Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans

Matrix metalloproteinases (MMPs) secreted from the leukemic macrophage cell-line THP-1 have been investigated. Under serum-free conditions, this cell-line synthesizes and secretes proMMP-9, which was detected in the culture medium as a monomer of 92 kDa, and in dimeric forms, including a homodimer of approximately 225 kDa. In addition, a new heterodimer complex is described, in which proMMP-9 is covalently linked to the core protein of chondroitin sulphate proteoglycan (CSPG) through one or more disulphide bridges. After SDS-PAGE electrophoresis, at least two forms of this complex were detected, a large form in the stacking gel and a smaller form with an estimated size of 300 kDa. When the CS chains were removed by chondroitin ABC lyase treatment, heterodimers of proMMP-9/CSPG core protein of approximately 145, 127 and 109 kDa were found, based on zymography and Western blots. Since as much as 10-15 % of the total proMMP-9 secreted from THP-1 cells was covalently linked to CSPG, this association may have important implications for transport, targetting and regulation of the enzyme activity.     

Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre-spina bifida splotch mouse embryos.

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.     

CSPGs在大鼠脊髓损伤后的表达及其与GFAP的关系

目的:观察脊髓损伤后CSPGs的表达及其与GFAP的关系。方法:成年雄性SD大鼠25只,随机分为对照组和损伤组,损伤组分脊髓挤压损伤后0h、72h、1w、4w组,运用免疫荧光双重染色方法观察CSPGs与GFAP的表达。结果:挤压伤后损伤部位的CSPGs和GFAP的表达均增高,但二者的变化趋势并不一样。其中CSPGs从损伤后表达开始增高,此后一直增加,并在1w至4w时逐渐稳定,主要分布逐渐集中于损伤部位;星形胶质细胞的免疫反应也逐渐增加,其分布逐渐集中于损伤区域的边缘,逐渐形成胶质瘢痕界膜。损伤1w至4w,损伤区域内几乎没有了星形胶质细胞表达,但仍留有大量的CSPGs。结论:早期抑制星形胶质细胞分泌CSPGs,可以防止在损伤部位沉积大量的CSPGs,从而减小其对再生纤维的抑制作用。     

Characterisation of chondroitin/dermatan sulphate proteoglycans synthesised by rat mammary myoepithelial and fibroblastic cell lines.

Chondroitin sulphate proteoglycans were isolated from the culture medium of rat mammary gland fibroblast (Rama 27) and myoepithelial (Rama 401) cell lines which had been labelled with [35S]sulphate. Chromatography on Sepharose CL-4B indicated that the Rama 401 proteoglycan was larger than the Rama 27 proteoglycan (Kav values 0.47 and 0.56, respectively). Treatment of the proteoglycans with alkaline NaBH4 yielded chondroitin sulphate chains with average M(r) values of 37,000 (Rama 401) and 21,000 (Rama 27). Structural analysis of the glycosaminoglycan chains indicated that both were co-polymers of chondroitin and dermatan sulphate although there were differences in the amounts and distribution of the disaccharide repeating units. The M(r) values of the core proteins, determined by immunoblotting, were about 43,000 and 46,000 (Rama 27) and 44,500 (Rama 401). Using an antibody to chondroitin sulphate proteoglycan in immunofluorescence experiments, the proteoglycan was demonstrated on the surface of both cell lines. Rama 27 cells additionally possessed an extensive fibrous extracellular matrix which also stained with the antibody. Staining of sections of lactating mammary gland suggested that the proteoglycan was present in the basement membrane as well as the stromal connective tissue. The presence of chondroitin sulphate proteoglycan in the basement membrane was confirmed by ultrastructural immunolocalisation.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Summary Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ''matrix'', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).     

