Hua Fan

Crystal Structure of Cd26/Dipeptidyl-Peptidase Iv in Complex with Adenosine Deaminase Reveals a Highly Amphiphilic Interface

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a β-propeller domain and an α/β-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-Å resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each β-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the β-propeller domain of DPPIV interact with two hydrophilic and heavily charged α-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.

GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response.

AIMS Ganoderma lucidum, a traditional Chinese medicine, is well known as a modulator of functions of the immune system as well as an anti-tumour agent. However, its active compounds and their molecular mechanisms of action are not well established. GLIS, a proteoglycan isolated from the fruiting body of G. lucidum, stimulates directly the activation of B lymphocytes. In this work, the immunoactivation capacities of GLIS as well as its anti-tumour effect were investigated in vitro and in vivo. MAIN METHODS Tumour-bearing mice were prepared by inoculation of mouse sarcoma S180 cells into BALB/c mice. Lymphocytes and bone marrow-derived macrophages were isolated from spleen and tibia/femurs, respectively. After stimulation with GLIS different immune responses of these cells were analysed. Anti-tumour effect of GLIS was determined. KEY FINDINGS After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice became activated, proliferated and produced large amounts of immunoglobulins. Bone marrow-derived macrophages from tumour-bearing mice also became activated after exposure to GLIS, and they produced important immunomodulatory substances, such as IL-1β, TNF-α and reactive nitrogen intermediates, like NO. GLIS markedly increased phagocytosis of macrophages, and very importantly, it markedly raised the macrophage-mediated tumour cytotoxicity. Treatment of mice with GLIS caused an inhibition of mouse sarcoma S180 tumour growth by 60% in vivo. SIGNIFICANCE These results indicate that GLIS exhibits a capacity to increase remarkably both humoral and cellular immune activities of tumour-bearing mice and inhibits tumour growth significantly. The anti-tumour effect of GLIS results from its capacity to increase the hosts immune activity.

The role of N-glycosylation in the stability, trafficking and GABA-uptake of GABA-transporter 1 Terminal N-glycans facilitate efficient GABA-uptake activity of the GABA transporter

Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft. The GABA transporter GAT1 belongs to the family of Na+‐ and Cl–‐coupled transport proteins which possess 12 putative transmembrane domains and three N‐glycosylation sites in the extracellular loop between transmembrane domain 3 and 4. To study the significance of N‐glycosylation, green fluorescence protein (GFP)‐tagged wild type GAT1 (NNN) and N‐glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells. Compared with the wild type, all N‐glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1‐deoxymannojirimycin (dMM). This indicates that N‐glycosylation, but not terminal trimming of the N‐glycans is involved in the attainment of a correctly folded and stable conformation of GAT1. All N‐glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA‐uptake activity. Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity. Further experiments showed that both N‐glycosylation mutations and dMM reduced the Vmax value, while not increasing the Km value for GABA uptake. Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na+ and slowed kinetics of the transport cycle. This indicates that N‐glycans, in particular their terminal trimming, are important for the GABA‐uptake activity of GAT1. They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding.

Crystal Structures of HIV-1 Tat-Derived Nonapeptides Tat-(1-9) and Trp2-Tat-(1-9) Bound to the Active Site of Dipeptidyl-Peptidase Iv (Cd26)

CD26 or dipeptidyl-peptidase IV (DPPIV) is engaged in immune functions by co-stimulatory effects on activation and proliferation of T lymphocytes, binding to adenosine deaminase, and regulation of various chemokines and cytokines. DPPIV peptidase activity is inhibited by both Tat protein from human immunodeficiency virus (HIV)-1 and its N-terminal nonapeptide Tat-(1–9) with amino acid sequence MDPVDPNIE, suggesting that DPPIV mediates immunosuppressive effects of Tat protein. The 2.0- and 3.15-Å resolution crystal structures of the binary complex between human DPPIV and nonapeptide Tat-(1–9) and the ternary complex between the variant MWPVDPNIE, called Trp2-Tat-(1–9), and DPPIV bound to adenosine deaminase show that Tat-(1–9) and Trp2-Tat-(1–9) are located in the active site of DPPIV. The interaction pattern of DPPIV with Trp2-Tat-(1–9) is tighter than that with Tat-(1–9), in agreement with inhibition constants (Ki) of 2 × 10–6 and 250 × 10–6 m, respectively. Both peptides cannot be cleaved by DPPIV because the binding pockets of the N-terminal 2 residues are interchanged compared with natural substrates: the N-terminal methionine occupies the hydrophobic S1 pocket of DPPIV that normally accounts for substrate specificity by binding the penultimate residue. Because the N-terminal sequence of the thromboxane A2 receptor resembles the Trp2-Tat-(1–9) peptide, a possible interaction with DPPIV is postulated.

Molecular mechanism and structural basis of interactions of dipeptidyl peptidase IV with adenosine deaminase and human immunodeficiency virus type-1 transcription transactivator

Dipeptidyl peptidase IV (DPPIV or CD26) is a multifunctional membrane glycoprotein. As an exopeptidase it regulates the activity of a series of biologically important peptides. Through its interaction with specific proteins and peptides, DPPIV is also involved in a wide range of biologically relevant processes such as cell adhesion, T cell activation and apoptosis. In this paper, we review our recent studies on the interactions of DPPIV with adenosine deaminase (ADA) and the transcription transactivator of the human immunodeficiency virus type-1 (HIV-1 Tat) as revealed by three-dimensional structure reconstructed by single particle analysis of cryo-electron microscopy (EM) and crystal structures of the human DPPIV-bovine ADA complex as well as the crystal structures of DPPIV in complex with HIV-1 Tat-derived nonapeptides. These results contribute importantly to the clarification of the molecular mechanisms of this multifunctional protein. The biological relevance of these interactions is discussed.

