Biomimetic Proteoglycans Mimic Macromolecular Architecture and Water Uptake of Natural Proteoglycans.

Aging and degeneration of human tissue come with the loss of tissue water retention and associated changes in physical properties partially due to degradation and subsequent loss of proteoglycans. We demonstrated a novel method of fabrication of biomimetic proteoglycans, which mimic the three-dimensional bottlebrush architecture and physical behavior of natural proteoglycans responsible for tissue hydration and structural integrity. Biomimetic proteoglycans are synthesized by an end-on attachment of natural chondroitin sulfate bristles to a synthetic poly(acryloyl chloride) backbone. Atomic force microscopy imaging suggested three-dimensional core-bristle architecture, and hydrodynamic size of biomimetic proteoglycans was estimated at 61.3 ± 12.3 nm using dynamic light scattering. Water uptake results indicated that biomimetic proteoglycans had a ∼50% increased water uptake compared to native aggrecan and chondroitin sulfate alone. The biomimetic proteoglycans are cytocompatible in the physiological ranges of concentrations and could be potentially used to repair damaged or diseased tissue with depleted proteoglycan content.

Role of keratan sulphate (sulphated poly -N-acetyllactosamine repeats) in keratoconic cornea, histochemical, and ultrastructural analysis.

AIMS: Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) present in the corneal stroma where it is thought to regulate collagen fibril diameter. In this study we investigated the distribution of KS in normal and keratoconic corneas. METHODS: Four normal, one mild, and four severe keratoconic corneas were used for the study. Distribution of keratan sulphate proteoglycans (KS-PG) was investigated using a primary monoclonal antibody (5-D-4) that recognizes disulphated disaccharides in the poly-N-acetyllactosamine repeats of KS. The immuno-reactivity of 5-D-4 was analyzed by immunohistochemistry and immuno-electron microscopy. RESULTS: Immuno-histochemistry showed diffuse 5-D-4 staining in keratoconic cornea compared to the punctuate staining in normal corneas. In the single cornea with mild keratoconus, immunogold microscopy revealed a very high density of KS-PG staining, especially in the posterior stroma, compared to severe keratoconic and normal cornea. The amount of KS-PG in the stroma in severe keratoconus was slightly less compared to the normal cornea. In the mild keratoconic cornea, a higher quantity of KS-PG was present around the keratocytes. In severe keratoconic corneas, a higher quantity of KS-PG was present within the keratocytes compared to normal cornea. CONCLUSIONS: The finding of an altered expression of KS in our keratoconic corneas, in particular the strong expression of KS in keratocytes, is in keeping with reports of an altered expression of proteoglycan metabolism in keratoconus. KS-PG plays an important role in stromal collagen fibril assembly and a dysregulation of KS-PG synthesis or catabolism could explain changes in collagen fibril spacing and diameter, which we have reported elsewhere.

Structural and biochemical aspects of keratan sulphate in the cornea.

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly- thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.

Differential relative sulfation of Keratan sulfate glycosaminoglycan in the chick cornea during embryonic development.

PURPOSE: To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency. METHODS: Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining. RESULTS: A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated. CONCLUSIONS: The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump.

Immunochemical localization of keratan sulfate proteoglycans in cornea, sclera, and limbus using a keratanase-generated neoepitope monoclonal antibody.

