Showing papers on "Keratan sulfate published in 2007"

Notes & Tips Thin-layer chromatography for the analysis of glycosaminoglycan oligosaccharides

Abstract: Glycosaminoglycans (GAGs) 1 are linear acidic polysaccharides found on cell surfaces and in the surrounding extracellular matrix. GAGs participate in and regulate many cellular events in physiological and pathophysiological processes, such as cell proliferation and differentiation, cell–cell and cell–matrix interactions, and viral infection, through their interaction with different proteins [1–3]. GAGs are divided into four main categories—hyaluronic acid (HA), chondroitin sulfate/dermatan sulfate (CS/DS), heparosan/heparan sulfate/heparin (HN/HS/HP), and keratan sulfate—based on monosaccharide composition and the configuration and position of the glycosidic bonds between their monosaccharides. The specificity of the interactions between GAGs and proteins results from the structural diversity of GAG type, size, saccharide composition, charge density, and sequence [4,5]. It often is necessary to determine the size and purity of GAG-derived oligosaccharides, analyze activity of enzymes acting on GAGs, and monitor the preparation of GAG-derived oligosaccharides. Polyacrylamide gel electrophoresis (PAGE) is a method used routinely for separation and analysis of GAGs and GAG-derived oligosaccharides, and the resulting gels usu

Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation.

Abstract: Corneal stromal cells (keratocytes) are responsible for the development and maintenance of the unique extra-cellular matrix of the transparent corneal stroma, which constitutes approximately 90% of the adult cornea. Although keratocytes are relatively quiescent in the normal adult cornea, they become activated to the fibroblast or myofibroblast phenotype after injury to the corneal stroma. Tissue culture models have been useful tools for studying keratocyte activation in vitro.1,2 When keratocytes isolated from the adult cornea are cultured in media without serum, they retain several features exhibited by the quiescent keratocytes in vivo.1,3– 8 However, when cultured in media with serum, growth factors, or cytokines, including TGF-β1, platelet-derived growth factor (PDGF), bFGF, insulinlike growth factor (IGF)-1, and IL-1α, they attain phenotypic characteristics exhibited by activated stromal cells in vivo. Changes in the characteristics of keratocytes on activation in vivo and in vitro include assembly of actin stress fibers, de novo expression of extracellular matrix proteins such as type III collagen,9–12 tenascin,13–15 and matrix metalloproteinases16–21 and significant decreases in the expression of keratan sulfate proteoglycans (KSPGs),1,3,22–25 prostaglandin D synthase,26 CD34,27 and corneal crystallins, namely, aldehyde dehydrogenase and α-transketolase.28 Although some phenotypic changes in activated stromal cells are transient, others may be permanent. When using in vitro tissue culture models for studying mechanisms of keratocyte activation, it is important to verify that the differential phenotypic characteristics exhibited by quiescent and activated keratocytes in vivo are recapitulated in vitro and to identify the extracellular factors that influence specific phenotypic changes in activated cells. It has become evident that macromolecules, which were once thought to be restricted to basement membranes (BMs) such as type IV collagen, laminin, and perlecan, are also expressed in connective tissues including corneal stroma.25,29 Six genetically distinct chains of type IV collagen—α1(IV)- α6(IV)— have been identified and shown to have different cellular interactions and tissue distributions.30,31 Immunohistochemical analysis has indicated the presence of α3(IV), α4(IV), and α5(IV) collagens in normal human corneal stroma, epithelial BM, and Descemet membrane.32 The expression of different type IV collagen isoforms is altered in regenerated stroma after radial keratotomy.10 Interestingly, α1(IV) and α2(IV) collagens, not detectable in the normal human corneal stroma or epithelial basement membrane, were expressed in regenerated corneal tissues after laser-assisted in situ keratomileusis (LASIK) and radial keratotomy. α3(IV) collagen was absent in regenerated BM. In the present study, we evaluated the expression of α1(IV) to α3(IV) collagen isoforms in the normal and regenerated rabbit stroma after photorefractive keratectomy (PRK). Differences were observed between expression patterns of type IV collagen isoforms in rabbit corneas and the reported findings in human corneas. An in vitro tissue culture model of corneal keratocyte activation was used to determine whether the phenotypic changes observed in vivo were recapitulated in vitro on the differentiation of keratocytes to fibroblasts or myofibroblasts.