Interaction of human dipeptidyl peptidase IV and human immunodeficiency virus type-1 transcription transactivator in Sf9 cells

BackgroundDipeptidyl peptidase IV (DPPIV) also known as the T cell activation marker CD26 is a multifunctional protein which is involved in various biological processes. The association of human-DPPIV with components of the human immunodeficiency virus type-1 (HIV1) is well documented and raised some discussions. Several reports implicated the interaction of human-DPPIV with the HIV1 transcription transactivator protein (HIV1-Tat) and the inhibition of the dipeptidyl peptidase activity of DPPIV by the HIV1-Tat protein. Furthermore, enzyme kinetic data implied another binding site for the HIV1-Tat other than the active centre of DPPIV. However, the biological significance of this interaction of the HIV1-Tat protein and human-DPPIV has not been studied, yet. Therefore, we focused on the interaction of HIV1-Tat protein with DPPIV and investigated the subsequent biological consequences of this interaction in Spodoptera frugiperda cells, using the BAC-TO-BAC baculovirus system.ResultsThe HIV1-Tat protein (Tat-BRU) co-localized and co-immunoprecipitated with human-DPPIV protein, following co-expression in the baculovirus-driven Sf9 cell expression system. Furthermore, tyrosine phosphorylation of DPPIV protein was up-regulated in Tat/DPPIV-co-expressing cells after 72 h culturing and also in DPPIV-expressing Sf9 cells after application of purified recombinant Tat protein. As opposed to the expression of Tat alone, serine phosphorylation of the Tat protein was decreased when co-expressed with human-DPPIV protein.ConclusionsWe show for the first time that human-DPPIV and HIV1-Tat co-immunoprecipitate. Furthermore, our findings indicate that the interaction of HIV1-Tat and human-DPPIV may be involved in signalling platforms that regulate the biological function of both human-DPPIV and HIV1-Tat.

Involvement of sialic acid in the regulation of γ-aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.

Triple helix conformation of β- d -glucan from Ganoderma lucidum and effect of molecular weight on its immunostimulatory activity

β-d-glucan (GLP20) isolated from Ganoderma lucidum fruiting bodies was successfully fractionated into five fractions with different weight-average molecular weights (Mw) through ultrasonic irradiation. The Mw, radius of gyration (Rg), hydrodynamic radius (Rh) and intrinsic viscosity ([η]) of these fractions in phosphate buffered saline (PBS) aqueous solution were determined using HPSEC-MALLS-RI-VS system. The results indicated that β-d-glucan displayed rigid chain conformations with ρ values lager than 2.06 for the five fractions. By applying the polymer solution theory, the exponent (ν and α) values of Rg = kMwν and [η] = kMwα were calculated as 0.72 and 1.05, respectively, which confirmed the rigid chain conformation of glucans with Mw from 2.9 × 105 to 2.42 × 106 g·mol-1. According to the known theory for chains, the molar mass per unit contour length ML, persistence length q and contour length h per main-chain glucose residue were estimated to be 2150 nm-1, 128 nm and 0.30 nm, respectively, which indicated that the glucan existed as triple-helical chains in PBS aqueous solution. Bioactivity study of glucan fractions on lymphocytes and THP-1 macrophages demonstrated that all these fractions could stimulate lymphocyte proliferation, promote macrophages to form pseudopodia, and enhance the levels of inflammatory cytokines IL-6 and TNF-α. Meanwhile, the glucan fractions with Mw higher than 1.82 × 106 g·mol-1 exhibited better activity on enhancing the release of inflammatory cytokines.

Anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum

Sulfated polysaccharides exhibit various biological properties, including anti-coagulant, anti-oxidant, anti-viral, anti-cancer, anti-inflammatory and immune regulatory activities. In the present study, the anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum formed by chemical sulfation, were investigated. We found that GLPss58 inhibited L-selectin/sTyr-sLeX binding significantly, blocked the binding of anti-l-selectin antibodies to L-selectin on the surface of human peripheral blood lymphocytes, and inhibited the secondary lymphoid tissue chemokine-induced chemotactic invasion of HPBLs. In vivo studies in mice showed that lymphocyte homing from peripheral blood to spleen and lymph nodes was significantly inhibited by GLPss58. Furthermore, GLPss58 also inhibited the activation of complement systems and blocked the binding of TNF-α and IFN-γ to their antibodies. These results indicate that GLPss58 is able to inhibit not only the L-selectin-mediated inflammation, but also the complement system- and cytokines mediated-inflammation. Our results suggest that GLPss58 is a favorable potential anti-inflammatory agent.

Structural elucidation of a polysaccharide from Flammulina velutipes and its immunomodulation activities on mouse B lymphocytes

A novel polysaccharide FVPB2 was purified from fruiting bodies of Flammulina velutipes. Its structure was elucidated by monosaccharide composition and methylation analyses, UV-Visible and FTIR spectroscopy as well as NMR. FVPB2 was a homogeneous heteropolysaccharide (molecular weight ~ 1.50 × 104 Da) containing D-galactose, D-mannose, L-fucose, and D-glucose at molar ratio of 1.9:1.2:1:2.5. In vitro immunomodulatory studies showed FVPB2 induced proliferation of mouse spleen lymphocytes in a dose-dependent manner. The levels of IgM and IgG, secreted by B cells, increased after FVPB2 treatment. So FVPB2 has potential to be a new important immunomodulatory nutraceutical.