Glycosaminoglycan—lipoprotein interactions: 2. Structure of heparin-releted variants with high affinity for low density lipoprotein

A particular heparan sulphate fraction which possessed the largest proportion of high affinity variants for human low density lipoprotein contained almost equal proportions of the repeating units l-iduronosyl(O-sulphate)N-sulphamidoglucosamine and d-glucoronosyl-N-acetylglucosamine. The heparan sulphate was fractionated on lipoprotein-agarose into three populations. Results of periodate oxidation—alkaline elimination indicated that the size of the completely N-sulphated block regions increased with increasing affinity. In contrast, the number of consecutive l-iduronosyl(O-sulphate)-containing repeats decreased with increasing affinity towards lipoprotein. After selective periodate oxidation—alkaline scission of d-glucoronic acid residues only a portion of the heparan sulphate fragments retained high affinity for lipoprotein. This portion consisted of fragments larger than dodecasaccharide which contained both l-iduronic acid-O-sulphate and non-sulphated uronic acid residues (−) 2:1). No affinity or little affinity was displayed by fragments (of comparable size) that contained only sulphated l-iduronic acid residues.     

Glycosaminoglycan-lipoprotein interactions: 3. Structure of dermatan sulphate variants with high affinity for low density lipoprotein

An iduronate-rich dermatan sulphate fraction was found to contain variants with high affinity for human low density lipoprotein. Digestion of a dermatan sulphate-lipoprotein complex with chondroitinase-ABC afforded fragments that contained between 10 and 20 disaccharide repeats. In this fragment half of the uronate residues were iduronate-sulphate. The non-sulphated iduronate-bearing repeats were interspersed among the repeats containing sulpho-iduronate. This feature is similar to that observed with heparin-related glycans having high affinity for lipoproteins.     

Interaction between heparan sulphate chains II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains

A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. ¹³C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N-acetyl-glucosamine than N-sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N-acetyl-to-N-sulphate ratio was approx. 2 : 1, the N-sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species.Both preparations were subjected to deaminative cleavage which produces fragments like uronate-(N-acetylglucosamine-uronate)_n-anhydromannose. Tetrasaccharides (n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate-N-acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate-N-acetylglucosamine-glucuronate-anhydromannose was the major sequence.In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-Å., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N-sulphated block regions and in regionsthat carry alternating N-acetyl- and N-sulphated glucosamine.     

A 4-deoxy analogue of N-acetyl-d-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro

Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -β1-4GlcA-α1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-d-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect. 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.     

Unique heparan sulfate from shrimp heads exhibits a strong inhibitory effect on infections by dengue virus and Japanese encephalitis virus

The structure and biological activities of a highly sulfated heparan sulfate (HS) extracted from shrimp (Penaeus brasiliensis) heads were characterized. Structurally the shrimp HS was more heterogenous than heparin, although it is still highly sulfated. The molecular mass of the shrimp HS preparation was determined to be 32.3 kDa by gel filtration HPLC. Analysis by surface plasmon resonance demonstrated that various growth/differentiation factors specifically bound to the shrimp HS with comparable affinity. Notably, the shrimp HS had a greater inhibitory effect against infections by dengue virus type 2 as well as Japanese encephalitis virus than heparin. Experiments on anticoagulant activity indicated that the shrimp HS exhibited significant anti-thrombin activity, but less than the commercial heparin. Hence, the HS preparation from shrimp heads, an industrial waste, is a prospective agent for a variety of clinical applications.     

Desulfated galactosaminoglycans are potential ligands for galectins: evidence from frontal affinity chromatography

Galectins, a group of β-galactoside-binding lectins, are involved in multiple functions through specific binding to their oligosaccharide ligands. No previous work has focused on their interaction with glycosaminoglycans (GAGs). In the present work, affinities of established members of human galectins toward a series of GAGs were investigated, using frontal affinity chromatography. Structurally-defined keratan sulfate (KS) oligosaccharides showed significant affinity to a wide range of galectins if Gal residue(s) remained unsulfated, while GlcNAc sulfation had relatively little effect. Consistently, galectins showed much higher affinity to corneal type I than cartilageous type II KS. Unexpectedly, galectin-3, -7, and -9 also exerted significant affinity to desulfated, GalNAc-containing GAGs, i.e., chondroitin and dermatan, but not at all to hyaluronan and N-acetylheparosan. These observations revealed that the integrity of 6-OH of βGalNAc is important for galectin recognition of these galactosaminoglycans, which were shown, for the first time, to be implicated as potential ligands of galectins.     

Separation of (R)- and (S)-tert-butyl 2-tert-butyl-4-methoxy-2,5-dihydro-1,3-imidazole-1-carboxylate (building block for amino acid synthesis) by preparative high performance liquid chromatography on a polysaccharide stationary phase

The preparative separation of the enantiomers of the title compound, a versatile chiral building block for the synthesis of unnatural amino acid esters, by high performance liquid chromatography on a chiral stationary phase (CSP), is reported for the first time. The CSP consists of amylose-(3,5-dimethylphenyl-carbamate), which has been coated onto the surface of macroporous aminopropyl-functionalized silica gel. The effect of mobile phase composition and the amount of amylose derivative on the silica gel has been thoroughly investigated. Using 2-propanol as organic modifier in hexane as mobile phase, on a semi-preparative column (200 mm × 40 mm ID, containing 192 g of stationary phase) about 200 mg of the racemate was separated per injection. Running the equipment under automatic conditions with repetitive injection mode allowed for the separation of 30 g per day. Both enantiomers were obtained with enantiopurities >99.75:0.25. Chirality 10:217–222, 1998. © 1998 Wiley-Liss, Inc.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.     

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: H and C NMR studies of oligosaccharides generated by nitrous acid depolymerisation

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH₄ and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete ¹H and ¹³C NMR data have been derived for the major components which were shown to have the structures: GlcA-(β1→3)-anTal6S-ol (I) and l-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-d-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.     

Synthesis of the C-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency.

In the process of developing a method for the synthesis of membrane proteins, the conditions for native chemical ligation, namely, detergent concentration and the chemical characteristics of the thiol additive were investigated in detail. The C-terminal region of the opioid receptor like 1, ORL1(288-370), which contains C-terminal intracellular and transmembrane domains, was chosen as a model. The building blocks, ORL1(329-370) and ORL1(288-328)-SR-Gly-Arg(5)-Leu (-SR- : -SCH(2)CH(2)CO-) were most effectively condensed slightly below the critical micelle concentration of SDS and in the presence of mercaptoethanesulfonic acid as a thiol additive. The results showed that the concentration of SDS and the charge on the thiol additive are crucial factors for the effective synthesis of a membrane protein by native chemical ligation.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.