Proteoglycan Core Protein Research Articles

Multiple Administration of Dexamethasone Possesses a Deferred Long-Term Effect to Glycosylated Components of Mouse Brain.

Glucocorticoids are used during glioblastoma treatment to prevent the cerebral edema effect surrounding normal brain tissue. The aim of our study was to investigate the long-term effects of multiple administrations of glucocorticoids onto the glycosylated components (proteoglycans and glycosaminoglycans) of normal brain extracellular matrix and the glucocorticoid receptor (GR, Nr3c1) in an experimental model in vivo. Two-month-old male C57Bl/6 mice (n = 90) were injected intraperitoneally with various doses of dexamethasone (DXM) (1; 2.5 mg/kg) for 10 days. The mRNA levels of the GR, proteoglycans core proteins, and heparan sulfate metabolism-involved genes were determined at the 15th, 30th, 60th, and 90th days by a real-time RT-PCR. The glycosaminoglycans content was studied using dot blot and staining with Alcian blue. A DXM treatment increased total GAG content (2-fold), whereas the content of highly sulfated glycosaminoglycans decreased (1.5-2-fold). The mRNA level of the heparan sulfate metabolism-involved gene Hs3St2 increased 5-fold, the mRNA level of Hs6St2 increased6-7-fold, and the mRNA level of proteoglycan aggrecan increased 2-fold. A correlation analysis revealed an association between the mRNA level of the GR and the mRNA level of 8 of the 14 proteoglycans-coding and 4 of the 13 heparan sulfate metabolism-involved genes supporting GR involvement in the DXM regulation of the expression of these genes. In summary, multiple DXM administrations led to an increase in the total GAG content and reorganized the brain extracellular matrix in terms of its glycosylation pattern.

Influence of feeding a soft diet on proteoglycan expression in rat temporomandibular joint discs

ObjectivesExtracellular matrix components play a significant role in maintaining tissue integrity and pathological processes of the temporomandibular joint (TMJ). This study aimed to evaluate the influence of a soft diet on the mRNA expression of proteoglycans and glycosaminoglycans (GAGs) linked to proteoglycan core proteins in rat TMJ discs. MethodsThirty 4-week-old male Wistar rats were assigned to one of two groups: a control group fed a regular pellet diet and a soft diet group fed a powdered diet for 4 weeks. The mRNA expression levels of 12 proteoglycans in TMJ discs were evaluated using real-time polymerase chain reaction (PCR). In addition, histomorphometric and biochemical analyses were performed to evaluate the thickness and deoxyribonucleic acid (DNA), GAG, and water content of the TMJ discs. ResultsThe TMJ disc thickness in the anterior, intermediate, and posterior bands decreased significantly in the soft diet group. The GAG content decreased significantly in the soft-diet group, whereas no significant differences in DNA content or water content ratio were observed between the groups. Real-time PCR indicated that the expression levels of aggrecan, versican, biglycan, decorin, fibromodulin, lumican, and chondroadherin decreased in the soft diet group. The expression levels of all versican isoforms decreased in the soft diet group. ConclusionsThese results indicate that the biomechanical environment of the TMJ caused by a soft diet is closely related to the expression of proteoglycans in TMJ discs, which may eventually increase the fragility of the TMJ discs.

Proteomics analysis on aortic smooth muscle cells reveals new potential targets for the treatment of Marfan syndrome-associated thoracic aortic aneurysm