Immunohistochemical evaluation of versican,in relation to chondroitin sulphate,in canine mammary tumours

The expression of increased amounts of versican, a chondroitin sulphate proteoglycan, in neoplastic tissues may play a role in promoting tumour cell proliferation and migration. This study investigated the immunolocalization of versican in normal and neoplastic canine mammary tissues, using antibodies 12C5 and 2B1, against different epitopes of the protein core of versican. Antibody CS56, recognising chondroitin sulphate (CS), was used to investigate the relation between versican and CS, which accumulates in canine mammary tumours. We found enhanced versican expression in both benign and malignant tumours, appearing in three main patterns: in periductal tissues, probably in association with basement membranes of ducts; in peripheral invasive areas of malignant tumours; and in spindle cell proliferations and myxoid areas of complex and mixed tumours. The 12C5 and 2B1 immunoreactivities co-localised in all types of tumours, and could be improved by chondroitinase digestion. The only exception was the abundant extracellular matrix (ECM) of spindle cell proliferations, particularly in myxoid areas of complex and mixed tumours, which displayed intense and diffuse 12C5 immunoreactivity and patchy or absent 2B1 and CS56 immunoreactivities; versican immunoreactivity could not be enhanced by chondroitinase digestion. The results indicate that versican is one of the extracellular matrix components characteristic of canine mammary tumours. It appears likely that in complex and mixed tumours versican exists in at least two forms, one of them lacking the CS attachment domain and the 2B1 epitope. Furthermore, the enhanced versican expression in the invasive areas of malignant tumours indicates the involvement of this proteoglycan in tumour cell invasion.     

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.     

Phosphorylation of chondroitin sulfate in proteoglycans from the swarm rat chondrosarcoma

Proteoglycans isolated from the Swarm rat chondrosarcoma were shown to contain 35 mol of phosphate/mol of proteoglycan. While 20% of this phosphate was released by digestion with dilute alkali in the presence of sodium borohydride and is presumably of the phosphoserine/phosphothreonine type, 78% of the phosphate copurified with the peptide-free chondroitin sulfate chains. When chondroitin sulfate chains purified by ethanol precipitation or Sephacryl S200 column chromatography were digested with chondroitinase AC and the digests chromatographed on Bio-Gel P-4, the phosphate co-migrated with a carbohydrate fragment that contained 2 glucuronic acid (one as delta 4,5-unsaturated sugar), 1-galactosamine, 2-galactose, and 1-phosphate residue/xylitol. A second fragment of similar composition but lacking phosphate was also recovered in a ratio of about 3 to 1 relative to the phosphorylated fragment. The phosphate in the chondroitin sulfate linkage region fragment had the alkaline phosphatase sensitivity as well as 31P NMR spectra of a monophosphate esterified to a secondary sugar alcohol. The phosphate was localized on the C-2 of the chain initiating xylose since these residues as xylitol showed a delayed release during acid hydrolysis and the xylitol was recovered intact after periodate oxidation. In the chondrosarcoma, 2-phosphoxylose appears to be a normal synthetic product since [32P]phosphate was readily incorporated into the proteoglycan and the incorporated isotope had similar biochemical properties as the unlabeled phosphate.     

Immunohistochemical localization of large chondroitin sulphate proteoglycan in porcine gingival epithelia.

In this study, we report the immunohistochemical localization of versican in healthy porcine gingival epithelia. The monoclonal antibody (mAb), 5D5, specifically recognizes core proteins of large chondroitin sulphate proteoglycans such as versican, neurocan and brevican, but not the core protein of aggrecan. Because neurocan and brevican appear to be specific to nervous tissue, the large chondroitin sulphate proteoglycans examined in this study is most likely versican. In the keratinized layer of the attached gingival epithelium, the basal and spinous cell surfaces showed intense staining for mAb 5D5. In the parakeratinized layer of the sulcus epithelium, the localization was restricted to the basal and lower spinous layers. In the junctional epithelium, intense staining was observed in one or two cell layers near the enamel surface. Immunoelectron microscopy revealed high-density depositions of 5D5 immunoreactivity on epithelial cell surfaces. At the enamel surface, 5D5 immunoreactivity was localized to the dental cuticle of the junctional epithelium but was not present in the internal basal lamina. These results suggest that versican, a large chondroitin sulphate proteoglycan, is involved in epithelial differentiation and downgrowth.     

The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans.

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.