Structure determination of Discoidin II from Dictyostelium discoideum and carbohydrate binding properties of the lectin domain

The social amoeba Dictyostelium discoideum adopts a cohesive stage upon starvation and then produces Discoidin I and II, two proteins able to bind galactose and N-acetyl-galactosamine. The N-terminal domain or discoidin domain (DS) is widely distributed in eukaryotes where it plays a role in extracellular matrix binding while the C-terminal domain displays sequence similarities to invertebrate lectins. We present the first X-ray structures of the wild-type and recombinant Discoidin II in unliganded state and in complex with monosaccharides. The protein forms a homotrimer which presents two binding surfaces situated on the opposite boundaries of the structure. The binding sites of the N-terminal domain contain PEG molecules that could mimics binding of natural ligand. The C-terminal lectin domain interactions with N-acetyl-D-galactosamine and methyl-beta-galactoside are described. The carbohydrate binding sites are located at the interface between monomers. Specificity for galacto configuration can be rationalized since the axial O4 hydroxyl group is involved in several hydrogen bonds with protein side chains. Titration microcalorimetry allowed characterization of affinity and demonstrated the enthalpy-driven character of the interaction. Those results highlight the structural differentiation of the DS domain involved in many cell-adhesion processes from the lectin activity of Dictyostelium discoidins.     

Unfolding energetics and stability of banana lectin

The unfolding pathway of banana lectin from Musa paradisiaca was determined by isothermal denaturation induced by the chaotrope GdnCl. The unfolding was found to be a reversible process. The data obtained by isothermal denaturation provided information on conformational stability of banana lectin. The high values of DeltaG of unfolding at various temperatures indicated the strength of intersubunit interactions. It was found that banana lectin is a very stable and denatures at high chaotrope concentrations only. The basis of the stability may be attributed to strong hydrogen bonds of the order 2.5-3.1 A at the dimeric interface along with the presence of water bridges. This is perhaps very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions.     

皖南尖吻蝮蛇中11个C型凝集素类似蛋白亚单位的cDNA克隆和序列分析

通过放射性标记DNA探针筛选和PCR克隆筛选 ,从皖南尖吻蝮蛇毒腺的λgt11cDNA文库中 ,克隆得到了 11个编码C型凝集素类似蛋白亚单位的cDNA .其中 2个克隆分别含有编码an tithrombinA的A链和B链的序列 .另外 6个则含有编码如下肽链的序列 :antithrombinC的A链和B链、agkisacutacin的A链和B链、抗凝血因素的A链和B链 ,其它 3个克隆含有编码未知蛋白质的序列 .有趣的是 ,在编码AntithrombinC的序列中 ,每条链中除了含有 7个与所有C型凝集素类似蛋白位置一致的Cys外 ,在B链上还含有一个额外的Cys3;2条B链上的Cys3可能形成一对新的链间二硫键 .氨基酸序列的比较及进化分析显示 ,C型凝集素类似蛋白B链起源于一个单系类群 ,A链至少有 4个进化起源     

Mannose-Binding Lectin Serum Levels are Low in Persons with Clinically Active Coccidioidomycosis

Background  Mannose-binding lectin (MBL) is a circulating collectin that is part of the innate immune response. We explored the serum levels of MBL in persons with different forms of coccidioidomycosis. Methods  Serum MBL was measured by ELISA from samples obtained from healthy donors with immunity to Coccidioides, and those with various forms of active coccidioidomycosis. Blood cell specimens from a subgroup of subjects with active coccidioidomycosis were examined for single nucleotide polymorphisms of the MBL gene and promoter regions. Results  The control group comprised 29 healthy immune subjects. Patient groups with active coccidioidomycosis consisted of 20 patients with symptomatic primary pulmonary coccidioidomycosis, 26 with non-meningeal disseminated coccidioidomycosis, and nine with coccidioidal meningitis. The group with active coccidioidomycosis was significantly older and more likely to be male than the control group (for both, P < 0.001). The mean ± SEM level of serum MBL in the healthy controls was 169.4 ± 28.6 ng/ml, significantly higher than the 79.2 ± 10.9 ng/ml for all active groups (P < 0.001). Moreover, the active coccidioidomycosis group was significantly more likely to have serum MBL level ≤70 ng/ml compared to the control group (P = 0.001). Genetic analysis in 27 subjects with active coccidioidomycosis revealed marked variation based on race and ethnicity. Among a subgroup of 10 white, non-hispanic men with active coccidioidomycosis, there was a significant association between the H and P haplotypes and MBL levels ≤70 ng/ml (P < 0.036 and P < 0.035, respectively). Conclusions  These data suggest that there is an association between low serum MBL levels and symptomatic coccidioidomycosis.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.     

从绿藻礁膜（Monostroma nitidum Wittr）分离半乳糖专一的凝集素

礁膜（Monostroma nitidum Wittr）经 25%～80%硫酸铵分级、DEAE-纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到纯化礁膜凝集素（Monostroma nitidum lectin,MNL）,在SDS-PAGE上显示单一蛋白染色带. 用Sephadex G-200层析测得其分子质量为66.6 kD, 用SDS-PAGE测得其分子质量为66.2 kD．该凝集素可以凝集人A、B、AB、O型红细胞,且凝集活性相同. 在对人（A、B、AB、O）、兔、鲤、鲫、鼠、羊、鸡、狗的红细胞凝集作用中,兔凝集作用最强．该凝集素在pH 4.00～10.53范围内均有活性,但在pH 5.20～9.40范围内活性最大．经100 ℃热处理30 min后,该凝集素对兔红细胞血凝活性保留25%,活性最大的温度范围为25～55 ℃．MNL被EDTA抑制,最小抑制浓度为3.13 mmol/L,但对 Ca^２＋和Mg^２＋不敏感．该凝集素凝集兔红细胞的作用不被D -果糖、D-甘露糖、D-葡萄糖、蔗糖、麦芽糖、γ-球蛋白、牛甲状腺球蛋白所抑制,但被D- 半乳糖和乳糖抑制,最小抑制浓度分别为5 mmol/L和2.5 mmol/L．     

Mannan-binding lectins in the coelomic fluid of various species of Far Eastern echinoderms

A mannan-binding lectin activity was revealed in the coelomic fluid of the following echinoderm species inhabiting the coastal areas of the Sea of Japan, the holothurian Eupentacta fraudatrio, sea urchins Echinocardium cordatum, Strongylocentrotus nudus and S. intermedius, brittle star Amphipholis kochii, sea stars Asterina pectinifera, Lethasterias fusca, Lysastrosoma anthosticta, and Distolasterias nipon. It was shown that, concurrently with the general pattern of lectin interaction with branched bacterial mannans, there were also distinctions caused by the fine carbohydrate specificity of lectins. The obtained data preconditioned the further study of physical and chemical properties and structural features of the echinoderm MBL and the revelation of their role in the formation of the adaptive immune response and in other biological processes.