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Glycobiology Advance Access originally published online on December 22, 2004
Glycobiology 2005 15(5):501-510; doi:10.1093/glycob/cwi031
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Glycobiology vol. 15 no. 5 © Oxford University Press 2004; all rights reserved.

Multiple and multivalent interactions of novel anti-AIDS drug candidates, sulfated polymannuronate (SPMG)-derived oligosaccharides, with gp120 and their anti-HIV activities

Haiying Liu1,3, Meiyu Geng1,2,3, Xianliang Xin3, Fuchuan Li3, Zhenqing Zhang3, Jing Li3 and Jian Ding2,4

3 Department of Pharmacology, Marine Drug and Food Institute, Ocean University of China, Qingdao 266003, People’s Republic of China, and 4 Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China

1 These authors contributed equally to this work.

2 To whom correspondence should be addressed; e-mail: gengmy{at}ouc.edu.cn; suozhang{at}mail.shcnc.ac.cn

Received on September 5, 2004; revised on December 14, 2004; accepted on December 15, 2004


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Sulfated polymannuronate (SPMG), a novel anti-AIDS drug candidate, combats HIV-1 infection mainly by binding to gp120 protein with high affinity. To explore the structural basis of this anti-HIV-1 action, size-defined oligosaccharides were prepared by semi-synthesis or separated from native SPMG. In this study, a series of homogeneously sized SPMG fragments are evaluated for their capacity to bind rgp120 using surface plasmon resonance (SPR) analysis. The minimum SPMG fragment size that interacts with rgp120 is a hexasaccharide. Additionally, binding capacity increases with the molecular size of oligosaccharides, with the affinity of large fragments (≥ 15–16 saccharides) approaching that of full-sized SPMG. Competitive inhibition and stoichiometric analyses disclose that SPMG oligos bind to multiple binding sites on gp120. Sugar chains longer than 15–16 saccharide residues (SPMG) display multivalent interactions, with one sugar chain binding to two or three gp120 molecules. Consistent with binding data, a positive correlation exists between the size of SPMG oligosaccharides and their anti-HIV activity. The octasaccharide is established to be the minimal active fragment inhibiting syncytium formation and lowering the P24 core antigen level in HIV-IIIB-infected CEM cells. Alternatively, about 50% anti-HIV activity was observed for 15–16 saccharides, whereas a 19–20-saccharide fragment displayed anti-HIV activity equivalent to native SPMG. The structures of the unique minimum hexasaccharide specifically recognized by gp120 and the minimum octasaccharide combating HIV-IIIB infection were representatively structured as [ManA (2s)ß1-4 ManA(2s/3s)]n.

Key words: anti-HIV-IIIB activity / rgp120 / SPMG-derived oligosaccharide / sulfated polymannuronate / surface plasmon resonance


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) plays key roles in virion–cell binding and virion–cell fusion stages during the entry of HIV-1 (Wyatt and Sodroski, 1998Go). Virus entry depends on sequential interactions of gp120 with the cellular CD4 receptor (and/or heparan sulfate [HS] proteoglycans on the cell surface) and one of the two coreceptors (CCR5 and CXCR4). Gp120 binds to the CD4 receptor, triggering conformational changes that consequently expose coreceptor binding sites. Subsequently, the CD4–gp120 complex interacts with the chemokine receptor, promoting additional conformational changes that lead to fusion between the viral and cellular membranes (Berson et al., 1996Go; Clapham et al., 1999Go; Wyatt and Sodroski, 1998Go).

Increasing evidence has indicated that sulfated polysaccharides, such as dextran sulfate and heparin, interact with gp120, blocking HIV-1 entry into target cells. The key determinants in these interactions include the principal neutralizing domains (PNDs), such as the V3, V1, V2, and C4 domains of gp120. Negatively charged sulfated polysaccharides bind the positively charged amino acids of these domains, consequently inhibiting HIV-1 replication in vitro and in vivo (Jagodzinski et al., 1996Go; Javaherian et al., 1994Go; Moulard et al., 2000Go). The biological functions of sulfated polysaccharides, including anti-HIV-1 activity, have been analyzed from their active units. However, the biological activities of these anionic polymers are unsuitable for clinical applications in view of the lack of target specificity and pharmacokinetic issues that arise due to the high intermolecular and intramolecular heterogeneity of the polymers caused by their polar nature (Spencer and Gideon, 2001Go). Moreover, nonspecific interactions with proteins other than target molecules lead to unexpected adverse effects, complicating their use as therapeutic agents. The efforts aimed at developing heparin oligosaccharide-based drugs, such as phosphosulfomannan (PI88), provide a good example. It is reasonable to assume that anti-HIV-1 activities of sulfated polysaccharides are a result of their specific interactions with gp120, particularly with a key requirement of defined chain length. (Javan et al., 1997Go; Nishimura et al., 1998Go; Rider et al., 1994Go).

