Articular cartilage functions as a cushion to cover and protect the joints between bones. The cartilage is formed by an extracellular matrix (ECM) of collagen II consisting of fibrils that are integrated within an abundant anionic network of proteoglycan aggregates. These proteoglycans and collagen fibres, along with glycoproteins and water, form a macromolecular network to produce a protective matrix that facilitates articular movement. Osteoarthritis (OA) is characterized by the triggering of events that lead to the cartilage ECM breakdown and loss of joint function [1
]. The susceptibility of cartilage to arthritic degradation highly depends on specific posttranslational modifications of ECM proteins. Glycosylation is the most frequent posttranslational modification of cell surfaces and ECM proteins [2
]. As a consequence, chondrocytes contain a dense coat of carbohydrates on their surfaces. These carbohydrate moieties mediate a wide variety of cell-to-cell and cell–matrix interactions that are critical for cartilage and bone development and function. Carbohydrate chains vary according to cell and tissue type and undergo modifications during a range of processes involving cellular differentiation, phenotypic changes, and oncogenesis [3
In particular, sialic acids are carboxylated sugars containing nine carbons that are usually located as terminal monosaccharides [7
]. Sialyltransferases (SiaTs) transfer sialic acids in α-2,3-, α-2,6- or α-2,8-configurations to the N- or O-linked oligosaccharides of glycoproteins. The α-2,3- and the α-2,6-SiaTs show a mutually exclusive expression pattern and a remarkable tissue specificity. Specific sialylation motifs lead to differential effects of glycoproteins on fundamental aspects of cell behaviour including growth, migration, intercellular communication, inflammation, ECM production [8
], and chondrocyte function [4
]. In fact, glycan-binding proteins (GBPs) and glycan–protein interactions are important for regulating a range of physiological or pathological processes that include inflammation and arthritis [6
]. GBPs are broadly classified into two major groups: lectins and glycosaminoglycan-binding proteins.
Carbohydrate recognition is one of the most sophisticated recognition mechanisms in biological systems [16
], and lectins are specialized in the recognition of specific glycan molecular patterns. Lectin–glycan interactions are undoubtedly a valuable system to control inflammatory pathways activation under pathological processes. For instance, galectins, beta-galactoside-binding animal lectins, bind and activate cell surface glycan receptors such as podoplanin (PDPN) [19
]. As an example, among this galectin family, galectin-1 has been reported to enhance secretion of effectors of degeneration by stimulating NF-kB and switching on an inflammatory response in OA [11
]. Interfering the interaction between galectins and glycosilated receptors, using plant lectins such as MASL, may in turn impede activation of downstream signalling pathways [21
]. Due to their specific characteristics, lectins have been used to differentiate malignant tumours (from benign) depending on the degree of glycosylation and have been proposed as alternative cancer therapeutics by their effect on cancer cell proliferation [23
]. In particular, the lectin MASL present in the seeds of the Maackia amurensis
binds to sialylated glycoproteins [24
] by recognition of terminal α-2,3-sialylated oligosaccharides [27
]. MASL has been reported to be composed of two molecular species, leucoagglutinin (MAL) and haemagglutinin (MAH) [28
], although recent studies have pointed to the presence of only single species [30
Specific modifications in lectin and glycan presentation underlie the contribution of glycobiology in the development and progression of several disorders. Expression of the α-2,3-sialylated glycoprotein PDPN receptor has been reported to trigger degenerative joint diseases including rheumatoid arthritis (RA) [31
]. In addition, PDPN participates in tissue development, repair, and inflammation [35
], e.g., the binding of the C-type lectin-like receptor 2 (CLEC-2) to the sialylated extracellular domain of PDPN has been implicated in the inflammatory response [38
], and the molecular binding characteristics for this interaction have been recently reported [42
]. Here, we present an extensive analysis of the effects of the lectin MASL on primary chondrocytes and cartilage structure using samples from healthy donors and patients with OA as well as animal models of arthritis. The results indicate that MASL preserves the structure and function of cartilage under diverse arthritic insults by interfering with the function of α-2,3-sialylated transmembrane receptors, such as the mucin-type transmembrane glycoprotein PDPN [36
]. These findings suggest that MASL inhibits the activation of signal transduction pathways mediated by NF-κβ that lead to progressive cartilage destruction during the pathogenesis of arthritis by increasing reactive oxygen species (ROS), inflammatory cytokines, and metalloproteinases [43
The ability to regulate the events of signalling cascades to protect cartilage from the catabolic effects that induce ECM degradation will undoubtedly help to avoid or delay invasive therapeutic methods such as total joint replacement and to ameliorate the negative effects of arthritis, one the most common causes of disability that impacts over 350 million people worldwide.
