Regulation of chondroitin sulfate synthesis. Effect of beta-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.     

Radioimmunological quantitation of urinary trypsin inhibitor

Using monospecific antibody to human urinary trypsin inhibitor, we developed a highly specific and sensitive radioimmunoassay (RIA) for measuring human urinary trypsin inhibitor. No cross-reactivity of the antibody with protein standard serum, which contained albumin, alpha 1-antitrypsin, haptoglobin, alpha 2-macroglobulin, transferrin, IgG and IgA, was observed. The sensitivity of the system was 10 ng of trypsin inhibitor per assay tube, and 5-10 microliters of urine was sufficient to determine the concentration of trypsin inhibitor in urine. The amounts excreted in the urine of 10 healthy men and 10 healthy women were 4.83 +/- 2.46 (mean +/- SD) and 3.86 +/- 1.35 mg/day, respectively. The correlation between estimates by RIA and those by enzymic assay was r = 0.96 (p less than 0.005). The method proposed here can be used to determine the concentration of urinary trypsin inhibitor in a small amount of biological fluids and cells.     

Plasmodium falciparum: adherence of the parasite-infected erythrocytes to chondroitin sulfate proteoglycans bearing structurally distinct chondroitin sulfate chains

Infection with Plasmodium falciparum during pregnancy leads to the selective adherence of infected red blood cells (IRBCs) in the placenta causing placental malaria. The IRBC adherence is mediated through the chondroitin 4-sulfate (C4S) chains of unusually low-sulfated chondroitin sulfate proteoglycans (CSPGs) in the placenta. To study the structural interactions involved in C4S-IRBC adherence, various investigators have used CSPGs from different sources. Since the structural characteristics of the polysaccharide chains in CSPGs from various sources differ substantially, the CSPGs are likely to differentially bind IRBCs. In this study, the CSPG purified from bovine trachea, a CSPG form of human recombinant thrombomodulin (TM-CSPG), two CSPG fractions from bovine cornea, and the CSPGs of human placenta, the natural receptor, were studied in parallel for their IRBC binding characteristics. The TM-CSPG and corneal CSPG fractions could bind IRBCs at significantly higher density compared to the placental CSPGs. However, the avidity of IRBC binding by TM-CSPG was considerably low compared to placental CSPGs. The corneal CSPGs have substantially higher binding strengths. The bovine tracheal CSPG bound IRBCs at much lower density and exhibited significantly lower avidity than the placental CSPGs. These data demonstrated that the bovine tracheal CSPG and TM-CSPG are not ideal for studying the fine structural interactions involved in the IRBC adherence to the placental C4S, whereas the bovine corneal CSPGs are better alternatives to the placental CSPGs for determining these interactions.     

The length of chondroitin sulfate chains in normal and degenerative cartilage

Content of chondroitin sulphate, the number and average length of its chains are studied in isolated glycosamineglycanes from unchanged and degeneratively changed cartilage in men of different age. The chondroitin sulphate amount in the cartilage tissue is shown to decrease without change in the number of its chains with the age and with the development of the degenerative process in the cartilage. It occurs as a result of shortening of the average length of chondroitin sulphate chains which can be induced either during atypical chondrocyte synthesis of proteoglycanes or under destruction of the latter by enzymes.     

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-alpha-trypsin inhibitor

cDNA studies have suggested that inter-alpha-trypsin inhibitor (ITI) is a complex of several different peptide chains; the sequence of the inhibitory part of ITI is in excellent agreement with that of the urinary trypsin inhibitor (UTI). The present report demonstrates that a compound immunologically related to UTI is released by digestion with porcine pancreatic elastase or human leucocyte elastase. Since UTI has been shown to be a proteoglycan, ITI has been treated by chondroitinase. In these conditions, ITI is dissociated and gives rise to two heavy chains (78 and 85 kDa) and one light chain (26 kDa) immunologically related to UTI and which in PAGE moves close to UTIc (produced by chondroitinase treatment of UTI). We suggest that ITI is a non-covalent complex comprising two heavy chains and one light chain immunologically related to UTI and which is also a proteoglycan.     

Immunochemical studies of human urinary trypsin inhibitor

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.     

Chondroitin 4-O-sulfotransferase-2 regulates the number of chondroitin sulfate chains initiated by chondroitin N-acetylgalactosaminyltransferase-1

Recently, it has been shown that a deficiency in ChGn-1 (chondroitin N-acetylgalactosaminyltransferase-1) reduced the numbers of CS (chondroitin sulfate) chains, leading to skeletal dysplasias in mice. Although these results indicate that ChGn-1 regulates the number of CS chains, the mechanism mediating this regulation is not clear. ChGn-1 is thought to initiate CS biosynthesis by transferring the first GalNAc (N-acetylgalactosamine) to the tetrasaccharide in the protein linkage region of CS. However, in vitro chondroitin polymerization does not occur on the non-reducing terminal GalNAc-linkage pentasaccharide structure. In the present study we show that several different heteromeric enzyme complexes composed of different combinations of four chondroitin synthase family members synthesized more CS chains when a GalNAc-linkage pentasaccharide structure with a non-reducing terminal 4-O-sulfation was the CS acceptor. In addition, C4ST-2 (chondroitin 4-O-sulfotransferase-2) efficiently transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of non-reducing terminal GalNAc-linkage residues, and the number of CS chains was regulated by the expression levels of C4ST-2 and of ChGn-1. Taken together, the results of the present study indicate that C4ST-2 plays a key role in regulating levels of CS synthesized via ChGn-1.     

