Biomedical subjects

Lai-Xi Wang

Publications and source records attributed to Lai-Xi Wang.

At least 19 recordsLinked to original sources

PubMed2006

A highly efficient chemoenzymatic approach toward glycoprotein synthesis.

[reaction: see text] A highly efficient endoglycosidase-catalyzed synthesis of homogeneous glycoproteins is described. By using ribonuclease B as a model system, it was demonstrated that Endo-A could efficiently attach a preassembled oligosaccharide to a GlcNAc-containing protein in a regio- and stereospecific manner, when the corresponding sugar oxazoline was used as the donor substrate. The method allows the synthesis of both natural and tailor-made N-linked glycoproteins in excellent yield.

Carbohydrate Sequence↗

PubMed2006

Glycopeptide synthesis through endo-glycosidase-catalyzed oligosaccharide transfer of sugar oxazolines: probing substrate structural requirement.

An array of sugar oxazolines was synthesized and tested as donor substrates for the Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A)-catalyzed glycopeptide synthesis. The experiments revealed that the minimum structure of the donor substrate required for Endo-A catalyzed transglycosylation is a Man beta1-->4-GlcNAc oxazoline moiety. Replacement of the beta-D-Man moiety with beta-D-Glc, beta-D-Gal, and beta-D-GlcNAc monosaccharides resulted in the loss of substrate activity for the disaccharide oxazoline. Despite this, the enzyme could tolerate modifications such as attachment of additional sugar residues or a functional group at the 3- and/or 6-positions of the beta-D-Man moiety, thus allowing a successful transfer of selectively modified oligosaccharides to the peptide acceptor. On the other hand, the enzyme has a great flexibility for the acceptor portion and could take both small and large GlcNAc-peptides as the acceptor. The studies implicate a great potential of the endoglycosidase-catalyzed transglycosylation for constructing both natural and selectively modified glycopeptides.

Amino Acid Sequence↗

PubMed2006

Toward oligosaccharide- and glycopeptide-based HIV vaccines.

The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS), and HIV type 1 (HIV-1) is responsible for the current global pandemic of HIV and AIDS. There is widespread agreement that the best hope to contain this pandemic is the development of an effective HIV-1 vaccine. The major difficulty in HIV vaccine design, however, is in identifying novel epitopes for inducing broadly neutralizing antibodies. Accumulating data have implied that the HIV-1 carbohydrates, which are a strong defense against host immune attack, can also serve as targets for vaccines. This notion was reinforced by the characterization of a novel oligomannose cluster on HIV-1 glycoprotein gp120 as the epitope of the broadly neutralizing antibody 2G12. This review describes the current status and prospective of the progress toward a carbohydrate-based HIV vaccine. Recent research on the design, synthesis and antigenicity of HIV-1 oligosaccharides and glycopeptide immunogens is also discussed.

AIDS Vaccines↗

PubMed2005

Chemoenzymatic synthesis of HIV-1 V3 glycopeptides carrying two N-glycans and effects of glycosylation on the peptide domain.

[structure: see text] A highly efficient chemoenzymatic synthesis of HIV-1 V3 domain glycopeptides carrying two N-linked core tri- and pentasaccharides was achieved. The synthesis consisted of two key steps: a solid-phase synthesis of the cyclic, 47-mer V3 domain peptide containing two GlcNAc residues and a novel endoglycosidase-catalyzed transglycosylation that simultaneously added two N-glycan moieties to the peptide precursor from the oligosaccharide oxazoline donor substrates. The availability of the synthetic glycopeptides allowed the probing of the effects of glycosylation on the HIV-1 V3 domain. It was demonstrated that glycosylation influenced the global conformations of the V3 domain and provided protection of the V3 domain against protease digestion.

Amino Acid Sequence↗

PubMed2005

Highly efficient endoglycosidase-catalyzed synthesis of glycopeptides using oligosaccharide oxazolines as donor substrates.

A highly efficient chemoenzymatic synthesis of N-glycopeptides was achieved. It was found that using synthetic oligosaccharide oxazolines, the mimics of the presumed oxazolinium ion intermediate formed in a retaining mechanism of substrate-assisted catalysis, as the donor substrates and GlcNAc-peptides as the acceptors, the endo-beta-N-acetylglucosaminidase (ENGase)-catalyzed transglycosylation gave a high yield (73-82%) of the corresponding glycopeptides in a regio- and stereospecific manner, regardless of the size of the peptide portions. The use of the oligosaccharide oxazolines as donor substrates not only expanded the substrate availability but also led to a substantial enhancement of the synthetic efficiency, compared to the use of natural N-glycans.

