Corneal Keratan Sulfate Proteoglycan Research Articles

Transcriptome and proteome revealed the differences in 3 colors of earlobe in Jiangshan Black-bone chicken

The earlobe is a featherless, exposed thickening located beneath the ear canal of chickens, which plays a visual signaling role in age, performance, mental vitality, reproduction, and other aspects. However, despite its importance, there have been few studies on the color differences and formation mechanisms of chicken earlobes, particularly the structurally blue earlobes characteristic of the Jiangshan black-bone chicken. In this study, we explored the physiological mechanisms that may influence the formation of differently colored earlobes using 3 types of earlobes from Jiangshan black-bone chickens: light peacock green (Green group), dark peacock green (Blue group), and dark reddish purple (Black group). All 3 earlobe colors exhibited positive melanin Masson-Fontana staining, and the thickness of collagen fibers in the dermis decreased in the order of Green, Blue, and Black groups. A total of 1,953 differentially expressed genes (DEGs) were detected in the 3 earlobes through mRNA sequencing, among which the GO term "collagen trimer" was significantly enriched in DEGs between groups. Additionally, 716 differentially expressed proteins (DEPs) were identified in the 3 earlobes using 4D-DIA proteomics, with the term "collagen fibril organization" being significantly enriched in DEPs between the Green and Black groups. Integrated analysis of transcriptome and proteome data revealed that 12 DEGs and DEPs were commonly differentially expressed between the Green and Black groups, including the gene LUM (corneal keratan sulfate proteoglycan), which was significantly enriched in the "collagen fibril organization" GO term. In conclusion, our study suggests that LUM plays a crucial role in the formation of peacock green earlobes in Jiangshan black-bone chickens. The high level of LUM in peacock green (Green and Blue groups) may affect collagen nanostructures, leading to a stronger effect of melanin-supported dermal collagen on the production of non-iridescent structural colors through coherent scattering, resulting in a bright structural blue color in Jiangshan black-bone chickens. In contrast, the low expression of LUM in dark reddish purple (Black group) reduces the reflection of non-iridescent structural colors, making the earlobe color appear almost black, similar to melanin.

Immunolocalisation and expression of keratocan in tendon

Short Communication Immunolocalisation and expression of keratocan in tendon S. G. Rees Ph.D.y*, A. D. Waggett Ph.D.z, B. C. Kerr Ph.D.z, J. Probert B.Sc.z, E. C. Gealy Ph.D.z, C. M. Dent M.D.x, B. Caterson Ph.D.z and C. E. Hughes Ph.D.z ySchool of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, UK zConnective Tissue Biology Laboratories, Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, UK xSchool of Medicine, Department of Orthopaedic and Trauma Surgery, Cardiff University, Llandough Hospital, Penlan Road, Penarth, Vale of Glamorgan, CF64 2XX, UK

Thyroxine Increases the Rate but Does Not Alter the Pattern of Innervation during Embryonic Chick Corneal Development

Embryonic chick corneal nerves reach limbal mesenchyme by embryonic day (E)5, encircle the cornea in several days, then defasciculate into the stroma simultaneously from all sides, while extracellular keratan sulfate proteoglycan (KSPG) accumulates from posterior to anterior stroma. Precocious thyroxine (T4)-induced increases in corneal thinning/transparency are blocked by 2-thiouracil (2-TU) inhibition of T3 synthesis. The hypothesis for this study was that precocious T4 exposure increases corneal innervation similarly. E8 embryos received T4, 2-TU, T4+2-TU, or buffer; corneas were harvested on E12. Corneal nerves were stained with neuronal beta-tubulin-specific TuJ1 antibody or chick nerve-specific CN antibody. Corneal thickness was determined from cryostat sections, and mRNA expression was measured by real-time PCR. Nerves avoided the cornea until E9, then entered the anterior stroma, extended toward and reached the cornea center by E14, and never invaded posterior stroma. E7 to E18 corneal expressions of nerve growth factor and neurotrophin-3 genes were unchanged; receptor gene expressions rose. E7 to E12 semaphorin 3A and 3F and ephrin A2 and A5 expressions did not change significantly; semaphorin and ephrin/eph expressions increased from E9 to E18. E8 T4 administration increased nerve extension by E11, but did not alter circumferential penetration, anterior-only penetration, or neurotrophin expressions. 2-TU prevented T4-induced precocious corneal thinning, but augmented T4 nerve stimulation. No changes in corneal neurotrophin or nerve pathfinding gene expressions accompany corneal transition to nerve growth cone permissiveness. T4 increases corneal nerve penetration rates by a non-T3-dependent mechanism. Results are consistent with possible roles for corneal KSPGs in regulating corneal nerve growth.

