Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy

In some patients with neuropathy and plasma cell dyscrasia, the serum IgM M-proteins are known to bind to the myelin associated glycoprotein and to peripheral nerve glycolipids. We have isolated two acidic glycolipids which bind to the M-protein from human cauda equina by DEAE-Sephadex, Iatrobeads, and high performance liquid column chromatographies. The major acidic glycolipid migrated between GM1 and GD1a and the minor acidic glycolipid migrated between GD1a and GD1b. Their structures were elucidated by sugar analysis, enzymatic digestion, mild acid hydrolysis, permethylation, fast atom bombardment mass spectrometry, and NMR studies. Their core structure was confirmed to be paragloboside by high performance thin-layer chromatography-immunostaining using anti-paragloboside monoclonal antibody. Both acidic glycolipids lacked sialic acid but contained sulfated glucuronic acid as their acidic moiety. The sulfate group in the glucuronic acid was established by periodate oxidation and permethylation studies to be attached to the 3 position. The structures of the two acidic glycolipids are therefore consistent with the following: IV3GlcUA(3-sulfate)nLcOse4Cer and VI3GlcUA(3-sulfate)nLcOse6Cer. Additionally, the free carboxyl group on the glucuronic acid residue was shown to be necessary to bind the IgM M-proteins from neuropathy patients.     

Preparation and Characterization of Antibodies Against a Sulfated Glucuronic Acid-Containing Glycosphingolipid

In some patients with demyelinating neuropathy there are immunoglobulin M paraproteins that react with carbohydrate determinants shared by myelin-associated glycoprotein (MAG) and two peripheral nerve acidic glycolipids, termed sulfoglucuronosylglycosphingolipids (SGGLs). To study the antigenicity of these glycolipids, we immunized three New Zealand white rabbits with sulfoglucuronosylparagloboside (SGPG), a major SGGL in peripheral nerve, emulsified in Freund's complete adjuvant and keyhole limpet hemocyanin. All three rabbits inoculated with SGPG showed weight loss and mild weakness, predominantly in their hind feet, 2-5 weeks postinoculation (PI). Two of the three rabbits again showed moderate weakness 3 and 8 months PI, respectively. Electrophysiological studies demonstrated a slowed nerve conduction velocity in the sciatic nerve. Anti-SGPG antibody titers in sera were detected at dilutions of 1:1,000 to 1:2,500 by an enzyme-linked immunosorbent assay. Although all three rabbit sera reacted with SGGLs, two reacted with a desulfated form of SGPG and the other did not, suggesting a fine heterogeneity in antigenic specificity. As with sera from patients with demyelinative paraproteinemia, all rabbit sera reacted with MAG in human CNS and PNS myelin. They also reacted with MAG from bovine CNS myelin as well as several low-molecular-weight glycoproteins in bovine peripheral nerve myelin. Thus, we demonstrated that the rabbit antisera generated against SGPG have the same or similar antigenic specificity as those of the anti-MAG M-proteins from patients with neuropathy. The results suggest that an autoimmune response against the sulfoglucuronosyl residue may participate in the immunopathogenesis of this type of neuropathy.     

Antibodies to L-periaxin in sera of patients with peripheral neuropathy produce experimental sensory nerve conduction deficits

L-Periaxin is a PDZ-domain protein localized to the plasma membrane of myelinating Schwann cells and plays a key role in the stabilization of mature myelin in peripheral nerves. Mutations in L-periaxin have recently been described in some patients with demyelinating peripheral neuropathy, suggesting that disruption of L-periaxin function may result in nerve injury. In this study, we report the presence of autoantibodies to L-periaxin in sera from two of 12 patients with diabetes mellitus (type 2)-associated neuropathy and three of 17 patients with IgG monoclonal gammopathy of undetermined significance (MGUS) neuropathy, an autoimmune peripheral nerve disorder. By comparison, anti-L-periaxin antibodies were not present in sera from nine patients with IgM MGUS neuropathy or in sera from 10 healthy control subjects. The effect of anti-L-periaxin serum antibody on peripheral nerve function was tested in vivo by intraneural injection. Sera containing anti-L-periaxin antibody, but not sera from age-matched control subjects, injected into the endoneurium of rat sciatic nerve significantly (p < 0.005, n = 3) attenuated sensory-evoked compound muscle action potential (CMAP) amplitudes in the absence of temporal dispersion. In contrast, motor-evoked CMAP amplitudes and latencies were not affected by intraneural injection of sera containing anti-L-periaxin antibody. Light and electron microscopy of anti-L-periaxin serum-injected nerves showed morphologic evidence of demyelination and axon enlargement. Depleting sera of anti-L-periaxin antibodies neutralized the serum-mediated effects on nerve function and nerve morphology. Together, these data support anti-L-periaxin antibody as the pathologic agent in these serum samples. We suggest that anti-L-periaxin antibodies, when present in sera of patients with IgG MGUS- or diabetes-associated peripheral neuropathy, may elicit sensory nerve conduction deficits.     