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Health Background Thoracic aortic aneurysm (TAA) is characterized by the abnormal enlargement of the initial segment of the aorta. Patients with genetic Marfan syndrome (MFS) develop TAA in young age, since mutations of fibrillin-1 contribute to thoracic aortic wall detrimental weakening. Presently, effective treatment options for this life-threatening condition are lacking, and existing pharmacological approaches are merely palliative, by postponing the need for surgical aortic replacement. To identify new treatments or MFS-related TAA (MFS-TAA), current research is concentrated on pinpointing specific biological targets and mechanisms underlying this aortic disorder. Purpose This study is aimed to unveil potential therapeutic targets that could be exploited for treating MFS-TAA by adopting an advanced proteomics technique. Material and Methods A label-free quantitative mass spectrometry technique was employed, utilizing a hybrid quadrupole-time of flight mass spectrometer coupled with a UPLC Mclass system and a nano-source, to analyze and compare the protein expression levels in vascular smooth muscle cells (VSMC) isolated from the aortas of MFS patients (MFS-VSMC) and healthy subjects (HC-VSMC). The proteomics findings were subsequently validated through qRT-PCR and Western blot assays. Results The proteomics investigation yielded 16 proteins displaying statistically significant differences. Among these, five proteins were notably more abundant in MFS-VSMC compared to HC-VSMC: nestin (NES, p<0.01), filamin B (FLNB, p<0.05), A-kinase anchor protein 12 (AKAP12, p<0.05), catenin delta-1 (CTNND1, p<0.05), and heparan sulfate proteoglycan core protein (HSPG2, p<0.05). The Gene Ontology (GO) analysis performed on cellular components of these 5 items using the clusterProfiler R package highlighted the enrichment of GO terms generally related to transmembrane proteins and anchoring junctions. Specifically, enriched GO terms included cell cortex (p<0.002), focal adhesion (p<0.002), cell-substrate junction (p<0.002), and intermediate filament binding (p<0.03). To verify these findings, two out of the five proteins with increased expression in MFS-VSMC (i.e., nestin and filamin B) were subjected to validation experiments. Results confirmed the elevated gene expression and protein levels of both nestin (p<0.05) and filamin B (p<0.05) in MFS-VSMC samples when compared with HC-VSMC. Conclusions Taken together, these results suggest that proteins with increased expression in MFS-VSMC may represent valuable biological target of MFS-TAA. This may encompass their potential both as diagnostic tools and/or mechanistic mediators of TAA in MFS subjects. In details, the pharmacological targeting of nestin and filamin B may represent a novel and exploitable therapeutic option for limiting/inhibiting TAA onset or progression in MFS pathological scenario.

Abstract 3001: Multiomics Analysis On Ascending Aorta Reveals New Potential Targets Of Marfan Syndrome-associated Thoracic Aortic Aneurysm

Thoracic aortic aneurysm (TAA) is defined as the pathological enlargement of the first aortic trait. Patients with genetic Marfan syndrome (MFS) develop TAA in young age, since mutations of fibrillin-1 contribute to aortic wall detrimental weakening. To date, effective treatment options for this life-threatening condition are lacking, and pharmacological approaches are merely palliative, postponing the surgical aortic replacement. To identify new treatments for MFS-related TAA (MFS-TAA), current research is concentrated on pinpointing specific biological targets and mechanisms underlying this aortic disorder. Aim of this study is to unveil new specific and potential targets of MFS-TAA. Spatial transcriptomics was performed on healthy subject and MFS patients’ thoracic aortas, whereas protein expression levels of vascular smooth muscle cells from MFS patients’ (MFS-VSMC) and healthy subjects (HC-VSMC) were analyzed by quantitative mass spectrometry. -Omics results were validated by qRT-PCR and Western blot assays. Spatial transcriptomic results revealed 2 different cell clusters in the aortic tunicae , best representing aortic VSMC. Structural proteins ( e.g. , NES , DES , CNN1 ), were the most transcribed genes in MFS-TAA in comparison with HC aorta. Notably, proteomics yielded five proteins, statistically more abundant in MFS-VSMC vs. HC-VSMC (p<0.05): nestin, filamin B, A-kinase anchor protein 12, catenin delta-1, and heparan sulfate proteoglycan core protein. The Gene Ontology (GO) analysis performed on cellular components of both transcripts and proteins more expressed in MFS aortic samples highlighted the enrichment of GO terms related to extracellular compartment, actin and cytoskeleton components, and cell adhesion. We validated the levels of nestin, which increased expression in MFS specimens has been observed by both -omics techniques. The results of validation experiments confirmed higher nestin gene expression and protein levels in MFS-VSMC samples (p<0.05) when compared with HC-VSMC. Results suggest a valuable biological target role for nestin, which pharmacological targeting may represent, in the future, a novel and exploitable therapeutic option for limiting/inhibiting TAA onset or progression in MFS scenario.

Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction.

Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFβ1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

A novel SPE-UPLC-MS/MS-based assay for the selective, simultaneous quantification of xylosyltransferase-I and -II activity

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan.The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results.Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.

Profiling of idiopathic macular hole vitreous proteome identifies the role of extracellular matrix remodelling, epithelial–mesenchymal transformation and unfolded protein-response pathways

Purpose: To analyze and describe the proteome of the vitreous humour in eyes with idiopathic macular holes. Methods: We performed mass spectrometry (MS)-based label-free quantitative analysis of the vitreous proteome of idiopathic macular hole (IMH) and control donor vitreous. Comparative quantification was performed using SCAFFOLD software which calculated fold changes of differential expression. Bioinformatics analysis was performed using DAVID and STRING software. Results: A total of 448 proteins were identified by LC-MS/MS in IMH and cadaveric eye vitreous samples, of which 199 proteins were common. IMH samples had 189 proteins that were unique and 60 proteins were present only in the control cadaveric vitreous. We found upregulation of several extracellular matrix (ECM) and cytoskeletal proteins, namely collagen alpha-1 (XVIII) chain, N-cadherin, EFEMP1/fibulin-3, basement membrane-specific heparan sulfate proteoglycan core protein, and target of Nesh-3. Several cytoskeleton proteins, namely tubulin, actin, and fibronectin levels, were significantly lower in IMH vitreous, probably reflecting increased ECM degradation. IMH vitreous also had a downregulation of unfolded protein response-mediated-mediated apoptosis proteins, possibly related to a state of increased cell survival and proliferation, along with a remodelling and aberrant production of ECM contents. Conclusion: The pathogenesis of macular holes may involve ECM remodelling, epithelial–mesenchymal transformation, downregulation of apoptosis, protein folding defects, and complement pathway. The vitreo-retinal milieu in macular holes contain molecules related to both ECM degradation and inhibition of the same, thereby maintaining a homeostasis.

Dexamethasone effects on the expression and content of glycosylated components of mouse brain tissue

Introduction. Glucocorticoids are actively used in the treatment of various diseases, however their long-term use leads to numerous negative side-effects, the molecular mechanisms of which remain poorly understood.Aim. Study of the short-term (1–10 days) effects of various doses of dexamethasone (Dex) (0,1–10 mg/kg) on the expression of the glucocorticoid receptor (GR, Nr3c1), core proteins of main proteoglycans and heparan sulfate metabolism-involved genes, as well as the content of carbohydrate macromolecules of glycosaminoglycans in the brain tissue of experimental animals.Materials and methods. In the study, C57Bl/6 mice were used. The expression of GR, proteoglycan core proteins and heparan sulfate metabolism-involved genes was determined by real-time polymerase chain reaction with reverse transcription. The content and localization of GR protein molecule were studied by Western blot and immunohistochemical analysis, and the glycosaminoglycan content was determined by dot-blot analysis and Alcian Blue staining.Results. It was shown that a single Dex administration leads to fast (1–3 days) short-term activation of GR expression (+1.5 times, p <0.05), proteoglycan’s genes (syndecan-3, Sdc3; perlecan, Hspg2; phosphacan, Ptprz1; neurocan, Ncan; +2–3-fold; p <0.05) and heparan sulfate-metabolism-involved genes (Ndst1, Glce, Hs2st1, Hs6st1, Sulf1 / 2; +1.5–2-fold; p <0.05) in the mouse brain, with a return to control values by 7–10 days after Dex administration. At the same time, the effect of Dex on carbohydrate macromolecules of glycosaminoglycans was more delayed and stable, increasing the content of low-sulfated glycosaminoglycans in the brain tissue in a dose-dependent manner starting from day 1 after Dex administration. Highly-sulfated glycosaminoglycans showed more delayed response to Dex administration, and an increase in their content was observed only at higher doses (2.5 and 10 mg/kg) and only on 7–10 days after its administration, apparently, mainly due to an increase in heparan sulfate content.Conclusion. In general, the effect of a single injection of Dex on the transcriptional activity of GR, proteoglycan core proteins and heparan sulfate metabolism-involved genes were short-termed, and the genes expression quickly returned to the normal levels. However, even a single use of Dex significantly increased the content of total as well as highly sulfated glycosaminoglycans in the mouse brain tissue, which can lead to the changes in the composition and structure of the brain tissue, as well as its functional characteristics.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Effect of boiling on texture of scallop adductor muscles and its mechanism based on label-free quantitative proteomic technique

