Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells.

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III. HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain. Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues. The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography. The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively. The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species. The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.     

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.     

Structural determination of novel tetra- and hexasaccharide sequences isolated from chondroitin sulfate H (oversulfated dermatan sulfate) of hagfish notochord

Oversulfated chondroitin sulfate H (CS-H) isolated from hagfish notochord is a unique dermatan sulfate consisting mainly of IdoAalpha1-3GalNAc(4S,6S), where IdoA, GalNAc, 4S and 6S represent L-iduronic acid, Nacetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. Several tetra- and hexasccharide fractions were isolated from CS-H after partial digestion with bacterial chondroitinase B to investigate the sequential arrangement of the IdoAalpha1-3GalNAc(4S,6S) unit in the CS-H polysaccharide. A structural analysis of the isolated oligosaccharides by enzymatic digestions, mass spectrometry and 1H NMR spectroscopy demonstrated that the major tetrasaccharides shared the common disulfated core structure delta4,5HexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc (4S) with 0 approximately 3 additional O-sulfate groups, where delta4,5HexA represents 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid. The major hexasaccharides shared the common trisulfated core structure delta4,5HexAalpha1-3 GalNAc(4S)beta1-4 IdoAalpha1-3 GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc(4S) with 1 approximately 4 additional O-sulfate groups. Some extra sulfate groups in both tetra- and hexasaccharides were located at the C-2 position of a delta4,5HexA or an internal IdoA residue, or C-6 position of 4-O-sulfated GalNAc residues, forming the unique disulfated or trisulfated disaccharide units, IdoA (2S)-GalNAc(4S), IdoA-GalNAc(4S,6S) and IdoA (2S)-GalNAc(4S,6S), where 2S represents 2-O-sulfate. Of the demonstrated sequences, five tetra- and four hexasaccharide sequences containing these units were novel.     

Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity

Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.     

Analysis of heparan sulfate oligosaccharides by nano-electrospray ionization mass spectrometry.

A highly sensitive method to identify and quantify heparan sulfate (HS) oligosaccharides by using nano-electrospray ionization mass spectrometry (nESI-MS) is described. The new approach allows us to detect approximately 50 nM of a chemically synthesized pentasaccharide with a structure of GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6SOMe (3-OH pentasaccharide). Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, and the remaining sample was recovered from the nanovial. The spectra shown were obtained by summing scans for 1--3 min. Hence, our data indicated that as little as 3 x 10(-15) mole of the pentasaccharide was consumed to obtain a reasonable spectrum at the concentration as low as 50 nM. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, demonstrating that this approach can be used to determine the amount of HS oligosaccharides. To this end, a 3-O-sulfated pentasaccharide was prepared by incubating the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3'-phosphoadenosine-5'-phospho[(35)S]sulfate. The resulting 3-O-sulfated pentasaccharide was purified and analyzed by nESI-MS. Based on the standard curve constructed with the 3-OH pentasaccharide, we calculated the concentration of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [(35)S]sulfate radioactivity. In conclusion, nESI-MS provides both high sensitivity and the capacity to quantify HSs. This approach is likely to become a very important tool for structural analysis and sequencing of HS and heparin oligosaccharides.     

Isolation of reducing oligosaccharide chains from the chondroitin/dermatan sulfate-protein linkage region and preparation of analytical probes by fluorescent labeling with 2-aminobenzamide

The glycosaminoglycan (GAG)-protein linkage regions of various proteoglycans share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residues in the core proteins. In previous analysis we demonstrated unique modifications by epimerization, sulfation and phosphorylation of the component sugars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. This will be useful for investigation of their biological roles, which are largely unknown, but they have been implicated in biosynthesis. A variety of linkage region-derived hexasaccharides was first prepared as reducing sugar chains from peptide chondroitin/dermatan sulfate of whale cartilage, shark cartilage, and bovine aorta by means of chondroitinase digestion in conjunction with beta-elimination in the absence of reducing reagents, but involving a mild alkali, 0.5 M LiOH, at 4 degrees C to prevent peeling reactions. The structures of these oligosaccharides were determined by the combination of HPLC, enzymatic digestion, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and (1)H NMR spectroscopy, which revealed eleven different hexasaccharides including a novel structure, DeltaHexAalpha1-3GalNAcbeta1-4IdoAalpha1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl (DeltaHexA and IdoA represent unsaturated hexuronic acid and L-iduronic acid, respectively). These oligosaccharides were labeled with a fluorophore, 2-aminobenzamide, to prepare analytical probes using the recently developed procedure [Kinoshita and Sugahara (1999) Anal. Biochem. 269, 367-378]. The fluorophore-tagged hexasacharides of low picomoles were well separated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry. The principle of the method should be applicable to the analysis of the linkage region oligosaccharides derived from heparin and heparan sulfate as well.     

