Yara M. Michelacci

Collagens and proteoglycans of the corneal extracellular matrix

The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV), and other nonfibrillar collagens (XIII and XVIII). FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils.Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.

Chondroitin sulfates and proteoglycans from normal and arthrosic human cartilage.

The structure of chondroitin sulfates and proteoglycans extracted from human normal young and adult cartilages and also from human arthrosic cartilages are reported. The adult articular cartilage contains almost exclusively chondroitin 6-sulfate, whereas the normal young and the arthrosic cartilage chondroitin sulfates are hybrid polymers, containing 4-sulfated and 6-sulfated disaccharide units, distributed in a quite random way along the molecules. The young cartilage proteoglycans also differ from the adult cartilage proteoglycans by their contents of keratan sulfate, the relative proportion of nonaggregating proteoglycans and electrophoretic migration in agarose gel slabs. The proteoglycans from arthrosic cartilages are very similar to those from young normal cartilages. Such changes in composition could lead to alterations in the proportion and size of the aggregates they form in the cartilages, furnishing the conditions for the processes of growth and calcification to occur.

A comparative analysis of structure and spatial distribution of decorin in human leiomyoma and normal myometrium.

Leiomyoma is a benign smooth muscle tumor of the uterus that affects many women in active reproductive life. It is composed by bundles of smooth muscle cells surrounded by extracellular matrix. We have recently shown that the glycosylation of extracellular matrix proteoglycans is modified in leiomyoma: increased amounts of galactosaminoglycans with structural modifications are present. The data here presented show that decorin is present in both normal myometrium and leiomyoma but tumoral decorin is glycosylated with longer galactosaminoglycan side chains. Furthermore, these chains contain a higher ratio D-glucuronate/L-iduronate, as compared to normal tissue. To determine if these changes in proteoglycan glycosylation correlates with modifications in the extracellular matrix organization, we compared the general structural architecture of leiomyoma to normal myometrium. By histochemical and immunofluorescence methods, we found a reorganization of muscle fibers and extracellular matrix, with changes in the distribution of glycoproteins, proteoglycans, and collagen. Thin reticular fibers, possibly composed by types I and III collagen, were replaced by thick fibers, possibly richer in type I collagen. Type I collagen colocalized with decorin both in leiomyoma and normal myometrium, in contrast to type IV collagen that did not. The relative amount of decorin was increased and the distribution of decorin and collagen was totally modified in the tumor, as compared to the normal myometrium. These findings reveal that not only decorin structure is modified in leiomyoma but also the tissue architecture changed, especially concerning extracellular matrix.

ISOLATION and PARTIAL CHARACTERIZATION of AN INDUCED CHONDROITINASE-B FROM FLAVOBACTERIUM-HEPARINUM

Summary A chondroitinase that degrades only chondroitin sulfate B was isolated from F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The chondroitinase B was separated from a constitutional chondroitinase AC also present in F. heparinum extracts. This enzyme acts only upon chondroitin sulfate B, producing oligo- and tetra- saccharides plus an unsaturated 4-sulfated disaccharide (Δ Di-4S). The oligosaccharide is in turn susceptible only to the chondroitinase AC, producing (Δ Di-4S) and other products. The action of these two enzymes confirms the hybrid nature of chondroitin sulfate B.

An improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases.

Chondroitinases are very important tools for the identification and structural analysis of proteoglycans. Enzymic analysis with Flavobacterium heparinum chondroitinases has shown that chondroitin sulphate and dermatan sulphate structures are modified in many human diseases, suggesting a diagnostic value for these enzymes. Furthermore, it was recently shown that F. heparinum chondroitinases AC and B inhibit tumoural cell growth, invasion and angiogenesis. Due to the increasing importance of F. heparinum chondroitinases, we investigated optimized conditions for preparation and assay of chondroitinases AC, B and C. The Dimethylmethylene Blue assay was modified and fully developed to measure the chondroitinase activities of crude extracts of F. heparinum . This method estimates chondroitin sulphate or dermatan sulphate depolymerization upon the digestion of chondroitinase, and was compared with A 232, which measures the unsaturated products formed. Trypticase was the best culture medium, both for bacterial growth and enzyme induction. The chondroitinases were solubilized by ultrasound under conditions that do not completely disrupt the cells, suggesting that they are located at the periplasmic space. Maximum chondroitinase induction occurred in the presence of 0.2–1.0 g/l chondroitin sulphate. Chondroitin sulphate‐degradation products were also inducers, but heparin and heparan sulphate were not. Chondroitinases AC, B and C were separated from each other by hydrophobic‐interaction chromatography on Phenyl‐Sepharose HP. When contaminant proteins were first removed from crude extract by Q‐Sepharose, the chondroitinases could be purified to homogeneity in this phenyl‐Sepharose chromatographic step.