Keratocan and Lumican Regulate Neutrophil Infiltration and Corneal Clarity in Lipopolysaccharide-induced Keratitis by Direct Interaction with CXCL1

Keratocan and lumican are keratan-sulfate proteoglycans (KSPG), which have a critical role in maintaining corneal clarity. To determine whether these KSPGs have a role in corneal inflammation, we examined Kera(-/-) and Lum(-/-) mice in a model of lipopolysaccharide (LPS)-induced keratitis in which wild-type mice develop increased corneal thickness and haze due to neutrophil infiltration to the corneal stroma. Corneal thickness increases caused by LPS mice were significantly lower in Kera(-/-) and Lum(-/-) than wild-type mice. Further, LPS-injected Lum(-/-) mice had elevated corneal haze levels compared with that of Kera(-/-) and wild-type. At 24 h post-injection, total enhanced green fluorescent protein-positive bone marrow-derived inflammatory cells in chimeric mice was significantly lower in Kera(-/-) mice and Lum(-/-) mice compared with wild-type mice. Neutrophil infiltration was inhibited in Kera(-/-) and Lum(-/-) mice at 6 and 24 h post-stimulation, with Lum(-/-) corneas having the most profound defect in neutrophil migration. Reconstitution of keratocan and lumican expression in corneas of Kera(-/-) and Lum(-/-) mice using adeno-keratocan and adeno-lumican viral vectors, respectively, resulted in normal neutrophil infiltration in response to LPS. Immunoprecipitation/Western blot analysis showed that lumican and keratocan core proteins bind the CXC chemokine KC during a corneal inflammatory response, indicating that corneal KSPGs mediate neutrophil recruitment to the cornea by regulating chemokine gradient formation. Together, these data support a significant role for lumican and keratocan in a corneal inflammatory response with respect to edema, corneal clarity, and cellular infiltration.

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.

An X-Ray Diffraction Study of Corneal Structure in Mimecan-Deficient Mice

Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.

Keratocan-deficient Mice Display Alterations in Corneal Structure

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.

Epithelial repair: roles of extracellular matrix.

To review studies of the roles of extracellular matrix (ECM) metabolism in corneal epithelium during wound repair. Methods. 1) Alterations in the structure and composition of epithelial basement membrane during corneal epithelial healing were examined histologically and immunohistochemically. 2) The effects of procollagen and hyaluronan synthesis inhibitors on the spread of rabbit corneal epithelium were determined in organ culture. 3) Expression of keratan sulfate proteoglycan (KSPG) proteins in corneal epithelium was examined during repair after injury in wild-type and lumican-null mice. 1) Corneal epithelial basement membrane was transiently degraded and reassembled during tissue repair. Patterns of type IV collagen immunoreactivity were also transiently altered. The system of matrix metalloproteinase-tissue inhibitors of metalloproteinases may play an important role in the disassembly and reorganization of epithelial basement membrane. 2) Inhibitors of procollagen secretion and hyaluronan biosynthesis disrupted the spread of a corneal epithelial sheet in situ. 3) Among the corneal KSPG proteins examined, lumican was transiently expressed in migrating murine corneal epithelial cells. Anti-lumican antibody inhibited corneal epithelial resurfacing in organ culture. The absence of lumican was found to delay corneal epithelial wound healing in mice. Extracellular matrix metabolism by the injured corneal epithelium is important in the repair process.