PURPOSE: To evaluate the use of neoepitope monoclonal antibody BKS-1, which recognizes keratanase-generated keratan sulfate (KS) stubs on keratan sulfate proteoglycans in human cornea, limbus, and sclera. METHODS: BKS-1 specifically recognizes a keratanase-generated neoepitope [N-acetyl-glucosamine-6-sulfate (GlcNAc-6-S)] at the nonreducing terminal of corneal and skeletal KS glycosaminoglycan chains. It was produced by using keratanase-digested KS peptides from bovine cartilage aggrecan as the immunizing antigen. BKS-1 was used in conjunction with 5D4 to analyze the KS distribution in human cornea, limbus, and sclera using Western blotting, immunohistochemistry, and electron microscopy. RESULTS: 5D4 Western blot analysis displayed a diffuse staining pattern, and it was difficult to distinguish differences among cornea, sclera, and limbus. However, BKS-1 showed differences in KS levels, with higher levels in the cornea and lower levels in the limbus and sclera. Ultrastructural studies showed that the monoclonal antibody (mAb) BKS-1 neoepitope was not observed in the epithelium or basement membrane; however, 5D4 was present in these layers. Large quantities of both antibodies were present in Bowman's layer, stroma, and Descemet's membrane, but the quantity of 5D4 was significantly higher (P < 0.001) than the quantity of BKS-1 in all these layers of the cornea. CONCLUSIONS: mAb 5D4 recognizes oversulfated structures within KS chains, whereas BKS-1 recognizes a single neoepitope on KS after keratanase digestion of monosulfated KS disaccharides. With the use of BKS-1, the authors identified a more clearly defined pattern for KS distribution in the cornea than was seen with 5D4. The presence of a large quantity of BKS-1 immunostaining in the cornea suggests that KS-substituted proteoglycans are more prevalent in the cornea than in the limbus or sclera.

Keratan sulfate glycosaminoglycan and the association with collagen fibrils in rudimentary lamellae in the developing avian cornea.

PURPOSE: Keratan sulfate (KS), through its association with fibrillar collagen as KS-substituted proteoglycan (KS PG), is thought to be instrumental in the structural development of the corneal stroma. The authors used two different sulfate motif-specific antibodies to identify the sequence of appearance, and the association with collagen, of sulfated KS during avian corneal morphogenesis. METHODS: Corneas from chicken embryos throughout the developmental period, from day 8 through day 18 of incubation, were examined by immunofluorescence and immunoelectron microscopy using monoclonal antibodies 5D4 and 1B4, which react with high- and low-sulfated epitopes on KS, respectively. RESULTS: KS was identified as punctate labeling at incubation day 8, the earliest stage examined, suggesting a cell-associated distribution. By day 10, labeling was more homogeneous, indicating that KS sulfation motifs were present in the stromal extracellular matrix. At day 12 through day 14, immunopositive sites were concentrated primarily in the anterior stroma but became more uniform throughout the full stromal thickness by day 18. From day 10 on, electron microscopy revealed a high-sulfated KS epitope closely associated with bundles of regularly arranged collagen fibrils, initially near cell surfaces in rudimentary lamellae. Individual cells, associated with collagen bundles with different fibril orientations, imply the potential for simultaneous deposition of multiple lamellae. CONCLUSIONS: During chick corneal morphogenesis, significant matrix deposition of high-sulfated KS epitope occurs by day 10, with accumulation subsequently proceeding in an anterior-to-posterior manner. High-sulfated KS likely serves to help define the regular spatial organization of collagen fibrils in bundles newly extruded into the extracellular milieu.

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase.

Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Ultrastructural immunolocalization of alpha-elastin and keratan sulfate proteoglycan in normal and scoliotic lumbar disc.

STUDY DESIGN: Comparative ultrastructural study of intervertebral discs from normal subjects and patients with scoliosis. OBJECTIVE: To identify ultrastructural relations among keratan sulfate (KS) proteoglycan, alpha-elastin, collagen fibers, and elastic fibers in normal and scoliotic discs. SUMMARY OF BACKGROUND: KS proteoglycans, elastic fibers, and collagen fibers play important mechanical roles in the intervertebral disc, but the distributions of KS proteoglycans and elastin in this tissue have received little attention. METHODS: Tissues were fixed in 4% paraformaldehyde. Monoclonal antibody 5-D-4 (which recognizes a KS epitope on aggrecan, fibromodulin, and lumican) and polyclonal anti-alpha-elastin were visualized with a 10-nm immunogold-conjugated secondary antibody. RESULTS: In a normal disc, a regular pattern of KS labeling occurred around collagen fibers, in the cell cytoplasm, and in the rough endoplasmic reticulum; the nucleus pulposus was more densely labeled for KS than was the anulus fibrosus. In scoliotic disc anulus fibrosus, KS labeling was weak throughout the matrix and pericellularly but abundant in lysosomes and on electron-dense material in degenerate cells. Degenerate collagen fibers in scoliotic tissue bore less KS than did normal fibers. KS labeling of the microfibrillar region of elastic fibers was strong in normal disc but weak in scoliotic disc. Elastin labeling of elastic fibers was weaker in scoliotic than in normal tissue. CONCLUSION: KS proteoglycans and elastic fibers are closely associated with the lamellar organization of the collagen fibers in a normal disc. In scoliosis, impaired regulation of collagen fibrillogenesis by lumican or fibromodulin may result in disruption of the lamellar structure.

