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PubMed2002

Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding.

The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with alpha 2-->6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLe(x) motif. Proximal alpha 1-->6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in > 90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively alpha 2-->3-linked N-acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin-Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans.

Amino Acid Sequence↗
PubMed1999

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1.

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.

Bacterial Toxins↗
PubMed1996

Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines.

The new unstable virescent seedling (vis) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene (nia) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1-like transposable element dTph1-3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4, is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1. Insertion of dTph1-3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1. In contrast to dTph1-like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1-3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.

Alleles↗
PubMed2002

Perchlorate reduction by a novel chemolithoautotrophic, hydrogen-oxidizing bacterium.

Water treatment technologies are needed that can remove perchlorate from drinking water without introducing organic chemicals that stimulate bacterial growth in water distribution systems. Hydrogen is an ideal energy source for bacterial degradation of perchlorate as it leaves no organic residue and is sparingly soluble. We describe here the isolation of a perchlorate-respiring, hydrogen-oxidizing bacterium (Dechloromonas sp. strain HZ) that grows with carbon dioxide as sole carbon source. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that was isolated from a gas-phase anaerobic packed-bed biofilm reactor treating perchlorate-contaminated groundwater. The ability of strain HZ to grow autotrophically with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from CO2. Chemolithotrophic growth with hydrogen was coupled with complete reduction of perchlorate (10 mM) to chloride with a maximum doubling time of 8.9 h. Strain HZ also grew using acetate as the electron donor and chlorate, nitrate, or oxygen (but not sulphate) as an electron acceptor. Phylogenetic analysis of the 16S rRNA sequence placed strain HZ in the genus Dechloromonas within the beta subgroup of the Proteobacteria. The study of this and other novel perchlorate-reducing bacteria may lead to new, safe technologies for removing perchlorate and other chemical pollutants from drinking water.

Biodegradation, Environmental↗
PubMed2002

Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan, a heparan sulfate proteoglycan.

In this study, the internalization mechanism of basic fibroblast growth factor (bFGF) at the blood-brain barrier (BBB) was investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin-resistant binding of [125I]bFGF to TM-BBB4 cells was found to be time-, temperature-, osmolarity- and concentration-dependent. Kinetic analysis of the cell-surface binding of [125I]bFGF to TM-BBB4 cells revealed saturable binding with a half-saturation constant of 76 +/- 24 nm and a maximal binding capacity of 183 +/- 17 pmol/mg protein. The heparin-resistant binding of [125I]bFGF to TM-BBB4 was significantly inhibited by a cationic polypeptide poly-L-lysine (300 micro m), and compounds which contain a sulfate moiety, e.g. heparin and chondroitin sulfate-B (each 10 micro g/mL). Moreover, the heparin-resistant binding of [125I]bFGF in TM-BBB4 cells was significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG-mediated endocytosis. RT-PCR analysis revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine-kinase receptors for bFGF) mRNA are expressed in TM-BBB4 cells. Moreover, immunohistochemical analysis demonstrated that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes.

Animals↗
PubMed1991

Simple determination of iodine in small specimens of thyroid tissue.

This work describes a simple photometric determination of the iodine concentration in thyroid tissue, a method based on the well-known catalytic Sandell-Kolthoff reaction. The modified ceric arsenite reaction is very sensitive and does not require complicated laboratory equipment. It can be performed in any routine clinical laboratory if instructions are followed strictly. By means of this method iodine concentration can be determined even in small specimens, for instance puncture samples of thyroid tissue or thyroid glands of small animals (mouse). Previous to the analysis, the tissue is digested in a mixture of sodium chlorate and perchloric acid at 100 degrees C. Using this manner of digestion between 94 and 110% of iodine in the sample were recovered. Comparison with neutron activation analysis showed excellent agreement of the obtained values.

Humans↗
PubMed1992

Stable heparin-producing cell lines derived from the Furth murine mastocytoma.

Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.

Animals↗
PubMed1995

Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth.

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of beta-migrating very low density lipoproteins (beta-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with beta-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of beta-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of beta-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimer's disease, may inhibit neuronal remodeling and contribute to the progression of the disease.

Alzheimer Disease↗
PubMed1996

Cell surface proteoglycans modulate net synthesis and secretion of macrophage apolipoprotein E.

Using a macrophage cell line that constitutively expresses a human apolipoprotein E (apoE) cDNA, we have investigated the post-translational metabolism of endogenously produced apoE. Inhibition of lysosomal or cysteine proteases led to significant inhibition of apoE degradation but did not increase apoE secretion, indicating that cellular degradation is not limiting for apoE secretion in macrophages. Treatment of macrophages with inhibitors of proteoglycan synthesis (4-methylumbelliferyl-beta-D-xyloside) or sulfation (sodium chlorate) enhanced the release of apoE from cells and significantly attenuated the increase in secretion produced by incubation with phosphatidylcholine vesicles (PV). These observations suggested that a significant fraction of the apoE retained by cells (and released by incubation with PV) was associated with proteoglycans. Treatment of cells with exogenous heparinase led to a greater than 4-fold increase in apoE secretion and similarly attenuated the response to PV, suggesting that apoE was trapped in an extracellular proteoglycan matrix. This conclusion was confirmed in studies showing that PV could enhance the release of apoE from cells during an incubation at 4 degrees C, but this enhanced release was abolished in proteoglycan-depleted cells. Incubation with lactoferrin at 4 or 37 degrees C produced a similar decrement in cellular apoE, again indicating the existence of a cell surface pool of apoE. Pulse-chase studies showed that the apoE trapped in the proteoglycan matrix was susceptible to rapid cellular degradation such that net synthesis of apoE (secreted plus cell-associated) was increased significantly in proteoglycan-depleted cells compared with control cells as early as 45 min during a chase period.

