Bovine Interphotoreceptor Matrix Research Articles

Interphotoreceptor matrix based biomaterial: Impact on human retinal progenitor cell attachment and differentiation.

While cell transplantation therapies show great promise as treatments for retinal degeneration, the challenge of low cell survival upon transplantation motivates exploration of materials that may serve as cell delivery vehicles and promote survival and differentiation. In this study, we explored the native matrix that surrounds the outer segments of photoreceptors and promotes their homeostasis, interphotoreceptor matrix (IPM), as a substrate for human retinal progenitor cells (hRPCs). Bovine IPM was characterized to determine its structure and biochemical composition, and processed to develop substrates for cells. Cell viability, morphology, proliferation and expression of photoreceptors marker genes were studied on IPM-based substrates in vitro. We explored different preparations of IPM as a scaffold. Lectin staining revealed that a distinct honeycomb structure of native IPM is lost during centrifugation to prepare a more concentrated suspension of matrix. Biochemical analysis of bovine IPM indicated presence of glycosaminoglycans and proteins. IPM mediated hRPC attachment and spreading with no signs of cytotoxicity. Cells proliferated more on native IPM substrates compared to IPM that was centrifuged to create a concentrated suspension. Cells cultured on IPM substrates expressed markers of photoreceptors: rhodopsin, NRL and ROM1. Together this data supports further exploration of IPM as a tool for retinal tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 891-899, 2018.

Association of matrix metalloproteinases with interphotoreceptor retinoid binding protein

PURPOSE. Interphotoreceptor retinoid binding protein (IRBP) is one of the major components of the interphotoreceptor matrix (IPM) where it may function in retinoid transport between the photoreceptor cells and the retinal pigment epithelium. In the course of studies of the metalloproteinases (MPs) of the IPM, we found that some MPs remain associated with IRBP through the standard purification scheme. We wished to report this finding as a caution to those working with IRBP prepared from fresh tissue. METHODS. IRBP was prepared from bovine IPM by procedures commonly used for its purification, including ion exchange, concanavalin A affinity and gel filtration chromatographies. The MPs were detected by zymography, both gelatin and casein, run with and without preactivation. RESULTS. Through each step of the purification both gelatinase and, especially, caseinase (stromelysin) activities were associated with IRBP. Inclusion of gelatin affinity chromatography did not totally remove the gelatinases. Much of this activity was latent and was only revealed following fractionation or by preactivation before zymography. Whether the fractionation steps remove an inhibitor or simply provide conditions appropriate for the activation of the latent zymogen forms is not known. CONCLUSIONS. The close association of the MPs and IRBP may suggest a functional role for this complex. As a practical consideration, it is likely that many preparations of IRBP may be contaminated with one or more MPs. We found no evidence for any other class of proteinase. Caution should thus be exercised in using an IRBP preparation purified from fresh tissue. It should be monitored for proteinase activity by zymography and/or prepared or stored in the presence of EDTA or dithiothreitol as much as possible.

Identification of Pigment Epithelium-Derived Factor in the Interphotoreceptor Matrix of Bovine Eyes

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-rPEDF, that immunoreacts in a specific, sensitive, and linear fashion with PEDF protein, and furthermore, blocks its neurotrophic activity. We show that Ab-rPEDF specifically recognizes a 49,500- M r polypeptide on Western transfers of a wash of the extracellular matrix between the retinal pigment epithelium and the neural retina-termed interphotoreceptor matrix (IPM). PEDF is present as approximately 1% of total soluble IPM protein. Starting with an IPM wash, PEDF protein is purified 164-fold to near homogeneity by ammonium sulfate fractionation and cation-exchange chromatography, with a recovery of 47%. The highly purified protein has an apparent M r of 49,500 ± 1,500 as assessed by SDS-polyacrylamide gel electrophoresis, and a native p I of 7.0-7.7. It elutes as a single peak on gel-filtration chromatography with a retention time immediately behind that of ovalbumin (43,000 M r ). N-glycosidase treatment indicates that each PEDF molecule has a 5% carbohydrate content attached to internal asparagine residue(s). Amino terminal sequence of the purified PEDF reveals removal of an amino-terminal peptide region for the mature protein. Purified PEDF has neurotrophic activity on human retino-blastoma cells, as previously observed for IPM. The neurotrophic activities of both PEDF and IPM are blocked by antiserum Ab-rPEDF. Altogether, PEDF is present in the bovine IPM as a soluble, extracellular, monomeric glycoprotein that by itself confers neurotrophic activity to the IPM. Thus, native PEDF isolated and purified as described here should prove useful for biochemical studies as well as other approaches.