Organization of glycosaminoglycan chains in a chondroitin sulfate-dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Production of chondroitin sulfate and chondroitin

The production of microbial polysaccharides has recently gained much interest because of their potential biotechnological applications. Several pathogenic bacteria are known to produce capsular polysaccharides, which provide a protection barrier towards harsh environmental conditions, and towards host defences in case of invasive infections. These capsules are often composed of glycosaminoglycan-like polymers. Glycosaminoglycans are essential structural components of the mammalian extracellular matrix and they have several applications in the medical, veterinary, pharmaceutical and cosmetic field because of their peculiar properties. Most of the commercially available glycosaminoglycans have so far been extracted from animal sources, and therefore the structural similarity of microbial capsular polysaccharides to these biomolecules makes these bacteria ideal candidates as non-animal sources of glycosaminoglycan-derived products. One example is hyaluronic acid which was formerly extracted from hen crests, but is nowadays produced via Streptococci fermentations. On the other hand, no large scale biotechnological production processes for heparin and chondrotin sulfate have been developed. The larger demand of these biopolymers compared to hyaluronic acid (tons vs kilograms), due to the higher titre in the final product (grams vs milligrams/dose), and the scarce scientific effort have hampered the successful development of fermentative processes. In this paper we present an overview of the diverse applications and production methods of chondroitin reported so far in literature with a specific focus on novel microbial biotechnological approaches.     

Impaired fertility in female mice lacking urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) is a serine proteinase inhibitor that is found in blood and urine. To investigate the physiological functions of UTI in vivo, we generated UTI-deficient mice by gene targeting. The mice showed no obvious abnormalities and appeared healthy. However, the females displayed a severe reduction in fertility. Wild-type embryos developed normally when transplanted into UTI-deficient female mice, suggesting that UTI-deficient females have a normal ability to maintain pregnancy. The number of naturally ovulated oocytes from UTI-deficient mice was greatly reduced compared with that from wild-type mice. Histologically, oocytes with disorganized corona radiata were frequently seen in the ovaries of UTI-deficient mice after hormonal stimulation. When ovaries from UTI-deficient mice were transplanted into wild-type mice, pups derived from the transplanted ovaries were obtained, suggesting that the ovary of UTI-deficient mice functions normally if UTI is supplied from the systemic circulation. These results demonstrate that UTI plays an important role in the formation of the stable cumulus-oocyte complex that is essential for oocyte maturation and ovulation.     

Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells.

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.     

Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans.

In the leukemic macrophage cell-line THP-1, a fraction of the secreted matrix metalloproteinase 9 (MMP-9) is linked to the core protein of chondroitin sulfate proteoglycans (CSPG). Unlike the monomeric and homodimeric forms of MMP-9, the addition of exogenous CaCl2 to the proMMP-9/CSPG complex resulted in an active gelatinase due to the induction of an autocatalytic removal of the N-terminal prodomain. In addition, the MMP-9 was released from the CSPG through a process that appeared to be a stepwise truncation of both the CSPG core protein and a part of the C-terminal domain of the gelatinase. The calcium-induced activation and truncation of the MMP-9/CSPG complex was independent of the concentration of the complex, inhibited by the MMP inhibitors EDTA, 1,10-phenanthroline and TIMP-1, but not by general inhibitors of serine, thiol and acid proteinases. This indicated that the activation and truncation process was not due to a bimolecular reaction, but more likely an intramolecular reaction. The negatively charged chondroitin sulfate chains in the proteoglycan were not involved in this process. Other metal-containing compounds like amino-phenylmercuric acetate (APMA), NaCl, ZnCl2 and MgCl2 were not able to induce activation and truncation of the proMMP-9 in this heterodimer. On the contrary, APMA inhibited the calcium-induced process, whereas high concentrations of either MgCl2 or NaCl had no effect. Our results indicate that the interaction between the MMP-9 and the core protein of the CSPG was the causal factor in the calcium-induced activation and truncation of the gelatinase, and that this process was not due to a general electrostatic effect.     

The major human urinary trypsin inhibitor is a proteoglycan

The major urinary trypsin inhibitor (Mr 44 000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride. This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (Mr 26 000) but still inhibits trypsin.     

Calcium-induced changes in thyroglobulin conformation

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.     

Multiple molecular forms of acid-stable plasmin inhibitor derived from human urinary trypsin inhibitor

It was found that cyanogen bromide (BrCN) treatment of the highly purified human urinary trypsin inhibitors (H-UTI; specific activity 1,897 U/mg protein, and L-UTI; specific activity 1,850 U/mg protein) readily produced new plasmin inhibitors with almost no loss of UTI activity. Five multiple forms of chemically cleaved inhibitors (UTIB-I, UTIB-II, UTIB-III, UTIB-IV and UTIB-V) could be isolated from BrCN-treated L-UTI by isoelectric focusing and gel filtration. These inhibitors were very acid-stable and their isoelectric points (pI) were 4.5, 4.6, 4.9, 5.1 and 6.4, respectively. The molecular weights by SDS-polyacrylamide gel electrophoresis were almost the same at about 23,000 +/- 3,000. Although these inhibitors showed both anti-plasmin and anti-trypsin activities, much higher anti-plasmin/anti-trypsin activities were observed in the cleaved inhibitors than in the parent UTI. They competitively inhibited human plasmin with Ki values of 3.0-4.1 X 10(-8) mol/l (H-D-Val-Leu-Lys-pNA substrate).