IOVS Archives of Disease in Childhood
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2005;46:4417-4423.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0883
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hindson, V. J.
Right arrow Articles by Bishop, P. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hindson, V. J.
Right arrow Articles by Bishop, P. N.

Opticin Binds to Heparan and Chondroitin Sulfate Proteoglycans

V. John Hindson,1 John T. Gallagher,2 Willi Halfter,3 and Paul N. Bishop1

1From the Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences and Academic Unit of Eye & Vision Science, School of Medicine, and the 2Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom; and the 3Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The extracellular matrix glycoprotein opticin is a small leucine-rich repeat proteoglycan/protein family member that was discovered associated with vitreous humor collagen fibrils. Opticin is present throughout the vitreous, but is particularly concentrated at the internal limiting lamina, where it colocalizes with type XVIII collagen. The present study investigated whether opticin interacts directly with the heparan sulfate (HS) proteoglycan type XVIII collagen.

METHODS. Solid-phase opticin binding assays were performed with immobilized type XVIII collagen and heparin albumin. Surface plasmon resonance (SPR) was used to investigate the binding of opticin to heparin and HS.

RESULTS. Opticin bound to type XVIII collagen via its HS chains. SPR showed that opticin bound to porcine intestinal mucosa HS and heparin with moderately high affinity (KD 73 and 43 nM, respectively). Binding inhibition studies showed that hexasaccharides of heparin had a lower affinity for opticin than larger oligosaccharides; the sulfate groups of heparin contributed variably to opticin binding, with the group at ring position two of iduronate contributing least; and chondroitin sulfate A and B bound to opticin, whereas binding to chondroitin sulfate C and hyaluronan was not observed.

CONCLUSIONS. Opticin binds to heparin, HS, chondroitin 4-sulfate, and dermatan sulfate, the binding affinity being dependent on sulfation pattern and oligosaccharide chain length. Opticin may provide a link between cortical vitreous collagen fibrils and the inner limiting lamina by binding HS proteoglycans and stabilize vitreous gel structure by binding chondroitin sulfate proteoglycans.

Opticin is an extracellular matrix glycoprotein that was initially identified as the major component in a pool of macromolecules that were isolated through their association with vitreous collagen fibrils.1 It is a member of the small leucine-rich repeat proteoglycan/protein (SLRP) family, characterized by tandem leucine-rich repeats flanked by capping motifs containing disulfide bonds. Many of the SLRPs are proteoglycans, but opticin is unique in that instead of possessing glycosaminoglycan (GAG) chains, it is substituted with a cluster of sialylated O-linked oligosaccharides near its amino terminus. Opticin and at least one other SLRP (i.e., decorin) are dimeric, and this dimerization is through association of the leucine-rich repeat (LRR) domains.2 3 4 The only function that has been ascribed to opticin to date is binding to growth hormone.5 Opticin is highly expressed by the nonpigmented ciliary epithelium,6 7 8 and our immunolocalization studies specifically identified opticin in the vitreous.9 Other authors, however, using a different opticin antibody, detected immunoreactivity in the cornea, iris, ciliary body, vitreous, retina, and choroid.10 Although in our studies opticin was localized throughout the vitreous cavity, it was particularly concentrated at the interfaces with surrounding basement membranes, i.e., in the internal limiting membrane (ILM) and adjacent to the lens capsule.9 Furthermore, opticin colocalized with the heparan sulfate (HS) proteoglycan type XVIII collagen in the ILM.9

Heparin and HS are GAGs that are synthesized attached to a core protein. Heparin is synthesized by mast cells, and HS is found on most cell surfaces and in the extracellular matrix, particularly in basement membranes. The internal limiting lamina (ILL), the basement membrane on the inner surface of the retina that is contained within the ILM, has been shown to contain HS proteoglycans including type XVIII collagen, perlecan, and agrin.11 Heparin and HS are linear polymers composed of alternating uronic acid and glucosamine residues that undergo modification after their initial synthesis. These modifications include N-deacetylation, sulfation, and epimerization of glucuronic acid to iduronic acid. Heparin is more highly sulfated than HS, although HS can contain highly sulfated domains that resemble heparin (S-domains) interspersed between areas containing less sulfation.12 Therefore, analysis of heparin binding can give insights into how interacting molecules bind to the S-domains of HS.

