Keratanase Digestion Research Articles

Distribution and processing of a disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae

To determine the expression and distribution of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), its substrates aggrecan and versican, and their binding partner hyaluronan in laminae of healthy horses. Laminae from the forelimb hooves of 8 healthy horses. Real-time quantitative PCR assay was used for gene expression analysis. Hyaluronidase, chondroitinase, and keratanase digestion of lamina extracts combined with SDS-PAGE and western blotting were used for protein and proteoglycan analysis. Immunofluorescent and immunohistochemical staining of tissue sections were used for protein and hyaluronan localization. Genes encoding ADAMTS-4, aggrecan, versican, and hyaluronan synthase II were expressed in laminae. The ADAMTS-4 was predominantly evident as a 51-kDa protein bearing a catalytic site neoepitope indicative of active enzyme and in situ activity, which was confirmed by the presence of aggrecan and versican fragments bearing ADAMTS-4 cleavage neoepitopes in laminar protein extracts. Aggrecan, versican, and hyaluronan were localized to basal epithelial cells within the secondary epidermal laminae. The ADAMTS-4 localized to these cells but was also present in some cells in the dermal laminae. Within digital laminae, versican exclusively and aggrecan primarily localized within basal epithelial cells and both were constitutively cleaved by ADAMTS-4, which therefore contributed to their turnover. On the basis of known properties of these proteoglycans, it is possible that they can protect the basal epithelial cells of horses from biomechanical and concussive stress.

Immunochemical Localization of Keratan Sulfate Proteoglycans in Cornea, Sclera, and Limbus Using a Keratanase-Generated Neoepitope Monoclonal Antibody

To evaluate the use of neoepitope monoclonal antibody BKS-1, which recognizes keratanase-generated keratan sulfate (KS) stubs on keratan sulfate proteoglycans in human cornea, limbus, and sclera. BKS-1 specifically recognizes a keratanase-generated neoepitope [N-acetyl-glucosamine-6-sulfate (GlcNAc-6-S)] at the nonreducing terminal of corneal and skeletal KS glycosaminoglycan chains. It was produced by using keratanase-digested KS peptides from bovine cartilage aggrecan as the immunizing antigen. BKS-1 was used in conjunction with 5D4 to analyze the KS distribution in human cornea, limbus, and sclera using Western blotting, immunohistochemistry, and electron microscopy. 5D4 Western blot analysis displayed a diffuse staining pattern, and it was difficult to distinguish differences among cornea, sclera, and limbus. However, BKS-1 showed differences in KS levels, with higher levels in the cornea and lower levels in the limbus and sclera. Ultrastructural studies showed that the monoclonal antibody (mAb) BKS-1 neoepitope was not observed in the epithelium or basement membrane; however, 5D4 was present in these layers. Large quantities of both antibodies were present in Bowman's layer, stroma, and Descemet's membrane, but the quantity of 5D4 was significantly higher (P < 0.001) than the quantity of BKS-1 in all these layers of the cornea. mAb 5D4 recognizes oversulfated structures within KS chains, whereas BKS-1 recognizes a single neoepitope on KS after keratanase digestion of monosulfated KS disaccharides. With the use of BKS-1, the authors identified a more clearly defined pattern for KS distribution in the cornea than was seen with 5D4. The presence of a large quantity of BKS-1 immunostaining in the cornea suggests that KS-substituted proteoglycans are more prevalent in the cornea than in the limbus or sclera.

Distinct Effects of N-Acetylgalactosamine-4-sulfatase and Galactose-6-sulfatase Expression on Chondroitin Sulfates

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

A novel keratanase‐generated keratan sulfate antibody and its application to structural analysis of skeletal and corneal keratan sulfate

