also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of 26 this study was to investigate the binding of opticin to the collagen II-containing fibrils found 27 in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated 28 that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the 29 gap region of the collagen D-period corresponding to the e2 stain band; this is the first 30 demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind 31 thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but 32 shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or 33 V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no 34 difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis 35 experiments showed that opticin did not affect fibril formation. We propose that when 36 opticin is bound to collagen fibrils, rather than influencing their morphology it instead 37 hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary 38 bridge linking the collagen fibrils to other non-collagenous molecules. 39 Introduction 42 Opticin is a member of the extracellular matrix small leucine-rich repeat 43 protein/proteoglycan (SLRP) family that was first identified in vitreous humour and 44 subsequently in cartilage [1, 2] . There are 18 members of the SLRP family which have been 45 divided into 5 classes based upon phylogeny [3] . Opticin is in class III and is therefore 46 closely related to the other class III SLRPs, epiphycan and osteoglycin/mimecan. Little is 47 known about the functions of class III SLRPs, but opticin has been shown to possess anti-48 angiogenic properties both in vitro and in vivo, and mechanistic experiments suggested that 49 its anti-angiogenic effects are elicited by opticin binding to collagen resulting in steric 50 inhibition of endothelial cell integrin-collagen interactions [4, 5] . However, this conclusion 51 was drawn from experiments using monomeric collagens, whereas in vivo these collagens 52 are in fibrillar structures. Furthermore, recent work has demonstrated that integrins that 53 bind collagen monomers do not bind collagen in fibrils directly, instead these integrins 54 interact with non-collagenous molecules associated with the surface of the fibrils [6] . It has 55 also been demonstrated that opticin deficient mice are protected against osteoarthritis 56 because the lack of opticin results in an alteration in the amounts of other SLRPs in cartilage 57 leading to altered fibril diameter and increased protection from proteolysis [7]. The above 58 studies prompted us to investigate interactions between opticin and collagen fibrils. 59 The vitreous humour contains a network of fine collagen fibrils of uniform thickness (10-20 60 nm depending upon species) that impart gel-like properties to the tissue [8], whereas in 61 human cartilage there are both thin ~ 18 nm diameter fibrils and thicker fibrils [9,10]. The 62 collagen fibrils from these two tissues are very similar in their collagen composition: They 63 are heterotypic (mixed in composition) fibrils in which fibril-forming type II collagen 4 Opticin binding to collagen fibrils 64 predominates, the fibrils also contain collagen IX and XI or V/XI. Collagen IX is a part-time 65 proteoglycan and a member of the FACIT (Fibril-Associated Collagens with Interrupted   66 Triple-helices) group of collagens that is located on the surface of the fibrils and has a role in 67 preventing fibril aggregation [9, 11] . Collagen XI and V/XI are very closely related fibril-68 forming collagens both containing an α1(XI) chain, in addition collagen XI contains α2(XI) 69 and α3(XI) chains and vitreous collagen V/XI contains an α2(V) chain, but the identity of the 70 third α-chain is uncertain [12] . Hybrid molecules containing α1(V) and α2(XI) collagen chains have 71 also been described in articular cartilage [13] . The collagen content of the fibrils determines 72 their morphology and it is possible to generate heterotypic collagen fibrils in vitro that have 73 identical morphology to their tissue counterparts [14] . Natural collagen II alone does not 74 form fibrils in vitro but rather forms short, tapered tactoids with D-periodic banding. 