Apolipoproteins E↗
PubMed1996

Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.

The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of the selenate reductase from Thauera selenatis.

Thauera selenatis is one of two isolated bacterial species that can obtain energy by respiring anaerobically with selenate as the terminal electron acceptor. The reduction of selenate to selenite is catalyzed by a selenate reductase, previously shown to be located in the periplasmic space of the cell. This study describes the purification of the enzyme from T. selenatis grown anaerobically with selenate. The enzyme is a trimeric alphabetagamma complex with an apparent Mr of 180,000. The alpha, beta, and gamma subunits are 96 kDa, 40 kDa, and 23 kDa, respectively, in size. The selenate reductase contains molybdenum, iron, and acid-labile sulfur as prosthetic group constituents. UV-visible absorption spectroscopy also revealed the presence of one cytochrome b per alphabetagamma complex. The Km for selenate was determined to be 16 microM, and the Vmax was 40 micromol/min/mg of protein. The enzyme is specific for the reduction of selenate; nitrate, nitrite, chlorate, and sulfate were not reduced at detectable rates. These studies constitute the first description of a selenate reductase, which represents a new class of enzymes. The significance of this enzyme in relation to cell growth and energy generation is discussed.

Amino Acid Sequence↗
PubMed1998

Functional analysis of diastrophic dysplasia sulfate transporter. Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans.

Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5'-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation.

Amino Acid Sequence↗
PubMed1999

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.

Animals↗
PubMed1999

Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.

Scavenger receptor, class B, type I (SR-BI) is a cell-surface glycoprotein that mediates selective uptake of high density lipoprotein cholesteryl ester (CE) without the concomitant uptake and degradation of the particle. We have investigated the endocytic and selective uptake of low density lipoprotein (LDL)-CE by SR-BI using COS-7 cells transiently transfected with mouse SR-BI. Analysis of lipoprotein uptake data showed a concentration-dependent LDL-CE-selective uptake when doubly labeled LDL particles were incubated with SR-BI-expressing COS-7 cells. In contrast to vector-transfected cells, SR-BI-expressing COS-7 cells showed marked increases in LDL cell association and CE uptake by the selective uptake pathway, but only a modest increase in CE uptake by the endocytic pathway. SR-BI-mediated LDL-CE-selective uptake exceeded LDL endocytic uptake by 50-100-fold. SR-BI-mediated LDL-CE-selective uptake was not inhibited by the proteoglycan synthesis inhibitor, p-nitrophenyl-beta-D-xylopyranoside or by the sulfation inhibitor sodium chlorate, indicating that SR-BI-mediated LDL-CE uptake occurs independently of LDL interaction with cell-surface proteoglycan. Analyses with subclones of Y1 adrenocortical cells showed that LDL-CE-selective uptake was proportional to the level of SR-BI expression. Furthermore, antibody directed to the extracellular domain of SR-BI blocked LDL-CE-selective uptake in adrenocortical cells. Thus, in cells that normally express SR-BI and in transfected COS-7 cells SR-BI mediates the efficient uptake of LDL-CE via the selective uptake mechanism. These results suggest that SR-BI may influence the metabolism of apoB-containing lipoproteins in vivo by mediating LDL-CE uptake into SR-BI-expressing cells.

Adrenal Cortex↗
PubMed1999

The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.

Amino Acid Sequence↗
PubMed2000

High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells.

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.

Amino Acid Sequence↗
PubMed2001

Characterization of Slit protein interactions with glypican-1.

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.

Amino Acid Sequence↗
PubMed2002

Oversulfated chondroitin/dermatan sulfates containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) interact with L- and P-selectin and chemokines.

We previously reported that versican, a large chondroitin/dermatan sulfate (CS/DS) proteoglycan, interacts through its CS/DS chains with adhesion molecules L- and P-selectin and CD44, as well as chemokines. Here, we have characterized these interactions further. Using a metabolic inhibitor of sulfation, sodium chlorate, we show that the interactions of the CS/DS chains of versican with L- and P-selectin and chemokines are sulfation-dependent but the interaction with CD44 is sulfation-independent. Consistently, versican's binding to L- and P-selectin and chemokines is specifically inhibited by oversulfated CS/DS chains containing GlcAbeta1-3GalNAc(4,6-O-disulfate) or IdoAalpha1-3GalNAc(4,6-O-disulfate), but its binding to CD44 is inhibited by all the CS/DS chains, including low-sulfated and unsulfated ones. Affinity and kinetic analyses using surface plasmon resonance revealed that the oversulfated CS/DS chains containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) bind directly to selectins and chemokines with high affinity (K(d) 21.1 to 293 nm). In addition, a tetrasaccharide fragment of repeating GlcAbeta1-3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines and oversulfated CS/DS chains containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) inhibit chemokine-induced Ca(2+) mobilization. Taken together, our results show that oversulfated CS/DS chains containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) are recognized by L- and P-selectin and chemokines, and imply that these chains are important in selectin- and/or chemokine-mediated cellular responses.

Base Sequence↗