Neuron-specific enolase: a neuronal survival factor in the retinal extracellular matrix?

To identify soluble proteins of the retinal interphotoreceptor matrix (IPM), we isolated IPM from the bovine eye by gentle lavage and subjected it to SDS-PAGE. In the resultant gel, a 46 kDa band was particularly prominent and appeared to be a single protein. This protein was electroblotted to nitrocellulose membrane, digested with trypsin, and selected peptides were isolated by HPLC and subjected to Edman microsequencing. The amino acid sequences of the peptides were found to be virtually identical to that of human neuron-specific enolase (NSE). A monoclonal antibody specific for human NSE confirmed the presence of this enzyme in the bovine IPM by both Western blotting and immunocytochemical analysis. Immunofluorescence microscopy demonstrated that NSE is mainly localized to the basal domain of the IPM surrounding photoreceptor cells but is also prominent in the inner segments of the cone photoreceptor neurons. When NSE was added to cultures of human retinoblastoma cells, no effect on morphology was observed. However, a positive effect on cell growth and/or survival was readily apparent. It thus seems that not only is NSE a significant component of the retinal extracellular matrix, but that it could function as a survival (neuronotrophic) factor for photoreceptor neurons.

Polarized Distribution of Metalloproteinases in the Bovine Interphotoreceptor Matrix

Previous studies from this laboratory had indicated that metalloproteolytic activity was present in the interphotoreceptor matrix. This report extends those observations by providing evidence for the presence of multiple forms of metalloproteinase and for their polarized distribution within the interphotoreceptor matrix as determined by zymogram analysis on substrate-loaded gels.Retinal pigment epithelium-associated interphotoreceptor matrix, both unfractionated as well as fractions separated by gel filtration, exhibited bands of proteolytic activity on gelatin-loaded gels at about 70-75 kDa and 90 kDa, possibly due to gelatinases A and B (MMP-2 and MMP-9, respectively). In contrast, no neutral proteolytic activity was seen in retina-associated interphotoreceptor matrix unless it was first fractionated by gel filtration, whereupon a diversity of forms was exhibited, including additional major bands of proteolytic activity at about 150 kDa and 100 kDa, on gelatin-loaded gels, and at about 180 kDa, on casein-loaded gels, along with many minor species. In all cases, all proteinase activity was inhibited by chelating agents.Since these enzymes may be involved in the turnover and remodeling of components present in the interphotoreceptor matrix, many of which are distributed in a compartmentalized fashion, this unequal distribution of metalloproteinases may be correlated with substrate specificity.

Characterization and Novel Activation of 72-kDa Metalloproteinase in Retinal Interphotoreceptor Matrix and Y-79 Cell Culture Medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M r gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification, typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

The presence of neutral metalloproteolytic activity and metalloproteinase inhibitors in the interphotoreceptor matrix.