Sulfated polymannuronate (SPMG), a novel sulfated polysaccharide rich in (1Æ 4) linked ß-D-mannuronate with an average molecular weight of 10 kDa, is prepared by sulfation modification of an alginate extract from brown algae (Figure 1). Our previous studies demonstrate significant inhibition of HIV and SIV replication by SPMG both in vitro and in vivo. The underlying anti-HIV-1 activity of SPMG is attributed to its antagonizing potency on HIV-1 entry, mainly via binding to the V3 domain of gp120 and subsequent blockage of viral attachment to target cells (Geng et al., 2003Go; Xin et al., 2000Go). Notably, SPMG is in Phase II clinical trials in China. Recent clinical data have disclosed that SPMG improves the status of AIDS patients by reversing viral load and increasing the CD4/CD8 ratio in infected individuals. This compound is therefore the first marine sulfated polysaccharide with the potential to become an effective anti-AIDS drug.



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  		Fig. 1. Schematic diagram of the repeating saccharide sodium units in SPMG. The monosaccharide is composed of D-mannuronate by ß(1Æ 4) glycosidic linkage, with sulfate modification of the hydroxyl group at the C2/C3 position.

 

In the present study, we investigated the capacity of size-defined, SPMG-derived oligosaccharides to bind to gp120 and their ability to affect syncytium formation and the P24 core antigen level in HIV-IIIB-infected CEM cells. Our binding studies reveal that a characterized hexasaccharide represents the minimum sequence for interactions with rgp120. The apparent binding affinity increases with the molecular size of the sugar chain, with larger fragments (≥ 15–16 saccharides) approaching that of native SPMG. Importantly, octasaccharide is the minimum size requirement for combating HIV infection, whereas a 19–20-saccharide fragment displays similar antagonizing potency as native SPMG.

A competitive inhibition assay, together with stoichiometric analyses, establish that two to three molecules of gp120 bind one SPMG. Alternatively, one SPMG binds to more than one binding site within a single gp120 molecule. This binding mode is postulated as favorable for combating HIV infection in pathological conditions.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Preparation and structural characterization of SPMG-derived oligosaccharide fragments
To determine the structure-based interactions between SPMG and gp120 and the minimum size antagonizing HIV infection, varying lengths of subunits retaining their parental repeating units were prepared from native SPMG.

To obtain smaller fractions, polymannuronate blocks were acid-hydrolyzed, separated (Figure 2), and effectively chemically sulfated. Molecular weights of fractions were determined with the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) assay (Table I). The molecular size increased with one mannuronate residue in turn embodied in the separate curve. Fractions a to h in Figure 2 represent mono- to octasaccharides. The 2- to 8-saccharide fragments were chemically sulfated, following the method used for parent SPMG. A schematic reaction for disaccharide sulfation is presented in Figure 3. Sulfate modification was further confirmed by IR spectral analysis (Figure 4). A new peak at ~1250 cm–1 was observed after sulfation in each oligomer mannuronate, which represented sulfate groups on mannuronate blocks (Huang et al., 2003Go). Significantly, the group frequencies of synthesized sulfated oligosaccharides were similar to that of native SPMG (Figure 4). The sulfate content of these oligosaccharides was measured quantitatively with chemical gelatin nephelometry (Table II). Synthesized oligosaccharides displayed the same sulfate content as parent SPMG, with three sulfate groups distributed equally on two mannuronate blocks. Furthermore, in 13C-nuclear magnetic resonance (NMR) spectra of the representative hexasaccharide, chemical shifts of both C-2 and partial C-3 were observed in the lower fields after sulfation, compared with those of its mannuronate precursor (Figure 5) (Heyraud et al., 1996Go). The data are suggestive of sulfate modification of the hydroxyl groups of C-2 and partial C-3.



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  		Fig. 2. Separation of polymannuronate-derived oligosaccharides by Bio-Gel P-6 chromatography. Mannuronate oligosaccharides degraded from polymannuronate by acidic hydrolysis were size-separated by application to a Bio-Gel P-6 (fine) column (1.6 x 180 cm) at a flow rate of 6 ml/h in 0.5 M NH4HCO3. Fractions (1 ml) were collected and analyzed using the carbazole reagent method.