As opticin appeared to colocalize with type XVIII collagen, we investigated whether there is a direct interaction between these macromolecules. We demonstrated that opticin does interact with the HS chains of type XVIII collagen and characterized in detail the opticin-HS interaction.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant type XVIII collagen was prepared as previously described.13 The HS-degrading enzyme K5 lyase12 was kindly provided by Ian Roberts (University of Manchester, Manchester, UK). Heparin albumin, heparin from porcine intestinal mucosa (PIM), HS from bovine kidney (BK), chondroitin sulfate from bovine trachea (CSA), dermatan sulfate from porcine intestinal mucosa (CSB), chondroitin sulfate from shark cartilage (CSC), hyaluronan from bovine vitreous humor (HA), and biotinylated albumin were all obtained from Sigma (St. Louis, MO). Biotinylated heparin and PIM HS were obtained from Celsus Laboratories (Cincinnati, OH); a proportion of the latter was biotinylated at the reducing end. Heparin oligosaccharides in the range dp2 (disaccharide) to 16 were prepared by partial heparinase scission of heparin and separation of the degradation products by high-resolution gel filtration as previously described.14 Heparin (from bovine lung) was used to produce oligosaccharides composed of ~24 monosaccharides (dp24) that had been selectively desulfated at three positions. Before desulfation the glucosamines were 98% N-sulfated and 92% 6-O-sulfated, whereas the iduronates were 89% 2-O-sulfated. Selective desulfation of glucosamine N-sulfates and de–N-sulfation followed by N-acetylation produced two molecules, denoted DNS and DNSRAc, respectively, that were 2% N-sulfated, 89% 6-O-sulfated, and 90% 2-O-sulfated. Similarly, de–2-O-sulfated heparin (DE2) contained 80% 6-O-sulfate groups, 91% N-sulfate groups, and a residual 2% of the 2-O-sulfates. De–6-O-sulfated heparin (DE6) contained 98% N-sulfate groups, 55% 2-O-sulfate groups, and a residual 4% of the 6-O-sulfates. CompDS was extensively desulfated at all three positions.

Polyclonal Antiserum
A rabbit polyclonal antiserum was raised against the bovine opticin sequence VLSLDNYDEVIDPSNYDELIDYGDQLPQVK. This antibody, called OPT-NB, was tested by Western blotting and ELISA and shown to bind opticin specifically (data not shown).

Production and Purification of Opticin
Recombinant bovine opticin was produced in 293-EBNA cells as previously described.2 However, the technique for purification from conditioned media was modified. Conditioned medium (~5 L) containing 5 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride was equilibrated overnight at 4°C by mixing with 100 mL of DEAE-Sepharose Fast Flow (Sigma) in 50 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl. The DEAE-Sepharose Fast Flow was then poured into a column and equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl before elution of the column with 0.7 M NaCl in the same buffer. The eluant was then diluted fivefold into 0.7 M (NH4)2SO4 containing 50 mM Tris-HCl (pH 7.5) (buffer A) and applied to a 5 mL fast-flow column (Phenyl Sepharose 6; Amersham Biosciences, Buckinghamshire, UK) equilibrated in the same buffer. The column was then washed with buffer A, followed by a linear gradient of 100% buffer A to 80% 50 mM Tris-HCl (pH 7.5) (buffer B). Opticin was then eluted isocratically with 80% buffer B. Opticin-containing fractions were loaded directly onto an anion exchange column (SAX-10; Dionex, Sunnyvale, CA), and the column was washed with 50 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl. Opticin was then eluted with a linear NaCl gradient (0.1–1 M) in 50 mM Tris-HCl (pH 7.4). Fractions containing purified opticin were collected and stored frozen (–70°C). Some of the opticin samples were biotinylated using a microbiotinylation kit (BiotinTag; Sigma). Biotinylation was carried out in PBS using a 70 M excess of biotin overnight at 4°C in darkness. The free biotin was removed by gel filtration chromatography (HiTrap column; Amersham Biosciences).

