycosaminoglycans and fucoidan in experimental glomerulonephritis. Future research should be aimed at the further identification of defined HS structures that have therapeutic potential in the treatment of glomerular diseases.

Keywords:  endothelial glycocalyx; fucoidan; glomerulonephritis; glycosaminoglycans; heparan sulfate; heparanase-1; proteinuria; sulodexide

DOI:  https://doi.org/10.3389/fmolb.2023.1223972
Exp Eye Res. 2023 Jul 15. pii: S0014-4835(23)00191-4. [Epub ahead of print]234 109570

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment.

H Hatami-Marbini, Md E Emu.

  The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Keywords:  CXL therapy; Glycosaminoglycans; Keratoconus; Pepsin degradation; Tensile properties

DOI:  https://doi.org/10.1016/j.exer.2023.109570