75 However, when mixed with sufficient type XI collagen, thin fibrils of uniform diameter are 76 formed. Here, using fibrils formed in vivo and in vitro, we investigated the binding of opticin 77 to collagen fibrils and the effects of opticin on fibril morphology. The vitreous collagen fibrils of opticin null and wild-type mice appeared morphologically 83 identical (Fig 1A,B) . The mean diameter of the fibrils from the opticin null mice was 9.99 nm 84 +/-2.12 nm (n = 286) and from wild-type mice was 10.77 nm +/-2.17 nm (n = 290); 5 Opticin binding to collagen fibrils 85 statistical analysis revealed no significant difference (p = 0.38); this and subsequent 86 statistical analyses used the two-sided Student`s t-test. Bovine vitreous collagen fibrils were analysed by immunoelectron microscopy using 100 polyclonal opticin antibodies and gold-conjugated anti-rabbit immunoglobulins. These 101 collagen fibrils showed extensive labelling ( Fig 1C) . Whilst opticin has been identified in 102 cartilage [2,7], we could not detect endogenous opticin by immunogold electron microscopy 103 in our preparations of cartilage fibrils preparations ( Fig 1D) . Therefore, we investigated the 104 localisation of opticin after it had been added to authentic cartilage fibril preparations that 105 had been digested with chondroitin ABC lyase (to prevent steric hindrance from 6 Opticin binding to collagen fibrils 106 glycosaminoglycans). We then observed that opticin localised to the thin fibrils, but not 107 thick collagen fibrils ( Fig 1E) . Binding of opticin to preformed fibrils and tactoids 163 Opticin bound to the surface of fibrils containing collagens II/XI or II/IX/XI when added after 164 reconstitution of fibrils (Fig. 3A,B) . Scant opticin binding was also observed when added to 165 reconstituted fibrils composed of just type XI collagen (Fig. 3C,D) . However, no binding was 166 observed to the thick, banded tactoids formed by type II collagen alone (Fig 3E,F) . vitro. Opticin did not bind tactoids formed from just type II collagen, and did not bind to 207 fibrils just composed of just collagen I (data not shown), despite binding to monomeric 208 collagens I and II. There was limited binding to fibrils composed of just collagen XI. 209 However, once copolymers were formed from collagens II/XI or II/IX/XI resembling vitreous 210 or thin cartilage collagen fibrils, extensive binding of opticin was observed. These data show 211 that collagen II must form a copolymer with collagen XI (and presumably collagen V/XI in 212 vitreous) to assemble into thin fibrils that then provide a specific opticin binding site. This is 213 the first time that a member of the SLRP family has been shown to require a particular 214 fibrillar collagen copolymer for binding, and this work raises the possibility that other 11 Opticin binding to collagen fibrils 215 members of the SLRP family have similarly exacting requirements for binding to collagen 216 fibrils. 217 The above data suggests that the function of opticin is to modulate the surface interactions 218 of vitreous and thin cartilage collagen fibrils rather than influence their morphology. The was then added at a concentration ranging between 1 µg/ml to 50 µg/ml dissolved in 100 µl 255 of TBS. After incubating for 1 h at room temperature the wells were washed with TBS-T. 256 Bound opticin was subsequently detected with the opticin antibody followed by a 13 Opticin binding to collagen fibrils 257 horseradish peroxidase-conjugated antibody raised against rabbit immunoglobulins. 258 Absorbance at 490 nm was measured using a microplate reader. 259 In Vitro Fibrillogenesis 260 Collagens II, IX, and XI from cultures of chick embryo sternal chondrocytes in agarose gels 261 were purified in their native and fibrillogenesis-competent states as previously described 262 [14] . Mixtures of collagens II and XI or collagens II, IX, and XI in were dissolved 0.1 M Tris- Electron microscopy with gold-conjugated opticin 288 Recombinant opticin was incubated with 6 nm gold sol following manufacturer's 289 instructions (Aurion). Isolated vitreous collagen fibrils were incubated with opticin gold 290 conjugate for 2 h at room temperature prior to absorbing onto carbon coated nickel grids. 291 The grids were washed several times with distilled water and negatively stained with 5% 292 uranyl acetate for 12 min. Electron micrographs were taken at 120 kV with an FEI Tecnai 293 transmission electron microscope. Densitometric scanning of the D-period was undertaken 294 as described previously [16] and the position of gold particles along the D-period was 295 analysed. The polarity of each fibril was determined by cross-correlation with a reference 296 average axial stain exclusion pattern (ASEP) and this also allowed the position of the gold 297 particle label within the D-period to be determined. 