Atypical composition and ultrastructure of proteoglycans in the mouse corneal stroma.

PURPOSE: Recently, gene-targeted strains of mice with null mutations for specific proteoglycans (PGs) have been used for investigations of the functional role of these molecules. In the present study, the corneal stroma of the mouse was examined to provide some baseline PG morphologies in this species. METHODS: Monoclonal antibodies to specific glycosaminoglycan (GAG) chain sulfation patterns were used to characterize PG composition in corneal extracts by SDS-PAGE and Western blot analysis and to identify their tissue distribution by immunofluorescence microscopy. PGs were also visualized by transmission electron microscopy after contrast enhancement with cationic dye fixation. RESULTS: Western blot analysis of pooled corneal extracts and immunofluorescence of tissue sections identified 4-sulfated, but not 6-sulfated, chondroitin sulfate/dermatan sulfate (CS/DS). Keratan sulfate (KS) was present only as a low-sulfated moiety. Electron microscopic histochemistry disclosed a complex array of corneal PGs present as (1) fine filaments radiating from collagen fibrils, and (2) elongate, straplike structures, running either along the fibril axis or weaving across the primary fibril orientation. These large structures were digested by chondroitinase ABC, but not by keratanase. CONCLUSIONS: KS in the mouse is predominantly undersulfated and generates an immunostaining pattern that differs from that observed in corneas of other mammalian species thus far investigated. The mouse cornea resembles other mammalian corneas in the presence of filamentous arrays of small, collagen-associated stromal PGs visualized by cationic dye staining. However, large dye-positive structures with a CS/DS component are also present and appear to be unique to the cornea of this species.

Experimental induction of anterior disk displacement of the rabbit craniomandibular joint: an immuno-electron microscopic study of collagen and proteoglycan occurrence in the condylar cartilage.

BACKGROUND: Results from our previous studies suggest that surgical induction of anterior disk displacement (ADD) in the rabbit craniomandibular joint (CMJ) leads to histopathological alterations consistent with osteoarthritis. In addition, molecular changes in collagens and glycosaminoglycans (GAGs) were observed using immunohistochemistry. The purpose of the present study was to further characterize those molecular changes in collagens and GAGs using immuno-electron microscopy. METHODS: The right joint of 15 rabbits was exposed surgically and all discal attachments were cut except for the posterior attachment (the bilaminar zone). The disc was then repositioned anteriorly and sutured to the zygomatic arch. The left joint was used as a sham-operated control. Ten additional joints were used as non-operated controls. Mandibular condyles were removed 2 weeks following surgery and processed for light and immuno-electron microscopy using colloidal gold-labeled antibodies against collagen type I, II, VI and IX and against keratan sulfate, chondroitin-4 and -6-sulfate, and link protein. RESULTS: Light microscopic results showed osteoarthritic changes. Immuno-electron microscopy of osteoarthritic cartilage demonstrated a decline in type II collagen, the abnormal presence of type I collagen and loss of type VI and IX collagens. Quantitative colloidal gold immuno-electron microscopy confirmed the depletion of keratan sulfate, chondroitin-4 and -6-sulfate, and link protein in osteoarthritic cartilage. CONCLUSION: Anterior disk displacement leads to molecular alterations in both the collagen and the proteoglycans of rabbit condylar cartilage characteristic of osteoarthritis in other synovial joints. These alterations are consistent with loss of the shock absorber function of the cartilage and injury of the underlying bone.