 

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  		Table I. MALDI-TOF MS (negative mode) spectra of polymannuronate-derived oligosaccharides

 


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  		Fig. 3. Synthesis of SPMG-derived oligosaccharides.

 


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  		Fig. 4. IR spectra of polymannuronate-derived oligosaccharides and sulfation modification derivatives. Spectra from top to bottom represent SPMG-derived disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, SPMG, and mannuronate, respectively.

 

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  		Table II. Degree of sulfation of synthesized oligosaccharides

 


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  		Fig. 5. 13C-NMR spectra of the hexmannuronate (A) and synthesized SPMG-derived hexasaccharide (B). Spectra were recorded in D2O at 25°C. Sulfated C-2 and C-3 were labeled with SC-2 and SC-3, respectively.

 

Higher size fractions (>8 saccharides) were prepared and purified by direct separation from native SPMG using Bio-Gel P10. Average molecular weights (Mw) of the fractions were estimated by high-performance gel permeation chromatography (HPGPC). Specifically, the molecular weights of SPMG-derived oligosaccharides were calculated using the standard curve, which was dependent on the elution times of standard low molecular weight heparin oligosaccharides. Our results confirm that the fragments separated and purified from native SPMG are 9-mer to 20-mer units (data not shown).

Characterization of interactions between SPMG and gp120
Previous studies by our group show that SPMG binds to rgp120 with high affinity, both at the molecular and cellular level (Geng et al., 2003Go). SPMG is a close structural homolog of endogenous glycosaminoglycans (GAGs), such as heparin, HS, chondroitin sulfate (CS) A and C, dermatan sulfate (DS), and hyaluronic acid (HA). Accordingly, we propose that GAGs compete with SPMG for binding to rgp120. To confirm this theory, we further examined the effects of soluble GAGs on SPMG-gp120 interactions using a competitive inhibition assay. As shown in Figure 6, free SPMG at a molar excessive concentration of 0.1 µM almost completely counteracted interactions between rgp120 and immobilized SPMG (91.2%), whereas the similar charge and sized heparin (also at 0.1 µM) significantly competed with SPMG for binding to rgp120 (67%). ID50 of SPMG and heparin for the competitive inhibition activity were 5.3 nM and 11.75 nM, respectively. These results suggested that the interaction of heparin with gp120 is similar to that of SPMG–gp120 interaction on one hand, and on the other hand heparin and SPMG to a great extent share the overlapping binding sites within gp120. In contrast, HS, DS, CS-A, CS-C, and HA showed little or no inhibition on the interaction, probably suggesting that they bind to gp120 with too relative lower binding avidity as compared with SPMG to compete SPMG–gp120 binding.



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  		Fig. 6. Inhibition of mono-rgp120 binding to SPMG immobilized onto the biosensor chip surface by SPMG and various soluble GAGs. Biotinylated SPMG was immobilized onto the streptavidin-CM5 sensor chip surface. Mono-rgp120 (3 µg/ml) was preincubated with free SPMG, heparin, CS-A, CS-C, DS, HA, and HS (1 µg/ml), respectively, and injected over the sensor chip surface. The experiment was performed at 25°C at a constant flow rate of 5 µl/min HBS-EP buffer. Here, 100% of the y-axis represents the binding response of immobilized SPMG to mono-rgp120 alone. The experiments were repeated two to three times with similar results.

 

Determination of the minimal binding sequence of SPMG to rgp120
To identify the minimum size of SPMG oligosaccharide required for rgp120 interactions, oligosaccharides of varying lengths were evaluated for competition inhibition of rgp120 binding to full-length SPMG immobilized on sensor chip surface with the surface plasmon resonance (SPR) assay. For this purpose, a large panel of oligosaccharides (1 µg/ml) was incubated with rgp120 (3 µg/ml), and injected over the sensor chip surface coated with SPMG. At this subsaturating concentration of gp120, excess oligosaccharide chains compete for binding to the limited number of gp120 molecules, possibly leading to the formation of 1:1 molar ratio of oligosaccharide–gp120 complexes. Full-length SPMG (~ 28 saccharide units) was used as a control. Under these experimental conditions, a six-saccharide unit was the smallest fragment displaying substantial inhibition capacity (equivalent to 56% that of parent SPMG). In contrast, smaller oligosaccharide fragments (≤5-mer) exhibited very low inhibition rates (less than 19%, compared to parent SPMG). Inhibitory effects increased with the molecular size of oligosaccharides, with maximal inhibition by 15–16-mer SPMG (equivalent to 94% that of parent SPMG) (Figure 7). The data indicate that the minimum size requirement for gp120 binding is hexasaccharide. Importantly, the SPMG-gp120 binding capacity strictly depends on the SPMG oligosaccharide sizes.