Introduction The objective of this study was to make monoclonal antibodies specific for keratanase‐generated neoepitopes in keratan sulfate (KS) and to use them along with existing KS monoclonal antibodies (e.g. 5D4, IB4) to investigate KS sulfation pattern motifs in connective tissue proteoglycans during development, ageing and disease.Methods Bovine nasal cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of GAG‐peptide fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS peptides from CS attachment domain fragments. Fractions were analysed by Western blotting for positive immunoreactivity for KS, then pooled and keratanase digested to generate ‘KS stub’ antigens. Immunization and fusions were carried out as previously described (Caterson et al. 1983; Hughes et al. 1992). Screenings involved the use of a range of antigens; including keratanase vs. keratanase II‐digested bovine cartilage aggrecan and bovine corneal KS‐PGs. A new monoclonal antibody, BKS‐I, was identified that specifically recognized a keratanase‐generated neoepitope on both skeletal and corneal KS. This novel monoclonal antibody was used along with existing KS monoclonal antibodies 5D4 and 1B4 to investigate KS structure.Results and discussion Bovine trypsin‐generated aggrecan KS‐peptides were chondroitinase ABC treated and either keratanase or keratanase II treated. The digests were run on SDS‐PAGE and immunolocated with monoclonal antibody 5D4 (that recognizes linear disulfated N‐acetyl lactosamine disaccharide‐containing segments in KS) and the new ‘KS‐stub’ monoclonal antibody BKS‐I. Our results indicated that there was reduced monoclonal antibody 5D4 immunostaining after keratanase pretreatment. However, keratanase II digestion completely removed all 5D4 structural epitopes. In contrast, BKS‐I showed no immunostaining on the untreated KS‐peptides but strong staining on keratanase treated samples and no staining after keratanase II digestion. Similar patterns of immunoreactivity were observed with Western blot analysis of untreated, keratanase treated and keratanase II treated corneal KS‐PGs.Conclusion These data indicate that monoclonal antibody BKS‐I recognizes a nonreducing terminal neoepitope‐containing sulfated N‐acetylglucosamine adjacent to a nonsulfated lactosamine disaccharide. We also conclude that skeletal KS must have a structure with four possible variations opposed to the generic structures, proposed as being made of disulfated disaccharides at the nonreducing end, followed by a series of monosulfated disaccharides at the middle and nonsulfated disaccharides nearer the linkage region. 5D4 staining, observed after keratanase digestion, indicates that there must be a minimum structure of a pentasulfated hexasaccharide remaining on the KS chain ‘stubs’ near the linkage region of skeletal and corneal KS. The BKS‐I monoclonal antibody can be used to demonstrate differential substitution of KS GAG chains in the CS attachment region of cartilage aggrecan with ageing. It has also proven useful for immunohistochemical analyses identifying the sites of KS–PG association with collagen lamellae of cornea.

Pathogenesis of abdominal aortic aneurysms: Possible role of differential production of proteoglycans by smooth muscle cells

Purpose: In vivo and in vitro observations strongly suggest that marked differences exist in the phenotype, growth, and matrix-producing capabilities of distinct smooth muscle cell subpopulations. An earlier study from our laboratory showed differences in matrix metalloproteinase expression patterns in cultures of medial smooth muscle cells from tissue affected by abdominal aortic aneurysm (AAA) or atherosclerotic occlusive disease and from normal arterial tissue. In this study we were interested in ascertaining whether smooth muscle cells from the same sample groups also synthesized different proteoglycan profiles that correlated with vascular disease. Methods: Proteoglycans from smooth muscle cell monolayer cultures from tissue affected by AAA or atherosclerotic occlusive disease and from normal arterial tissue were examined by means of immunoblotting and affinity-blotting composite agarose polyacrylamide gel electrophoresis (CAPAGE) and sodium dodecyl sulphate PAGE. Enzyme-linked immunosorbent assay (ELISA) was used to quantitate perlecan levels in smooth muscle cell monolayer media samples. Results: Versican, perlecan, and biglycan levels were significantly elevated in AAA smooth muscle cell cultures. Two populations of smooth muscle cell versican were identified by means of CAPAGE-immunoblotting and by means of a novel affinity-blotting technique with biotinylated hyaluronan. A small keratan sulfate–substituted proteoglycan was present in similar levels in all smooth muscle cell cultures. This proteoglycan had a free core protein of about 55 kd after keratanase digestion and had a relatively high charge-to-mass ratio, as was evident from its electrophoretic mobility in CAPAGE; this proteoglycan was tentatively identified as keratocan. Immunoblotting with monoclonal antibodies 3-G-10 (anti- D heparan sulfate, heparan sulfate stubs generated by heparitinase treatment) and 10-E-4 (anti–native heparan sulfate chains) helped identify several smooth muscle cell heparan sulfate–substituted proteoglycans. Elevated levels of intact and processed perlecan core protein were identified in AAA cultures by means of immunoblotting with a monoclonal antibody to perlecan core protein (A76). ELISA measurements confirmed that perlecan levels were significantly higher in AAA smooth muscle cell cultures compared with the normal arterial tissue and tissue affected by atherosclerotic occlusive disease. Conclusions: Because heparan sulfate proteoglycans can bind growth factors, their elevated synthesis by AAA smooth muscle cells in combination with an increased expression of matrix metalloproteinases may at least partly explain the differential proliferative capacity of the AAA smooth muscle cells examined and may govern the pattern of abnormal cellular proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease. (J Vasc Surg 1998;28:676-86.)