Neutral proteolytic activity, having a pH optimum of about 7, was present in the high molecular weight fraction of bovine interphotoreceptor matrix (IPM) separated by gel filtration on Sephacryl S-500. The enzyme(s) was active toward a number of exogenous substrates, including albumin, Azocoll, and gelatin. However, it was inactive toward a synthetic substrate for bacterial collagenase. Proteolytic activity was proportional to protein; however, the time course of the reaction was nonlinear, suggesting that "activation" of a precursor form might be necessary. Of a number of specific inhibitors tested, those directed toward metalloproteinases (1,10-phenanthroline greater than EDTA greater than EGTA) proved most effective. While activity was also inhibited by sulfhydryl reagents and dithiothreitol, inhibitors specific for cysteine proteinases were ineffective. Higher specific activity was present in IPM obtained from retinal pigment epithelium (RPE) than from retina. An endogenous proteinase inhibitor(s) was also found in IPM from both RPE and retina. It was effective against the endogenous metalloproteolytic activity of IPM and also against thermolysin, but not against trypsin or papain. Fractionation of IPM on Sephacryl S-500 revealed a broad peak of inhibitory activity at molecular weights of less than 10(5) daltons. This is the first report of the presence of neutral proteolytic activity and metalloproteinase inhibitor(s) in bovine IPM. These materials may function in concert to maintain the proper level of various components within this matrix.

High molecular weight mucin-like glycoproteins of the bovine interphotoreceptor matrix

A very high molecular weight mucin-like glycoprotein was isolated by gel filtration of interphotoreceptor matrix (IPM) from fresh bovine eyes and purified to apparent homogeneity by cesium chloride/guanidine hydrochloride (GuHCl) equilibrium density gradient centrifugation. Although a molecular weight in excess of 10 7 Da is suggested by gel filtration, the presence of SDS or GuHCl did not alter its elution position, indicating that the large size was not simply due to aggregation. Treatment of this material with disulfide reagents, however, led to a decrease in molecular size. On a relative basis, substantially more of this glycoprotein is present in IPM prepared from retina than from retinal pigment epithelium. While the carbohydrate and amino acid composition are not those of a true ‘mucin’, the large size and many other properties are quite ‘mucin-like’. The carbohydrate composition suggests the presence of both N- and O-glycosidically linked sugar chains. The presence of a mucin-type O-glycosidic linkage is indicated by its susceptibility to alkaline cleavage, with concomitant loss of serine and threonine and increase in 240 nm absorbance; production of a fluorescent product upon reaction with cyanoacetamide; lectin binding properties; and production of N-acetylgalactosaminitol upon alkaline borohydride elimination. This glycoprotein was digested by pronase and trypsin, confirming its protein nature, but was resistant to digestion with chondroitin ABC lyase, hyaluronidase and heparinase, as well as RNAase, indicating that these components were not present to any appreciable extent. ELISA for cartilage keratan sulfate was also negative. Centrifugation in CsCl GuHCl gradients indicated a density much lower than that of a proteoglycan or nucleic acid as well. In vitro biosynthetic studies suggest that both retina and retinal pigment epithelium may be major sources of material in the IPM. The elution patterns of radioactivity were strikingly similar to the UV elution patterns of IPM. The medium from retinal incubations contained very high molecular weight material which was resistant to enzymes which hydrolyse glycosaminoglycans, suggesting that retina may be the source of this high molecular weight, mucin-like glycoprotein.

Effect of light on endogenous ligands carried by interphotoreceptor retinoid-binding protein