Solid-Phase Binding Assays
Ninety-six–well plates (Costar, Cambridge, MA) were coated (25 µL/well) with 0.5 µg/mL type XVIII collagen overnight at 4°C. After rinsing three times with TBST (50 mM Tris-HCl, pH 7.4; 0.15 M NaCl; 0.05% Tween 20) and blocking with 5% BSA in TBST for 1 hour, the wells were incubated for 2 hours at 35°C in 50 nM Tris-acetate buffer (pH 8) with or without K5 lyase (5 ng/mL). After washing with TBST, the wells were incubated with biotinylated bovine opticin (with or without unbiotinylated opticin) in TBST for 90 minutes. After washing, the plates were incubated with ExtraAvidin-Peroxidase (Sigma) diluted 1:1000 in TBST for 40 minutes before washing and incubating with ABTS-(NH4)2 solution (Sigma) according to the manufacturer’s instructions. Absorbance was read at 405 nm.

For heparin binding studies, the procedure was as above, with the following exceptions. The plates were coated overnight with heparin albumin (10 µg/mL). Competitive inhibitors were mixed with (unbiotinylated) opticin 30 minutes before application to the wells. The bound opticin was detected using OPT-NB (1:500), followed by horseradish peroxidase–conjugated mouse anti-rabbit IgG before applying the ABTS-(NH4)2. All data points were collected in triplicate (presented as means ± SD) and were blanked against BSA.

Surface Plasmon Resonance (SPR)
The SPR measurements were performed using a commercially available instrument (BIAcore 3000; BIAcore AB, Uppsala, Sweden). Biotinylated albumin (150 µg/mL), biotinylated heparin (50 µg/mL), or biotinylated PIM HS (50 µg/mL) in HBS-P buffer (BIAcore) was injected over streptavidin SA chips for 1 to 2 minutes at a flow rate of 10 µL/min at 25°C. This resulted in ~1000 resonance units of biotinylated albumin and ~300 resonance units of biotinylated heparin or HS being immobilized on the respective chips. Binding assays were performed at 25°C in HBS-P buffer. Association was monitored over 1 to 2 minutes and dissociation over 1.5 minutes at a flow rate of 20 to 30 µL/min. The surface was then regenerated with sequential pulses (flow rate 30 to 40 µL/min) of 6 M GuHCl and then 1 M NaCl, both in 50 mM Tris-HCl (pH 7.5). All data were blanked against biotinylated albumin (no binding was observed with either biotinylated albumin or streptavidin alone) and double referenced against HBS-P buffer. The kinetic parameters ka and kd (association and dissociation rate constants, respectively) were analyzed simultaneously using a global fit. The software (BIAevaluation Version 3.1; BIAcore) simultaneously fitted the sensorgrams obtained at different concentrations of opticin, fixing each kinetic parameter to a single value for each set of experimental data. Apparent equilibrium dissociation constants (KD) were calculated as the ratio kd/ka with the maximal capacity (Rmax) of the surface floated during the fitting procedure. The mean square of the signal noise {chi}2 was determined to give an indication of the goodness of the fits of the experimental data. For competition studies, a fixed concentration of opticin was preincubated (>30 minutes) with each putative inhibitor before injection at 10 to 20 µL/minute. The response in resonance units at the end of the association phase was recorded, blanked against the control flow cell, and double referenced against a control buffer injection. Data were presented as percentage inhibition relative to the value obtained without inhibitor.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Interaction between Opticin and Type XVIII Collagen
Opticin bound to the immobilized type XVIII collagen in a concentration-dependent and saturable manner (Fig. 1A) . The binding was not due to biotinylation, as it could be effectively competed with unbiotinylated opticin (Fig. 1B) . Prior digestion of the type XVIII collagen with the purified heparanase K5 lyase greatly decreased the binding. K5 lyase acts in the nonsulfated regions of HS that are found at regular intervals along the polymer chain12 ; a K5 lyase site is present near the GAG-protein linkage region in HS proteoglycans, and its action efficiently removes HS from core proteins. Therefore, opticin bound to type XVIII collagen largely through interactions with its HS chains.



