Chondroitin sulfate proteoglycan in the extracellular matrix of the canine superior olivary nuclei

The perineuronal extracellular matrix of the canine superior olivary nuclei was examined by the histochemical method. The extracellular matrix was stained with Alcian blue (pH 1.0 and 2.5), high iron diamine and ruthenium red. The staining intensity of Alcian blue in the extracellular matrix was remarkably reduced after chondroitinase ABC digestion but not after that of heparitinase or hyaluronidase. These results indicate that the extracellular matrix consists of proteoglycans and contains the chondroitin sulfate proteoglycan.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. I. Characterization of the culture system with emphasis on stromal proteoglycan biosynthesis

Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.     

Structure of outer hair cell stereocilia links in the chinchilla

The structure of side, tip, and “attachment” links of chinchilla outer hair cell (OHC) stereocilia was studied by transmission and scanning electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. Tannic acid, which interacts with many different types of proteins and glycoproteins irrespective of their electrical charge, showed strong reactivity for the central area of the side links and weak reactivity for the marginal area of these links adjacent to the stereocilia membrane. Tannic acid treatment revealed the tip links as thin strands, about 5 nm thick. Attachment links were poorly visualized after tannic acid treatment and appeared as sparse filamentous strands at tips of the tallest OHC stereocilia. Cuprolinic blue, at a high critical electrolyte concentration, reacted with strongly negative, primarily sulfated, carbohydrate residues of glycoconjugate macromolecules. In contrast to the tannic acid treatment, the central portions of the OHC stereocilia side links were unstained after Cuprolinic blue treatment; however, membrane-associated ends of these links were darkly stained. The tip links showed a similar appearance as after tannic acid treatment; however, Cuprolinic blue revealed an electron-dense substructure at both ends of its insertion into the stereocilia. Cuprolinic blue reactive structures were also observed as attachment links only at the tips of the OHC stereocilia of the tallest row in each bundle. These structures formed a crown-like array around the tip of each stereocilium. Their primary function appears to be attachment of type B fibrils of the tectorial membrane to the tallest OHC stereocilia. Cuprolinic blue reactive structures of the side, tip, and attachment links appear to contain acidic, sulfated residues of proteoglycans or glycoproteins. These structures may function as connective elements between the stereocilia links and the hair cell cytoskeleton.     

Formation of the tectorial membrane of the cochlear canal during the embryogenesis of birds

By means of electron microscopy formation of the tectorial membrane of the cochlear canal and differentiation of the cells participating in the process (supporting cells of the basilar papilla and anterior homogeneous cells--AHC) have been studied in chick embryos. The AHC, to which the tectorial membrane is fixed, produce fine fibrillar material, included into the composition of the tectorial membrane. The cells mentioned form a number of cytoskeletal structures connected with the mechanical function of the tectorial membrane. Besides the network of the tonofilaments, gradually filling cytoplasm of the AHC, some peculiar attachings in the form of collagenous fibrillar bundles are revealed, they reach the AHC from the sublying connective tissue and have a direct contact with the basal membrane of the cells. The beginning of the tectorial membrane formation precedes the formation of the cytoskeletal structures. The latter appear only when the mass of the tectorial membrane, and hence, the mechanical loading on the AHC is great enough.     

New role of glycosaminoglycans on the plasma membrane proposed by their interaction with phosphatidylcholine

Glycosaminoglycan side chains of membrane proteoglycans have been claimed to be located at the outermost layer of the glycocalyx surrounding the cell. In this study measurements by surface plasmon resonance and solid-phase assay have shown that both chondroitin sulfate and keratan sulfate but not heparin associate with phosphatidylcholine under physiological conditions. Spectrophotometric measurements also showed that chondroitin sulfate restricts the lateral diffusion of phosphatidylcholine in liposomes. These findings indicate that chondroitin sulfate and/or keratan sulfate chains of membrane proteoglycans crouch on the surface of the membrane while heparan sulfate chains stretch outward from the membrane surface as postulated traditionally.     

Scanning electron microscope studies of the papilla basilaris of some turtles and snakes

The papillae basilares of three species of turtles and four species of snakes were studied by SEM. The papillae of turtle are relatively large among reptiles and are characterized by a long, horizontal middle section resting on wide basilar membrane. Both terminal ends of the papilla extend onto the surrounding limbus in the form of a forked or "T" -shaped end or as a curved, "hook"- like processes. Details vary with the species. In the three species of turtles studied, there were between 1,100 and 1,400 hair cells on a papilla. The tectorial membrane covering the horizontal portion of the papilla is heavy in appearance and tightly attached to the kinocilial bulbs. The terminal ends of the papilla are covered by a thin gelatinous material. In addition, mat-like tectorial network covers the supporting cells and extends from the microvilli of the supporting cells to the overlying tectorial membrane. All hair cells are unidirectionally and abneurally oriented. The supporting cell surfaces form a large part of the papilla and, thus, hair cell density is low. The papillae of the two boid snake species studied are moderately long among snakes and contain a moderate number of hair cells (574 in Epicrates and 710-780 in Constrictor). Papillar form is elongate, avoid, or canoe-shaped. The tectorial membrane may be either highly fenestrated or moderately dense and covers all but a few of the terminal hair cells. A tectorial-like mat covers all but a few of the terminal hair cells. Most hair cells are unidirectionally and abneurally oriented. A few terminal cells in boids may show reverse orientation. Hair cell density is similar to that of turtles.     