Amniotic mesenchymal stem cells display neurovascular tropism and aid in the recovery of injured peripheral nerves

Recently, we reported that human amniotic membrane‐derived mesenchymal stem cells (AMMs) possess great angiogenic potential. In this study, we determined whether local injection of AMMs ameliorates peripheral neuropathy. AMMs were transplanted into injured sciatic nerves. AMM injection promoted significant recovery of motor nerve conduction velocity and voltage amplitude compared to human adipose‐derived mesenchymal stem cells. AMM implantation also augmented blood perfusion and increased intraneural vascularity. Whole‐mount fluorescent imaging analysis demonstrated that AMMs exhibited higher engraftment and endothelial incorporation abilities in the sciatic nerve. In addition, the higher expression of pro‐angiogenic factors was detected in AMMs injected into the peripheral nerve. Therefore, these data provide novel therapeutic and mechanistic insights into stem cell biology, and AMM transplantation may represent an alternative therapeutic option for treating peripheral neuropathy.     

Detection of the L2/HNK-1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.     

Glycolipid-dependent interaction between human migration-inhibitory factor and mononuclear phagocytes

It has been previously established in the guinea pig that the response of peritoneal macrophages to migration inhibitory factor (MIF) is enhanced by a macrophage glycolipid and that gangliosides reversibly bind MIF. This suggests that glycolipids function as cell surface receptors for MIF. In this report, it is demonstrated that the response of human peripheral blood monocytes to human MIF is augmented by preincubation of these cells with glycolipidenriched material extracted from the human macrophage-like cell line U937 or human peripheral blood monocytes and with a purified glycolipid from guinea pig peritoneal macrophages. In addition, a mixed ganglioside preparation from bovine brain shows the same effect. In contrast, the pure gangliosides, G_M1 and G_D1a, and glycolipids from the HL-60 cell line, which is a MIF-unresponsive cell line, were not able to enhance the response to human MIF. The specificity of enhancement by particular glycolipids could not be attributed to an increased uptake of only enhancing glycolipids since there was no significant difference between the association of monocytes with radioactive liposomes containing biologically active or inactive glycolipids. Pronase treatment did not affect the enhancing activity of the U937 glycolipidenriched material. Incubation of cells with glycolipids results in enhancement only if done at 37 °C and not at 4 °C. Therefore, the association of lipid with the monocyte surface appears to be dependent on temperature.Further evidence for the receptor nature of these enhancing glycolipids is provided by experiments involving affinity purification experiments. Coupling of bovine brain mixed gangliosides to agarose resulted in a matrix capable of reversibly binding MIF. G_D1a-agarose was inactive in this respect.     

Sulfated Glucuronyl Paragloboside in Rat Brain Microvessels

In patients with neuropathy associated with para-proteinemia, there are monoclonal immunoglobulin M antibodies reacting with myelin-associated glycoprotein and sulfated glucuronyl glycolipids. There are indications that the monoclonal antibodies may be responsible for these neuropathies. However, the mechanism by which the antibodies gain access to the nervous tissue, which is separated by the blood-brain barrier or blood-nerve barrier, is still unknown. In this study, we examined the presence of the sulfated glucuronyl glycolipid antigens on brain endothelial cells. Micro-vessels were isolated from adult Lewis rat brain cortex. Sulfated glucuronyl paragloboside (SGPG) was detected in the acidic lipid fraction by a TLC immunostaining method. Immunofluorescence studies showed positive staining on the surface of microvessels. In addition, SGPG could be detected in the cultured endothelial cells of human umbilical vein. These findings suggest that the endothelial cells contain an-tigenic sites for interaction with the autoantibodies. This type of interaction may result in damages to the endothelial cell function and may be responsible for changes in the blood-brain barrier permeability and the ensuing penetration of large molecules, such as immunoglobulins, into the endo-neurial space.     