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  		Fig. 7. Effects of a panel of SPMG-derived oligosaccharides and native SPMG on interactions between monomeric-rgp120 and SPMG. Preincubation of gp120 with a large panel of SPMG-derived oligosaccharides (from 2-mer to 20-mer) or free SPMG were injected over the SPMG immobilized sensor chip surface. The experiment was performed at 25°C with a constant flow rate of 5 µl/min HBS-EP buffer. Histograms of inhibition rates of these SPMG-derived oligosaccharides and native SPMG on interactions between rgp120 and SPMG. 100% of the y-axis represents the binding response of immobilized SPMG to rgp120 alone. The experiment is representative of two independent experiments with similar results.

 

Determination of the binding mode of hexasaccharide-rgp120 and SPMG-rgp120
To further investigate the appropriate length of saccharide length chain that displays multivalent interactions with gp120, gp120 was injected over the sensor chip surface immobilized with a selective 6-saccharide and native SPMG. The real-time binding sensograms were recorded. Using BIAeval 3.1 software, the simultaneous fitting and kinetic binding affinities were performed and calculated. Results indicated that both the binding of gp120 to 6-mer and to native SPMG is dose-dependent (Figure 8A, 8B). The simultaneous fitting curve for the interaction of gp120 with immobilized 6-mer oligosaccharide (including both association and dissociation phases) fitted well to simple 1:1 (Langmurie) binding model and giving a {chi}2 value to 1.6 (which describes the closeness of the fit). Kinetic analyses were then calculated and gave rise to a KD value of 0.452 µM. Furthermore, the Scatchard plot analysis of the equilibrium binding data released a straight line for 6-mer oligosaccharide–gp120 interaction, yielding a KD value (0.484 µM) similar to the constant derived from the kinetic parameters using the software.



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  		Fig. 8. SPR analysis of the binding capacity of gp120 to hexasaccharide or SPMG immobilized onto the biosensor chip surface. Different concentrations of gp120 were injected at a flow rate of 10 µl/min over biotinylated hexasaccharide or SPMG immobilized on the streptavidin-coated sensor chip surface. The x-axis stands for flow time, whereas the y-axis represents the binding resonance unit. The experiment was performed at 25°C. (A) Binding response curves of interactions between gp120 and hexasaccharide immobilized on the sensor chip. (B) Binding response curves of interactions between gp120 and SPMG immobilized on the sensor chip. (C) Scatchard plot of the hexasaccharide–gp120 equilibrium binding data directly measured on the sensorgrams. (D) Scatchard plot of SPMG–gp120 equilibrium binding data directly measured on the sensorgrams. The concentrations are (from top to bottom) 0.83, 0.42, 0.21, 0.11, 0.05 µM in A; 0.53, 0.26, 0.13, 0.07 and 0.03 µM in B. The experiments shown are representative of two to three independent experiments with similar results.

 

Because native SPMG-gp120 binding fitting yielded an unreasonable {chi}2 value using simple 1:1 binding model, the bivalent analyte model was then selected. The global fitting of the data produced a {chi}2 value < 2.0, implicating that the fitting procedure by the bivalent analyte model considerably improved and better described the kinetic data. This binding model gave Ka1 at 2.54 x 104 M–1s–1 indicative of a relatively high association rate constant, and Kd1 at 1.71 x 10–3ns–1 a relatively stable dissociation rate constant, suggesting a high-affinity interaction between native SPMG and gp120. Although the second binding event produces dissociation rate constant at 1.31 x 10–4 s–1, suggestive of the further stabilization of binding complex. In addition, results from Scatchard plot analysis also displays a two-component binding curve, further confirming the bivalent binding model between SPMG and gp120. Importantly, the notion that the calculated data approach the experimental data in turn implicated that a correct fitting model was selected. Stoichiometric value was further calculated, and results demonstrated that one SPMG interacted with two to three molecules of gp120, permitting the bivalent binding.