View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 1. Solid-phase binding assays with immobilized type XVIII collagen. (A) Biotinylated opticin binds to type XVIII collagen in a concentration-dependent and saturable manner. (B) The addition of a 10-fold excess of nonbiotinylated opticin effectively competes the interaction. Digestion of the type XVIII collagen with K5 lyase (an enzyme that removes the HS side-chains) decreases binding by ~80%.

 
Solid-Phase Binding Assays with Immobilized Heparin
Opticin bound to heparin albumin in a concentration-dependent and saturable manner (Fig. 2A) . Inhibition studies showed that heparin, PIM HS, and to a lesser extent BK HS in solution competed with these interactions and that the biotinylation of heparin and PIM HS did not affect their ability to inhibit the binding of opticin to immobilized heparin (Fig. 2B) . Therefore, opticin bound to heparin and HS and preferably bound highly sulfated HS (see Discussion).



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. Solid-phase assays with immobilized heparin albumin. (A) Opticin binds to heparin albumin in a concentration-dependent and saturable manner. (B) Inhibition of the binding of opticin to heparin albumin using BK HS, PIM HS, biotinylated PIM HS (PIM bHS), heparin, and biotinylated heparin (bheparin), all at 0.2 mg/mL.

 
Kinetic Studies of Opticin-Heparin and -HS Interactions
As the biotinylation of heparin and PIM HS did not influence the binding to opticin, biotinylated forms were immobilized on streptavidin SA chips for kinetic studies. The SPR analyses were then performed by injecting opticin at varying concentrations over the immobilized biotinylated heparin and PIM HS (Fig. 3) . The dissociation rates of the complexes of opticin with heparin and HS were not influenced by the contact time (1–8 minutes, data not shown), indicating that linked reactions did not take place. Kinetic data were fitted to both a 1:1 Langmuir binding model and a heterogeneous ligand model involving parallel reactions between opticin and heparin or HS; the results were similar, so the kinetic parameters for the Langmuir binding model are presented (Table 1) . Fits were not improved by incorporating a term that takes into account mass transfer, suggesting that this is not a limiting factor under these experimental conditions. The calculated KD for opticin binding to heparin and to PIM HS was 43 and 73 nM, respectively.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. Overlays of SPR sensorgrams showing binding of opticin to heparin and HS. (A) Opticin at (from top to bottom) 1000, 700, 450, 300, 200, 100, and 50 nM was injected over biotinylated heparin. (B) Opticin at (from top to bottom) 1300, 1000, 700, 450, 300, and 200 nM was injected over immobilized biotinylated PIM HS. All injections were performed in duplicate and at a flow rate of 20 µL/min.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Langmuir 1:1 Binding Model Data for Interaction between Opticin and Biotinylated Heparin or PIM HS

 
Competition Assays Using SPR and Solid-Phase Assays
Heparin oligosaccharides of different fixed lengths were used in competition assays to evaluate the contribution of oligosaccharide length to binding using SPR and solid-phase assays (Fig. 4) . Similar results were obtained for heparin (in solid-phase assays and SPR) and PIM HS (using SPR). These analyses all showed that dp6 heparin oligosaccharides had a lower apparent affinity for opticin than larger oligosaccharides (dp8–16). There was a significant difference in the inhibitory activities between dp6 and 8, but little further increase with oligosaccharides of size dp10 and above, suggesting that a sulfated octamer fully occupies the GAG-binding site of opticin.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. Inhibition of opticin binding to heparin and HS by heparin oligosaccharides. (A) Solid-phase binding assays with immobilized heparin albumin showing inhibitory effect of 0.2 mM size-defined heparin oligosaccharides. (B) Opticin was preincubated with size-defined heparin oligosaccharides (8.8 µM), then injected onto heparin- and PIM HS–coated streptavidin SA chip surfaces. Chart records the level of opticin bound to the surface at the end of the association phase, presented as percentage of inhibition.