Antibodies to glycolipids in demyelinating diseases of the human peripheral nervous system

Antibodies to complex glycolipids occur in patients with a variety of diseases of the peripheral nervous system. Many patients with demyelinating neuropathy occurring in association with IgM paraproteinemia have a monoclonal antibody that reacts with a carbohydrate determinant shared between sulfate-3-glucuronyl paragloboside (SGPG), the myelin-associated glycoprotein and other glycoproteins of peripheral nerve. Other patients with neuropathy in association with IgM paraproteinemia have monoclonal antibodies reacting with carbohydrate determinants on various gangliosides. More than 80% of the IgM monoclonal antibodies from patients of this type that have been screened in our laboratory react with SGPG or ganglioside antigens. High levels of antibodies reacting with ganglioside antigens are also found in some patients with inflammatory neuropathies such as Guillain-Barré Syndrome and chronic relapsing inflammatory polyneuropathy. The pathogenetic significance of these antibodies reacting with acidic sphingoglycolipids remains to be established.     

Isolation and partial characterization of a sulfogalactoglycerolipid from rat brain.

The brain of adult rats were analyzed for the presence of 35-SO4-containing glycolipids following intraventricular injection of Na2-35SO4. Radiochromatographic analyses revealed the presence of two minor 35-SO4-containing glycolipids, in addition to sulfogalactosylceramide. One of these two minor sulfolipids was isolated and tentatively identified as a 1-O--alkyl-2-0-acyl-3-(3'-sulfogalactosyl)-glycerol, a compound recently demonstrated to be the major glycolipid of mammalian testis. The alkyl and acyl compositions of the compound from rat brain are more heterogeneous than those from rat testis. The non-sulfated form of the galactoglycerolipid was also detected in rat brain. The amount of the sulfogalactoglycerolipid in rat brain is 0.19 mumol per gram wet weight, approximately one-third of the amount in rat testis (per gram wet weight), and is approximately one-fifteenth that of sulfogalactosylceramide in rat brain. The possible significance of the common occurrence in brain and testis of sulfated and non-sulfated galactolipids is discussed.     

Polarity of neutral glycolipids, gangliosides, and sulfated lipids in MDCK epithelial cells

Confluent monolayers of MDCK (Madin-Darby canine kidney) cells provide a widely used model system for studying epithelial cell polarity. We determined the polarity of epithelial cell plasma membrane glycolipids and sulfated lipids by analyzing the lipids released from both sides of monolayers of metabolically labeled MDCK cells. These lipids were released either as endogenously shed material or in budding viruses. All of the glycolipids were detected in both the apical and basolateral domains of the plasma membrane. However, galactosylceramide was more basally oriented than any of the other glycolipids; thus, the ratio of glucosylceramide to galactosylceramide was more than twice as great in the apical domain as in the basolateral domain. A sulfated sterol, which comigrated with cholesterol sulfate, was released in a more basally polarized manner than any of the glycolipids. These results indicate the presence of mechanisms which can produce different degrees of polarity for specific lipids in polarized epithelial cells.     

Sulfatide-binding proteins

Sulfatides (galactosyl ceramide-I3-sulfate) and other sulfated glycolipids are found in many tissues. The cell adhesion proteins laminin, thrombospondin, and von Willebrand factor bind specifically to sulfated glycolipids. Methods for characterizing the specificity of these interactions using surface-adsorbed glycolipids are reviewed. The three proteins do not bind to other anionic lipids, including gangliosides, phospholipids, or cholesterol 3-sulfate. Binding to sulfatides is saturable and of relatively high affinity. Relative binding avidity depends on the oligosaccharide structure of the glycolipids. Binding to sulfatides in erythrocyte membranes can account for the hemagglutinating activities of the three proteins and may play a role in the interactions of these proteins with other cell types.     