A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland

The monoclonal antibody 5-D-4 recognizes heavily sulphated forms of keratan sulphate epitope. It reacted strongly with the cell surfaces of most thyroid papillary carcinomas from all the individuals examined, independently of the blood group of the patients. Cells of follicular variants of papillary carcinomas were also labelled by 5-D-4. In contrast, no labelling with this antibody was observed in other types of thyroid neoplasms, or in normal tissues. The reactivity of 5-D-4 with papillary carcinomas was markedly reduced or abolished by prior digestion with endo-beta-galactosidase, keratanase II, or N-glycosidase F. Although keratanase digestion had no effect on 5-D-4 labelling, it revealed the binding sites of Griffonia simplicifolia agglutinin II (GSA-II), which recognizes terminal N-acetylglucosamine in a limited number of carcinoma cells from some individuals. Blood group ABH antigens, which are simultaneously expressed together with keratan sulphate epitope in cancer cells, were eliminated by digestion with endo-beta-galactosidase and N-glycosidase F, but were resistant to keratanase and keratanase II treatment. These results indicate that keratan sulphate oligosaccharides are cancer-associated and are probably oncofoetal antigens, as are the blood group antigens in human thyroid glands. The results suggests that poly-N-acetyllactosamine, which is ubiquitously and consistently produced in papillary carcinomas, is modified in two different ways: sulphation on the 6-position of at least some units of either galactose or N-acetylglucosamine or both, and decoration of non-reducing termini with the blood group antigens. Along with the endo-beta-galactosidase-GSA-II labelling procedure, labelling with 5-D-4 may be a useful diagnostic means for distinguishing papillary carcinoma from other types of thyroid neoplasms.

6B4 proteoglycan/phosphacan is a repulsive substratum but promotes morphological differentiation of cortical neurons.

6B4 proteoglycan/phosphacan is one of the major phosphate-buffered saline-soluble chondroitin sulfate proteoglycans of the brain. Recently, this molecule has been demonstrated to be an extracellular variant of the proteoglycan-type protein tyrosine phosphatase, PTPzeta (RPTPbeta). The influence of the 6B4 proteoglycan, adsorbed onto the substratum, on cell adhesion and neurite outgrowth was studied using dissociated neurons from the cerebral cortex and thalamus. 6B4 proteoglycan adsorbed onto plastic tissue culture dishes did not support neuronal cell adhesion, but rather exerted repulsive effects on cortical and thalamic neurons. When neurons were densely seeded on patterned substrata consisting of a grid-like structure of alternating poly-L-lysine and 6B4 proteoglycan-coated poly-L-lysine domains, they were concentrated on the poly-L-lysine domains. However, 6B4 proteoglycan did not retard the differentiation of neurons but rather promoted neurite outgrowth and development of the dendrites of cortical neurons, when neurons were sparsely seeded on poly-L-lysine-conditioned coverslips continuously coated with 6B4 proteoglycan. This effect of 6B4 proteoglycan on the neurite extension of cortical neurons was apparent even on coverslips co-coated with fibronectin or tenascin. By contrast, the neurite extension of thalamic neurons was not modified by 6B4 proteoglycan. Chondroitinase ABC or keratanase digestion of 6B4 proteoglycan did not affect its neurite outgrowth promoting activity, but a polyclonal antibody against 6B4 proteoglycan completely suppressed this activity, suggesting that a protein moiety is responsible for the activity. 6B4 proteoglycan transiently promoted tyrosine phosphorylation of an 85x10(3) Mr protein in the cortical neurons, which correlated with the induction of neurite outgrowth. These results suggest that 6B4 proteoglycan/phosphacan modulates morphogenesis and differentiation of neurons dependent on its spatiotemporal distribution and the cell types in the brain.

Phenotypic modulation of newly synthesized proteoglycans in human cartilage and chondrocytes

The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic articular cartilage age-matched to the osteoarthritic cartilage specimens was studied in explant cultures and in chondrocytes generated by explant outgrowth from the cartilages. Twenty-four hours after explanation, both nonarthritic articular cartilage and osteoarthritic cartilage synthesized principally one large proteoglycan core protein that migrated on 3-5% acrylamide gels with an apparent molecular mass (M(r)) of approximately 520 kDa after enzymatic digestion with chondroitinase ABC and keratanase. The proteoglycan was found in both the explant itself and in the medium compartment of the culture as well. This proteoglycan contained chondroitin-6-sulfate, keratan sulfate and the hyaluronan binding region as evidenced by immunoblotting with murine anti-proteoglycan monoclonal antibodies indicating that the proteoglycan was aggrecan. To a much lesser extent two additional proteoglycan core proteins were also found in the explant but were not seen in the culture medium compartment. These proteoglycans possessed apparent M(r)'s of approximately 480 kDa and approximately 390 kDa on 3-5% acrylamide gels after chondroitinase ABC and keratanase digestion. The medium compartment contained principally the approximately 520 kDa proteoglycan core protein. In osteoarthritic cartilage explants, the pattern of newly synthesized proteoglycans recovered from the tissue as assessed on 3-16% polyacrylamide gradient gels remained relatively the same from day 1 after explantation up to 36 days of culture. By contrast, the proteoglycans recovered from the culture medium contained chondroitin sulfate and keratan sulfate after 1, 7, and 21 days in culture but by 36 days appeared to contain only chondroitin sulfate. Chondrocytes generated from osteoarthritic cartilage and age-matched nonarthritic articular cartilage synthesized different patterns of large (greater than 200 kDa) proteoglycan. Whereas chondrocytes derived from osteoarthritic cartilage continued to synthesize principally the approximately 520 kDa proteoglycan core protein, the chondrocytes derived from nonarthritic cartilage synthesized in addition to this proteoglycan, abundant amounts of the other two proteoglycan core proteins as well.

Keratanase digestion of keratan sulphates: characterization of large oligosaccharides from the N-acetyllactosamine repeat sequence and from the non-reducing terminal chain caps