 
Inhibition studies were undertaken with specifically desulfated ~24dp heparin oligosaccharides to evaluate the contribution of the different sulfate groups to opticin binding to heparin and HS (Fig. 5) . Similar results were obtained for heparin (using solid-phase assays and SPR) and PIM HS (using SPR). Removal of the N-sulfates (DNS and DNSRAc) or the sulfates at ring position 6 of glucosamine (DE6) substantially lowered the ability of the oligosaccharides to compete binding to heparin and HS, whereas removal of the sulfates at ring position 2 of iduronate (DE2) had a lesser effect on the ability of the oligosaccharides to compete this interaction. This was particularly notable in the SPR analyses (Fig 5B) in which the de–2-sulfated heparin reduced opticin binding to heparin and HS by only ~20%. Therefore, the N-sulfates (DNS and DNSRAc) or the sulfates at ring position 6 of glucosamine (DE6) contribute more to opticin binding than the sulfates at ring position 2 of iduronate (DE2). Oligosaccharides which had all three sulfates removed (CompDS) had a similar affinity to opticin as DE6, DNS, and DNARAc, suggesting that the removal of all three sulfate groups did not lower binding affinity substantially more than just removing the N-sulfates (DNS and DNSRAc) or the sulfates at ring position 6 of glucosamine (DE6).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of opticin binding to heparin and HS by selectively desulfated heparins. (A) Opticin was preincubated with selectively desulfated heparin oligosaccharides (32 µM), and the level of binding to immobilized heparin albumin was determined by ELISA. (B) Opticin was preincubated with selectively desulfated heparin oligosaccharides (8 and 12 µM), then injected onto heparin- and PIM HS–coated strepatvidin SA chip surfaces. Chart records the level of opticin bound to the surface at the end of the association phase, presented as percentage of inhibition.

 
SPR and solid-phase inhibition were used to analyze the inhibitory effects of various other GAGs on the interactions between opticin and heparin or PIM HS (Fig. 6) . Again the results were similar for heparin (using solid-phase assays and SPR) and PIM HS (using SPR). CSA (which is mainly chondroitin 4-sulfate) and CSB (which is mainly dermatan sulfate) were much more effective at competing the interactions than CSC (which is mainly chondroitin 6-sulfate) and hyaluronan. Therefore, CSA and CSB bound to opticin, but CSC and hyaluronan did not show significant binding in these assays. It can be implied that the 4-sulfate group of chondroitin (and possibly dermatan) sulfate is important for opticin binding, whereas the 6-sulfate group contributes less.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 6. Inhibition of opticin binding to heparin and HS binding by various GAGs. (A) Opticin was preincubated with 0.2 mg/mL heparin, CSA, CSB, CSC, or hyaluronan, and the level of binding to immobilized heparin albumin was determined by ELISA. (B) Opticin was preincubated with 0.01 mg/mL heparin, CSA, CSB, CSC, or hyaluronan, then injected onto heparin- or PIM HS–coated streptavidin SA chip surfaces. Chart records the level of opticin bound to the surface at the end of the association phase, presented as percentage of inhibition.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study showed for the first time that opticin binds to type XVIII collagen through its HS chains. Binding was not completely eliminated by K5 lyase digestion, and the residual binding may have been due to the recombinant form of type XVIII collagen being partially substituted with chondroitin sulfate instead of HS; however, the natural form is only substituted with HS.13 As the binding of opticin to the recombinant type XVIII collagen was predominantly through interactions with HS, a detailed analysis of opticin binding to HS and heparin was then undertaken using solid-phase binding assays and SPR. The Langmuir 1:1 binding model of the SPR data gave a good fit for both heparin and HS, suggesting that the binding involves an interaction between a distinct heparin or HS binding site on opticin and a specific sulfated region of heparin or HS. The heterogenous ligand parallel reaction model was evaluated (data not shown), but this did not give a better fit, suggesting that opticin does not recognize multiple sites on heparin and HS of distinct affinity. The fact that opticin bound less strongly to BK HS than PIM HS suggests that opticin binds preferentially to highly sulfated forms: BK HS contains on average <1 sulfate group per disaccharide and ~10% N-unsubstituted GlcN residues; by contrast, PIM HS has 1.5 sulfates per disaccharide and <2% N-unsubstituted amino sugars.15 Furthermore, the finding that opticin bound PIM HS (KD 73 nM) with similar affinity to heparin (KD 43 nM) suggests that opticin preferentially binds to S-domains, highly sulfated regions within HS that resemble heparin.