2. Materials and Methods
2.1. Cartilage Processing and Primary Culture
Cartilage was collected and processed as previously reported [44
]. The Institutional Ethics Committee for human research approved this study (Registration Code CAEIG: 2012/094—PI13/00591). All patients signed informed consent forms. The cartilage samples were immediately frozen in situ in Cryomold®
Standard using the Tissue-Tek®
compound and isopentane in liquid nitrogen and stored at −80 °C. Healthy cartilage was obtained from donors with no history of joint disease who suffered a hip or knee fracture. Medical record data and histological analysis were used to confirm healthy samples. The modified Mankin score method [45
] was used to grade the histological samples (healthy and arthritic with radiologic diagnosis). Samples from the normal/healthy, early arthritic, and moderate grade II and III groups were selected to perform this study following previously reported methodologies [44
]. For treatments, MASL was purchased from Sigma-Aldrich (St. Louis, MO, USA) or kindly provided by Sentrimed. Human primary chondrocytes were isolated and cultured as follow: fresh cartilage was rinsed with saline, and cells were isolated as previously described [45
]. For this, 2.5 million chondrocytes were plated into 162-cm2
flasks and incubated at 37 °C in 5% CO2
and 100% humidity in DMEM containing 100 µg/mL of Primocin (InvivoGen PrimocinTM
) and 15% foetal calf serum (FCS) (Life Technologies Gibco, New York, NY, USA) until 80–90% confluence was reached. After 3 or 4 weeks of primary culture, these chondrocytes were used for experiments. Synovial tissue of human donors was collected and synoviocytes were isolated by using the explant method; synovial tissue was cut into small pieces (explants) and cultured at 5% CO2
and 37 °C. Synovial cells were attached to the 100-mm dish and were cultured in RPMI 1640 with 20% FBS and a 0.1% insulin solution (Sigma-Aldrich). TC28a2 (human chondrocytes cell line) were kindly donated by Dr. Mary Goldring and cultured in DMEM containing 100 µg/mL of Primocin and 15% FCS.
2.2. Tissue Culture
Immediately after surgery, cartilage punches of 4 mm size were prepared from cartilage explants, which were cut using the Biopsy Punch BP-40F (Kai Corporation, Tokyo, Japan). The punches were cultured in 48-well culture plates overnight in DMEM without serum. The medium was changed to DMEM with 0.1% FCS containing MASL and/or oligomycin, and the cells were then incubated for 7 days. MASL (400 nM) was added to the medium 40 min before the addition of 5 µg/mL of oligomycin. Medium and drugs were replaced every 2 days. At the end of the experiments, every punch was cut in two parts, with half being used for RNA isolation and the other half being frozen in Cryomold Standard and Tissue-Tek O.C.T. compound and stored at −80 °C for immunohistochemistry and immunofluorescence assays.