Contribution of hyperglycemia on diabetic complications in obese type 2 diabetic SDT fatty rats: effects of SGLT inhibitor phlorizin

The spontaneously diabetic torii (SDT) fatty rat is a new model of type 2 diabetes showing overt obesity, hyperglycemia and hyperlipidemia. With early onset of diabetes mellitus, diabetic microvascular complications, including nephropathy, peripheral neuropathy and retinopathy, are observed at young ages. In the present study, blood glucose levels of female SDT fatty rats were controlled with phlorizin, a non-selective SGLT inhibitor, to examine whether and how these complications are caused by hyperglycemia. Phlorizin treatment adequately controlled plasma glucose levels during the experiment. At 29 weeks of age, urinary albumin excretion considerably increased in SDT fatty rats. Glomerulosclerosis and tubular pathological findings also indicate diabetic nephropathy. These renal parameters tended to decrease with phlorizin; however, effects were partial. Sciatic nerve conduction velocities were significantly delayed in SDT fatty rats compared with Sprague-Dawley (SD) rats. Intraepidermal nerve fiber density, an indicator of subclinical small nerve fiber neuropathy, significantly decreased in SDT fatty rats. Retinal dysfunction (prolongation of peak latency for oscillatory potential in electroretinograms) and histopathological eye abnormalities, including retinal folding and mature cataracts were also observed. Both nerve and eye disorders were prevented with phlorizin. These findings indicate that severe hyperglycemia mainly causes diabetic complications in SDT fatty rats. However, other factors, such as hyperlipidemia and hypertension, may affect diabetic nephropathy. These characteristics of diabetic complications will become helpful in evaluating new drugs for diabetic complications using SDT fatty rats.     

Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6×Histidine-fused EF-Tu (His₆-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His₆-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His₆-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His₆-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.     

ACTH/MSH like peptides in the treatment of cisplatin neuropathy.

The neurological toxicity seen in patients treated with cisplatin in most cases concerns ototoxicity and peripheral neuropathy. Thus far, the pathogenesis of cisplatin neuropathy remains obscure. Yet the fact that cisplatin affects mainly the sensory peripheral nerve fibers points towards an involvement of the dorsal root ganglia. In a rat model of cisplatin neuropathy, following a cumulative dose of approx. 12 mg/kg cisplatin the sensory nerve conduction velocity began to slow as compared to age-matched controls. Peptides derived from ACTH and MSH are known to exert neurotrophic effects. In vivo they facilitate postlesion repair mechanisms in the peripheral nervous system by enhancing the early sprouting response of the damaged nerve. Surprisingly, chronic treatment with a synthetic ACTH4-9 analog not only prevented cisplatin neurotoxicity following a low or high dose regimen, but also counteracted already existing cisplatin-induced neurotoxicity. Stimulated by these findings a randomized, double blind, placebo-controlled study was performed to assess the efficacy of the peptide in the prevention of cisplatin neuropathy in women suffering from ovarian cancer. The threshold of vibration perception (VPT) was used as the principal measure of neurotoxicity. Following 6 cycles of chemotherapy the VPT had increased more than 8-fold in women receiving placebo as co-medication. Whereas the VPT in women receiving 1 mg/m2 body surface ACTH4-9 analog before and after each cisplatin cycle only increased less than 2-fold. No side effects of the peptide treatment were observed and the clinical response to the chemotherapy was similar in all treatment groups. Collectively these preclinical and clinical data suggest that treatment based on non-endocrine fragments of ACTH/MSH may be a therapeutic option in the treatment of cisplatin neuropathy.     