Fibroblast Growth Factor-2 Promotes Keratan Sulfate Proteoglycan Expression by Keratocytes in Vitro

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

Expression of the Keratan Sulfate Proteoglycans Lumican, Keratocan and Osteoglycin/Mimecan During Chick Corneal Development

The corneal proteoglycans belong to the Leu-rich proteoglycan (LRP) gene family and contain chondroitin/dermatan (CS/DS) or keratan sulfate (KS) chains. These proteoglycans play a critical role in generating and maintaining a transparent matrix within the corneal stroma. Decorin which has CS/DS chains and lumican which has KS chains, were first to be identified in the cornea. Two other corneal KS proteoglycans (KSPGs), keratocan and osteoglycin/mimecan were recently identified in bovine corneas. We cloned and sequenced chick osteoglycin/mimecan and found it to contain a stretch of 60 amino acids that showed no identity to the presumed mammalian homolog. The 177 base pair DNA coding for this unique sequence shows 47% identity to an 189 base pair sequence between exons 4 and 5 of the bovine osteoglycin/mimecan gene. This indicates that this cDNA represents an alternatively spliced form of osteoglycin/mimecan containing a unique N-terminal sequence.The expression of each of the three corneal KSPGs in the developing and mature chick cornea was investigated by competitive PCR and immuno-biochemical analysis of corneal extracts. Competitive PCR was used to determine the message levels for chick lumican, keratocan and osteoglycin in embryonic day 9, 12, 15, 18 and adult corneas. Results showed that lumican mRNA fluctuated during development but remained at a relatively high level while keratocan and osteoglycin message levels declined steadily from day 9 to adult. Additionally, lumican mRNA was present at higher levels, during all stages of corneal development, than keratocan and at much higher levels than osteoglycin. Antibodies shown to be specific for each KSPG were used to characterize proteoglycans isolated from embryonic and adult chick corneas. KSPGs from embryonic corneas eluted 1–2 fractions earlier on Q-Sepharose than KSPG from adult corneas. Additionally, Western blot analysis showed that embryonic KSPGs were more keratanase-resistant, endo-β-galactosidase sensitive than adult KSPGs. The results of this study indicate an alteration in sulfation or the fine structure of the glycosaminoglycan chains occurs during corneal maturation for the 3 KSPGs.

Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.

Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.

The Bovine Mimecan Gene: MOLECULAR CLONING AND CHARACTERIZATION OF TWO MAJOR RNA TRANSCRIPTS GENERATED BY ALTERNATIVE USE OF TWO SPLICE ACCEPTOR SITES IN THE THIRD EXON

Mimecan is a proteoglycan expressed by many connective tissues. It was originally isolated in a truncated form as a bone-associated glycoprotein, osteoglycin, and was considered an osteoinductive factor. Recently, we demonstrated that the full-length translation product of the cDNA encoding mimecan is a corneal keratan sulfate proteoglycan present in other tissues without keratan sulfate chains. We also described multiple mimecan mRNA transcripts generated by differential splicing and alternative polyadenylation. In this study, we isolated genomic clones and determined the genomic organization of the bovine mimecan gene. The gene is spread over >33 kilobases of continuous DNA sequence and contains eight exons. The newly discovered first exon, identified by 5'-rapid amplification of cDNA ends, consists of a 5'-untranslated region and is enriched in C+G nucleotides. Two transcription initiation sites starting at the first and at the second exons were determined by primer extension. Molecular characterization shows that alternatively spliced RNA isoforms are generated by the use of two distinct splice acceptor sites in the third exon situated 278 base pairs apart. We determined a partial genomic structure of the human mimecan gene and demonstrated two alternatively spliced RNA transcripts that are generated likewise. Despite the diversity of mimecan transcripts, the primary structure of the core protein is encoded from exons 3 to 8 and remains unchanged, indicating its functional importance. Using ribonuclease protection assay, we analyzed the patterns of spliced RNA expressed in cultured bovine keratocytes. We demonstrated that their expression is differentially modulated in a temporal manner by basic fibroblast growth factor.

Cloning, characterization and tissue-specific expression of the gene encoding bovine keratocan, a corneal keratan sulfate proteoglycan.