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood-vessel development in the chick chorioallantoic membrane

This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.     

Endothelial glycocalyx of blood circulation system. I. Detection,components, and structural organization

The luminal surface of a blood vessel accommodates a complex multicomponent system of mainly carbohydrates and proteins called glycocalyx. According to the concept of the double protective layer, glycocalyx is the first protection barrier of the vascular wall. The structure of glycocalyx is determined by a group of proteoglycans, glycoproteins, and glycosaminoglycans. Two groups of molecules are distinguished within the glycocalyx constituents, that is, membrane proteoglycans (syndecans and glypicans bound to endothelial cell membranes) and soluble proteoglycans (perlecan, biglycan, versican, decorin, and mimecan). There are five types of glycosaminoglycan chains; these are heperan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. There is a dynamic equilibrium between the soluble components of glycocalyx and flowing blood, which allows for separation of the endothelial surface layer. Due to its complexity and location at the interface of blood circulation system, glycocalyx is involved in the maintenance of vascular homeostasis. Here, the molecular composition of glycocalyx, properties of its components, biosynthesis, and common structural features are discussed.     

Morphological and fine structural features of the basilar papilla in ambystomatid salamanders (Amphibia; Caudata)

The morphology and fine structure of the basilar recess and basilar papilla were investigated in four species of salamanders from the family Ambystomatidae. The otic relationships of the recess and papilla to the proximal part of the lagena and saccule are described, and new terminology is suggested for the periotic relationships of the basilar recess to a diverticulum of an intracapsular periotic sac. The basilar papilla consists of supporting cells united laterally by gap junctions, capped by microvilli uniformly arranged around a short, central cilium, and hair cells that typically show several synapses with a single afferent nerve fiber, each marked by a rounded synaptic body surrounded by vesicles. In contrast to anuran basilar papillae, efferent nerve terminals were observed in synapse with hair cells and, rarely, upon afferent fibers. The distal half of the ambystomatid papilla contained hair cells capped by tall ciliary bundles, with kinocilia that show swellings near their tips with delicate attachments to adjacent tall stereocilia. A tectorial body covers only this region of the papilla. Hair cells with shorter stereocilia, situated in the proximal half and at the papillar margins, are related only to filamentous extensions of the tectorial body. The ambystomatid basilar recess and papilla are compared to auditory end-organs in other vertebrates, and it is suggested that a basic distinction can be made between aural neuroepithelia in amniotes versus that in nonamniotic vertebrate ears.     

Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development

In this study, we describe the distribution of various classes of proteoglycans and their potential matrix ligand, hyaluronan, during neural crest development in the trunk region of the chicken embryo. Different types of chondroitin and keratan sulfate proteoglycans were recognized using a panel of monoclonal antibodies produced against specific epitopes on their glycosaminoglycan chains. A heparan sulfate proteoglycan was identified by an antibody against its core protein. The distribution of hyaluronan was mapped using a biotinylated fragment that corresponds to the hyaluronan-binding region of cartilage proteoglycans. Four major patterns of proteoglycan immunoreactivity were observed. (1) Chondroitin-6-sulfate-rich proteoglycans and certain keratin sulfate proteoglycans were absent from regions containing migrating neural crest cells, but were present in interstitial matrices and basement membranes along prospective migratory pathways such as the ventral portion of the sclerotome. Although initially distributed uniformly along the rostrocaudal extent of the sclerotome, these proteoglycans became rearranged to the caudal portion of the sclerotome with progressive migration of neural crest cells through the rostral sclerotome and their aggregation into peripheral ganglia. (2) A subset of chondroitin/keratan sulfate proteoglycans bearing primarily unsulfated chondroitin chains was observed exclusively in regions where neural crest cells were absent or delayed from entering, such as the perinotochordal and subepidermal spaces. (3) A subset of chondroitin/keratan sulfate proteoglycans was restricted to the perinotochordal region and, following gangliogenesis, was arranged in a metameric pattern corresponding to the sites where presumptive vertebral arches form. (4) Certain keratan sulfate proteoglycans and a heparan sulfate proteoglycan were observed in basement membranes and in an interstitial matrix uniformly distributed along the rostrocaudal extent of the sclerotome. After gangliogenesis, the neural crest-derived dorsal root and sympathetic ganglia contained both these proteoglycan types, but were essentially free of other chondroitin/keratan-proteoglycan subsets. Hyaluronan generally colocalized with the first set of proteoglycans, but also was concentrated around migrating neural crest cells and was reduced in neural crest-derived ganglia. These observations demonstrate that proteoglycans have diverse and dynamic distributions during times of neural crest development and chondrogenesis of the presumptive vertebrae. In general, chondroitin/keratan sulfate proteoglycans are abundant in regions where neural crest cells are absent, and their segmental distribution inversely correlates with that of neural crest-derived ganglia.     