Interphotoreceptor retinoid-binding protein (IRBP) is a vitamin A carrier present only in the extracellular material lying between the neural retina and the retinal pigment epithelium of vertebrate eyes. The amount of retinol bound endogenously by IRBP in this interphotoreceptor space is known to increase upon illumination. This finding led to the hypothesis that IRBP may act as a shuttle for vitamin A during the visual cycle that regenerates rhodopsin. In the present work, we separated IRBP from other retinoid-binding proteins in bovine interphotoreceptor matrix preparations by means of size-exclusion chromatography. IRBP's endogenous ligands were retained during this procedure and were then extracted into hexane and analysed by normal-phase HPCL. We found that IRBP carries, in a light-dependent manner, all the retinoid isomers involved in the visual cycle. For dark-adapted eyes the amounts of bound ligands are (in nmol per eye) 0·09 all- trans retinol, 0·11 11- cis retinol, 0·04 all- trans retinal, 0·16 11- cis retinal, and 0·07 retinyl esters. For light-adapted eyes the amount of all- trans retinol was found to increase by a factor of five, and that of 11- cis retinal to decrease by a factor of four. (These eyes contain 3·1 nmol of IRBP, which does not change in amount with lighting conditions.) Thus, the major endogenousligand of IRBP is 11- cis retinaldehyde in the dark and all- trans retinol in the light. The data are consistent with a role for IRBP as a non-selective scavenger and stabilizer of retinoids released from photoreceptors and pigment epithelial cells. However, it cannot be concluded from these data that IRBP is involved in directed transport of retinoids across the interphotoreceptor matrix, since there is no evidence for the appropriate spatial gradients in the ligands bound to the protein.

Selective presence of acid hydrolases in the interphotoreceptor matrix

Adler and Martin (1983, Curr. Eye Res. 2, 359-66) found cathepsin D to be present in crude preparations of bovine interphotoreceptor matrix (IPM). The purpose of the present study was to determine, by investigating several acid hydrolases in purer IPM samples, whether hydrolytic enzymes abundant in RPE lysosomes were present also as normal components of the IPM. IPM was prepared from bovine eyes by the introduction of a small bleb of buffer between the neural retina and the RPE. These IPM samples were free from significant contamination by surrounding tissues; they contained IRBP as their only major protein, and had negligible amounts of lactate dehydrogenase and ROS-specific proteins. Most acid hydrolases were assayed fluorometrically by measuring the 4-methylumbelliferone released upon hydrolysis of appropriate derivatives; the substrate for cathepsin was hemoglobin. The amounts of the enzymes found in the IPM were far from uniform and could not be correlated with enzyme activities in either RPE or retina homogenates. The hydrolases in the IPM varied in amount from beta-galactosidase (28% of the RPE level), through N-acetyl-beta-glucosaminidase (20%), alpha-fucosidase (15%), beta-glucuronidase (12%), alpha-glucosidase (8%), cathepsin D (7%), alpha-mannosidase (7%), down to beta-glucosidase, acid phosphatase, and acid lipase (trace amounts, less than 1%). These results agree with the relative amounts of enzymes found by Wilcox (1987) to be secreted into the medium by cultured human RPE cells. Furthermore, the rank order of hydrolases in the IPM is the same as that for hydrolases secreted (but not recaptured) by human fibroblasts in I-cell disease. The conclusion from these correlations is that lysosomal enzymes are probably secreted, as a normal process, by the RPE into the IPM, where they may have a role in digesting shed outer segments and in catabolizing IPM components.

Phosphorylation of interphotoreceptor retinoid-binding protein (IRBP)

The phosphorylation of interphotoreceptor retinoid-binding protein (IRBP), the major soluble (glycolipo) protein of the interphotoreceptor matrix (IPM) and a putative intercellular retinoid-transport vechicle, has been examined in a crude bovine IPM wash using [γ- 32P]ATP. SDS-polyacrylamide gel electrophoresis and autoradiography, size-exclusion high-performance liquid chromatography (HPLC) and ion-exchange HPLC all showed IRBP to be phosphorylated in this system. The phosphorylation probably is of serine and/or threonine residues rather than of tyrosine. Interestingly, phosphorylated IRBP was bound tightly to concanvalin A (Con A)-Sepharose and was not eluted by 50 mM α-methyl- d-mannoside indicating a marked alteration in binding characteristics upon phosphorylation.