Proteoglycans from the cartilage of young hammerhead shark Sphyrna lewini

Abstract 1. 1. The majority of proteoglycans (84% of uronic acid) were extracted from shark fin cartilage with 3 M GuHCl. Conventional methods with either 4 M GuHCl or 8 M urea extracted only 36% and 12%, respectively. 2. 2. The proteoglycans have shown higher hydrodynamic sizes than those observed for mammalian hyaline cartilages. 3. 3. Several chondroitin sulphate ( MW = 43,000 ) and keratan sulphate ( MW = 18,000 ) chains, in a ratio of 4:1, are attached to the same protein core. 4. 4. We were unable to show the presence of hyaluronic acid in shark fin cartilage and the shark cartilage proteoglycans appear to lack the ability to form complexes with hyaluronic acid. 5. 5. These data suggest that the proteoglycans in shark fin cartilage present a structural organization which is different from that of mammalian cartilages.

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparium

Abstract During the investigation of alternative methods for the large sclae preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast tot he chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashiom, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chontroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of Δ-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30°C and at pH 6.0–7.5. Only 15% of the activity was observed at 37°C, indicating that the enzyme is very sensitive to thermal denaturation. It is stronly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.

Proteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats

In the renal glomerulus, two extracellular matrices have been identified, the glomerular basement membrane and the mesangial matrix. Accumulation of glomerular extracellular matrix is a conspicuous feature of most forms of progressive glomerular disease, including diabetic nephropathy. Since proteoglycans are prominent components of the extracellular matrix, we examined the glycosaminoglycans and proteoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. A mixture of dermatan sulfate and heparan sulfate was recovered. Dermatan sulfate was the predominant glycosaminoglycan synthesized and most of it was released to the culture medium, in contrast to heparan sulfate which was found to be cell associated to a higher degree. The dermatan sulfate chains are composed by D-glucuronic and L-iduronic acid-containing disaccharides and are highly sulfated. Mesangial cells from diabetic rats produce much more glycosaminoglycans than mesangial cells from normal rats, especially dermatan sulfate and this increase was proportional to the duration of diabetes. In contrast, exposure of mesangial cell from normal rats to elevated glucose did not lead to any changes in glycosaminoglycan synthesis, indicating that this short-term culture conditions may not adequately simulate diabetes mellitus. Other factors related to diabetes environment may be responsible for the observed alterations. The dermatan sulfate was secreted to the medium as proteoglycan. Two dermatan sulfate proteoglycans were identified, with molecular weights of 120 and 85 kDa respectively. The proteoglycan core protein M(r) was 45 kDa and the dermatan sulfate chains were 35 kDa. It is possible that the two proteoglycans represent two populations, one with two dermatan sulfate side chains (120 kDa) and the other with only one side chain (85 kDa), presumably fitting in the decorin/biglycan family of small proteoglycans.

Effect of epithelial debridement on glycosaminoglycan synthesis by human corneal explants.

The purpose of the present work was to investigate the effects of mechanical epithelial debridement upon glycosaminoglycan synthesis by human corneal explants. Corneal explants were maintained under tissue culture conditions for 2-72 days and the glycosaminoglycans synthesized in 24 h were metabolically labeled by addition of 35S-sulfate to the culture medium. These compounds were isolated from the tissue explants and analyzed by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. The glycosaminoglycans synthesized by isolated epithelial cells and by corneas previously submitted to epithelial cell debridement were compared to controls. Keratan sulfate (26 kDa) and dermatan sulfate (43 kDa) were the main corneal glycosaminoglycans, each one corresponding to about 50% of the total. Nevertheless, the main 35S-labeled glycosaminoglycan was 35S-dermatan sulfate (73%), with smaller amounts of 35S-keratan sulfate (15%) and 35S-heparan sulfate (12%), suggesting a lower synthesis rate for keratan sulfate. The main glycosaminoglycan synthesized by isolated epithelial cells was heparan sulfate. The removal of epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by stromal cells. This increased synthesis of dermatan sulfate suggests a relationship between epithelium and stroma and could be related to the corneal opacity that may appear after epithelial cell debridement.

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide C indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.