2.3. Cell Viability Assay
Human articular chondrocytes were plated in a 96-well plate and treated with 0, 400, and 720 nM MASL (Sentrimed, Inc., Voorhees, NJ, USA) with and without 5 µg/mL of oligomycin (Sigma Aldrich, Darmstadt, Germany), an ATP synthase blocker, for 1 and 17 h. The cytotoxicity of these drugs was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay (Cell Proliferation Kit I from Roche, Grenzach-Wyhlen, Germany). Absorbance was measured with a NanoQuant Microplate Reader (Tecan Trading AG, Switzerland) at 570 nm.
2.4. Adhesion Assay
Human chondrocytes were seeded onto fibrinogen-coated wells in the presence of 400 or 720 nM MASL for 1 h. Wells coated with BSA were used as negative controls. Cell adhesion was evaluated using the CytoSelectTM Cell Adhesion Assay Kit (Cell Biolabs, Inc., San Jose, CA, USA) and measured with a NanoQuant Microplate Reader (Tecan Trading AG, Männedorf, Switzerland) at a wavelength of 560 nm.
2.5. Cell Growth and Migration
Chondrocytes were cultured to confluence in 24-well culture plates containing an insert that forms a 0.9 mm gap on the monolayer (CytoSelectTM Wound Healing Assay Ki. Cell Biolabs, Inc. San Jose, CA, USA). After the insert was removed, the cells were treated with 720 or 400 nM MASL during 24 h (TC28a2 cells) or 10 days (primary chondrocytes) in DMEM supplemented with 1% FCS. Cells were imaged under an inverted light microscope (Nikon Eclipse Ti and NIS-Elements software).
2.6. Animals, Treatments, and Histological Analysis
We used BALB/c mice in this study. All mice were 12 weeks old. Equal number of males (26–29 g) and females (20 g) were used and housed in conventional conditions. The studies were approved by the local ethics committee. Cartilage degeneration and joint inflammation were induced by three intra-articular injections of 25 µL of a sterile PBS solution containing 5 µg of LPS and 0.1% BSA in the left joint (Sigma-Aldrich, St. Louis, MO, USA). The right knee was injected with PBS-BSA alone. The control group received PBS-BSA only in the right joint. The joints were injected three times per week during the first week and the final week prior to the mice being sacrificed. The lectin was orally administered to each mouse in the form of a food supplement as indicated [30
]. The mice were fed dried pellets (100 mg) containing 1 mg of MASL in sterile water (100 µL) for 7 weeks and 3 days prior to the LPS injection. The controls were fed a dried pellet containing the same volume of sterile water without MASL. Before feeding, the food was removed from the cages and each mouse remained in a separate cage for the treatment. Mice were returned to their regular cages after eating the individual piece of pellet with or without MASL. The knee joint diameter was measured 24 h after each injection with a digital calibre (S_Cal WORK, Sylvac, Swiss, Switzerland). Animals were sacrificed, and the isolated joints (knees) were fixed in 4% formaldehyde, decalcified, rinsed with 70% ethanol, and embedded in paraffin (Merck) for 17 h. Serial sections (4 μm) were stained with haematoxylin–eosin, Safranin-O Fast Green, and toluidine blue to analyse cartilage damage as previously reported [45
2.7. Immunohistochemistry and Immunofluorescence
Human articular chondrocytes were cultured on chamber slides and fixed with 4% formaldehyde (PFA) for 10 min at room temperature. Frozen cartilage sections were sequentially sectioned (4 µm) and processed as previously described with minor variations [45
]. The samples were counterstained with haematoxylin–eosin or DAPI (Sigma Aldrich, Darmstadt, Germany). Anti-Podoplanin (18H5) antibody was supplied by Merck Millipore and anti-NF-κβ (sc-8008) was supplied by Santa Cruz Biotechnology. Negative controls without primary antibody were performed to test the specificity of each antibody. Haematoxylin–eosin, Safranin-O Fast Green, Masson’s trichrome, and toluidine blue were used to stain the cartilage sections. Images were taken on an Olympus BX61 microscope using a DP71 digital camera (Olympus); the AnalySISD
5.0 software (Olympus Biosystems, Hamburg, Germany) was used for image calibration and quantification.