Levels of alpha- and gamma-tocopherol in human eyes: evaluation of the possible role of IRBP in intraocular alpha-tocopherol transport

Alpha-tocopherol was distributed almost equally between the retina and its underlying pigmented layers (pigment epithelium and choroid). Only 8.4% of the total alpha-tocopherol occurred in the iris and ciliary body. Alpha-tocopherol content was expressed as amount per eye, per cm2, and per 100 g wet weight. The combined retina and pigment epithelium-choroid contained 2.9 +/- 1.0 mg/100 g wet weight (means +/- SD, n = 30 donors). Gamma-tocopherol represented 20.9 +/- 12.2 mol % of the alpha-tocopherol. The anterior tissues contained 0.4 +/- 0.2 mg/100 g (n = 19 donors). No significant correlation with age was found. Purified bovine interstitial retinol-binding protein (IRBP) bound exogenous 3H-alpha-tocopherol, which could be displaced by unlabeled all-trans retinol (KD = 10(-6) M). Much higher concentrations of unlabeled alpha-tocopherol were required to achieve a partial displacement of bound 3H-all-trans retinol. No endogenous alpha-tocopherol could be detected in bovine interphotoreceptor matrix.

Interphotoreceptor matrix glycosaminoglycans in bovine eye.

The glycosaminoglycans of bovine interphotoreceptor matrix were analyzed biochemically and compared with those of neural retina and retinal pigment epithelium. Chondroitin sulfates extracted from these three tissues were electrophoretically low-sulfated and composed of various kinds of chondroitin sulfate isomers. Interphotoreceptor matrix and retinal pigment epithelium contained hyaluronic acid as another glycosaminoglycan. With regard to neural retina, chemical characteristics of another component identified on the membrane are still unknown.

Two-dimensional gel electrophoretic analysis of lectin receptors in the bovine interphotoreceptor matrix

Proteins and glycoproteins of the bovine interphotoreceptor matrix (IPM) with or without neuraminidase treatment was analysed by two-dimensional gel electrophoresis combined with Western blotting and staining with seven horseradish peroxidase-labeled lectins. More than 80 spots of proteins and glycoproteins were revealed on the gel. Nineteen spots (or groups of spots) were revealed by staining with five lectins [concanavalin A, wheat germ agglutinin (WGA), peanut agglutinin (PNA), Ricinus communis agglutinin-1 (RCA-1) and soybean agglutinin (SBA)]; some of those spots were specific for one lectin and others reacted with several lectins. We could not detect distinct spots reacting with Dolichos biflorus agglutinin or Ulex europaeus agglutinin-1. Neuraminidase digestions of the IPM increased and unmasked the binding spots for PNA, RCA-1 and SBA. The spots of WGA-receptors without neuraminidase treatment were mostly identical to the receptors for PNA, RCA-1 and SBA, which became prominent after the digestion. Spots reacting with RCA-1 were mostly identical to the spots of SBA-receptors. The spots reacting with PNA coincided only partially with the spots reacting with RCA-1 and SBA.

12] Purification of interstitial retinol-binding protein from the eye

This chapter describes the purification of interstitial retinol-binding protein (IRBP) from the eye. IRBP is synthesized and secreted by cells of the neural retina, which may therefore have an important function in regulating its amount in the subretinal space. Purified bovine IRBP binds approximately two molecules of all-trans- retinol and contains 8.4% by weight of carbohydrate that consists of sialic acid, neutral hexoses (mannose, fucose, galactose), and glucosamine in the molar ratio of approximately 1:3:2. The purification of IRBP to homogeneity by high-performance size-exclusion chromatography of crude bovine interphotoreceptor matrix (IPM) is described. Sufficient IRBP is obtained by this method to demonstrate that it consisted of a single concanavalin A (Con A)-binding polypeptide with apparent M r of 140–145K by SDS–polyacrylamide gel electrophoresis. The anion-exchange chromatography on DEAE-cellulose (DE-52) followed by gel-filtration chromatography on Sepharose CL-4B can be used to obtain purified preparations of bovine and human IRBPs so permitting their partial characterization. For rat IRBP used, Con A-Sepharose can be followed by anion-exchange chromatography on DEAE-cellulose. The preparation of bovine IRBP in milligram quantities is described in the chapter.

Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina.

A retinol-binding glycoprotein ( IRBP ) was purified in milligram quantities from the extracellular matrix ( interphotoreceptor matrix) that occupies the subretinal space in bovine eyes. IRBP binds 2.2 molecules of all-trans retinol with a KD of approximately 10(-6) M. The holoprotein has lambda max at 280 nm (E1%1 cm = 10.99) and at 330 nm (E1%1 cm = 7.88). When freshly isolated from light-exposed eyes, IRBP contains up to 0.6 molecule of all-trans retinol, together with small amounts of the 11-cis and 13-cis isomers. IRBP also binds exogenous cholesterol, alpha-tocopherol, and all-trans retinoic acid, all of which are completely displaced by all trans retinol. The affinity of alpha-tocopherol for IRBP was at least several orders of magnitude less than that of all-trans retinol. IRBP contains 8.4% by weight of carbohydrate, which consists of sialic acid, neutral hexoses, and glucosamine in the molar ratio of approximately 1:3:2. No galactosamine was detected. Observations on the binding of 125I-labeled lectins to IRBP in sodium dodecyl sulfate-polyacrylamide gels before and after desialosylation suggest that at least one oligosaccharide chain is of the sialated biantennary complex type and contains fucose. The Mr of IRBP on calibrated size-exclusion columns averaged 249,000; on sodium dodecyl sulfate-polyacrylamide gels (with or without dithiothreitol) the apparent Mr was 144,000. IRBP exists in at least four isoelectric forms that bind concanavalin A and have pI values ranging from 4.4 to 4.8. Rabbit anti-bovine IRBP antiserum gave a single precipitin line against purified bovine IRBP , which showed a line of complete identity with crude bovine interphotoreceptor matrix and a line of partial identity with human interphotoreceptor matrix. The human material contains a prominent protein with lectin-binding properties similar to bovine IRBP but with a somewhat faster electrophoretic mobility. When isolated bovine neural retinas were incubated with 3H-labeled fucose, glucosamine, or leucine, a solitary labeled protein identified as IRBP was secreted into the medium. Labeled IRBP could not be detected in the medium when retinal pigment epithelium was incubated with these precursors under the same conditions. Neural retinas incubated with 3H-labeled leucine in the presence of tunicamycin secreted a form of IRBP that did not bind concanavalin A and had an Mr reduced by approximately 5,000.

Retinol-binding proteins in bovine interphotoreceptor matrix

Interphotoreceptor matrix (IPM) material from bovine eyes contains 1.8 nmoles per eye of vitamin A in light-adapted eyes and 0.7 in dark-adapted eyes, nearly all as unesterified retinol. This constitutes a significant fraction of the vitamin A in the retina-IPM-retinal pigment epithelium functional complex. Two distinct, non-serum retinol-carrying proteins are abundant in the IPM. One of these, of MW 17000, may be the cellular retinol-binding protein present in retina and pigment epithelium. Even more retinol is bound, in the light-adapted IPM, to a protein of MW ∼ 290,000; this binding is greatly reduced in the dark. These retinol-binding proteins may function in transporting vitamin A through the IPM for the visual cycle.

Proteins and glycoproteins of the bovine interphotoreceptor matrix: Composition and fractionation

Extracellular interphotoreceptor matrix material from adult bovine eyes was isolated by rinsing the apposing surfaces of neural retina and pigment epithelium. As a first step towards examining their structure and function, the matrix proteins and glycoproteins were separated into classes by means of ion exchange and lectin affinity chromatography. These groups of proteins were characterized by their molecular weight distributions (through gel electrophoresis) and carbohydrate compositions (through gas chromatography). The principal interphotoreceptor matrix glycoprotein, isolated by concanavalin A—Sepharose chromatography, has molecular weight about 140 000 and a sugar composition characteristic of an asparagine-linked glycoprotein with complex side chain. Carbohydrate analysis of ion-exchange chromatographic fractions confirmed that the uronic acid found in the matrix is a constituent of a small amount of proteoglycan, equivalent in weight to about two percent of the macromolecules present. There is evidence for mucin-like glycoproteins in the interphotoreceptor matrix, but no indication of either fibronection or collagen.

