Hyaluronan and chondroitin sulfate proteoglycans are colocalized to the ciliary zonule of the rat eye: a histochemical and immunocytochemical study

 In previous studies, chondroitin sulfate proteoglycans have been localized to the periphery of the zonular fibers and the individual zonular fibrils (or microfibrils) after Cuprolinic blue staining in conjunction with chondroitinase digestions and immunogold labelling with 2-B-6 antibody. In the present study, we wished to determine if these proteoglycans are linked to hyaluronan to form a large multimolecular aggregate. To accomplish this, we localized the hyaluronan using a biotinylated hyaluronan-binding protein fragment of chondroitin sulfate proteoglycan, containing also the link protein, purified from bovine nasal cartilage. The results showed that the ciliary zonule of the rat eye was reactive with the biotinylated hyaluronan-binding probe as demonstrated by streptavidin-peroxidase-diaminobenzidine staining and streptavidin-gold labelling. Hyaluronan-gold labelling showed that the gold particles were mostly localized on the periphery of the zonular fibers, which was similar to the localization pattern of the zonule associated-proteoglycans. This hyaluronan-binding probe also strongly labelled the sites of zonule insertion over the basement membrane of the inner ciliary epithelium at the pars plana and the lens capsule at the equatorial region, which suggests its probable role in the attachment of ciliary zonule to the basement membranes. To demonstrate whether these two molecules are linked to one another, ultrastructural colocalization of both hyaluronan and chondroitin sulfate proteoglycans was performed on the same sections by double-gold labelling, and combined Cuprolinic blue staining and hyaluronan-gold labelling. Gold particles of 15 and 10 nm in sizes labelling both hyaluronan and chondroitin 4-sulfate, were colocalized to the surface of the zonular fibers. The combined Cuprolinic blue staining and hyaluronan-gold labelling showed that the gold particles were localized towards the ends of the Cuprolinic blue-stained rodlets, which strongly suggests that these chondroitin sulfate proteoglycans are linked to the hyaluronan chain to form a large aggregate surrounding the periphery of the zonular fibers. These ciliary zonule-associated proteoglycan-hyaluronan aggregates may play a role in organizing the individual zonular fibrils (microfibrils) into bundles of zonular fibers. Accepted: 5 November 1996     

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.     

Immunohistochemical localization of glycosaminoglycans and proteoglycans in predentin and dentin of rat incisors.

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aldehyde fixation of rat incisors using (a) four monoclonal antibodies (1-B-5,9-A-2,3-B-3, and 5-D-4) which recognize epitopes in unsulfated chondroitin (C0-S), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatment of tissue sections with chondroitinase ABC localized the majority of stainable PG (C4-S, KS, DSPG, C0-S, and C6-S) in predentin and, to a lesser extent (C4-S and small DSPG), in the dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a definite difference between PG of predentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules during dentinogenesis and mineralization.     

Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage

The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4-sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component

Immunohistochemical methods were used for the detection of the amyloid P component in the microfibrils of two regions: the zonule of the eye and the connective tissue of the foot pad in 20- to 50-gm mice. Following fixation by immersion in 4% formaldehyde, the eyes and foot pads were embedded in paraffin, and sections were immunostained for light microscopy by using antiamyloid P component antiserum followed by peroxidase-antiperoxidase procedure. For electron microscopy, formaldehyde-fixed tissues were immunostained for the amyloid P component with protein A-gold by using either thin Lowicryl sections or frozen sections which were then embedded in Epon for thin sectioning. In the zonule of the eye, the light microscope showed that zonular fibers were strongly immunostained for the amyloid P component; there was also weak staining of the nonpigmented ciliary epithelium at the distal end of the fibers and of the zonular lamella at their proximal end. The electron microscope revealed clear-cut immunolabeling of the microfibrils making up zonular fibers as well as of individual microfibrils. In the foot pad, the light microscope detected a weak diffuse staining of connective tissue, whereas the electron microscope showed immunolabeling restricted to microfibrils. It was concluded that the amyloid P component was present in, or associated with, microfibrils. Purified amyloid P component was prepared and examined in the electron microscope after either negative staining or routine processing. After negative staining, it appeared as flat pentagonal units, frequently associated into columns. After routine processing, the units looked like cross sections of microfibrillar tubules. The dimensions of the units matched those of the hypothetical segments of the tubules. It was concluded that this tubule consisted of a column of amyloid P units. The cohesion of the units within the column was likely to be reinforced by the bands present at the surface of microfibrils.     

Chondroitin sulfate proteoglycans in the rat thalamus: expression during postnatal development and correlation with calcium-binding proteins in adults

Postnatal expression of chondroitin sulfate proteoglycans was studied in the rat thalamus by immunocytochemistry and Western immunoblotting techniques with monoclonal antibodies that recognize carbohydrate epitopes (clones CS-56, 1-B-5, 2-B-6). The complex of the results shows that these antibodies recognize mostly nonoverlapping molecules whose expression is regulated during postnatal development. Chondroitin sulfate proteoglycans, recognized by antibody CS-56, and hyaluronan, identified by antibody 1-B-5 after hyaluronidase digestion, are abundant in the neuropil of most thalamic nuclei at the perinatal stage and progressively decrease during the second week of life, attaining levels barely detectable by immunocytochemistry at the end of the third week. In adult thalamus, chondroitin sulfate proteoglycans of high molecular mass, bearing glycosaminoglycans unsulfated in the linking region, and recognized by antibody 1-B-5 are confined to perineuronal nets around neurons chiefly localized in thalamic reticular nucleus. The immunoreactvity for antibody 2-B-6, specific for chondroitin-4-sulfate, is low at the perinatal stage and is not detectable in adult thalamus. Double-immunolabeling has shown that, along the rostrocaudal extension of reticular nucleus, the most developed perineuronal nets are associated with a subset of neurons expressing calretinin, and not with parvalbumin-positive neurons, which represent the largest neuronal population of the nucleus. The distribution of perineuronal nets supports the presence, in thalamic reticular nucleus, of neuronal subpopulations with different morphological and physiological features.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural and immunohistochemical analysis of proteoglycans in mouse pubic symphysis

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase.The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.     

Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT).

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.     

Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT)

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.     

Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

Summary The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S, still exist in mineralized cartilage matrix and lamina limitants.     

Association of fibronectin with the microfibrils of connective tissue

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.     

Further characterization of a large proteoglycan in human lung alveoli

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, heavily staining, generally large, filaments have been demonstrated in human lung alveoli. In some lung specimens they are abundant, while in others they are very scanty. The filaments are seen: around bundles of collagen fibrils, at places which seem electron microscopically almost empty, associated with basement membranes around elastin, and sometimes associated with individual collagen fibrils. After poststaining tiny threads--connecting the filaments--could sometimes be observed. The filaments are resistant to treatment with nitrous acid, heparitinase or pronase after prefixation. After digestion with chondroitinase ABC, chondroitinase AC or pronase without prefixation, the filaments are no longer detectable. The tiny threads are chondroitinase ABC resistant. It is concluded that the Cuprolinic Blue-positive filaments represent proteoglycans which contain chondroitin sulfate and/or glucuronic acid-rich dermatan sulfate. The possible role of these proteoglycans in tissue repair is discussed.     

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.     

Synthesis and structural organization of zonular fibers during development and aging.

Zonular fibers are a specific form of extracellular matrix composed mainly of fibrillins. The purpose of this study was to determine which cells secrete fibrillin-1 during development and aging. A specific guinea pig fibrillin-1 mRNA probe was designed and cloned in order to identify fibrillin-secreting cells in guinea pig eye, using in situ hybridization. Immunofluorescence, with a specific guinea pig monoclonal antibody, was used to compare protein levels at different stages from birth to 35 months of age. Electron microscopy and immunolabeling were used to investigate the organization of zonular microfibril bundles. We identified the cells of non-pigmented epithelium of the ciliary body as the main source of fibrillin secreted into the zonule. Moreover, while mRNA expression decreased during aging, there was no decrease in fibrillin immunoreactivity, as previously described in human aorta. These data indicate a very slow turnover of the zonular microfibrils which can be correlated with the appearance during aging of a new periodic fibrillar structure. This new structure may reflect an increased cross-linking in the long-lived zonular microfibrillar bundles.     

Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.     

The amino-terminal region of a proteochondroitin core protein, secreted by aortic smooth muscle cells, shares sequence homology with the pre-propeptide region of the biglycan core protein from human bone

Smooth muscle cells, isolated from rat and bovine aortae and grown in vitro, synthesize chondroitin sulfate proteoglycans which are secreted into the growth media. Analysis of metabolically [35S]-labeled macromolecules, employing ion-exchange chromatography, revealed a single peak of radioactivity, upon elution with a linear salt gradient. Treatment of the material with enzymes that specifically degrade chondroitin sulfate demonstrated that chondroitin-4-sulfate was the predominant species isolated from rat smooth muscle cells and that chondroitin-4-sulfate and dermatan sulfate were the predominant species isolated from bovine aortic smooth muscle cells. Treatment of the native proteoglycans with chondroitinase ABC and subsequent SDS-PAGE analysis of the digestion products resulted in the appearance of a band with an apparent molecular weight of 45,000. Electrotransfer of the core protein to Immobilon-P membrane and gas phase sequencing of the amino-terminal region revealed striking homology between the core proteins of the rat and bovine proteochondroitin with the pre-propeptide region of human bone biglycan.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage.

From the carbohydrate-protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4-sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondroitinase ABC, and subsequent beta-elimination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500-MHz 1H-NMR spectroscopy. The nonsulfated compound (A) had the following conventional structure: delta GlcA(beta 1-3)-GalNAc(beta 1-4)GlcA(beta 1-3)Gal(beta 1-4)Xylol, where GlcA, delta GlcA and GalNAc are glucuronic acid; 4,5-unsaturated glucuronic acid and 2-deoxy-2-N-acetylamino-D-galactose, respectively. The other compounds were sulfated derivatives of compound A. Two monosulfated compounds (B and C) had an ester sulfate on C4 or C6 of the GalNAc residue, respectively and the disulfated compound (D) had two ester sulfate groups, namely, one on C4 of the GalNAc and the other on C4 of the Gal residue substituted by GlcA. The molar ratio of A/B/C/D was 0.21:0.16:0.36:0.27. The compound containing Gal-4-O-sulfate was previously isolated by us in the form of a sulfated glycoserine [delta GlcA(beta 1-3)GalNAc(4-O- sulfate)(beta 1-4)GlcA(beta 1-3)Gal(4-O-sulfate)(beta 1-3)-Gal(beta 1- 4)Xyl beta 1-O-Ser] from the carbohydrate-protein linkage region of rat chondrosarcoma chondroitin-4-sulfate proteoglycans [Sugahara K., Yamashina, I., DeWaard, P., Van Halbeek, H. & Vliegenthart, J.F.G. (1988) J. Biol. Chem. 263, 10,168-10,174]. The discovery of this structure in the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans from nontumorous cartilage indicates that it is not a tumor-associated product but rather a physiological biosynthetic product since it represents a significant proportion. The biological significance of this structure is discussed in relation to glycosaminoglycan biosynthesis.