The mechanism behind textural changes in scallop adductor muscle during boiling was investigated through proteomic analysis, determination of water holding capacity (WHC) and oxidative indices, as well as observation with scanning electron microscopy and multiphoton nonlinear optical microscopy. The hardness and shear force showed the trend of first rising and then falling in 45 min-boiling time. The results suggested that short-time boiling caused the oxidation, denaturation and aggregation of proteins, resulting in the transverse contraction of myofibers and lateral cross-linked aggregation of muscle fibers and a rise in WHC, which led to the increase in hardness and shear force. While long-time boiling caused the progressive degradation of structural proteins such as fibrillin-1, collagen alpha-2(I) chain, myosin heavy chain, basement membrane-specific heparan sulfate proteoglycan core protein, and paramyosin, resulting in a loose myofibril network and the decrease in WHC, which led to the decrease in hardness and shear force.

Separation and Identification of Native Proteoglycans by Composite Agarose-Polyacrylamide Gel Electrophoresis and Immunoblotting.

Composite agarose-polyacrylamide gel electrophoresis (CAPAGE) in gels of 1.2% w/v polyacrylamide and 0.6% w/v agarose can be used to examine the heterogeneity of full-length native proteoglycan populations and their fragments in crude tissue extracts, and when used in conjunction with immunoblotting and specific antibodies to proteoglycan core protein and glycosaminoglycan, side chain epitopes can provide significant information on the level of proteoglycan polydispersity/heterogeneity and a number of proteoglycan populations present in tissue samples. This can be a technically difficult technique, but it reveals significant information on proteoglycans from small tissue samples not possible by any other separation methodology. Native full-length and proteoglycan fragments are examined in this technique something which cannot be done in the popular SDS-PAGE format unless the glycosaminoglycan side chains are first removed. Furthermore, since proteoglycans do not require renaturation from SDS-protein complexes, the proteoglycan populations separated by native electrophoresis are highly reactive with antibodies in immunoblotting procedures. Despite the massive sizes of proteoglycans, transfer conditions have been determined which provide close to quantitative transfer to nitrocellulose membranes without exceeding the binding capacity of such membranes, avoiding bleed-through of the transferred proteoglycans. Development of biotinylated hyaluronan and its application in an affinity blotting procedure has also yielded significant information on aggregatable proteoglycan populations separated by CAPAGE from a number of cartilages and vascular tissues in health and disease. While the CAPAGE system can be a technically demanding technique to master particularly in gel preparation, all other steps are straightforward, and the method yields invaluable information on proteoglycan populations extracted from connective tissues in health and disease that cannot be ascertained by any other technique. Further improvements in the detection of proteoglycan features with the development of novel bio-affinity probes or new antibody preparations are expected to further improve the utility of CAPAGE separation methodology.

Chondroitin sulfate is not digested at all in the mouse small intestine but may suppress interleukin 6 expression induced by tumor necrosis factor-α

Salmon nasal cartilage proteoglycan (PG) was orally administered to mice. The PG digest was recovered from the small intestine, and its sugar chain size and unsaturated disaccharide content were examined. The elution position of the PG digest following Sepharose CL-4B chromatography was consistent with that of actinase-digested PG prior to administration. The PG digest was incubated with chondroitinase ABC, which resulted in the elution pattern of the unsaturated disaccharides being identical to that of the degraded product of actinase-digested PG. The core protein of PG was digested in the mouse small intestine, but chondroitin sulfate, which is the sugar chain of PG, was not degraded at all. Then, the effects of chondroitin 4- and 6-sulfates on human colon cancer cells were examined. These chondroitin sulfates were found to suppress the expression of interleukin-6 induced by TNF-α. Overall, the chondroitin sulfate chain may act on the intestinal epithelium and suppress inflammation of the intestinal tract.