Determination of the minimal active sequence of SPMG for anti-HIV activity
Syncytium formation and P24 core antigen levels are important indicators of HIV infection. Therefore, we determined the effects of SPMG oligosaccharides of selectively sized oligosaccharides on syncytium formation and P24 core antigen levels in HIV-IIIB-infected CEM cells (Table III). HIV-IIIB-infected CEM cells (1000TCID50) were incubated in the absence or presence of 10 µg/ml oligosaccharides for 6 days after the appearance of SI in mock group. As shown in Table III, small saccharides (3-mer and 6-mer) exhibited no antisyncytium activity, whereas 8-mer, 10-mer, and 15–16-mer SPMG displayed inhibition to a certain extent. Consistent with data from the binding experiment, 19–20-mer SPMG displayed the strongest antisyncytium activity, similar to native SPMG. Azidothymidine (AZT) (10 µg/ml) inhibited syncytium formation to some extent, as both SPMG and 19–20-mer.


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  		Table III. Inhibitory effects of SPMG oligosaccharides and SPMG on anti-syncytium-inducing phenotype (SI) in HIV-IIIB-infected CEM cells (n = 4)

 

Next the effects of selectively sized oligosaccharides were investigated on the P24 core antigen protein level in HIV-IIIB-infected CEM cells (Table IV). The 3-mer and 6-mer saccharide residues had no effects on the P24 core antigen level in HIV-IIIB-infected CEM cells. The 8-mer, 10-mer, and 15–16-mer units decreased the P24 core antigen level to 39.6%, 43.8%, and 53.1%, respectively. The 19–20-mer inhibited the antigen protein level by 92.7%, equivalent to native SPMG (94%). At the same concentration, AZT induced 100% inhibition of HIV infection. Based on the data, we propose that the octasaccharide is the minimum size requirement for anti-HIV activity, whereas the 19–20-saccharide unit is sufficient to induce maximum inhibition of HIV infection.


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  		Table IV. Inhibition by oligosaccharides and SPMG on P24 core antigen protein levels in HIV-IIIB-infected CEM cells (mean ± SD, n = 4)

 


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
In this study, we initially focus on the chemical synthesis of novel low-molecular-weight sulfate oligosaccharides (2 to 8 saccharides) and separation of higher-molecular-weight sulfate oligosaccharides (>8 saccharides) from native SPMG to obtain extensive size-defined oligosaccharides. We further evaluate their binding capacities and modes with gp120 and the specific size of the active component against HIV infection. These homogeneous fragments share the same mannuronate backbone with a specific degree of sulfation at the C2 and/or C3 position, similar to SPMG (3.0 sulfate groups within two pyranohexose A) but differ with respect to chain length. Most important, these resulted SPMG-derived oligosaccharides are first established to be structurally novel, displaying specific and size-dependent binding to gp120 and consequently anti-HIV activities.

2-Mer to 5-mer SPMG fragments exhibited similar low binding capacity to recombinant gp120. However, a three- to fourfold increase in capacity was observed from 6-mer to 8-mer SPMG. Beyond a length of 15–16-mer, oligosaccharides exhibited similar capacity as integral SPMG. Although the minimal binding fragment is hexasaccharide, suboptimal binding was evident at longer sizes, starting from octasaccharide. In general, the inhibition capacity increased with molecular size from 6-mer to 15–16-mer SPMG. These different binding capacity patterns among oligosaccharides of varying lengths may be attributed to the involvement of distinct binding sites within gp120 for SPMG and its oligosaccharides.

Our SPR data that 8-mer SPMG initially exhibited potent competitive inhibition on native SPMG-gp120, particularly with the fact that 8-mer partially inhibited the neutralization of anti-V3 monoclonal antibody (0.5ß) to the V3 peptide using enzyme-linked immunosorbent assay (data not shown) suggested that V3 domain is at least in part involved in the interactions between gp120 and 8-mer SPMG. In fact, in addition to the V3 region acting as the preferential site for sulfated polysaccharide binding, other regions within gp120 also exert an influence on gp120-polysaccharide interactions (Batinic and Robey, 1992Go; Moulard et al., 2000Go; Witvrouw et al., 1994Go). The notion that the 6-mer fails to trigger inhibitory potency to the same extent as 8-mer did, we hypothesized, is due to the fact that 6-mer does not provide sufficient length to tightly interact with V3 binding site. In fact, 6mer exhibited lower affinity for gp120 with KD values in the micromolar range. Significantly, the 15–16-mer affording similar inhibition as native SPMG did, putatively suggested that this is the optimal length required for occupying all binding sites involved within one gp120 molecule.