Altered collagen fibril formation in the sclera of lumican-deficient mice.

PURPOSE: To better understand the role of lumican (corneal keratan sulfate proteoglycan) in the scleral extracellular matrix, collagen fibril size, shape, and organization were evaluated in the sclera of wild-type mice and in mice homozygous or heterozygous for a null mutation in the lumican gene. METHODS. Anterior and posterior sclera from 6-month-old wild-type (lum+/lum+) and lumican-deficient mice (lum+/lum- and lum-/lum-) were analyzed by transmission electron microscopy. In addition, lumican was characterized in the sclera of wild-type and lumican-deficient mice by Western blot analyses. RESULTS: Lumican was present in the mouse sclera as an approximately 48-kDa core protein containing short glycosaminoglycan side chains consisting of moderate- to low-sulfated keratan sulfate. The wild-type mouse sclera consisted of irregularly arranged lamellae of collagen fibrils with an average diameter of 47.37 +/- 0.648 nm in the anterior sclera and 54.68 +/- 0.342 nm the posterior sclera. Collagen fibrils in the sclera of lumican mutant mice (lum+/lum- and lum-/lum-) were significantly larger in diameter in anterior (72.61 +/- 0.445 and 84.47 +/- 0.394 nm, respectively) and posterior (75.92 +/- 0.361 and 80.90 +/- 0.490 nm, respectively) scleral regions compared with wild-type mice (P < 0.001). CONCLUSIONS: The results of the present study indicate that null mutations in one or both alleles of the lumican gene result in significant defects in scleral collagen fibril formation that could lead to alterations in ocular shape and size and severely affect vision.

Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy.

PURPOSE: We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using energy-filtering transmission electron microscopy (EF-TEM) and atomic force microscopy (AFM). METHODS: Human cornea and sclera were digested with keratanase to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observation, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed. RESULTS: With CB staining, numerous CB-positive short filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic collagen fibrils in keratanase-treated corneal collagens were deeper and more regular than in untreated ones. Moreover, the depth and periodicity of keratanase-untreated corneal collagens were shallow and irregular in comparison with keratanase-untreated scleral collagens. On the other hand, there was no difference in depth or regularity between keratanase-treated and -untreated scleral collagen fibrils. Using AFM imaging, additional thin grooves sub-bands were detected on the surface of keratanase-treated corneal collagen fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestion. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands. CONCLUSION: Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with proteoglycans, we conclude that KSPG is found in ample amounts in the human cornea in comparison with sclera. Moreover, we topologically detected KSPG attached to a and c bands of collagen fibrils.

Immunohistochemical and charge-specific localization of anionic constituents in pseudoexfoliation deposits on the central anterior lens capsule from individuals with pseudoexfoliation syndrome.

BACKGROUND: Pseudoexfoliation (PSX) syndrome is a degenerative systemic disorder that is characterized primarily by deposits of distinct fibrillar material on the surface lining the anterior and posterior chambers of the eye and is often associated with cataract and glaucoma. Although some components of the PSX material have been identified, the precise composition is obscure. METHODS: High-resolution scanning electron microscopy in conjunction with colloidal cationic gold labeling was used to localize anionic constituents at the surface of PSX aggregates. Transmission electron microscopy was applied for the immunocytochemical detection of glycosaminoglycans, and to monitor the charge-specific distribution of colloidal thorium dioxide and ferritin in PSX material. The specific binding of antibodies was confirmed by immunohistological staining of paraffin-embedded specimens. RESULTS: Paraffin-embedded tissue sections revealed immunoreactivity for keratan sulfate and dermatan sulfate proteoglycan within PSX material deposited on the surface of the anterior lens capsule. Post-embedding immunogold labeling of keratan sulfate demonstrated an intense label of PSX aggregates primarily associated with mature PSX fibrils, whereas dermatan sulfate proteoglycon appeared to be present in low quantities. Additionally, keratan sulfate was found at the humoral periphery of the lens capsules. To further investigate the distribution of anionic sites in PSX material, we used cationic colloidal tracers of different size, such as gold, thorium dioxide and ferritin. PSX aggregates exhibited a strong negative charge, resulting very likely from glycosaminoglycan chains of proteoglycans. The density of anionic sites was higher at the interfibrillar matrix. Lens capsules associated with PSX material revealed a diminished accumulation of cationic ferritin at the humoral surfaces. CONCLUSIONS: Increased amounts of different glycosaminoglycans identified in PSX material suggest an important role of proteoglycans for the pathogenic pathway in PSX.