As well as preferentially binding highly sulfated forms of HS, opticin showed distinct binding preferences for oligosaccharides of certain length and sulfation pattern. Opticin bound preferentially heparin oligosaccharides composed of eight or more monosaccharides. The sharp increase in apparent binding strength from dp6 to 8 (Fig 4) with little enhancement of binding by longer saccharides indicates that the GAG-recognition site accommodates eight sugar residues, corresponding to an approximate length of 3.6 nm.16 Heparin adopts a helical conformation in solution, with clusters of sulfate groups in each repeating disaccharide disposed on opposite sides of the helical axis.16 The higher affinity for a dp8 fragment is unlikely to be due to two opticin molecules binding on opposite faces of the helix because the biosensor data fit with a 1:1 binding stoichiometry. It seems more likely that an octamer sequence fits into a binding groove on the opticin surface that interacts with sulfate and COO– groups on both sides of the helix. The dp6 fragment probably fails to make all the ionic and hydrogen bonds that are readily formed with longer sequences, ≥dp8. The S-domains of HS are commonly between 6 and 12 sugars in length, so providing the sulfation requirements are met, it is highly probable that the HS of extracellular matrix proteoglycans such as type XVIII collagen will contain binding sites for opticin.

The competition assays with selectively desulfated heparin oligosaccharides indicated that 2-O-sulfates, 6-O-sulfates and N-sulfates are all involved in binding, but that 2-O-sulfates contribute least to the interaction, thereby demonstrating selectivity in the molecular recognition between opticin and heparin or HS. Thus, opticin belongs to the group of heparin- and HS-binding proteins that require a well-defined length and sulfation pattern for optimum binding, which also includes endostatin, fibronectin, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, and antithrombin III; by contrast, others, such as thrombin and lactoferrin, bind less specifically to multiple sites. Direct comparisons can be made with published data on the binding of endostatin to heparin and HS,17 18 as the same reagents were used in the present study. Opticin binds ~20 times more strongly to heparin and HS than endostatin. Endostatin interacts optimally with heparin or HS oligosaccharides composed of 12 or more sugar units, whereas opticin preferentially binds to oligosaccharides containing 8 or more sugars. Endostatin and opticin interact with N-, 2-O-, and 6-O- sulfates, and in both cases, the N- and 6-sulfates are the most important functional groups. By contrast, the widely studied extracellular matrix protein fibronectin, which binds to HS and heparin through its hep11 domain, shows a preference for 2-sulfates over 6-sulfates.19 Therefore, these different proteins prefer specific but distinct heparin and HS binding sites.

Opticin and type XVIII collagen colocalize at the ILM,9 a light microscopic structure containing cortical vitreous, the ILL, and Müller cell footplates. Within the ILM, the vitreous collagen fibrils of the posterior vitreous cortex are generally orientated parallel to the ILL surface, suggesting that intermediary molecules provide a link between them, thus maintaining vitreoretinal adhesion.20 Type XVIII collagen is a basement membrane component, and recent electron microscopic studies have located this molecule mainly on the vitreal side of the ILL.21 Opticin is associated with the vitreous collagen fibrils, so by binding the HS of type XVIII collagen, opticin may link the vitreous collagen fibrils to the ILL, thus fulfilling the role of a "molecular glue" that contributes toward vitreoretinal adhesion. Type XVIII collagen has been implicated in vitreoretinal adhesion because type XVIII collagen null mice tend to have vitreoretinal disinsertion,21 and patients with Knobloch syndrome (caused by mutations in type XVIII collagen) have early posterior vitreous detachments.22 Equally, opticin could bind to the HS chains of other ILL proteoglycans, including agrin and perlecan. If the interaction between opticin and the ILL HS proteoglycans is important in vitreoretinal attachment, this interaction could provide a new target for pharmacological vitreoretinal disinsertion.23

