Differential relative sulfation of Keratan sulfate glycosaminoglycan in the chick cornea during embryonic development.
ABSTRACT To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency.
Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining.
A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated.
The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump.
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ABSTRACT: The chemical composition of tissues can influence their form and function. As a prime example, the lattice-like arrangement of collagen fibrils required for corneal transparency is controlled, in part, by sulfated proteoglycans, which, via core proteins, bind to the collagen at specific locations along the fibril axis. However, to date, no studies have been able to directly identify and characterize sulfur (S) in the cornea as a function of tissue location. In this study, X-ray absorption near-edge structure spectroscopy and micro-beam X-ray fluorescence (μ-XRF) chemical contrast imaging were employed to probe the nature of the mature (bovine) cornea as a function of position from the anterior sub-epithelial region into the deep stroma. Data indicate an inhomogeneity in the composition of S species in the first ≈50 μm of stromal depth. In μ-XRF chemical contrast imaging, S did not co-localize with phosphorous (P) in the deep stroma where sulfates are prominent. Rather, P is present only as isolated micrometric spots, presumably identifiable as keratocytes. This study lends novel insights into the elemental physiology of mature cornea, especially in relation to its S distribution; future studies could be applied to human tissues. Moreover, it defines an analytical protocol for the interrogation of S species in biological tissues with micrometric resolution.Analytical and Bioanalytical Chemistry 06/2013; 405(21). DOI:10.1007/s00216-013-7120-x · 3.66 Impact Factor
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ABSTRACT: Glycosaminoglycans (GAGs) are distributed in the whole body and play a variety of important physiological roles associated with inflammation, growth, coagulation, fibrinolysis, lipolysis, and cell-matrix biology. Accumulation of undegraded GAGs in lysosomes gives rise to a distinct clinical syndrome, mucopolysaccharidoses. Measurement of each specific GAG in a variety of specimens is urgently required to understand GAG interaction with other molecules, physiological status of patients, and prognosis and pathogenesis of the disease. We established a highly sensitive and accurate tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and quantified by negative ion mode of multiple reaction monitoring. Subclasses of HS and GAGs with identical molecular weights can be separated using a Hypercarbcolumn (2.0 mm×50 mm, 5 μm) with an aectonitrile gradient in ammonium acetate (pH 11.0). We also developed a GAG assay by RapidFire with tandem mass spectrometry (RF-MS/MS). The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds. RF-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods. However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights. Both methods can be applied to analysis of dried blood spots, blood, and urine specimens. In this article, we compare the assay methods for GAGs and describe their potential applications.
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ABSTRACT: Keratan sulfate (KS) is a glycosaminoglycan (GAG) type consisted of a sulfated poly-N-acetyl lactosamine chain. Besides acting as a constitutive molecule of the extracellular matrices, this GAG also plays a role as a hydrating and signaling agent in cornea and cartilage tissues. Inasmuch, KS is widely explored in the pharmaceutical industry. This review will cover the major achievements described in the literature of 2010-2014 concerning this GAG. Discussion about KS' roles in physiopathological conditions, as target or therapeutic molecule in diseases, methods of analysis and detection as well as KS-related enzymes, metabolism and developmental biology is properly provided.International Journal of Biological Macromolecules 08/2014; 72. DOI:10.1016/j.ijbiomac.2014.08.029 · 3.10 Impact Factor