The chemokine IL-8 is critically important in inflammatory processes in human tissues, and IL-8 interactions with sulfated glycosaminoglycans have been implicated in modification of inflammatory responses in bronchial epithelium. To determine the role of chondroitin-4-sulfate (C4S) in mediating effects of IL-8, we silenced the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B [ASB]) that removes the 4-sulfate group from C4S, in the IB3-1 and C38 bronchial epithelial cell lines and in normal primary bronchial epithelial cells. When ASB was silenced and IL-8 production stimulated by exposure to TNF-alpha, ASB activity declined by roughly 75%, cellular C4S content increased by over 7.5 microg/mg protein, cell-bound IL-8 increased by over 530 pg/mg protein, and secreted IL-8 declined by over 520 pg/mg protein in all cell lines (P < 0.001). When cell lysates were immunoprecipitated with C4S antibody after ASB silencing and TNF-alpha, the IL-8 content of the immunoprecipitate was approximately 500 pg/mg protein, indicating that most of the cell-bound IL-8 was associated with C4S. Cell fractionation demonstrated that the IL-8 content associated with the cell membranes was about twice that of the cytosolic fraction. Also, ASB appeared to localize in the cell membrane, as well as in lysosomes. Neutrophil attraction to the cell lysates increased after ASB silencing, consistent with increased attraction to the cell-bound IL-8. These findings provide evidence for the influential role of ASB and C4S in the regulation of IL-8 secretion, and suggest that changes in ASB activity and C4S content may have a significant impact on IL-8-mediated inflammatory responses.

Seven commercial heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physico-chemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (MN 10–11 kDa, MW 13–14 kDa, polydispersity (PD) 1.3–1.4) and by size exclusion chromatography (MN 14–16 kDa, MW 21–25 kDa, PD 1.4–1.6). 1D 1H- and 13C-NMR evaluation of the heparin samples was performed and peaks were fully assigned using 2D NMR. The percentage of glucosamine residues with 3-O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8–7.9, 78–82 and 13–14 %, respectively. There was substantial variability observed in the disaccharide composition with, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of heparin lyase I–III digested heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with heparin lyase I, II and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these heparins. The binding affinities of these heparins to antithrombin III and thrombin were evaluated by using a SPR competitive binding assay. This study provides the physico-chemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered heparin.

Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive, and neural tissues. Corneal KS glycosaminoglycan is found N-linked to lumican, keratocan, and mimecan proteoglycans and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited due to restricted the shipment of bovine central nervous system by-products across international borders in efforts to prevent additional cases of mad cow disease. We report a simple method for the purification of multi-milligram quantities of bovine corneal KS and characterize its structural properties. We also examined its protein-binding properties and discovered that corneal KS bound with high affinity to fibroblast growth factor-2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing.

The elucidation of the structure of glycosaminoglycan has proven to be challenging for analytical chemists. Molecules of glycosaminoglycan have a high negative charge and are polydisperse and microheterogeneous, thus requiring the application of multiple analytical techniques and methods. Heparin and heparan sulfate are the most structurally complex of the glycosaminoglycans and are widely distributed in nature. They play critical roles in physiological and pathophysiological processes through their interaction with heparin-binding proteins. Moreover, heparin and low-molecular weight heparin are currently used as pharmaceutical drugs to control blood coagulation. In 2008, the health crisis resulting from the contamination of pharmaceutical heparin led to considerable attention regarding their analysis and structural characterization. Modern analytical techniques, including high-performance liquid chromatography, capillary electrophoresis, mass spectrometry, and nuclear magnetic resonance spectroscopy, played critical roles in this effort. A successful combination of separation and spectral techniques will clearly provide a critical advantage in the future analysis of heparin and heparan sulfate. This review focuses on recent efforts to develop hyphenated techniques for the analysis of heparin and heparan sulfate.

Chondroitin sulfate (CS) has an important role in cell division, in the central nervous system, and in joint related pathologies such as osteoarthritis. Due to the complex chemical structure and biological importance of CS, simple, sensitive, high-resolution, and robust analytical methods are needed for the analysis of CS disaccharides and oligosaccharides. An ion pairing, reversed-phase, ultraperformance liquid chromatography (IPRP-UPLC) separation, coupled to electrospray ionization mass spectrometry with ion trap mass analyzer was applied for the analyses of CSderived disaccharides. UPLC separation technology utilizes small particle diameter, short column length, and elevated column temperature to obtain high resolution and sensitivity. Hexylamine (15 mM) was selected the optimal ion-pairing reagent.