Arthritis and Duchenne muscular dystrophy: the role of chondroitin sulfate and its associated proteoglycans in disease pathology and as a diagnostic marker.

Chondroitin sulfate (CS) is a ubiquitous glycosaminoglycan covalently attached to the core proteins of cell surface, extracellular, and intracellular proteoglycans. The multistep and highly regulated biosynthesis of chondroitin sulfate and its degradation products give rise to a diverse species of molecules with functional regulatory properties in biological systems. This review will elucidate and expand on the most recent advances in understanding the role of chondroitin sulfate and its associate proteoglycans, in arthritis and Duchenne muscular dystrophy (DMD), two different and discrete pathologies. Highlighting not only the biodiverse nature of this family of molecules but also the utilization of CS proteoglycans, CS, and its catabolic fragments as biomarkers and potential therapeutic targets for disease pathologies.

Presence of chondroitin sulphate and requirement for heparan sulphate biosynthesis in the developing zebrafish inner ear.

Epithelial morphogenesis to form the semicircular canal ducts of the zebrafish inner ear depends on the production of the large glycosaminoglycan hyaluronan, which is thought to contribute to the driving force that pushes projections of epithelium into the lumen of the otic vesicle. Proteoglycans are also implicated in otic morphogenesis: several of the genes coding for proteoglycan core proteins, together with enzymes that synthesise and modify their polysaccharide chains, are expressed in the developing zebrafish inner ear. In this study, we demonstrate the highly specific localisation of chondroitin sulphate to the sites of epithelial projection outgrowth in the ear, present before any morphological deformation of the epithelium. Staining for chondroitin sulphate is also present in the otolithic membrane, whereas the otoliths are strongly positive for keratan sulphate. We show that heparan sulphate biosynthesis is critical for normal epithelial projection outgrowth, otolith growth and tethering. In the ext2 mutant ear, which has reduced heparan sulphate levels, but continues to produce hyaluronan, epithelial projections are rudimentary, and do not grow sufficiently to meet and fuse to form the pillars of tissue that normally span the otic lumen. Staining for chondroitin sulphate and expression of versican b, a chondroitin sulphate proteoglycan core protein gene, persist abnormally at high levels in the unfused projections of the ext2 mutant ear. We propose a model for wild-type epithelial projection outgrowth in which hyaluronan and proteoglycans are linked to form a hydrated gel that fills the projection core, with both classes of molecule playing essential roles in zebrafish semicircular canal morphogenesis.

Proteolysis: a key post-translational modification regulating proteoglycans.

Proteoglycans are composite molecules comprising a protein backbone, i.e., the core protein, with covalently attached glycosaminoglycan chains of distinct chemical types. Most proteoglycans are secreted or attached to the cell membrane. Their specialized structures, binding properties, and biophysical attributes underlie diverse biological roles, which include modulation of tissue mechanics, cell adhesion, and the sequestration and regulated release of morphogens, growth factors, and cytokines. As an irreversible post-translational modification, proteolysis has a profound impact on proteoglycan function, abundance, and localization. Proteolysis is required for molecular maturation of some proteoglycans, clearance of extracellular matrix proteoglycans during tissue remodeling, generation of bioactive fragments from proteoglycans, and ectodomain shedding of cell-surface proteoglycans. Genetic evidence shows that proteoglycan core protein proteolysis is essential for diverse morphogenetic events during embryonic development. In contrast, dysregulated proteoglycan proteolysis contributes to osteoarthritis, cardiovascular disorders, cancer, and inflammation. Proteolytic fragments of perlecan, versican, aggrecan, brevican, collagen XVIII, and other proteoglycans are associated with independent biological activities as so-called matrikines. Yet, proteoglycan proteolysis has been investigated to only a limited extent to date. Here, we review the actions of proteases on proteoglycans and illustrate their functional impact with several examples. We discuss the applications and limitations of strategies used to define cleavage sites in proteoglycans and explain how proteoglycanome-wide proteolytic mapping, which is desirable to fully understand the impact of proteolysis on proteoglycans, can be facilitated by integrating classical proteoglycan isolation methods with mass spectrometry-based proteomics.