Multivalency, a well-known binding pattern, greatly enhances the affinity of protein–carbohydrate interactions. In fact, multivalent binding is dependent on the sugar length (Rathore et al., 2001Go; Rusnati et al., 1999Go; Spencer and Gideon, 2001Go; Takagaki et al., 2002Go), which exhibits tighter binding as the size of sugar chain gets large enough and otherwise represents low-affinity binding (Shenoy et al., 2002Go). These notions are highly consistent with our finding that increase in sugar size lead to further improvement in binding capacity, with full length SPMG reaching to the maximal potency.

Notably, native SPMG significantly inhibits SPMG–gp120 binding (92% inhibition rate, ID50 of 5.3 nM). Likewise, heparin at given concentrations also potently inhibited the interaction of SPMG with gp120 (67% inhibition rate, ID50 of 11.7 nM). These data suggested that both SPMG and heparin displayed the similar competitive inhibition profile, sharing the overlapping binding sites to a great extent, particularly V3 domain. Dealing with other GAGs (HS, CS, DS, HA), they afford a different fashion on SPMG–gp120 interaction. They all exhibit little or no inhibition on monomeric gp120–SPMG interaction, indicating that they might display rather low binding affinity for V3 motif comparable with SPMG and heparin. Because sugar–protein interactions are predominately dependent on saccharide composition and extent and distribution of sulfation of the sugar backbone (Javan et al., 1997Go; Katsuraya et al., 1999Go; Nakashima et al., 1995Go; Yoshida et al., 2001Go), therefore the relative high affinity of SPMG and heparin for gp120 may benefit particularly from its defined sulfate groups and specific saccharide composition. It is true that both SPMG and heparin are highly sulfated, in comparison with HS, DS, CS-C, CS-A, and HA. On the other hand, even though it bears a similar degree of sulfation as SPMG, heparin still exhibits subtle difference in binding potency from SPMG, which alternatively suggests a contribution of saccharide composition to such difference.

Having established hexasaccharide residues as the minimum binding sequence and the binding mode between oligosaccharide–gp120 interactions, we designed additional experiments and attempted to characterize the minimum bioactive saccharide unit against HIV infection. The effects of SPMG-derived oligosaccharides on HIV-IIIB-infected CEM cells were evaluated by detecting both syncytia formation (SI) and the P24 core antigen level of infected cells. The 3-mer and 6-mer SPMG units failed to inhibit syncytium formation and the P24 protein level in CEM cells. Anti-HIV activities started from 8-mer, and 10–20-saccharide residues or higher units confer to potential antagonists, with 8-mer oligosaccharide the minimum size requirement for combating HIV infection. The 19–20-mer unit displayed comparable potency to full-length SPMG. In view of the finding that units up to 19–20-mer saccharide display similar anti-HIV activity to native SPMG, we propose that the 19–20-mer sugar chain length is sufficient to permit multivalent interactions of trimeric gp120–gp41 and subsequent virion–cell fusion.

Current views hold that on the surface of the virion, each gp120 is noncovalently associated with a molecule of the transmembrane glycoprotein, gp41. These heterodimers are organized into trimers, which trigger virion–cell fusion (Chan et al., 1997Go; Weissenhorn et al., 1996Go, 1997Go). Importantly, the interaction of gp120 in the functional heterotrimers (expressed on transfected COS-1 cell) with SPMG and oligos and the interaction of SPMG and its oligos with oligomeric gp120 induced by CaCl2 in cell free system both exhibit the similar binding profiles as monomeric gp120 did (data not shown). All these findings, particularly with the potent anti-HIV activity of SPMG and its oligos in HIV-IIIB-infected CEM cells, strongly support the theory that the binding mode of SPMG and its oligos with monomeric gp120 can greatly unravel what happens in pathological conditions.

The nonnegligible discrepancy in different size requirement for binding gp120 and for biological activities against HIV infection, we assumed, is due to the hypothesis that size constraint of hexasaccharide, though sufficient enough to bind gp120, is insufficient to induce the proper conformational change and thus failed to elicit the antagonizing potency against HIV-IIIB infection. This is often observed in GAG–protein interactions, such as those of the growth factors (Ornitz et al., 1992Go; Pye et al., 1998Go; Takagaki et al., 2002Go; Walker et al.,1994Go).