Synthesis of small proteoglycans substituted with keratan sulfate by rabbit articular chondrocytes

35S-Labeled proteoglycans produced by chondrocytes from immature and mature rabbits were fractionated on associative CsCl gradients. In all cultures, greater than 85% of the incorporated radioactivity was present in the A1 fraction (rho 1.60) as chondroitin sulfate/keratin sulfate-substituted aggregating proteoglycan monomer; the remainder was present in small proteoglycans in the A2, A3, and A4 fractions of low buoyant densities (rho 1.53, 1.45, 1.37, respectively). Detailed glycosaminoglycan analysis of the A2, A3, and A4 fractions showed dermatan sulfate-rich species were present throughout. However, in both immature and mature cultures, 30-45% of the glycosaminoglycans in the A3/A4 combined fractions were present as keratan sulfate, as shown by insensitivity to digestion with chondroitinase ABC, specific digestion with endo-beta-galactosidase, and reactivity with antibody 5D4. Immature and mature chondrocytes synthesized very similar amounts of the low buoyant density keratan sulfate proteoglycan on a per cell basis. Moreover, 51 and 37% of the total keratan sulfate produced by immature and mature chondrocytes, respectively, were present in the low buoyant density proteoglycan. Pulse-chase experiments indicated that the low buoyant density keratan sulfate was not derived from the large aggregating proteoglycan by proteolysis in the extracellular space. The small keratan sulfate proteoglycans appear to be present as a species distinct from the small dermatan sulfate proteoglycans in these cultures in that they can be separated on Q-Sepharose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent size (40-60 kDa), composition, and heterogeneity of the keratan sulfate proteoglycans suggest that they may be related to the small keratan sulfate proteoglycans of cornea.     

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. II. Binding sites of proteoglycans on the cell surface

Exogenous proteoglycans stained for electron microscopy with colloidal gold and/or cuprolinic blue bind to the surface of cultured arterial smooth muscle cells at two different sites. (I) About 20% of the proteoglycans adsorbed to the cells from the culture medium interact as monomeric and multimeric proteoglycans with smooth or coated membrane areas. (II) The bulk of exogenous proteoglycans exhibits high affinity binding to cell membrane-associated 10 nm fibrils containing or being closely associated with fibronectin and to collagen. It is suggested that the self association of proteoglycans and their binding to the cell membrane and to cell surface-associated fibronectin and collagen are important for maintaining an appropriate micro-environment for the cultured cells.     

Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

Potential role for heparan sulfate proteoglycans in regulation of transforming growth factor-beta (TGF-beta) by modulating assembly of latent TGF-beta-binding protein-1

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-beta in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-beta into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-beta into the ECM, with increased amounts of soluble latent TGF-beta. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by beta-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-beta availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.     

Localization of basement membrane glycoproteins in rat kidney during foetal development

The distribution of basement membrane glycoproteins (type IV collagen, laminin, fibronectin, and proteoglycans) was studied in foetal rat kidney by immunohistochemical techniques using polyclonal antibodies. From the first stages of nephron differentiation, all these glycoproteins were detectable by immunofluorescence in the tubular and glomerular basement membranes and in the mesangial matrix. As differentiation proceeded, labelling of glycoproteins progressively intensified, except for that of fibronectin, which gradually decreased in the glomerular basement membrane (GBM) and was barely observable at full differentiation. With immunoperoxidase staining in electron microscopy, all glycoproteins were seen to be widely dispersed in the spaces between the epithelial and endothelial glomerular cells so long as the GBM remained a loose structure. However, after it became a compact, 3-layered formation, type IV collagen and laminin were distributed throughout the GBM, whereas proteoglycans and anionic sites appeared as 2 rows of granules confined to the laminae rarae.     

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.     

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.     

The tectorial membrane of the lizard ear: types of structure

This study is concerned with the forms of the tectorial membrane in the lizard ear and its manner of attachment to the ciliary tufts of the hair cells. These structures and their variations were observed in 20 species representing eight families of lizards. Three forms of tectorial membrane were found, a continuous form that extends throughout the length of the auditory papilla, an abbreviated form that reaches the papilla only in one region, and a dendritic form that is particularly narrow at first and then branches extensively to supply all the hair cells. Occasionally the lower edge of the tectorial membrane makes direct connections with the hair tufts. More often there are special connecting structures between the membrane and the hair tufts. Seven types of these structures were identified, as follows: (1) simple fibers, (2) open network, (3) heavy network, (4) fiber plate, (5) finger processes, (6) sallets, and (7) remote connections. These types of tectorial connections are described and illustrated.