The present study showed that the GAG-binding site in opticin is not specific for HS and heparin, as it also interacts with chondroitin 4-sulfate and dermatan sulfate, whereas binding to chondroitin-6-sulfate was not clearly demonstrable in the assays used. There are at least two chondroitin sulfate proteoglycans in the vitreous with which opticin could interact, including type IX collagen and versican.24 25 26 Chondroitin 4-sulfate is the predominant form in bovine type IX collagen.24 However, the overall CS content of vitreous from different species showed variations in sulfation patterns, with 6-sulfated chondroitin sulfate predominating in human vitreous, but the 4-sulfated form predominating in pig, goat, and sheep vitreous.27 As opticin is associated with vitreous collagen fibrils, its ability to bind chondroitin 4-sulfate may allow the collagen fibrils to be indirectly linked to versican. Furthermore, adjacent vitreous collagen fibrils could be linked together by the chondroitin sulfate chains of type IX collagen on one collagen fibril extending across to bind opticin on an adjacent fibril. This type of morphology has been observed in electron microscopy studies of the vitreous, labeling the chondroitin sulfate chains with Cupromeronic blue.28 29 Thus opticin, through its interactions with GAGs, could play a role both in the maintenance of vitreoretinal adhesion and in the structural organization of the vitreous gel.


   Acknowledgements
 
The authors thank Diana Ruiz Nivia for technical assistance and Sylvie Ricard-Blum (Institut de Biologie Structurale, Grenoble, France) for assistance with analysis of the SPR data.


   Footnotes
 
Supported by The Wellcome Trust. PNB is a Wellcome Trust Senior Research Fellow in Clinical Science.

Submitted for publication July 11, 2005; revised August 11, 2005; accepted September 29, 2005.