This is the first study to disclose that SPMG-derived hexasaccharide residues represent the minimum sequence for binding to the gp120 molecule and an octasaccharide is required for minimum anti-HIV activity (representative structure of [ManA (2s)ß1-4 ManA(2s/3s)]n). We additionally show multiple and multivalent binding modes between SPMG and gp120 interactions. Our data should facilitate elucidation of the structure–activity relationship of sulfated polysaccharides in combating HIV-1 infection. Thus the SPMG-derived octasaccharide as prototype may be potentially used for developing promising sugar-based therapeutics against AIDS.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Materials and equipments
SPMG and polymannuronate were provided by Marine Drug and Food Institute (Ocean University, China). SPMG with an average molecular weight of 10 kDa was prepared by acidic hydrolysis of alginate extracted from brown algae, followed by chemical sulfation and fractionation. Polymannuronate, a precursor of SPMG, is composed of pure mannuronate residues. Heparin, HS, CS-A and CS-C, DS, and HA were obtained from Sigma (St. Louis, MO). Bio-Gel P-6 (fine), Bio-GelP-10 (fine), and Sephadex G-10 were purchased separately from Bio-Rad (Hercules, CA) and Pharmacia (Uppsala, Sweden). IR spectra were obtained using a Nicolet Nexus 470 spectrophotometer with a KBr disk. The CM5 biosensor chip was from BIAcore (Uppsala, Sweden). Recombinant gp120 (rgp120, Mw = 120 kDa, strain MN) was purchased from ViroStat (Portland, OR). Sulfo-N-hydroxysuccinimide(NHS)-biotin, streptavidin, and other chemical agents employed were from Sigma.

Preparation of SPMG oligosaccharides with different chain lengths
Smaller oligosaccharides were prepared by chemical sulfation of mannuronate, following the protocol for native SPMG synthesis. The 1% (w/v) carbohydrate matrix of polymannuronate blocks was hydrolyzed at pH 4.0, 110°C for 4 h. Mixtures were separated using the Bio-Gel P6 column (1.6 x 180 cm). Samples were eluted at a flow rate of 6 ml/h in 0.5 M ammonium bicarbonate, and 1-ml fractions were collected. Fractions were analyzed by the carbazole reagent method (Bitter and Muir, 1962Go), and peaks were pooled and freeze-dried for further sulfation modification.

The pooled peak was subjected to sulfate modification by reacting mannuronate with ClSO3H in formamide. Briefly, 50-mg fractions were added to sulfating reagents containing 500 µl formamide and 125 µl ClSO3H, and reacted at 67°C for 3 h. The pH of the products was adjusted to 7.0 with 4 mol/L NaOH and desalted using Sephadex G-10. The product peak was pooled and freeze-dried.

Larger oligosaccharides were prepared by separation from native SPMG. Native SPMG was separated using Bio-Gel P-10 column (1.6 x 180 cm), Samples were eluted at a flow rate of 6 ml/h in 0.5 M ammonium bicarbonate, and 1-ml fractions were collected and analyzed with the carbazole reagent method as described. The molecular weights of these fractions were analyzed by HPGPC, using a G3000PWxl column (300 mm x 7.8 mm) (Tosoh, Japan). Samples (5 g/L, 20 µl) were eluted with 2.84% Na2SO4 (w/v) at a flow rate of 0.5 ml/min at 40°C, and monitored with a RI 2410 refractive index detector (Waters, Milford, CT). The standard curve was obtained from theelution times of molecular weights of low-molecular-weight heparin standards. The Mw of obtained samples were calculated according to the standard curve using GPCw software (Longzhida Company, China).

MALDI-TOF spectroscopy
Mass spectra were recorded using a MALDI-TOF mass spectrometer, BIFLEX III (Bruker Daltonics, Billerica, MA). For mass spectrometry, the fragment was dissolved in distilled water. The sample (0.5 µl) was applied on to a plate. Subsequently, 0.5 µl matrix solution (10 mg/ml 2,5-dihydroxybenzonic acid in 50% acetonitrile) was mixed with the aliquot and dried. The analyzer was used in the negative ion linear mode.

NMR studies
13C-NMR was performed on a JEOL JNM-ECP600 spectrometer equipped with a 5 mm field gradient tunable probe. Oligosaccharides were exchanged three times with 99.9% D2O (Cambridge Isotope Laboratories, Cambridge, MA). Samples were analyzed at 25°C. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate was used as the internal standard.