Keratan sulfate (KS) chains prepared from both bovine tracheal rings and bovine femoral head cartilage were digested with the enzyme keratanase from Pseudomonas species; large repeat-sequence and non-reducing terminal oligosaccharides were fractionated and purified using high-performance ion-exchange chromatography. The main, β-linked pentasulfated hexasaccharide repeat segment, [ R6], GlcNAc(6S)1- 1–3Gal(6S) 1–4GlcNAc(6S) 1–3Gal(6S) 1–4GlcNAc(6S) 1–3Gal-ol and the asialo β-linked capping pentasulfated heptasaccharide, [ C7], Gal 1–4GlcNAc(6S) 1–3Ga1(6S) 1–4GlcNAc(6S) 1–3Gal(6S) 1–4GlcNAc(6S) 1–3Gal-ol have been completely characterized by high-field NMR spectroscopy using one- and two-dimensional methods. Partial 1H assignments are summarized for three homologous series of higher oligosaccharides: GlcNAc(6S)[ 1–3 Gal(6S) 1–4GlcNAc(6S)] 3–5 1–3Gal-ol [ R8,R10,R12] Gal 1–4GlcNAc(6S)[ 1–3Gal(6S) 1–4GlcNAc(6S)] 3–5 1–3Gal-ol [ C9,C11,C13] NeuAcα 2–3 Gal 1–4 G1cNAc(6 S)[ 1–3 Gal(6 S) 1–4 GlcNAc(6 S)] 2–4 1–3Gal-ol [ C8,C10,C12] obtained from keratan sulfate by keratanase cleavage. The first shows that the unsulfated galactose residues within the repeat sequence region of KS may be separated by fully sulfated segments which have a wide distribution of lengths. The others, viz. those with sialylated caps, and the related galactose capped asialo-segments (derived from a KS digestion in which the keratanase also exhibited sialidase activity) represent an homologous series of epitopes in which the first internal unsulfated galactose is located at a position which may be up to five or more fully sulfated N-acetyllactosamine disaccharide repeat units along from the non-reducing terminus of the KS polymer.

Spectroscopic Characterisation of Disaccharides Derived from Keratan Sulfates

Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy. The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate. (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion. (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously [Brown, G. M., Huckerby, T. N., Morris, H. G., Abram, B. L. & Nieduszynski, I. A. (1994) Biochemistry 33, 4836-4846; Brown, G. M., Huckerby, T. N. & Nieduszynski, I. A. (1994) Eur. J. Biochem. 224, 281-308], GlcNAc(6S)beta(1-6)GalNAc-ol, [formula: see text], [formula: see text], all isolated after keratanase II digestion. (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion. In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy. The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Spectroscopic characterisation of disaccharides derived from keratan sulfates.

Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy. The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate. (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion. (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously [Brown, G. M., Huckerby, T. N., Morris, H. G., Abram, B. L. & Nieduszynski, I. A. (1994) Biochemistry 33, 4836-4846; Brown, G. M., Huckerby, T. N. & Nieduszynski, I. A. (1994) Eur. J. Biochem. 224, 281-308], GlcNAc(6S)beta(1-6)GalNAc-ol, [formula: see text], [formula: see text], all isolated after keratanase II digestion. (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion. In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy. The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage. Oligosaccharides generated by keratanase digestion.

The structure of the repeat region and chain caps of the N-linked keratan sulphate chains attached to bovine tracheal cartilage fibromodulin has been examined. The chains were fragmented by keratanase digestion, the resultant oligosaccharides isolated by strong anion-exchange chromatography, and their structures determined using high-field 1H-n.m.r. spectroscopy. The chains were found to possess the following general structure: [formula: see text] All of the capping oligosaccharides isolated terminate with alpha(2-3)-linked N-acetylneuraminic acid. No alpha(2-6)-linked N-acetylneuraminic acid chain terminators, nor any fucose, alpha (1-3)-linked to N-acetylglucosamine along the repeat region, were detected. This work demonstrates that the structure of the repeat region and chain caps of N-linked keratan sulphate attached to fibromodulin isolated from bovine tracheal cartilage is identical with that of O-linked keratan sulphate chains attached to aggrecan derived from non-articular cartilage.

600 MHz 1H NMR study of a fucose-containing heptasaccharide derived from a keratanase digestion of bovine articular cartilage keratan sulphate

600 MHz 1H NMR study of a fucose-containing heptasaccharide derived from a keratanase digestion of bovine articular cartilage keratan sulphate

N.m.r. spectroscopic studies of fucose-containing oligosaccharides derived from keratanase digestion of articular cartilage keratan sulphates. Influence of fucose residues on keratanase cleavage