Keratan sulfate glycosaminoglycan and the association with collagen fibrils in rudimentary lamellae in the developing avian cornea.

Abstract: PURPOSE. Keratan sulfate (KS), through its association with fibrillar collagen as KS-substituted proteoglycan (KS PG), is thought to be instrumental in the structural development of the corneal stroma. The authors used two different sulfate motif-specific antibodies to identify the sequence of appearance, and the association with collagen, of sulfated KS during avian corneal morphogenesis. METHODS. Corneas from chicken embryos throughout the developmental period, from day 8 through day 18 of incubation, were examined by immunofluorescence and immunoelectron microscopy using monoclonal antibodies 5D4 and 1B4, which react with high- and low-sulfated epitopes on KS, respectively. RESULTS. KS was identified as punctate labeling at incubation day 8, the earliest stage examined, suggesting a cell-associated distribution. By day 10, labeling was more homogeneous, indicating that KS sulfation motifs were present in the stromal extracellular matrix. At day 12 through day 14, immunopositive sites were concentrated primarily in the anterior stroma but became more uniform throughout the full stromal thickness by day 18. From day 10 on, electron microscopy revealed a high-sulfated KS epitope closely associated with bundles of regularly arranged collagen fibrils, initially near cell surfaces in rudimentary lamellae. Individual cells, associated with collagen bundles with different fibril orientations, imply the potential for simultaneous deposition of multiple lamellae. CONCLUSIONS. During chick corneal morphogenesis, significant matrix deposition of high-sulfated KS epitope occurs by day 10, with accumulation subsequently proceeding in an anterior-toposterior manner. High-sulfated KS likely serves to help define the regular spatial organization of collagen fibrils in bundles newly extruded into the extracellular milieu. (Invest Ophthalmol Vis Sci. 2007;48:3083‐3088) DOI:10.1167/iovs.06-1323

Influence of estradiol on vascular endothelial growth factor expression in bone: a study in Göttingen miniature pigs and human osteoblasts.

Abstract: Ovariectomy (OVX) in animal models is an accepted method to simulate postmenopausal osteoprosis. Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation, hypertrophic cartilage remodeling, ossification, and angiogenesis. We hypothesized that reduced VEGF expression in bone contributes to OVX-induced bone loss and tested it in a miniature pig model and in vitro using human osteoblasts. Seventeen primiparous sows (Gottingen miniature pigs) were allocated to two experimental groups when they were 30 months old: a control group (n = 9) and an OVX group (n = 8). After 15 months, VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay and verified by Western blot analysis. VEGF and its receptor (VEGFR) were localized by immunohistochemistry. Expression of VEGF mRNA was analyzed by real-time reverse-transcription polymerase chain reaction. Differently sulfated glycosaminoglycans were localized in subchondral bone histochemically. Osteoblasts were immunopositive for VEGF. VEGF concentration in the vertebra was 27% lower in OVX miniature pigs. VEGFR-2 could be immunostained on osteoblasts. VEGF mRNA and protein were detectable in the lumbar vertebrae of all animals. In subchondral trabecular bone of OVX animals, significantly more islands of mineralized cartilage containing chondroitin 4- and 6-sulfate or keratan sulfate occurred compared to the control group. The occurrence of remnants of mineralized cartilage in subchondral bone of the OVX group may be caused by a delayed bone turnover due to low VEGF levels. In vitro experiments revealed an increase of VEGF in the supernatant of osteoblasts after incubation with estradiol. In conclusion, estrogen seems to be a key factor for regulation of VEGF expression in bone. Loss of VEGF due to menopause may be a reason for reduction of bone density.

Differential expression of the keratan sulphate proteoglycan, keratocan, during chick corneal embryogenesis

Abstract: Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency.

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae)

Abstract: The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to ∼80% and 20% of the total GAGs, respectively. In the fish eye, KS represents ∼ 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by ΔUA-1→3-GalNAc(4SO4) (59.4%), ΔUA-1→3-GalNAc(6SO4) (23.1%), and ΔUA-1→3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism.

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle.

Abstract: The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl 2 (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.

Localization of glycosaminoglycans (GAGs) in pleomorphic adenoma (PA) of salivary glands: an immunohistochemical and histochemical evaluation.

