Mapping the differential distribution of glycosaminoglycans in the adult human retina, choroid, and sclera.

PURPOSE. To map the distribution of different classes of glycosaminoglycans (GAGs) in the healthy human retina, choroid, and sclera. METHODS. Frozen tissue sections were made from adult human donor eyes. The GAG chains of proteoglycans (PGs) were detected with antibodies directed against various GAG structures (either directly or after pretreatment with GAG-degrading enzymes); hyaluronan (HA) was detected using biotinylated recombinant G1-domain of human versican. The primary detection reagents were identified with FITC-labeled probes and analyzed by fluorescence microscopy. RESULTS. Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and HA were present throughout the retina and choroid, but keratan sulfate (KS) was detected only in the sclera. HS labeling was particularly strong in basement membrane-containing structures, the nerve fiber layer (NFL), and retinal pigment epithelium (RPE)-for example, intense staining was seen with an antibody that binds strongly to sequences containing 3-O-sulfation in the internal limiting membrane (ILM) and in the basement membrane of blood vessels. Unsulfated CS was seen throughout the retina, particularly in the ILM and interphotoreceptor matrix (IPM) with 6-O-sulfated CS also prominent in the IPM. There was labeling for DS throughout the retina and choroid, especially in the NFL, ganglion cell layer, and blood vessels. CONCLUSIONS. The detection of GAG chains with specific probes and fluorescence microscopy provides for the first time a detailed analysis of their compartmentalization in the human retina, by both GAG chain type and sulfation pattern. This reference map provides a basis for understanding the functional regulation of GAG-binding proteins in health and disease processes.

Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions.

The interactions of glycosaminoglycans (GAGs) with proteins underlie a wide range of important biological processes. However, the study of such binding reactions has been hampered by the lack of a simple frontline analysis technique. Previously, we have reported that cold plasma polymerization can be used to coat microtiter plate surfaces with allyl amine to which GAGs (e.g., heparin) can be noncovalently immobilized retaining their ability to interact with proteins. Here, we have assessed the capabilities of surface coats derived from different ratios of allyl amine and octadiene (100:0 to 0:100) to support the binding of diverse GAGs (e.g., chondroitin-4-sulfate, dermatan sulfate, heparin preparations, and hyaluronan) in a functionally active state. The Link module from TSG-6 was used as a probe to determine the level of functional binding because of its broad (and unique) specificity for both sulfated and nonsulfated GAGs. All of the GAGs tested could bind this domain following their immobilization, although there were clear differences in their protein-binding activities depending on the surface chemistry to which they were adsorbed. On the basis of these experiments, 100% allyl amine was chosen for the generation of a microtiter plate-based "sugar array"; X-ray photoelectron spectroscopy revealed that similar relative amounts of chondroitin-4-sulfate, dermatan sulfate, and heparin (including two selectively de-sulfated derivatives) were immobilized onto this surface. Analysis of four unrelated proteins (i.e., TSG-6, complement factor H, fibrillin-1, and versican) illustrated the utility of this array to determine the GAG-binding profile and specificity for a particular target protein.

Efficient production of human beta-defensin 2 (HBD2) in Escherichia coli.

Human beta-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 beta-isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed (1)H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa, C. albicans and S. aureus.

Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells.

We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFalpha-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.