Keratan sulphate chains from bovine articular cartilage were fully digested with keratanase from Pseudomonas sp. and the products were reduced with alkaline borohydride. The resultant fragments were fractionated on a Nucleosil 5SB column and the earliest eluting fucose-containing oligosaccharides were isolated. Structural analysis using 1H n.m.r. spectroscopy (600 MHz) showed the two least-charged species to have the following structure: GlcNAc(6S) beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S) beta 1- 3Gal beta 1-4GlcNAc(6S) beta 1-3Gal-ol and GlcNAc(6S) beta 1-3Gal beta 1- 4(Fuc alpha 1-3)GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S) beta 1-6(Gal beta 1- 3)GalNAc-ol. Both galactoses adjacent to the fucosylated N-acetylglucosamine residue are unsulphated. Therefore, it can be deduced from these structures that the presence of fucose on N-acetylglucosamine residues in keratan sulphates protects both of the adjacent unsulphated galactose residues from keratanase cleavage. This result has implications for the interpretation of keratanase fingerprints, because in articular cartilage keratan sulphates the keratanase-resistant blocks are not solely those with fully sulphated galactose residues, but also include the fucosylated sequences, which have unsulphated galactoses. It is, therefore, not possible to estimate their galactose sulphation or the size of the fully sulphated disaccharide-repeat sequences from keratan sulphates that contain fucose.

13C NMR spectroscopy of keratan sulphates: Assignments for the reduced form of a repeat unit tetrasaccharide derived from keratan sulphate by keratanase digestion and partial assignments for two fucosylated pentasaccharides

AbstractA full assignment of the 60°C 13C NMR spectrum for the reduced form of a repeat unit β‐linked tetrasaccharide derived by keratanase digestion from keratan sulphate, GlcNAc(6SO4)β1–3Gal(6SO4)β1–4GlcNAc(6SO4)β1–3‐Gal‐ol, was achieved via two‐dimensional C/H correlation and 1H COSY experiments, and a complete first‐order analysis of the 600 MHz proton spectrum. Partial 13C assignments for two fucosylated keratan sulphate pentasc‐charides derived using hydrazinolysis and nitrous acid treatment are discussed.

The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.

A CNS specific proteoglycan associated with astrocytes in rat optic nerve

A novel CNS specific keratan sulfate proteoglycan is identified by a monoclonal antibody (mAb) TED15. The antibody recognizes a carbohydrate epitope that is removed from the core protein following keratanase digestion. The relative molecular weight of the glycoprotein is not altered following digestion with heparinase, heparitinase or chondroitinase ABC. By immunoblot analysis, the TED15 antigen is differentially distributed in diencephalic derivatives, being present in the optic nerve but absent from the retina. Within the optic nerve, the TED15 antigen is associated with astrocytes, while in the retina no labeling of astrocytes or Müller cells is observed.

1-C-6 epitope in cartilage proteoglycan G2 domain is masked by keratan sulphate

The presence of the protein epitope recognized by monoclonal antibody 1-C-6 was investigated on the globular G1 and G2 domains of pig cartilage proteoglycan core protein. After reduction of disulphide bonds and removal of keratan sulphate chains with keratanase, both G1 and G2 domains were shown to contain the epitope. However, without keratanase digestion the epitope on the G2 domain was poorly detected. The results suggest that a keratan sulphate chain substituted close to the epitope sequence in the G2 domain prevents antibody access to the epitope and thus masks its detection. This shows the 1-C-6 epitope to be a conserved protein sequence in the G2 domain of proteoglycans from different species, but its detection may be masked by glycosylation.

Full proton NMR assignment of the reduced form of a repeat unit tetrasaccharide derived by keratanase digestion of keratan sulphate

AbstractThe full assignment of the ambient‐temperature high‐field 1H NMR spectrum for the reduced form of a repeat‐unit β‐linked tetrasaccharide derived by keratanase digestion from keratan sulphate, GlcNAc(6‐SO4)13Gal(6‐SO4)14GlcNAc(6‐SO4)13Gal‐ol, was achieved using two‐dimensional COSY and RELAYED‐COSY experiments. The problem of the choice of the measurement temperature for 1H NMR spectra from keratan sulphate oligomers and polymers is discussed. A modified protocol for the preparation of keratanase‐derived oligosaccharides is proposed.