Biosynthesis of gastric mucus glycoprotein of the rat

We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.     

N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.     

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Isolation and structural determination of novel sulfated hexasaccharides from squid cartilage chondroitin sulfate E that exhibits neuroregulatory activities

Squid cartilage chondroitin sulfate E (CS-E) exhibits various biological activities, including anticoagulant activities, lymphoid regulatory activities, and neuroregulatory activities [Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413]. These activities are expressed through molecular interactions with specific proteins, including heparin cofactor II, selectins, CD44, chemokines, and the heparin-binding growth factor midkine. Hence, the sugar sequence information is essential for a better understanding of the CS-E functions. Previously, several novel tetrasaccharides containing the unreported 3-O-sulfated glucuronic acid (GlcA) were isolated after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, hexasaccharides were isolated to obtain more detailed sequence information, especially around the GlcA(3-O-sulfate) residue, and were characterized by fast atom bombardment mass spectrometry and 500 or 600 MHz (1)H NMR spectroscopy. The findings demonstrate one tetrasulfated and five pentasulfated hexasaccharide sequences, five of them being novel. They were composed of three disaccharide building units of either A [GlcA(beta1-3)GalNAc(4-O-sulfate)], E [GlcA(beta1-3)GalNAc(4,6-O-disulfate)], K [GlcA(3-O-sulfate)(beta1-3)GalNAc(4-O-sulfate)], L [GlcA(3-O-sulfate)(beta1-3)GalNAc(6-O-sulfate)], or M [GlcA(3-O-sulfate)(beta1-3)GalNAc(4,6-O-disulfate)], forming E-A-A, M-A-A, K-L-A, E-E-A, K-K-A, and A-M-A hexasaccharide sequences. The K-L tetrasaccharide sequence is to date unreported. The isolated sequences appear to indicate the occurrence of an unreported GlcA 3-O-sulfotransferase specific for chondroitin sulfate. The obtained sequence information will be useful for investigating the structure-function relationship and biosynthesis of CS-E.     

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.     

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.     

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.     

Structural requirements of heparan sulfate for the binding to the tumor-derived adhesion factor/angiomodulin that induces cord-like structures to ECV-304 human carcinoma cells

Tumor-derived adhesion factor/angiomodulin (AGM) is accumulated in tumor blood vessels and on the endothelial cell surface (Akaogi, K., Okabe, Y., Sato, J., Nagashima, Y., Yasumitsu, H., Sugahara, K., and Miyazaki, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8384-8389). In cell culture, it promotes cell adhesion and morphological changes to form cord-like structures of the human bladder carcinoma cell line ECV-304. The cord formation is prevented by heparin, which inhibits the binding of AGM to ECV-304 cells. This observation suggests that AGM interacts with cell surface heparan sulfate (HS) proteoglycans. In this study, HS glycosaminoglycans and core proteins of integral transmembrane proteoglycans, syndecan-1 and -4, were identified by immunocytochemistry on ECV-304 cells, and the structural requirements for the interaction of HS with AGM were characterized. Inhibition experiments with sulfated polysaccharides and chemically modified heparin derivatives indicated that sulfate groups were essential for both AGM-HS binding and cord-like structure formation and that the rank order of the different sulfate groups in terms of their contribution was N-sulfate > 6-O-sulfate > 2-O-sulfate. The minimum size of heparin, a chemical analog of HS, required for the binding to AGM was a dodecasaccharide as determined by competition experiments using size-defined heparin oligosaccharides. Thus, a specific sulfation pattern in the HS of cell surface syndecans of ECV-304 cells is required for AGM binding and the morphological changes.     

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.     

Molecular orbital study of complexes of zinc(II) with sulphide, thiomethanolate, thiomethanol, dimethylthioether, thiophenolate, formiate, acetate, carbonate, hydrogen carbonate, iminomethane and imidazole. Relationships with structural and catalytic zinc in some metallo-enzymes.