Vitamin a transport between retina and pigment epithelium—an interstitial protein carrying endogenous retinol (interstitial retinol-binding protein)

We have demonstrated and partially characterized an interstitial retinol-binding protein (IRBP) confined to bovine interphotoreceptor matrix (IPM). The native protein is a concanavalin A-binding glycoprotein with a mol. wt of 260 k as measured by gel-filtration and size-exclusion high-performance liquid chromatography. On SDS-gels, its mol. wt is 140–145 k. Since the protein is glycosylated, this value is probably too high. Hence the native protein may be a dimer consisting of two identical subunits. The endogenous ligand has been analyzed by high-performance liquid chromatography—it consists mainly of all- trans retinol. Occasionally, retinal and 11- cis retinol are also associated with it. The amount of retinol bound to IRBP increases when the eyes are illuminated. The total binding capacity was estimated to represent 4–5% of the retinol released from a total rhodopsin bleach. We have established that, like serum retinol-binding protein, IRBP can also bind retinoic acid, although it has not been established that retinoic acid is an endogenous ligand. The fluorescence emission λ max for IRBP with its native ligand is at 470 nm and the excitation λ max for this fluorescence is at 333 nm. Other retinoid carriers in the interphotoreceptor matrix have molecular weights of about 15 and 33 k. These probably correspond to cellular retinol- and retinal-binding proteins, respectively. Since both proteins have been identified in the pigment epithelium and retina cytosols, their presence in the IPM could be a result of cell damage. We conclude that interstitial retinol-binding protein is the best candidate for a transport protein carrying retinol between the rod outer segments and the pigment epithelium.

Proteins of the bovine interphotoreceptor matrix: Tissues of origin

Soluble material from the interphotoreceptor matrix of adult bovine eyes was isolated by gentle washing of the retina and of the apical pigment epithelium surface. Sugar and protein analyses of the macromolecules present in this extracellular layer showed that proteins outweigh glycosaminoglycans by a factor of about 50. (Glycosaminoglycans are the only matrix components previously examined.) The matrix proteins were characterized with respect to their molecular weights, tissues of origin and relationship to the subretinal fluid formed in retinal detachment. SDS-polyacrylamide gel electrophoresis of dithiothreitol-reduced bovine interphotoreceptor matrix revealed a limited and specific set of proteins: a major Coomassie blue-staining band at 140 000 daltons (a glycoprotein) plus several proteins between 21 000 and 64 000 with the most prominent band at 47 000. This pattern was completely different from those of serum, vitreous or pathological subretinal fluid; even serum albumin was totally absent from the matrix. Furthermore, the matrix proteins bore little resemblance to supernatants from tissue homogenates of retina or pigment epithelium. Thus the proteins found in the matrix do not appear to arise from general decomposition of surrounding tissues or from serum infusion. However, most of the matrix proteins can be accounted for as products of either the retina or pigment epithelium, as seen by their similarity to proteins retrieved from the conditioned culture media when bovine retinas or pigment epithelial cells were incubated overnight. These proteins were newly synthesized, as shown by incorporation of labeled leucine and glucosamine. Subretinal fluid contained none of these product proteins. The findings are discussed with respect to the physiological state of ocular tissues during retinal detachment. A procedure is described for obtaining bovine pigment epithelium cells with good yield and viability. The method involves trypsin treatment of the eyecup, without brushing out the cells.