2.8. Western Blot
Chondrocytes were pelleted and lysed in 200 µL of chilled home-made lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8, 0.1% (w/v) SDS, 0.5% v/v Nonidet P-40, 0.5% (v/v) sarkosyl), and supplemented with 5 µg/mL protease inhibitors cocktail and 1 mM PMSF. Bradford protein assay was used to determine total protein. Precisely, 15 µg of protein were separated in a 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Co., Bedford, MA, UK). Transference was confirmed by staining the membrane with ATX Ponceau S red solution (Sigma-Aldrich, Darmstadt, Germany), continued by 1 h blocking using 5% milk in TBS (Tris-Buffered-Saline; 20 mM Tris and 150 mM NaCl) and 0.05% Tween-20 (Sigma-Aldrich, Darmstadt, Germany). Primary antibody incubation was performed O/N at 4 °C, and HRP-secondary probing at RT for 1 h. PierceTM ECL Western Blotting Substrate in a LAS-300 Imager (Fujifilm, Tokyo, Japan) was used. Mouse anti-α-tubulin antibody (T9026, 1:10.000 in 5% milk in TBS) was supplied from Sigma-Aldrich (Darmstadt, Germany); mouse monoclonal anti-NF-κβ (sc-8008, 1:500 in 5% milk in TBS) and mouse monoclonal anti-p-Iκβ-α (sc-8404, 1:100 in 5% milk in TBS) antibodies were supplied from Santa Cruz Biotechnology.
2.9. Lectin-Binding Analysis
HiLyte Fluor TR (red) was used to conjugate MASL and to study its affinity to α2–3-linked sialic acid-modified glycoproteins in cultured cells and cartilage. Chamber slides of primary cultures or tissue sections were exposed to a solution that contained 200 µg/mL of conjugated MASL in PBS for 20 min at RT. Samples were washed with PBS and processed for microscopic analysis.
2.10. Quantitative RT-PCR
TRIzol reagent was used to isolate total RNA from chondrocytes according to manufacturer’s instructions (Invitrogen). Frozen cartilage was pulverized with a prechilled mortar and recovered in 1 mL of QIAzol Lysis Reagent (74804, Qiagen). The samples were incubated on ice for 5 min. Next, 200 µL of chloroform was added to all samples, which were then vigorously agitated for 15 s and then incubated for 3 min at RT. RNA was isolated using QIAcube following the manufacturer’s instructions (Qiagen). DNase treatment (RNase-free DNase, Invitrogen) was performed to total RNA to ensure the degradation of the DNA in the samples. A total of 1 µg of total RNA per sample was used to synthesize cDNA with the SuperScript® VILO™ cDNA Synthesis Kit as instructed by the manufacturer (Invitrogen). Quantitative PCR was performed with the LightCycler 480 SYBR Green I Master from Roche on a real-time PCR machine (LightCycler® 480 System, Roche, Grenzach-Wyhlen, Germany) with the primers GAATCCTCAACCCATATTTCATCC and CACTGCCACACTGCCAAG for ST3Gal3 and ATTCCTGAGTGCTGTCTTCC and ATCTTATTTCTCCGTTTCATTTCC for ST3Gal6. HPRT1 was used as reference gene with the primers TTGAGTTTGGAAACATCTGGAG and GCCCAAAGGGAACTGATAGTC.