Sulfate content assay
The sulfate content assay was performed using chemical gelatin nephelometry (Zhang, 1994Go). Sulfate groups were released from polysaccharides by hydrolysis to produce BaSO4 with Ba2+ in solution. Briefly, 1 mg sulfate oligosaccharide was dissolved in 1 ml HCl (1 M) and subjected to airproofed hydeolysis at 100°C for 6 h. The sulfate content was measured from the standard curve. The standard curve was calculated as follows: 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 ml of 100 µg/ml standard K2SO4 was diluted to 2 ml. Next, 1 ml diluted HCl and 0.5 ml BaCl2-gelatin solution were added. Absorbance was detected at 500 nm.

Binding studies by SPR assay
Competitive inhibition and kinetics of binding of different-sized sulfated oligosaccharides to gp120 were investigated using SPR (BIAcore). For this purpose, native SPMG or selective size-defined saccharides were biotinylated at the reducing end, and a flow cell of a CM5 sensor chip was activated with streptavidin. Biotinylated native SPMG or selective size-defined saccharides were allowed to react with the streptavidin-coated sensor chip (Li et al., 2003Go). Briefly, 5 µM SPMG and selective oligosaccharides were dissolved in 30 µl deionized distilled water, followed by the simultaneous addition of 5 µM 1-Etyl-3-(3-Dimethylaminopropyl)carbodiimide and 5 µM NHS. The mixture was incubated at room temperature overnight.

Each desired oligosaccharide was immobilized on the CM5 sensor chip surface at 25°C with a constant flow rate of 5 µl/min HBS-EP buffer (HBS-EP buffer: 0.01 M HEPES, 0.15 M NaCl, 3 mM ethylenediamine tetra-acetic acid, 0.005% polysorbate 20 [v/v], pH 7.4). To assess real-time binding capacity, 35 µl of rgp120 and soluble GAGs or oligosaccharides or rgp120 alone was injected over the sensor chip surface with the immobilized oligosaccharide, followed by 5 min washing with HBS-EP buffer. The sensor chip surface was regenerated using 60 µl 2 M NaCl. All binding experiments were performed at 25°C with a constant flow rate of 10 µl/min HBS-EP. For binding affinity assessment, the association phase was allowed to proceed to equilibrium. To correct for nonspecific binding and bulk refractive index change, a blank channel (FC2) without oligosaccharide was employed as a control for each experiment. Sensorgrams for all binding interactions were recorded in real time and analyzed after subtracting that from the blank channel. Changes in mass due to the binding response were recorded as resonance units (Ru).

Binding kinetics and affinities were determined by SPR using BIACORE software 3.1. Stoichiometry was calculated according to the following formula:

Anti-HIV assay
Human T-lymphoma CEM cells were incubated with 200 µl HIV-1-IIIB (1000TCID50/2 x 106 cells) at 37°C for 1.5 h. Cells were washed three times with serum-free IMDM (Gibco, Grand Island, NY) medium, and the supernatant discarded after centrifugation. Infected CEM cells (2 x 105 cells/ml, 100 µl) were placed in 96-well microtiter plates and cultured in IMDM medium with 10% fetal bovine serum. Oligosaccharides at a concentration of 10 µg/ml were added to the cell. After incubation for 6 days, syncytium-inducing phenotype (SI) of infected CEM cells was observed. The P24 core antigen protein content (ng/ml) in the supernatant of CEM cells was detected with a P24 kit (ZeptoMetrix, USA). Mock and uninfected CEM cells were used as control. AZT, the first-line anti-AIDS therapy, was used as positive control. Inhibition (%) was calculated as follows:


   Acknowledgements
 
This work was funded by the Natural Science Foundation of China (A30070694, F30130200), Chinese High-tech Project 863 (2001AA620405) andNational Basic Research Program Grant (2003CB716400). We are grateful to Professor Jiandong Jiang and Dr. Jiannong Li for help with antiviral evaluation.


   Abbreviations
 
AZT, azidothymidine; CEM cells, human T-lymphoma cells; CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; gp120, envelope glycoprotein 120; HA, hyaluronic acid; HIV-1, human immunodeficiency virus type I; HPGPC, high-performance gel permeation chromatography; HS, heparan sulfate; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; NMR, nuclear magnetic resonance; SPMG, sulfated polymannuronate; SPR, surface plasmon resonance


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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T. Ghosh, K. Chattopadhyay, M. Marschall, P. Karmakar, P. Mandal, and B. Ray
Focus on antivirally active sulfated polysaccharides: From structure-activity analysis to clinical evaluation
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