Amino Acid Sequence↗

PubMed2005

Modification and structure-activity relationship of a small molecule HIV-1 inhibitor targeting the viral envelope glycoprotein gp120.

This paper describes selected modification and structure-activity relationship of the small molecule HIV-1 inhibitor, 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity, probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806 moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1 infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not show inhibitory activity at up to 200 nM.

Anti-HIV Agents↗

PubMed2005

A novel fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases.

A fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases (ENGases) was developed. The assay was based on the findings that a coupled chitinase can specifically capture and hydrolyze the fluorogenic intermediate that is formed by the ENGase-catalyzed transglycosylation to release a fluorophore, but does not hydrolyze the donor asparagine-linked N-glycan and the acceptor 4-methylumbelliferyl N-acetylglucosaminide. The assay method was verified by detecting the transglycosylation activities of the known ENGases. Its application for assessing the effects of organic solvents on transglycosylation activity was demonstrated. The novel coupled assay provides a highly sensitive, easy, and quantitative method for screening endo-beta-N-acetylglucosaminidases with transglycosylation activities useful for glycoconjugate synthesis.

Chitinases↗

PubMed2005

Chemoenzymatic synthesis of CD52 glycoproteins carrying native N-glycans.

A facile synthesis of homogeneous CD52 glycoproteins carrying native N-glycans was achieved using an endolycosidase-catalyzed oligosaccharide transfer as the key step. The synthesis consists of two steps: the solid phase synthesis of GlcNAc-CD52 and the transfer of a high-mannose type or complex type N-glycan from Man(9)GlcNAc(2) Asn or a sialglycopeptide to the GlcNAc-CD52, under the catalysis of the endo-beta-N-acetylglucosaminidases from Arthrobacter (Endo-A) and Mucor hiemalis (Endo-M), respectively.

Antigens, CD↗

PubMed2005

Chemoenzymatic synthesis of HIV-1 gp41 glycopeptides: effects of glycosylation on the anti-HIV activity and alpha-helix bundle-forming ability of peptide C34.

C34 is a 34-mer peptide derived from the C-terminal ectodomain of HIV-1 envelope glycoprotein, gp41. The C34 region in native gp41 carries a conserved N-glycan at Asn637 and the sequence is directly involved in the virus-host membrane fusion, an essential step for HIV-1 infection. This paper describes the synthesis of glycoforms of C34 which carry a monosaccharide, a disaccharide, and a native oligosaccharide moiety. The synthesis of the glycopeptide which carries a native high-mannose type N-glycan was achieved by a chemoenzymatic approach by using an endoglycosidase-catalyzed oligosaccharide transfer as the key step. The effects of glycosylation on the inhibitory activity and the helix-bundle forming ability of C34 were investigated. It was found that glycosylation moderately decreases the anti-HIV activity of C34 and, in comparison with C34, glyco-C34 forms less compact six-helix bundles with the corresponding N-terminal peptide, N36. This study suggests that conserved glycosylation modulates the anti-HIV activity and conformations of the gp41 C-peptide, C34.

Amino Acid Sequence↗

PubMed2005

Differential expression of endogenous sialidases of human monocytes during cellular differentiation into macrophages.

Sialidases are enzymes that influence cellular activity by removing terminal sialic acid from glycolipids and glycoproteins. Four genetically distinct sialidases have been identified in mammalian cells. In this study, we demonstrate that three of these sialidases, lysosomal Neu1 and Neu4 and plasma membrane-associated Neu3, are expressed in human monocytes. When measured using the artificial substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (4-MU-NANA), sialidase activity of monocytes increased up to 14-fold per milligram of total protein after cells had differentiated into macrophages. In these same cells, the specific activity of other cellular proteins (e.g. beta-galactosidase, cathepsin A and alkaline phosphatase) increased only two- to fourfold during differentiation of monocytes. Sialidase activity measured with 4-MU-NANA resulted from increased expression of Neu1, as removal of Neu1 from the cell lysate by immunoprecipitation eliminated more than 99% of detectable sialidase activity. When exogenous mixed bovine gangliosides were used as substrates, there was a twofold increase in sialidase activity per milligram of total protein in monocyte-derived macrophages in comparison to monocytes. The increased activity measured with mixed gangliosides was not affected by removal of Neu1, suggesting that the expression of a sialidase other than Neu1 was present in macrophages. The amount of Neu1 and Neu3 RNAs detected by real time RT-PCR increased as monocytes differentiated into macrophages, whereas the amount of Neu4 RNA decreased. No RNA encoding the cytosolic sialidase (Neu2) was detected in monocytes or macrophages. Western blot analysis using specific antibodies showed that the amount of Neu1 and Neu3 proteins increased during monocyte differentiation. Thus, the differentiation of monocytes into macrophages is associated with regulation of the expression of at least three distinct cellular sialidases, with specific up-regulation of the enzyme activity of only Neu1.