An ultrastructural investigation into proteoglycan distribution in human corneas.

PURPOSE: We report an investigation into the distribution of proteoglycans (PGs) in normal, organ-cultured and dextran-treated human corneas. METHODS: Immunogold labeling was carried out at the electron microscope level to localize keratan sulphate (KS), chondroitin sulphate (CS), and heparan sulphate (HS) PGs. RESULTS: High levels of labeling for CS was found in the epithelium, endothelium, and keratocytes, with light labelling present in the basement membranes and the corneal stroma. Labeling for HS was present in the epithelium, endothelium, and keratocytes, with intense labeling present at the endothelium/Descemet's membrane interface and the epithelium/Bowman's layer interface. Large filaments were also observed in these regions in cuprolinic blue-stained specimens. Keratan sulphate was present at high levels in the stroma and the basement membranes with low levels present within the keratocytes, epithelium, and endothelium. The pattern of KS labeling along the collagen fibrils in the stroma sometimes showed evidence of periodicity. Organ-cultured corneas had extensive collagen-free "lakes," the interior of which immunolabeled positively for KS and showed staining with cuprolinic blue. The lakes were greatly reduced in the dextran-treated samples. CONCLUSION: This investigation determined the ultrastructural distribution of KS, CS, and HS PGs in human cornea and showed that organ culture is associated with a change in distribution of stromal PGs.

MR-microscopic visualization of anisotropic internal cartilage structures using the magic angle technique.

NMR microscopic studies of articular cartilage at 7.1 T are presented. Using a special experimental design, T2-weighted spin-echo images of cartilage-bone plugs were taken under variable angles with respect to the static magnetic field B0 to visualize the angular-dependent representation of internal matrix structures mediated by the collagen network arrangement. To quantify the observed orientational effect in the MR images, exact measurements of the transverse relaxation time T2 were taken using the CPMG sequence. The NMR experiments show the strong influence of the cartilage orientation with respect to the static magnetic field on the inhomogeneous appearance of the articular cartilage in the MR image. Additionally performed polarization light microscopic investigations demonstrate the direct relation between the oriented collagenous structures and the anisotropic regions observed in the MR images. A simple cartilage matrix model derived from the experimental findings is proposed, and consequences for the clinical assessment of the articular joint are discussed.

Proteoglycans contain a 4.6-A repeat in corneas with macular dystrophy: II. Histochemical evidence.

PURPOSE: Synchrotron x-ray diffraction experiments indicate that corneas with macular corneal dystrophy (MCD) contain unusual 4.6-A periodic repeats thought to reside in proteoglycans or glycosaminoglycans. Recently the 4.6-A x-ray reflection was found to be significantly diminished after incubation of MCD specimens in buffer containing chondroitinase ABC or N-glycanase. We examined the sulfated proteoglycans in these glycosidase-digested MCD corneas. METHODS: Transmission electron microscopy was used in conjunction with cuprolinic blue-staining for sulfated proteoglycans. RESULTS: Incubation of an MCD specimen in enzyme buffer left both small and large proteoglycan filaments in the stromal matrix, whereas incubation in the presence of chondroitinase ABC removed these molecules from the tissue. Incubation in buffer containing N-glycanase, on the other hand, removed the large proteoglycan filaments from the MCD stroma but left unaffected the small collagen-associated proteoglycans. CONCLUSION: These results are consistent with the interpretation that 4.6-A periodic repeats in MCD corneas reside in large sulfated proteoglycan filaments (or aggregates thereof) that may contain chondroitin/dermatan sulfate and keratan sulfate or keratan components.