2.11. Computational Modelling of the MASL Proteins (MAL and MAH)
MASL is reported to be a tetramer formed by a MAH protein [28
] of 32 kD and a MAL protein of 37 kD. An X-ray crystallographic structure of MAL in complex with Neu5Acα2–3Galβ1–4Glc is available at the Protein Data Bank [www.rcsb.org
, accessed April 16, 2020 (PDB ID 1DBN) [27
] at a resolution of 2.75 Å. Chain A was used, the co-factors of crystallization and the N-Acetyl-D-Glucosamine (GlcNAc) and Neu5Acα(2–3)Galβ(1–4)Glc (sialyllactose) ligands were removed, and cap termini were inserted with the Protein Preparation Wizard within the Maestro suite 8 (version 9.3, Schrödinger, LLC, New York, NY, USA, 2012). Manganese and calcium ions were included. All of the water molecules were removed, except for those involved in the coordination of manganese and calcium, and hydrogen ions were added with Epik at a physiological pH. The calculated protonation by Epik state was maintained. The protein model was minimized and charges were calculated with OPLS 2005 using water as an implicit solvent [46
]. Thus, 3D structure of MAL was obtained by homology modelling (see Appendix A
2.12. Molecular Dynamics (MD) Simulations
Both MASL protein structures (MAL and MAH) were refined by means of MD simulations. The complexes with the tetrasaccharide Neu5Acα(2–3)Galβ-(1–3)[Neu5Acα(2–6)GalNAc from the best docked poses in MAH (an MAL) were also submitted to MD simulations. For all the MD simulations, GLYCAM06, gaff, and ff14SB were used as force fields, and were run using Amber 14 [47
counterions were added to neutralize the system. Each system was then solvated by using TIP3P waters in a cubic box with at least 10 Ǻ of distance around the complex. The shake algorithm was applied to all hydrogen containing bonds, and 1 fs integration step was used. Periodic boundary conditions were applied, as well as the smooth particle mesh Ewald method to represent the electrostatic interactions, with a grid space of 1 Ǻ. Each system was gently annealed from 100 to 300 K over a period of 25 ps. The systems were then maintained at temperature of 300 K during 50 ps with a solute restraint and progressive energy minimizations, gradually releasing the restraints of the solute followed by a 20 ps heating phase from 100 to 300 K, where restraints were removed. Production simulation for each system lasted 40 ns. Coordinate trajectories were recorded each 2 ps throughout production runs, yielding an ensemble of 5000 structures for each complex. The root mean square deviation (RMSD) as a function of time with respect to the starting structure for the α-C atoms was computed using CPPTRAJ [42
2.13. Computational Modelling of the Ligand Neu5Acα(2–3)Galβ-(1–3)[Neu5Acα(2–6)]GalNAc
The tetrasaccharide Neu5Acα(2–3)Galβ-(1–3)[Neu5Acα(2–6)]GalNAc was generated by the Carbohydrate Builder from GLYCAM (www.glycam.com
). Part of this tetrasaccharide is also present in the X-ray crystallographic structure containing CLEC-2 and sialylated podoplanin (PDB ID 3WSR). In this X-ray complex, the terminal Neu5Acα(2–3) unit was exposed to the solvent; for this reason, the corresponding electron densities were missing [48
]. Eight conformations were generated by the Carbohydrate Builder, but only two structures fit with the X-ray crystallographic pose. The final structures were minimized with MacroModel (version 10.2, Schrödinger, LLC, New York, NY, USA, 2013) using the MM3* force field, and the structure with the lowest potential energy was used for docking purpose.
2.14. Docking Calculations
A set of 122 possible conformations of the tetrasaccharide 2
Neu5Acα(2–3)Galβ-(1–3)[Neu5Acα(2–6)]GalNAc was generated with MacroModel 8 version 10.6 (Schrödinger, LLC, New York, NY, USA, 2014) by performing a conformational search with OPLS2005 in the implicit solvent [46
], and the conformations were charged with the same force field. The docking was performed by means of the Glide program (version 6.5, Schrödinger, LLC, New York, NY, USA, 2014), generating a cubic grid box of 253
defining the centre as the centre of mass between Tyr249, Tyr164, Tyr73, and Ser134 for MAL. A standard precision docking was applied.