Abstract: The tumor matrix of salivary pleomorphic adenoma (PA) is characteristically rich in glycosaminoglycans (GAGs), which contribute to its complex histoarchitecture. This study evaluated the microscopic localization of various GAGs in 17 PAs, using a panel of anti-GAG monoclonal antibodies and biotinylated hyaluronic acid (HA)-binding protein. Both epithelial and mesenchymal-like tissues were confirmed to contain GAGs. Luminal epithelial cells mostly lacked GAGs, whereas GAGs were seen both in the cytoplasm and cell membrane of non-luminal epithelial cells. In addition, small intercellular accumulations of GAGs were often present in solid epithelial areas, implying the epithelial origin of GAGs. GAGs did not appear to be a main component of the hyaline matrix. The myxoid region was consistently stained for both chondroitin 6-sulfate (CS-6) and HA but variably for chondroitin 4-sulfate (CS-4), dermatan sulfate (DS) and keratan sulfate (KS); heparan sulfate (HS) was not detected. The chondroid region showed increased staining for CS-6 but reduced staining for HA when compared with the myxoid region. In addition, CS-4, DS and KS were seen both in chondroid cells and the territorial matrix, whereas HS was present only in the cells. It is suggested that GAGs in PA are mainly produced by non-luminal cells and influence the proliferation, differentiation, secretory activity and shape of tumor cells, thus contributing to the morphological diversity of this tumor.

Significant decrease in α1,3-linked fucose in association with increase in 6-sulfated N-acetylglucosamine in peripheral lymph node addressin of FucT-VII-deficient mice exhibiting diminished lymphocyte homing

Abstract: Lymphocyte homing is mediated by binding of L-selectin on lymphocytes with L-selectin ligands present on highendothelial venules (HEV) of peripheral and mesenteric lymph nodes. L-selectin ligands are specific O-linked carbohydrates, 6-sulfo sialyl Lewis X, composed of sialylated, fucosylated, and sulfated glycans. Abrogation of fucosyltransferase-VII (FucT-VII) results in almost complete loss of lymphocyte homing, but structural analysis of carbohydrates has not been carried out on FucT-VII null mice. To determine whether functional losses seen in FucT-VII null mice are caused by structural changes in carbohydrates, we elucidated the carbohydrate structure of GlyCAM-1, a major L-selectin counter-receptor. Our results show that most a1,3-fucosylated structures in 6-sulfo sialyl Lewis X are absent and 6-sulfo N-acetyllactosamine is increased in the mutant mice. Surprisingly, the amount of 6 0 -sulfated galactose (Gal) that bound to Sumbucus nigra agglutinin column was also increased. We found that structures of those oligosaccharides containing 6 0 -sulfated Gal are almost identical to those synthesized by keratan sulfate sulfotransferase (KSST). We then showed that overexpression of KSST suppresses the expression of sialyl Lewis X on Chinese hamster ovary (CHO) cells engineered to express sialyl Lewis X. Moreover, KSST expression in those cells suppressed lymphocyte rolling compared with mock-transfected CHO cells expressing 6-sulfo sialyl Lewis X. 6 0 -Sulfo sialyl Lewis X can neither be found in GlyCAM-1 from CHO cells expressing both KSST and FucT-VII nor be found in GlyCAM-1 from HEV of mice. These results combined together suggest that KSST competes with FucT-VII for the same acceptor substrate and downregulates the synthesis of L-selectin ligand by inhibiting a1,3-fucosylation.

Asexual growth of babesia bovis is inhibited by specific sulfated glycoconjugates

Abstract: In the present study, inhibitory effects of several sulfated and nonsulfated glycoconjugates were evaluated on the in vitro asexual growth of Babesia bovis. Among the selected sulfated glycoconjugates, dextran sulfate, heparin, heparan sulfate, fucoidan, and chondroitin sulfate B strongly inhibited the parasitic growth, and all but chondroitin sulfate B induced a significant accumulation of extracellular merozoites in culture. In contrast, chondroitin sulfate A, keratan sulfate, and protamine sulfate, as well as nonsulfated dextran and hyaluronic acid, did not influence the growth. These findings indicate that the asexual growth of B. bovis merozoites is inhibited by specific sulfated glycoconjugates, possibly providing us with an important insight into the molecular interaction(or interactions) during the process of the erythrocyte invasion by B. bovis merozoites.