Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.     

A proteomic approach for identification and localization of the pericellular components of chondrocytes

Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

A Mechano-chemical Model for the Passive Swelling Response of an Isolated Chondron under Osmotic Loading

The chondron is a distinct structure in articular cartilage that consists of the chondrocyte and its pericellular matrix (PCM), a narrow tissue region surrounding the cell that is distinguished by type VI collagen and a high glycosaminoglycan concentration relative to the extracellular matrix. We present a theoretical mechano-chemical model for the passive volumetric response of an isolated chondron under osmotic loading in a simple salt solution at equilibrium. The chondrocyte is modeled as an ideal osmometer and the PCM model is formulated using triphasic mixture theory. A mechano-chemical chondron model is obtained assuming that the chondron boundary is permeable to both water and ions, while the chondrocyte membrane is selectively permeable to only water. For the case of a neo-Hookean PCM constitutive law, the model is used to conduct a parametric analysis of cell and chondron deformation under hyper- and hypo-osmotic loading. In combination with osmotic loading experiments on isolated chondrons, model predictions will aid in determination of pericellular fixed charge density and its relative contribution to PCM mechanical properties.     

Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose

Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.     

Molecular characterization of two novel alternative spliced variants of the KLRF1 gene and subcellular distribution of KLRF1 isoforms

Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.     

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1–3 ·m in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4–6 weeks in culture and progressively developed a broad territorial region up to 12 ·m wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly This revised version was published online in November 2006 with corrections to the Cover Date.     

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1–3 ·m in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4–6 weeks in culture and progressively developed a broad territorial region up to 12 ·m wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly This revised version was published online in November 2006 with corrections to the Cover Date.     

The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules

In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis

In articular cartilage, chondrocytes are surrounded by a pericellular matrix (PCM), which together with the chondrocyte have been termed the "chondron." While the precise function of the PCM is not know there has been considerable speculation that it plays a role in regulating the biomechanical environment of the chondrocyte. In this study, we measured the Young's modulus of the PCM from normal and osteoarthritic cartilage using the micropipette aspiration technique, coupled with a newly developed axisymmetric elastic layered half-space model of the experimental configuration. Viable, intact chondrons were extracted from human articular cartilage using a new microaspiration-based isolation technique. In normal cartilage, the Young's modulus of the PCM was similar in chondrons isolated from the surface zone (68.9 +/- 18.9 kPa) as compared to the middle and deep layers (62.0 +/- 30.5 kPa). However, the mean Young's modulus of the PCM (pooled for the two zones) was significantly decreased in osteoarthritic cartilage (66.5 +/- 23.3 kPa versus 41.3 +/- 21.1 kPa, p < 0.001). In combination with previous theoretical models of cell-matrix interactions in cartilage, these findings suggest that the PCM has an important influence on the stress-strain environment of the chondrocyte that potentially varies with depth from the cartilage surface. Furthermore, the significant loss of PCM stiffness that was observed in osteoarthritic cartilage may affect the magnitude and distribution of biomechanical signals perceived by the chondrocytes.     

Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan

Three antibodies reacting with corneal keratan sulfate proteoglycan were used to detect antigenically related molecules in 11 bovine and 13 embryonic chick tissues. Two monoclonal antibodies recognized sulfated epitopes on the keratan sulfate chain and a polyclonal antibody bound antigenic sites on the core protein of corneal keratan sulfate proteoglycan. Competitive immunoassay detected core protein and keratan sulfate antigens in guanidine HCl extracts of most tissues. Keratan sulfate antigens of most bovine tissues were only partially extracted with guanidine HCl, but the remainder could be solubilized by CNBr treatment of the guanidine-extracted residue. Keratan sulfate and core protein antigens co-eluted with purified corneal keratan sulfate proteoglycan on ion exchange high-performance liquid chromatography (HPLC). Endo-beta-galactosidase digestion of the HPLC-purified keratan sulfate antigens eliminated the binding of monoclonal anti-keratan sulfate antibodies in enzyme-linked immunosorbent assay. Extracts of all 11 bovine tissues, except those from brain and cartilage, could bind both anti-keratan sulfate monoclonal antibodies and anti-core protein polyclonal antibody simultaneously. Binding was sensitive to competition with keratan sulfate and to digestion with endo-beta-galactosidase. These results suggest widespread occurrence of a proteoglycan or sulfated glycoprotein bearing keratan sulfate-like carbohydrate and a core protein resembling that of corneal keratan sulfate proteoglycan.     

Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilage

The pericellular matrix (PCM) is a narrow region of tissue that completely surrounds chondrocytes in articular cartilage. Previous theoretical models of the "chondron" (the PCM with enclosed cells) suggest that the structure and properties of the PCM may significantly influence the mechanical environment of the chondrocyte. The objective of this study was to quantify changes in the three-dimensional (3D) morphology of the chondron in situ at different magnitudes of compression applied to the cartilage extracellular matrix. Fluorescence immunolabeling for type-VI collagen was used to identify the boundaries of the cell and PCM, and confocal microscopy was used to form 3D images of chondrons from superficial, middle, and deep zone cartilage in explants compressed to 0%, 10%, 30%, and 50% surface-to-surface strain. Lagrangian tissue strain, determined locally using texture correlation, was highly inhomogeneous and revealed depth-dependent compressive stiffness and Poisson's ratio of the extracellular matrix. Compression significantly decreased cell and chondron height and volume, depending on the zone and magnitude of compression. In the superficial zone, cellular-level strains were always lower than tissue-level strains. In the middle and deep zones, however, tissue strains below 25% were amplified at the cellular level, while tissue strains above 25% were decreased at the cellular level. These findings are consistent with previous theoretical models of the chondron, suggesting that the PCM can serve as either a protective layer for the chondrocyte or a transducer that amplifies strain, such that cellular-level strains are more homogenous throughout the tissue depth despite large inhomogeneities in local ECM strains.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.     

Visualization of the large heparan sulfate proteoglycan from basement membrane

Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length means = 52 nm) and extended following negative staining (length means = 83 nm) or rotary shadowing (length means = 87 nm; 400-kDa core length means = 80 nm; P200 length means = 44 nm). Two or three glycosaminoglycan side chains (length means = 146 +/- 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.     

Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein II

A keratan sulfate proteoglycan was isolated under denaturing conditions from the mineral compartment of rabbit cortical bone. This small proteoglycan (Kd = 0.39 on Superose 6, Mr approximately 20,000 on sodium dodecyl sulfate gels) contained small keratan sulfate chains that were distinctly bimodal in size. The keratanase and endo-beta-galactosidase digestible glycosaminoglycan chains were O-linked to a core protein of Mr approximately 80,000. This core protein had several properties in common with the bone sialoprotein II molecule of bovine and human bone including: a closely spaced doublet band on sodium dodecyl sulfate electrophoresis gels; a high staining intensity with Stains All that was greatly diminished by neuraminidase; a significant amount of small O-linked oligosaccharides; and an amino-terminal amino acid sequence that was nearly identical to human bone sialoprotein II. (In contrast, bone sialoprotein II in human, bovine, and rat bone does not appear to have any keratan sulfate chains.) Antiserum made against the keratan sulfate proteoglycan reacted with its core protein on electrotransfers from sodium dodecyl sulfate-polyacrylamide gels.     

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.