Characterization of human nerve basal lamina for their binding properties of anti-MAG antibodies

The functional importance of the basal lamina in Schwann cell development and in adult peripheral nerve fibers is well known. We have demonstrated previously by confocal microscopy that IgM deposits are present on the basal lamina of myelinating Schwann cells of nerve biopsies from patients with an anti-MAG IgM neuropathy. Therefore, the basal lamina was postulated to represent an early target for the uptake of autoantibodies on the surface of myelinated nerve fibers. In this study, the preparation of cell- and myelin-free basal lamina from human peripheral nerves, using a detergent-dependent method is described and characterized by immunohistochemical and biochemical analysis. Using these methods we demonstrated that an enrichment of basal lamina components of Schwann cells with extraction of myelin could be achieved. Western blot analysis and immunohistochemical characterization showed that anti-MAG IgM antibodies did not recognize an epitope on the basal lamina of normal nerves. The established method will allow in situ investigations of basal lamina components from human peripheral nerves in health and in disease, e.g. peripheral neuropathies of infectious or inflammatory origin.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.     

Sulfatide binding properties of murine and human antiganglioside antibodies

Antiganglioside antibodies form an important component of the innate and adaptive B cell repertoire, where they provide antimicrobial activity through binding encapsulated bacterial glycans. In an aberrant role, they target peripheral nerve gangliosides to induce autoimmune nerve injury. An important characteristic of antiganglioside antibodies is their ability to selectively recognize highly defined glycan structures. Since sialylated and sulfated glycans often share lectin recognition patterns, we here explored the possibility that certain antiganglioside antibodies might also bind 3-O-sulfo-beta-D-galactosylceramide (sulfatide), an abundant constituent of plasma and peripheral nerve myelin, that could thereby influence any immunoregulatory or autoimmune properties. Out of 25 antiganglioside antibodies screened in solid phase assays, 20 also bound sulfatide (10(-5) to 10(-6) M range) in addition to their favored ganglioside glycan epitope ( approximately 10(-7) M range). Solution inhibition studies demonstrated competition between ganglioside and sulfatide, indicating close proximity or sharing of the antigen binding variable region domain. Sulfatide and 3-O-sulfo-beta-D-galactose were unique in having this property amongst a wide range of sulfated glycans screened, including 4- and 6-O-sulfo-beta-D-galactose analogues. Antiganglioside antibody binding to 3-O-sulfo-beta-D-galactose was highly dependent upon the spatial presentation of the ligand, being completely inhibited by conjugation to protein or polyacrylamide (PAA) matrices. Binding was also absent when sulfatide was incorporated into plasma membranes, including myelin, under conditions in which antibody binding to ganglioside was retained. These data demonstrate that sulfatide binding is a common property of antiganglioside antibodies that may provide functional insights into, and consequences for this component of the innate immune repertoire.     

Steroid sulfatase mediated growth Sof human MG-63 pre-osteoblastic cells

Estrogen plays an important role in maintaining bone density. Postmenopausal women have low plasma estrogen, but have high levels of conjugated steroids, particularly estrone sulfate (E₁S) and dehydroepiandrosterone sulfate (DHEAS). Conversion of these precursors to active estrogens may help maintain bone density in postmenopausal women. The enzyme steroid sulfatase (STS) converts sulfated steroids into active forms in peripheral tissues. STS occurs in bone, but little is known about its role in bone function. In this study, we investigated STS activity and expression in the human MG-63 pre-osteoblastic cell line. We also tested whether sulfated steroids can stimulate growth of these cells. MG-63 cells and microsomes both possessed STS activity, which was blocked by the STS inhibitors EMATE and 667 Coumate. Further evidence for STS in these cells was provided by RT-PCR, using STS specific primers, which resulted in cDNA products of the predicted size. We then tested for growth of MG-63 cells in the presence of estradiol-17β, E₁S and DHEAS. All three steroids stimulated MG-63 cell growth in a steroid-free basal medium. We also tested whether the cell growth induced by sulfated steroids could be blocked using a STS inhibitor (667 Coumate) or using an estrogen receptor blocker (ICI 182,780). Both compounds inhibited E₁S-induced cell growth, indicating that E₁S stimulates MG-63 cell growth through a mechanism involving both STS and the estrogen receptor. Finally, we demonstrated using RT-PCR that MG-63 cells contain mRNA for both estrogen receptor alpha and estrogen receptor beta. Our data reveal that STS is present in human pre-osteoblastic bone cells and that it can influence bone cell growth by converting inactive sulfated steroids to estrogenic forms that act via estrogen receptor alpha or beta.     

Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy

Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base.