A chondroitin sulfate/keratan sulfate proteoglycan, PG-1000, forms complexes which are concentrated in the reticular laminae of electric organ basement membranes.

Previously, we identified PG-1000 as part of a disulfide-linked complex of two large proteoglycans (PG-1000 and the beta component) and three smaller proteins purified from the extracellular matrix of elasmobranch electric organ (Iwata and Carlson, 1991, J. Biol. Chem. 266: 323-333). PG-1000 is a chondroitin sulfate/keratan sulfate proteoglycan with a molecular mass of about 1.2 x 16(6) daltons. When visualized in the electron microscope, PG-1000 has the typical "bottle-brush" appearance expected for a proteoglycan with an average total length of about 345 nm and about 20 chains of approximately 110 nm (Carlson and Wight, 1987, J. Cell Biol. 105: 3075-3086). Using immunocytochemical methods, we now demonstrate that PG-1000 is a component of the interstitial extracellular matrix of the electric organ. PG-1000 immunoreactivity is found throughout the interstitial matrix, but it is highly concentrated in that region of the matrix immediately adjacent to the basal lamina, the reticular lamina. The reticular and basal laminae together form the basement membrane. PG-1000 immunoreactivity is especially apparent on basal laminae that surround nerve fibers and nerve terminals. When the disulfide-linked PG-1000 complexes are purified and examined in the electron microscope following rotary shadowing, they appear as bottle-brush structures which are often attached at a central region and radiate like spokes of a wheel. These aggregates contain two to six proteoglycan monomers. We hypothesize that the PG-1000 complexes are disulfide-stabilized parts of an extended network of linked proteoglycans in the reticular lamina.

Proteodermatan and proteokeratan sulfate (decorin, lumican/fibromodulin) proteins are horseshoe shaped. Implications for their interactions with collagen.

The small proteoglycans proteodermatan and proteokeratan sulfates organize collagen fibrils in extracellular matrix [Scott, J. E. (1992) FASEB J. 6, 2639-2645], thus helping to maintain tissue shape. Their interaction with fibrils is probably via the protein. They have been examined by rotary shadowing-electron microscopy, which showed that these leucine-rich-repeat proteins are horseshoe shaped. Morphometry and comparison with polypeptide sequences suggest ways in which decorin could interact with tissue collagen fibrils. It is proposed that decorin is a bidentate ligand attached to two parallel neighboring collagen molecules in the fibril, helping to stabilize fibrils and orient fibrillogenesis.

Cell surface-associated keratan sulfate on normal and migrating corneal endothelium.

PURPOSE: To investigate cell surface-associated keratan sulfate on the corneal endothelium. METHODS: Immunolabeling techniques were used at the light, scanning, and transmission electron microscopic level to localize keratan sulfate on the corneal endothelium. The investigation included human, bovine, and rabbit corneal endothelia. A quantitative study of the relationship between cell size and keratan sulfate levels was conducted on normal bovine corneal endothelium. Changes in the distribution of keratan sulfate and chondroitin sulfate on endothelial cell surfaces were investigated on organ cultured bovine corneas during endothelial wound healing. Changes in the levels of keratan sulfate during endothelial wound healing were investigated in organ cultured human corneas and in vivo in rabbit corneas. Inhibition-enzyme-linked immunosorbent assay also was used to detect keratan sulfate in the aqueous humor. RESULTS: A variegated distribution of keratan sulfate was revealed on normal human, bovine, and rabbit corneal endothelia. Some cells had high levels of keratan sulfate on their surfaces whereas others, sometimes immediately adjacent, had little or none. Wound healing experiments resulted in changes of keratan sulfate levels on the migrating endothelial cells in bovine, human, and rabbit. In wounded organ cultured bovine corneas, there was a decrease in keratan sulfate levels and an increase in chondroitin sulfate levels on migrating endothelial cells. Keratan sulfate was detected in bovine aqueous humor. CONCLUSIONS: The pattern of occurrence of keratan sulfate and chondroitin sulfate on the corneal endothelial cells in normal and wounded cornea suggests that these glycosaminoglycans have differing roles in endothelial adhesion and migration.