Chondroitinase ABC Administration Facilitates Serotonergic Innervation of Motoneurons in Rats With Complete Spinal Cord Transection.

Chondroitinase ABC (ChABC) is an enzyme that degrades glycosaminoglycan side-chains of chondroitin sulfate (CS-GAG) from the chondroitin sulfate proteoglycan (CSPG) core protein. Previous studies demonstrated that the administration of ChABC after spinal cord injury promotes nerve regeneration by removing CS-GAGs from the lesion site and promotes the plasticity of spinal neurons by removing CS-GAGs from the perineuronal nets (PNNs). These effects of ChABC might enhance the regeneration and sprouting of descending axons, leading to the recovery of motor function. Anatomical evidence, indicating that the regenerated axons innervate spinal motoneurons caudal to the lesion site, however, has been lacking. In the present study, we investigated whether descending axons pass through the lesion site and innervate the lumbar motoneurons after ChABC administration in rats with complete spinal cord transection (CST) at the thoracic level. At 3 weeks after CST, 5-hydroxytryptamine (5-HT) fibers were observed to enter the lesion in ChABC-treated rats, but not saline-treated rats. In addition, 92% of motoneurons in the ventral horn of the fifth lumbar segment (L5) in saline-treated rats, and 38% of those in ChABC-treated rats were surrounded by chondroitin sulfate-A (CS-A) positive structures. At 8 weeks after CST, many 5-HT fibers were observed in the ventral horn of the L5, where they terminated in the motoneurons in ChABC-treated rats, but not in saline-treated rats. In total, 54% of motoneurons in the L5 ventral horn in saline-treated rats and 39% of those in ChABC-treated rats were surrounded by CS-A-positive structures. ChABC-treated rats had a Basso, Beattie, and Bresnahan (BBB) motor score of 3.8 at 2 weeks, 7.1 at 3 weeks, and 10.3 at 8 weeks after CST. These observations suggest that ChABC administration to the lesion site immediately after CST may promote the regeneration of descending 5-HT axons through the lesion site and their termination on motoneurons at the level of caudal to the lesion site. ChABC administration might facilitate reinnervation by degrading CS-GAGs around motoneurons. Motor function of the lower limbs was significantly improved in ChABC-treated rats even before the 5-HT axons terminated on the motoneurons, suggesting that other mechanisms may also contribute to the motor function recovery.

Excavating proteoglycan structure-function relationships: modern approaches to capture the interactions of ancient biomolecules.

Proteoglycans are now well regarded as key facilitators of cell biology. Although a majority of their interactions and functions are attributed to the decorating glycosaminoglycan chains, there is a growing appreciation for the roles of the proteoglycan core protein and for considering proteoglycans as replete protein-glycan conjugates. This appreciation, seeded by early work in proteoglycan biology, is now being advanced and exalted by modern approaches in chemical glycobiology. In this review, we discuss up-and-coming methods to unearth the fine-scale architecture of proteoglycans that modulate their functions and interactions. Crucial to these efforts is the production of chemically defined materials, including semisynthetic proteoglycans and the in situ capture of interacting proteins. Together, the integration of chemical biology approaches promises to expedite the dissection of the structural heterogeneity of proteoglycans and deliver refined insight into their functions.

A Simple Quality Evaluation Method for Proteoglycan after Addition to Beverages.

Over the past 10 years, many products utilizing the functionality of salmon cartilage proteoglycan have come on the market, and consumer awareness of proteoglycan has increased. During this period, the biggest issue has been how to evaluate the amount and quality of proteoglycan in the cartilage extract blended in the products. In this study, we propose an immunological method that can easily evaluate the amount and quality of proteoglycan in the proteoglycan-containing compositions. By the present method, it is possible to evaluate not only the retention of the functional domains of the core protein of proteoglycan, but also that of chondroitin sulfate chains linked to the core protein. Furthermore, the binding activity of proteoglycan to hyaluronan can be evaluated if hyaluronan is used as a probe instead of an antibody. This method is expected to be useful for proteoglycan quality evaluation during the manufacturing process and product storage.

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.