Geometry optimization and energy calculations have been performed at the density functional B3LYP/LANL2DZ level on hydrogen sulfide (HS-), dihydrogensulfide (H2S), thiomethanolate (CH3S-), thiomethanol (CH3SH), thiophenolate (C6H5S-), methoxyde (CH3O-), methanol (CH3OH), formiate (HCOO-), acetate (CH3COO-), carbonate (CO3(2-)), hydrogen carbonate (HCO3-), iminomethane (NH=CH2), [ZnS], [ZnS2]2-, [Zn(HS)]+, [Zn(H2S)]2+, [Zn(HS)4]2-, [Zn(CH3S)]+, [Zn(CH3S)2], [Zn(CH3S)3]-, [Zn(CH3S)4]2-, [Zn(CH3SH)]2+, [Zn(CH3SCH3)]2+, [Zn(C6H5S)]+, [Zn(C6H5S)2], [Zn(C6H5S)3]-, [Zn(HS)(NH=CH2)2]+, [Zn(HS)2(NH=CH2)2], [Zn(HS)(H2O)]+, [Zn(HS)(HCOO)], [Zn(HS)2(HCOO)]-, [Zn(CH3O)]+, [Zn(CH3O)2], [Zn(CH3O)3]-, [Zn(CH3O)4]2, [Zn(CH3OH)]2+, [Zn(HCOO)]+, [Zn(CH3COO)]+, [Zn(CH3COO)2], [Zn(CH3COO)3]-, [Zn(CO3)], [Zn(HCO3)]+, and [Zn(HCO3)(Imz)]+ (Imz, 1,3-imidazole). The computed Zn-S bond distances are 2.174A for [ZnS], 2.274 for [Zn(HS)]+, 2.283 for [Zn(CH3S)]+, and 2.271 for [Zn(C6H5S)]+, showing that sulfide anion forms stronger bonds than substituted sulfides. The nature of the substituents on sulfur influences only slightly the Zn-S distance. The optimized tetra-coordinate [Zn(HS)2(NH=CH2)2] molecules has computed Zn-S and Zn-N bond distances of 2.392 and 2.154A which compare well with the experimental values at the solid state obtained via X-ray diffraction for a number of complex molecules. The computed Zn-O bond distances for chelating carboxylate derivatives like [Zn(HOCOO)]+ (1.998A), [Zn(HCOO)]+ (2.021), and [Zn(CH3COO)]+ (2.001) shows that the strength of the bond is not much influenced by the substituent on carboxylic carbon atom and that CH3- and HO- groups have very similar effects. The DFT analysis shows also that the carboxylate Ligand has a preference for the bidentate mode instead of the monodentate one, at least when the coordination number is small.     

Characterization of proteoglycans synthesized by a rat parathyroid cell line

The structure, biosynthesis, and distribution of cell-associated proteoglycans in a clonal line of parathyroid cells, which exhibit differentiated characteristics such as calcium-regulated hormone secretion and cell growth, were studied by metabolic labeling with [3H] glucosamine and [35S]sulfate as precursors. Proteoglycans were isolated by two consecutive ion exchange chromatography steps and then analyzed by gel filtration, polyacrylamide gel electrophoresis, and specific enzyme and chemical reactions. The cells synthesize almost exclusively (greater than 95%) heparan sulfate (HS) proteoglycans with a glycosaminoglycan synthesis rate of approximately 0.5 micrograms/10(6) cells/24 h. Two major HS proteoglycan species were identified. HS proteoglycan-I has a mass of approximately kDa with a single HS chain (approximately 12 kDa) and a core protein of approximately 150 kDa including oligosaccharides. HS proteoglycan-II has a mass of approximately 170 kDa with 3-4 HS chains (approximately 30 kDa) and a core protein of 70-80 kDa including oligosaccharides. In the medium with low ionized calcium (0.05 mM), HS proteoglycan-I is synthesized at approximately 1.6 times the rate and HS proteoglycan-II at a similar rate as for cells cultured in the medium with high ionized calcium (2.1 mM). The distribution of proteoglycans, examined by the accessibility of the molecules to trypsin, was dramatically influenced by environmental calcium concentration; at low calcium levels 70-80% of the HS proteoglycans are trypsin-accessible while only 20-30% are accessible at high calcium levels. This suggests that the proteoglycans are primarily on the cell surface in low calcium and in trypsin-inaccessible compartments in high calcium conditions.     

Characterization of glycopeptides labelled from D-[2-3H]mannose and L-[6-3H]fucose in intestinal epithelial cell membranes during differentiation.

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.     

Alpha-pyrones and a 2(5H)-furanone from Hyptis pectinata

Three pyrones and a 2(5H)-furanone, designated pectinolides D-G, have been isolated from the dichloromethane extract of Hyptis pectinata. The metabolites were characterized on the basis of 1D and 2D NMR spectroscopic techniques. The pyrones were identified as 6S-[3S,6S-(diacetoxy)-5R-hydroxy-1Z-heptenyl]-5S-hydroxy-5,6-dihydro-2H-pyran-2-one (1)- pectinolide D, 6S-[3S,5R,6S-(triacetoxy)-1Z-heptenyl]-5S-acetoxy-5,6-dihydro-2H-pyran-2-one (2)- pectinolide E and 6S-[3S,5R,6S-(triacetoxy)-1Z-heptenyl]-5S-acetoxy-4R-methoxy-3,4,5,6-tetrahydro-4H pyran-2-one (3)- pectinolide F. The furanone was identified as [2'Z,5(1')Z] 5-(4'S,6'R,7'S-triacetoxy-2-octenylidene)-2(5H)-furanone (4)-pectinolide G.