Animals↗

PubMed2004

Synthesis, conformation, and immunogenicity of monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178.

Several monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178 were synthesized. Conformational studies showed that multivalent assembly enhanced the alpha-helical content of the peptide. Therefore, 2-, 3-, or 4-alpha-helix bundles of peptide DP178 could be obtained by assembling the peptide on a suitable bi-, tri-, or tetravalent template. Immunization studies indicated that while peptide DP178 alone was poorly immunogenic, the tetravalent peptide MVP-1 raised high titers of antibodies in mice that recognize not only peptide DP178 but also the native HIV-1 glycoprotein gp41, even in the absence of a carrier protein or adjuvant. The study suggests that carbohydrate-centered multivalent peptides provide not only a model for mimicking protein alpha-helix-bundle structure, but also an effective immunogen for raising high-titer antibodies against HIV-1 envelope glycoprotein gp41.

Amino Acid Sequence↗

PubMed2004

Synthesis and anti-HIV-1 activity of 4-[4-(4,6-bisphenylamino-triazin-2-ylamino)-5-methoxy-2-methylphenylazo]-5-hydroxynaphthalene-2,7-disulfonic acid and its derivatives.

A structure-based design approach has been used to optimize a lead HIV-1 entry inhibitor targeted to the envelope glycoprotein gp41. The docking study on this lead compound revealed important structural requirements that need to be preserved as well as structural non-requirements that could be eliminated to substantially reduce the molecular size of the lead compound. Based on the results from docking study, a limited number of analogues were designed and synthesized. This approach yielded a new analogue (compound 4) that retained the anti-HIV-1 activity with reduced molecular size approaching towards more drug-like character.

Anti-HIV Agents↗

PubMed2004

Design and synthesis of alpha Gal-conjugated peptide T20 as novel antiviral agent for HIV-immunotargeting.

An efficient chemo-enzymatic synthesis of alpha Gal-conjugated peptide T20 as novel HIV-immuno-targeting agent is described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized alpha Gal epitope and its chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide alpha Gal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system. The experimental data suggest that the synthetic alpha Gal-T20 might be valuable for in vivo HIV-immuno-targeting via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and HIV-infected cells, thus providing an additional dimension of HIV intervention.

Anti-HIV Agents↗

PubMed2004

Design and synthesis of a template-assembled oligomannose cluster as an epitope mimic for human HIV-neutralizing antibody 2G12.

The synthesis and antibody-binding affinity of a novel template-assembled oligomannose cluster as an epitope mimic for human anti-HIV antibody 2G12 are described. Cholic acid was chosen as the scaffold and three high-mannose type oligosaccharide (Man(9)GlcNAc(2)Asn) moieties were selectively attached at the 3alpha, 7alpha, and 12alpha-positions of the scaffold through a series of regioselective transformations. Binding studies revealed that the synthetic oligosaccharide cluster is 46-fold more effective than the subunit Man(9)GlcNAc(2)Asn in inhibiting 2G12-binding to immobilized gp120. The scaffold approach described in this paper provides an avenue to designing more effective epitope mimics for antibody 2G12 in the hope of developing a carbohydrate-based vaccine against HIV-1.

Antibodies, Monoclonal↗

PubMed2004

Resveratrol glucuronides as the metabolites of resveratrol in humans: characterization, synthesis, and anti-HIV activity.

Resveratrol is a natural product with diverse biological activities. We have previously reported that resveratrol possesses potent synergistic inhibitory activity against human immunodeficiency virus (HIV)-1 infection in combination with nucleoside analogs (Heredia et al. 2000. J Acquir Immune Defic Syndr 25:246-255). As a part of our program in developing resveratrol as a component for anti-HIV chemotherapy, we describe in this article the characterization, chemical synthesis, and biological effects of the human metabolites of resveratrol. We found that resveratrol was metabolized in humans into two metabolites, which were characterized as resveratrol-3-O- and 4'-O-glucuronides. For further biological studies, we reported two simple, alternative methods for the synthesis of the metabolites. The cytotoxic and antiviral activities of resveratrol and its metabolites were compared in cell culture experiments using human peripheral blood mononuclear cells. Whereas resveratrol was cytotoxic at > or =30 microM, no cytotoxicity was observed for the metabolites at concentrations as high as 300 microM. However, resveratrol showed strong synergistic anti-HIV activity with didanosine at 10 microM, but no synergistic effects were observed for either of the metabolites at up to 300 microM. Nevertheless, the in vitro activity of the metabolites (resveratrol glucuronides) may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human beta-glucuronidase could convert the metabolites back to resveratrol locally or systematically in vivo. The present studies have implications for future development of resveratrol and/or its derivatives as a chemotherapeutic agent.