2.15. Statistical Analysis
GraphPad Prism software (version 5.00) was used to analyse the data. Data are presented as the mean ± S.E.M. or mean ± S.D. Mann–Whitney test was used to assess significant differences between the groups. * p < 0.05 was considered significant. * p < 0.05; ** p < 0.01, and *** p < 0.001.
Dysregulation of glycosylation in chondrocytes has been suggested as a critical regulator of inflammatory response and cartilage degeneration [4
]. Previous reports indicate that osteoarthritic cartilage degradation is promoted by factors that shift the expression of α-2,6-sialylated to α-2,3 sialylated glycoproteins in chondrocytes [4
]. The results presented here indicate that MASL can target these α-2,3-sialylated glycoproteins, such as PDPN, in cartilage and protect articular chondrocytes from the detrimental effects of inflammatory and catabolic events activated, among others, by the canonical signalling pathway NF-kB.
PDPN is induced during oncogenesis and inflammatory processes [31
]. The PDPN receptor consists of an extracellular domain, a transmembrane domain, and an intracellular tail. While the intracellular tail can be modified by protein kinases [37
], the majority of the protein consists of a highly glycosylated extracellular domain that impacts PDPN signalling and can be effectively targeted by antibodies and lectins [30
]. In particular, the lectin MASL shows dynamic abilities to target PDPN and normalize the morphology and phenotype of tumour cells [30
]. In addition, PDPN increases MMP activity in tumour cells [74
] and MASL, by targeting PDPN, blocks ECM degradation that is required for malignant cell invasion [30
]. Our results in arthritis models indicate that MASL can protect and maintain cartilage extracellular matrix structure in vivo in the presence of damaging insults that would otherwise lead to cartilage degradation by shifting the sialylation patterns in chondrocyte glycoproteins. Changes in the expression of sialyltransferases can reshape the arthritic cartilage glycophenotype reactivity contributing to activation of inflammatory pathways. Additionally, previous studies have reported that TNF blockers inhibit PDPN expression [77
], which is upregulated in synovial cells from RA patients [31
]. Our results for OA are consistent with these previous observations for tumour cells (regarding PDPN and MMP activity) and for RA (regarding PDPN and inflammation).
In traditional medicine, MASL has been used to treat inflammatory disorders including arthritis. In the present study, the in vitro effect of MASL in the articular cartilage was studied by using primary chondrocytes isolated from healthy donors and OA patients after joint replacement. Further studies will be necessary to analyse the effect of MASL in other cell types and tissues from the whole joint such as synovial membrane or subchondral bone which are also involved in OA progression. The ex vivo experiments using human cartilage punches support the data obtained in articular chondrocytes in primary culture. The results were further confirmed in an in vivo model when MASL was administered orally. However, we only analysed the effect of MASL at cartilage at the joint site. Overall, the results obtained in vitro and in vivo are consistent enough, however, complementary studies analysing the effect of MASL in different tissues and its presence in the plasma will help for the development of new therapeutic strategies in order to move to the clinic.
Terminal sialic acids are involved in many cellular functions, and changes in their biosynthesis or degradation are involved in degenerative disorders, such as diabetes, inflammatory disorders, or Alzheimer’s disease, by affecting ligands, masking antigenic sites, controlling signalling pathways, or regulating immunological and inflammatory functions [79
]. There is a growing interest in the targeting of catabolic and inflammatory signalling pathways for the prevention of cartilage and joint degeneration in OA and RA and, in general, in age-associated degenerative diseases. Here, we focus on the lectin MASL that holds promise for drug discovery research for the treatment of arthritis. The increased levels of the α-2,3-SiaT isoforms together with increase in the levels of α-2,3-sialylated glycoproteins in chondrocytes from osteoarthritic patients might shed mechanistic light on the pathophysiology of OA. The ability of MASL to target sialylated glycoproteins such as PDPN, and to attenuate NF-kB activation might offer new possibilities for new therapeutic strategies to target sialylation during acute disease stages in order to avoid cartilage degradation and joint degeneration in OA patients.