Keratocan is one of three major keratan sulfate proteoglycans characteristically expressed in cornea. We reported previously the sequence of bovine Kera cDNA. In this study, the complete bovine Kera gene was cloned and sequenced, and its expression pattern was determined. The Kera gene is composed of three exons and two introns that span 8.830kb of the bovine genome. The first exon contains 287 nucleotides of 5'-UTR sequence. Both of the two large introns of 1322 and 4178bp contain (CA)n repeats. The bovine Kera gene has a TATA box that is located 28bp upstream from tsp. Primer extension and S1 nuclease protection analyses were used to determine the major tsp. RPA indicate that cornea and sclera are the two tissues with the highest expression of Ktcn mRNA. This restricted expression in eye tissues, as well as the unique modification of keratocan with long keratan sulfate chains in cornea, suggests that this molecule may be important in developing and maintaining corneal transparency.

Cloning and chromosomal localization of mouse keratocan, a corneal keratan sulfate proteoglycan.

The corneal stroma consists of a highly structured extracellularmatrix that is maintained by specialized fibroblasts called kerato-cytes. The transparency of the cornea is dependent on the presenceof highly sulfated proteoglycans (PG) that can bind collagen andcan regulate collagen fibril diameter (Rada et al. 1993). The cor-neal proteoglycans that have been identified contain numerousLeu-rich repeats and all belong to the same gene family, the Leu-rich proteoglycans (LRP). These include a chondroitin/dermatansulfate proteoglycan (CS/DSPG), decorin, and three keratan sul-fate proteoglycans (KSPG), lumican, keratocan, and osteoglycin(Funderburgh et al. 1991, 1993; Blochberger et al. 1992; Corpuz etal. 1996). The corneal KSPGs are glycoproteins exclusively duringearly corneal development before corneal transparency (Cornuet etal. 1994) and in macular corneal dystrophy type I, an inheriteddisease that is characterized by corneal opacity and deposits(Klintworth et al. 1977, 1983; Nakazawa et al. 1984). These pre-vious studies show a direct correlation between corneal transpar-ency and the presence of keratan sulfate. The core protein andpoly-lactosamine side chains alone appear to be insufficient extra-cellular matrix components for sustaining a transparent structure inthe cornea.The primary structure of keratocan, based on the deducedamino acid sequence from bovine and chick cDNA sequences,shows N- and C-terminal globular domains containing four andtwo Cys residues forming two and one disulfide bond, respec-tively, and 11 highly conserved Leu-rich repeats located mainlybetween the two globular domains (Corpuz et al. 1996; Dunlevy etal. 1998). The two-dimensional model of chick keratocan, basedon the X-ray crystallography structure of ribonuclease inhibitor, amolecule comprised entirely of Leu-rich repeats, (Kobe et al. 1993,1995), shows the Leu-rich repeats coiled in a spiral with theseregions in close proximity to each other, forming a horseshoestructure (Dunlevy et al. 1998). This model also shows two out ofthree KS chains extended outward from the outer surface of thehorseshoe with the inner surface more likely to be involved incollagen binding.On the basis of mRNA levels, keratocan is most abundant incornea and sclera, but is also found at substantially lower levels inligament, artery, cartilage, skeletal muscle, and skin (Corpuz et al.1996), and keratocan is more specifically expressed in cornea thanis lumican. Consequentially, we are interested in gaining furtherinsights into its tissue-specific regulation of expression, its role indevelopment and maintenance of corneal transparency. In the pre-sent study, we report the cDNA sequence of mouse keratocan andlocalization of the gene to distal Chromosome (Chr) 10 by segre-gation analysis of restriction fragment length variants (RFLVs) inrecombinant inbred (RI) strains of mice.Total RNA was isolated from mouse whole eyes by use of aguanidinium thiocyanate-phenol-chloroform procedure (Chomc-zynski and Sacchi 1987). An mRNA purification kit (PharmaciaBiotech) was used to further purify the RNA, and ∼10 mgofmRNA was used to prepare a cDNA library (Stratagene CustomLibrary Department) in the Uni-Zap XR vector system. The librarywas screened with a high-specific-activity