Disclosure: V.J. Hindson, None; J.T. Gallagher, None; W. Halfter, None; P.N. Bishop, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Paul N. Bishop, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK; paul.bishop{at}manchester.ac.uk.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reardon AJ, Le Goff M, Briggs MD, et al. Identification in vitreous and molecular cloning of opticin, a novel member of the family of leucine-rich repeat proteins of the extracellular matrix. J Biol Chem. 2000;275:2123–2129.[Abstract/Free Full Text]
  2. Le Goff MM, Hindson VJ, Jowitt TA, Scott PG, Bishop PN. Characterization of opticin and evidence of stable dimerization in solution. J Biol Chem. 2003;278:45280–45287.[Abstract/Free Full Text]
  3. Scott PG, Grossmann JG, Dodd CM, Sheehan JK, Bishop PN. Light and X-ray scattering show decorin to be a dimer in solution. J Biol Chem. 2003;278:18353–18359.[Abstract/Free Full Text]
  4. Scott PG, McEwan PA, Dodd CM, Bergmann EM, Bishop PN, Bella J. Crystal structure of the dimeric protein core of decorin, the archetypal small leucine-rich repeat proteoglycan. Proc Natl Acad Sci USA. 2004;101:15633–15638.[Abstract/Free Full Text]
  5. Sanders EJ, Walter MA, Parker E, Aramburo C, Harvey S. Opticin binds retinal growth hormone in the embryonic vitreous. Invest Ophthalmol Vis Sci. 2003;44:5404–5409.[Abstract/Free Full Text]
  6. Takanosu M, Boyd TC, Le Goff M, et al. Structure, chromosomal location, and tissue-specific expression of the mouse opticin gene. Invest Ophthalmol Vis Sci. 2001;42:2202–2210.[Abstract/Free Full Text]
  7. Bishop PN, Takanosu M, Le Goff M, Mayne R. The role of the posterior ciliary body in the biosynthesis of vitreous humour. Eye. 2002;16:454–460.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  8. Frolova EI, Fokina VM, Beebe DC. The expression pattern of opticin during chicken embryogenesis. Gene Expr Patterns. 2004;4:335–338.[CrossRef][Medline][Order article via Infotrieve]
  9. Ramesh S, Bonshek RE, Bishop PN. Immunolocalisation of opticin in the human eye. Br J Ophthalmol. 2004;88:697–702.[Abstract/Free Full Text]
  10. Friedman JS, Faucher M, Hiscott P, et al. Protein localization in the human eye and genetic screen of opticin. Hum Mol Genet. 2002;11:1333–1342.[Abstract/Free Full Text]
  11. Halfter W, Dong S, Schurer B, et al. Composition, synthesis, and assembly of the embryonic chick retinal basal lamina. Dev Biol. 2000;220:111–128.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  12. Murphy KJ, Merry CL, Lyon M, Thompson JE, Roberts IS, Gallagher JT. A new model for the domain structure of heparan sulfate based on the novel specificity of K5 lyase. J Biol Chem. 2004;279:27239–27245.[Abstract/Free Full Text]
  13. Dong S, Cole GJ, Halfter W. Expression of collagen XVIII and localization of its glycosaminoglycan attachment sites. J Biol Chem. 2003;278:1700–1707.[Abstract/Free Full Text]
  14. Goger B, Halden Y, Rek A, et al. Different affinities of glycosaminoglycan oligosaccharides for monomeric and dimeric interleukin-8: a model for chemokine regulation at inflammatory sites. Biochemistry. 2002;41:1640–1646.[CrossRef][Medline][Order article via Infotrieve]
  15. Wei Z, Lyon M, Gallagher JT. Distinct substrate specificities of bacterial heparinases against N-unsubstituted glucosamine residues in heparan sulfate. J Biol Chem. 2005;280:15742–15748.[Abstract/Free Full Text]
  16. Mulloy B, Forster MJ, Jones C, Davies DB. N.M.R. and molecular-modelling studies of the solution conformation of heparin. Biochem J. 1993;293:849–858.
  17. Blackhall FH, Merry CL, Lyon M, et al. Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates. Biochem J. 2003;375:131–139.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  18. Ricard-Blum S, Feraud O, Lortat-Jacob H, et al. Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations. J Biol Chem. 2004;279:2927–2936.[Abstract/Free Full Text]
  19. Lyon M, Rushton G, Askari JA, Humphries MJ, Gallagher JT. Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin. J Biol Chem. 2000;275:4599–4606.[Abstract/Free Full Text]
  20. Bishop PN. Structural macromolecules and supramolecular organisation of the vitreous gel. Prog Retin Eye Res. 2000;19:323–344.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  21. Fukai N, Eklund L, Marneros AG, et al. Lack of collagen XVIII/endostatin results in eye abnormalities. EMBO J. 2002;21:1535–1544.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  22. Duh EJ, Yao YG, Dagli M, Goldberg MF. Persistence of fetal vasculature in a patient with Knobloch syndrome: potential role for endostatin in fetal vascular remodeling of the eye. Ophthalmology. 2004;111:1885–1888.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  23. Sebag J. Is pharmacologic vitreolysis brewing?. Retina. 2002;22:1–3.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  24. Bishop PN, Crossman MV, McLeod D, Ayad S. Extraction and characterization of the tissue forms of collagen types II and IX from bovine vitreous. Biochem J. 1994;299:497–505.
  25. Reardon A, Heinegard D, McLeod D, Sheehan JK, Bishop PN. The large chondroitin sulphate proteoglycan versican in mammalian vitreous. Matrix Biol. 1998;17:325–333.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  26. Theocharis AD, Papageorgakopoulou N, Feretis E, Theocharis DA. Occurrence and structural characterization of versican-like proteoglycan in human vitreous. Biochimie (Paris). 2002;84:1237–1243.
  27. Noulas AV, Skandalis SS, Feretis E, Theocharis DA, Karamanos NK. Variations in content and structure of glycosaminoglycans of the vitreous gel from different mammalian species. Biomed Chromatogr. 2004;18:457–461.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  28. Scott JE. The chemical morphology of the vitreous. Eye. 1992;6:553–555.
  29. Bishop PN, Holmes DF, Kadler KE, McLeod D, Bos KJ. Age-related changes on the surface of vitreous collagen fibrils. Invest Ophthalmol Vis Sci. 2004;45:1041–1046.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Br. J. Ophthalmol.Home page
D. C Assheton, E. P Guerin, C. M Sheridan, P. N Bishop, and P. S Hiscott
Neoplastic transformation of ciliary body epithelium is associated with loss of opticin expression
Br. J. Ophthalmol., February 1, 2007; 91(2): 230 - 232.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
W. Halfter, U. Winzen, P. N. Bishop, and A. Eller
Regulation of eye size by the retinal basement membrane and vitreous body.
Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3586 - 3594.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hindson, V. J.
Right arrow Articles by Bishop, P. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hindson, V. J.
Right arrow Articles by Bishop, P. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS