Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate.

By analyzing outer membrane proteins of Bacteroides thetaiotaomicron on two-dimensional polyacrylamide gels, we were able to identify 10 protein spots that were associated with growth on chondroitin sulfate but not with growth on glucuronic acid or other monosaccharides. These proteins were distinct from the outer membrane polypeptides that were associated with growth on two other negatively charged polysaccharides, polygalacturonic acid and heparin. Of the 10 protein spots that were associated with growth on chondroitin sulfate, 4 could be detected on immunoblots with antiserum that had been raised against outer membranes from bacteria grown on chondroitin sulfate and then cross-adsorbed with membranes from bacteria grown on glucose. Synthesis of these four proteins appeared to be regulated coordinately with synthesis of the two enzymes that degrade chondroitin sulfate, chondroitin lyase I and II. Although one of the four proteins (Mr 110,000) was similar in molecular weight to the chondroitin lyases, the cross-adsorbed antiserum which detected this outer membrane protein did not cross-react with either of these two enzymes.     

Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.     

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species.

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.     

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.     

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.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.     

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.     

Testican-1 inhibits attachment of Neuro-2a cells.

Testican-1 is a highly conserved, multidomain, chondroitin sulfate proteoglycan that is most abundantly transcribed in the brain by neurons. This testican messenger RNA is not detected in normal quiescent astrocytes, but is up regulated when these cells are activated in response to injury such as cerebral stroke. Other chondroitin sulfate proteoglycans found in glial scars, including neurocan, have been shown to inhibit neural cell attachment and neurite extensions and may thus impede axonal regeneration. Here we report the expression and purification of a proteoglycan form of recombinant testican and its effects on neuron-derived cells in culture. We demonstrate that testican inhibits attachment of Neuro-2a cells and their ability to form neurite extensions. Both testican proteoglycan and the core glycoprotein that has been depleted of chondroitin sulfate inhibit cell attachment. Pre-treatment of the culture substratum with testican inhibits Neuro-2a attachment, but pre-treatment of the cells with testican does not inhibit their attachment. Testican, therefore, blocks attachment sites on cultureware and may also block attachment sites in the extracellular matrix of the brain.     

Chondroitin sulfate at the plasma membranes of cultured fibroblasts

We have previously shown that in confluent human fibroblast cultures chondroitin sulfate proteoglycan is a component of the fibronectin-containing pericellular matrix fibers. In the present work the distribution of chondroitin sulfate was studied in subconfluent cell cultures using antibodies that bind to a chemically defined carbohydrate fragment of chondroitinase ABC-modified chondroitin sulfate proteoglycan. Using immunofluorescence microscopy, we observed, in addition to the fibrillar matrix staining, chondroitin sulfate diffusely distributed at the cell surface. In indirect immunoferritin electron microscopy this staining corresponded to patchy binding of ferritin close (24 nm) to the outer aspect of the plasma membrane. The patchy organization appeared uniform in all cell surfaces. The cell surface chondroitin sulfate could not be removed from the plasma membrane by agents that dissociate electrostatic interactions. These data show that in fibroblasts chondroitin sulfate is a component of the outer aspect of the plasma membrane, and raise the possibility of an integral plasma membrane chondroitin sulfate proteoglycan.     

Isolation and expression in Escherichia coli of cslA and cslB, genes coding for the chondroitin sulfate-degrading enzymes chondroitinase AC and chondroitinase B, respectively, from Flavobacterium heparinum

In medium supplemented with chondroitin sulfate, Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB (chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of F. heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. Truncated cslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F. heparinum.     

Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate.

Based on sequence homology with the recently cloned human chondroitin synthase, we identified a novel beta1,4-N-acetylgalactosaminyltransferase, which consisted of 532 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 27% identity to that of human chondroitin synthase. The expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc not only to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUAbeta1--3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein linkage region of chondroitin sulfate. Hence, the enzyme is involved in the biosynthetic initiation and elongation of chondroitin sulfate and is the key enzyme responsible for the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The coding region of this enzyme was divided into seven discrete exons and localized to chromosome 8. Northern blot analysis revealed that the chondroitin GalNAc transferase gene exhibited a ubiquitous but markedly differential expression in human tissues and that the expression pattern was similar to that of chondroitin synthase. Thus, more than two distinct enzymes forming the novel gene family are required for chain initiation and elongation in chondroitin/dermatan sulfate as in the biosynthesis of heparin/heparan sulfate.     

Digestion of proteoglycan by Bacteroides thetaiotaomicron

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.     

Regulatory aspects of glycosaminoglycan biosynthesis

The control of glycosaminoglycan biosynthesis was investigated by studying the kinetic and regulatory properties of some enzymes involved in the formation of UDP-sugar precursors: UDP-N-acetylglucosamine 4'-epimerase, catalyzing the interconversion of hexosamine precursors and UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase, utilizing UDP-glucose for the formation of uronic acid and galactose precursors. The study was carried out in tissues with different glycosaminoglycan production: bovine cornea, producing both chondroitin sulfate and keratan sulfate, and newborn-pig epiphysial-plate cartilage, producing mostly chondroitin sulfate. The biosynthesis of hexosamine precursors appeared to be regulated by the value of the NAD/NADH ratio. This control mechanism regulated also the activities of both UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase and, therefore, it could correlate the biosynthesis of glycosaminoglycan precursors with the redox activity of the cell. At the level of UDP-glucose utilization two other control mechanisms were demonstrated: the different affinities of UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase for UDP-glucose in tissues with different glycosaminoglycan production and the cellular concentration of UDP-xylose. This sugar-nucleotide inhibited UDP-glucose dehydrogenase, but did not affect the UDP-glucose 4'-epimerase activity; therefore, and increase of its cellular concentration may result in a decreased chondroitin sulfate synthesis and in an increased keratan sulfate formation.     

Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.     

Importance of mucopolysaccharides as substrates for Bacteroides thetaiotaomicron growing in intestinal tracts of exgermfree mice.

We used two approaches to determine whether the mucopolysaccharide chondroitin sulfate is an important source of carbon and energy for Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice. First, we tested the ability of three mutants that grew poorly or not at all on chondroitin sulfate to colonize the intestinal tract of a germfree mouse and to compete with wild-type B. thetaiotaomicron in this model system. One mutant (CG10) was rapidly outcompeted by the wild type. However, since this mutant was unable to grow on chondroitin sulfate because it could not grow on N-acetyl-galactosamine, one of its monosaccharide components, this mutant might also be unable to utilize glycoprotein mucins. Two mutants (46-1 and 46-4) were isolated that grew poorly on chondroitin sulfate but normally on both component sugars. One of them was outcompeted by the wild type, but the percent wild type increased more slowly than with CG10. In one experiment, the percent wild type never reached 100%. The other (46-4) was not outcompeted by the wild type. These results indicate that, although chondroitin sulfate may be a carbon source in the animal, it is not of major importance. Our second approach was to determine by immunoblot analysis whether a 28-kilodalton outer membrane protein that is produced by B. thetaiotaomicron only when it is grown on chondroitin sulfate or hyaluronic acid was being produced at induced level by B. thetaiotaomicron growing in the ceca of exgermfree mice. There was no evidence for induction of this protein in vivo. Thus, the immunoblot results are consistent with results of the mutant competition experiments.     

Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.     

A chondroitin synthase-1 (ChSy-1) missense mutation in a patient with neuropathy impairs the elongation of chondroitin sulfate chains initiated by chondroitin N-acetylgalactosaminyltransferase-1

Background

Previously, we identified two missense mutations in the chondroitin N-acetylgalactosaminyltransferase-1 gene in patients with neuropathy. These mutations are associated with a profound decrease in chondroitin N-acetylgalactosaminyltransferase-1 enzyme activity. Here, we describe a patient with neuropathy who is heterozygous for a chondroitin synthase-1 mutation. Chondroitin synthase-1 has two glycosyltransferase activities: it acts as a GlcUA and a GalNAc transferase and is responsible for adding repeated disaccharide units to growing chondroitin sulfate chains.

Methods

Recombinant wild-type chondroitin synthase-1 enzyme and the F362S mutant were expressed. These enzymes and cells expressing them were then characterized.

Results

The mutant chondroitin synthase-1 protein retained approximately 50% of each glycosyltransferase activity relative to the wild-type chondroitin synthase-1 protein. Furthermore, unlike chondroitin polymerase comprised of wild-type chondroitin synthase-1 protein, the non-reducing terminal 4-O-sulfation of GalNAc residues synthesized by chondroitin N-acetylgalactosaminyltransferase-1 did not facilitate the elongation of chondroitin sulfate chains when chondroitin polymerase that consists of the mutant chondroitin synthase-1 protein was used as the enzyme source.

Conclusions

The chondroitin synthase-1 F362S mutation in a patient with neuropathy resulted in a decrease in chondroitin polymerization activity and the mutant protein was defective in regulating the number of chondroitin sulfate chains via chondroitin N-acetylgalactosaminyltransferase-1. Thus, the progression of peripheral neuropathies may result from defects in these regulatory systems.

General significance

The elongation of chondroitin sulfate chains may be tightly regulated by the cooperative expression of chondroitin synthase-1 and chondroitin N-acetylgalactosaminyltransferase-1 in peripheral neurons and peripheral neuropathies may result from synthesis of abnormally truncated chondroitin sulfate chains.     

Importance of mucopolysaccharides as substrates for Bacteroides thetaiotaomicron growing in intestinal tracts of exgermfree mice

We used two approaches to determine whether the mucopolysaccharide chondroitin sulfate is an important source of carbon and energy for Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice. First, we tested the ability of three mutants that grew poorly or not at all on chondroitin sulfate to colonize the intestinal tract of a germfree mouse and to compete with wild-type B. thetaiotaomicron in this model system. One mutant (CG10) was rapidly outcompeted by the wild type. However, since this mutant was unable to grow on chondroitin sulfate because it could not grow on N-acetyl-galactosamine, one of its monosaccharide components, this mutant might also be unable to utilize glycoprotein mucins. Two mutants (46-1 and 46-4) were isolated that grew poorly on chondroitin sulfate but normally on both component sugars. One of them was outcompeted by the wild type, but the percent wild type increased more slowly than with CG10. In one experiment, the percent wild type never reached 100%. The other (46-4) was not outcompeted by the wild type. These results indicate that, although chondroitin sulfate may be a carbon source in the animal, it is not of major importance. Our second approach was to determine by immunoblot analysis whether a 28-kilodalton outer membrane protein that is produced by B. thetaiotaomicron only when it is grown on chondroitin sulfate or hyaluronic acid was being produced at induced level by B. thetaiotaomicron growing in the ceca of exgermfree mice. There was no evidence for induction of this protein in vivo. Thus, the immunoblot results are consistent with results of the mutant competition experiments.