Differential Splicing and Alternative Polyadenylation Generate Multiple Mimecan mRNA Transcripts

We previously showed the 25-kDa corneal keratan sulfate proteoglycan to be a translation product of the gene producing osteoglycin and proposed the name mimecan for this gene and its product. We also demonstrated three mimecan RNA transcripts using Northern blot analysis. In this report, we investigate the mechanisms accounting for these transcripts. Ribonuclease protection analysis and reverse transcription-polymerase chain reaction of bovine corneal mRNA detected a mimecan transcript that lacked 278 base pairs of the 5'-untranslated region between residues 62 and 340. This splice variant represents the predominant form of mimecan mRNA in bovine cornea and sclera. It was also detectable in other bovine tissues as a minor transcript. Two additional cDNA clones that were isolated contained 398 bases of nucleotide sequence at the 3'-end of mimecan cDNA, not present in the published sequence. Ribonuclease protection analyses with the 3'-probe, which included the new sequence, allow detection of three RNA transcripts while 5'-probes recognized only two. These results indicate that the three canonical polyadenylation sites in the 3'-untranslated region of mimican mRNA are alternatively selected. Possible roles for this previously undetected degree of diversity of mimecan RNA isoforms transcribed in the same tissue are discussed.

Mimecan, the 25-kDa Corneal Keratan Sulfate Proteoglycan, Is a Product of the Gene Producing Osteoglycin

Bovine cornea contains three unique keratan sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with keratan sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal keratan sulfate proteoglycan, also present in many non-corneal tissues without keratan sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.

Synthesis of Corneal Keratan Sulfate Proteoglycans by Bovine Keratocytes in Vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.

Conformation, stability and interactions of corneal keratan sulfate proteoglycan

We have monitored the molecular conformation, stability, interaction and dynamics of keratan sulfate proteoglycan, the major structural protein component of the cornea, in solution, by studying the fluorescence spectral features of its tryptophan residues as component-specific intrinsic spectral probes (collagen, the other structural component of the cornea, has no tryptophans). Our study suggests that the Trp region of the molecule is in a motionally restricted environment as it exhibits a fluorescence red-edge effect and shows dipole relaxation. The extrinsic spectral probe 8-anilinonaphthalene 1-sulfonate reveals keratan sulfate proteoglycan to possess significant surface hydrophobicity. This dual character of keratan sulfate proteoglycan allows us to label it as an ‘ambidextran’ proteoglycan. The molecule is stable between pH 5–8 and has a T m value of 72°C. Disulfide bonds play a role in the stability of the molecule. KSPG is seen to interact with collagen and the model compound, poly( l-proline). Interaction of the proteoglycan with unilamellar vesicles appears to be more interfacial than penetrative. This dual interaction displayed by KSPG with collagen and with lipid assemblages suggests that it plays the role of a ‘filler’ in the extracellular matrix of the cornea.

Keratan sulfate and dermatan sulfate proteoglycans associate with type VI collagen in fetal rabbit cornea.

Keratan sulfate proteoglycan (KSPG) and dermatan sulfate proteoglycan (DSPG) are associated with collagen fibrils in adult rabbit cornea. Because certain cytochemical data suggested that proteoglycans are associated with type VI collagen in the fetal rabbit cornea, we developed polyclonal antibodies specific to the core proteins of rabbit corneal KSPG (lumican and/or fibromodulin) and DSPG (decorin and/or biglycan) and used the antibodies as immunocytochemical probes to determine proteoglycan ultrastructural location. Immunogold particles were associated with Type VI collagen filaments but not with collagen fibrils in fetal and neonate rabbit cornea. Association of corneal KSPG and DSPG with Type VI collagen was immunocytochemically confirmed with monoclonal antibodies to low-sulfated keratan sulfate glycosaminoglycan (GAG) and chondroitin-4-sulfate GAG of DSPG. The monospecificity of the polyclonal and monoclonal antibodies and exclusive binding of antibodies to Type VI collagen filaments, together with previous chemical identification of low-sulfated KSPG and DSPG in developing rabbit cornea, support our contention that corneal proteoglycans in developing rabbit cornea are associated with Type VI collagen.

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.