Administration, Oral↗

PubMed2004

Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design.

Human antibody 2G12 broadly neutralizes human immunodeficiency virus type 1 (HIV-1) isolates and shows protective activity against viral challenge in animal models. Previous mutational analysis suggested that 2G12 recognized a novel cluster of high-mannose type oligosaccharides on HIV-1 gp120. To explore the carbohydrate antigen for HIV-1 vaccine design, we have studied the binding of 2G12 to an array of HIV-1 high-mannose type oligosaccharides by competitive ELISAs and found that Man9GlcNAc is 210- and 74-fold more effective than Man5GlcNAc and Man6GlcNAc in binding to 2G12. The results establish that the larger high-mannose oligosaccharide on HIV-1 is the favorable subunit for 2G12 recognition. To mimic the putative epitope of 2G12, we have created scaffold-based multivalent Man9 clusters and found that the galactose-scaffolded bi-, tri-, and tetra-valent Man9 clusters are 7-, 22-, and 73-fold more effective in binding to 2G12 than the monomeric Man9GlcNAc2Asn. The experimental data shed light on further structural optimization of epitope mimics for developing a carbohydrate-based HIV-1 vaccine.

AIDS Vaccines↗

PubMed2003

Cholic acid as template for multivalent peptide assembly.

Cholic acid, an amphiphilic steroid containing several selectively addressable functionalities, was exploited as a rigid template for multivalent peptide assembly. Thus, cholic acid-based templates suitable for chemoselective peptide ligation were synthesized, in which maleimide or bromoacetyl moieties were selectively introduced at the 3alpha, 7alpha, 12alpha-positions of cholic acid with varied length of linkers. Three peptides were chosen and tested for the chemoselective ligation. These include the HIV-1 peptide inhibitor DP178, the universal T-helper epitope derived from tetanus toxoid (830-844), and the minimum epitope sequence of the HIV-neutralizing antibody 2F5. It was found that the maleimide-functionalized templates are highly efficient for the ligation of all the peptides, while bromoacetyl templates led to low yield of ligation. Circular dichroism (CD) spectroscopic studies of the multivalent peptides (10a and 11a) containing three strands of peptide DP178 indicate that the template-assembled peptides form three alpha-helix bundles with significantly enhanced alpha-helix contents than the single peptide. The results suggest that cholic acid is a valuable template for constructing alpha-helix bundles that may be useful as mimics of conformational epitopes for vaccine development.

Amino Acid Sequence↗

PubMed2003

Specific activation of ERK pathways by chitin oligosaccharides in embryonic zebrafish cell lines.

Chitin oligosaccharides (COs) play a role in plant development and are presumed to affect body plan formation during vertebrate embryogenesis. The mechanisms of COs recognition and cellular processes underlying embryonic development are still not understood. We analyze the possible link with the mitogen-activated protein kinase pathway that is conserved in evolution through the plant and animal kingdom and has been implicated in diverse cellular processes, including cell growth, proliferation, differentiation, survival, and vertebrate development. We show that in vivo stimulation of embryonic zebrafish cells ZF13 and ZF29 with chitin tetrasaccharides at 10-9 M concentration transiently induced activation/phosphorylation of extracellular regulated kinases (ERKs), with a maximum after 15 min. Furthermore the biological specificity of chitin tetrasaccharides and various derivatives was examined. The replacement of one or two GlcNAc residues of the chitin backbone by glucose and fucosylation of chitin tetrasaccharides at the reducing terminus caused a complete loss of their activity. We also tested a chitin tetrasaccharide analogue in which the oxygen atoms in glycosidic linkages were replaced by sulfur atoms. This analog, which could not be enzymatically hydrolyzed, was as potent an inducer as chitin tetrasaccharide. These results suggest that the observed activation of ERKs is chitin tetrasaccharide-specific and does not require further enzymatic processing. We examined possible signaling pathways leading to ERK activation by COs by use of phosphospecific antibodies and inhibitors. We conclude that a high-affinity CO receptor system exists that links to the Raf, MEK, and ERK pathway in zebrafish cells.

Animals↗