Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins.     

糖链结构不同的纤连蛋白与HT1080细胞结合研究

人血浆纤连蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）与人胎盘纤连蛋白两者在肽链结构上基本相同,但人血浆Ｆｎ的Ｎ－糖链结构为二天线结构,而人胎盘Ｆｎ不仅Ｎ－糖链的数量增加,同时还含有多天线结构,分别用~（１２５）Ｉ标记这两种具有不同糖链结构的Ｆｎ,观察两者与ＨＴ１０８０细胞的饱和结合的亲和性,结果发现,在４℃,人血浆Ｆｎ与ＨＴ１０８０细胞的饱和结合为１２９ｎｇ／１０~５细胞,解离常数为２．８３×１０~（－８）ｍｏｌ／Ｌ,人胎盘Ｆｎ与ＨＴ１０８０细胞的饱和结合为１３３ｎｇ／１０~６细胞,解离常数为２．６４×１０~（－８）ｍｏｌ／Ｌ．因而,人血浆Ｆｎ与人胎盘Ｆｎ上Ｎ－糖链的不同并未影响其与受体的结合．     

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca²⁺-bound LipL32 was determined at 2.3 Å resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca²⁺ binding. A significant conformational change was observed for the Ca²⁺-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca²⁺ binding. Based on the crystal structure of Ca²⁺-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca²⁺ binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.     

内源性逆转录病毒长末端重复序列在嗜酸性粒细胞增多症的基因表达及核苷酸序列分析

探索人内源性逆转录病毒长末端重复序列(LTR)基因及表达与嗜酸性粒细胞增多症发生的关系。PCR法检测嗜酸性粒细胞增多症患者外周血中内源性逆转录病毒长末端重复序列基因,RT-PCR法检测内源性逆转录病毒基因表达。变性高效液相分析和序列测定LTR片段核苷酸序列,对不同株基因序列作同源性的比较分析。PCR结果显示:20例嗜酸性粒细胞增多症患者细胞中均获得内源性逆转录病毒长末端重复序列扩增产物,嗜酸性粒细胞增多症组中长末端重复序列基因有高的表达,而正常人表达为阴性。与HERV-K家族LTR基因相应区域核苷酸序列比较;嗜酸性粒细胞增多组长末端重复序列U3、R、U5区同源性分析有核苷酸的改变,与淋巴瘤对照比较没有大片段的缺失。人类基因组中普遍存在逆转录病毒长末端重复序列。正常人和嗜酸性粒细胞增多症患者中长末端重复序列有不同程度核苷酸碱基的变异,但是,二者比较,这种改变与嗜酸性粒细胞的增多没有明显的相关性。在嗜酸性粒细胞增多症患者中有高的基因表达而正常人中没有可检出的病毒基因的表达,嗜酸粒细胞的增多可能与逆转录病毒基因表达水平有关,其诱导嗜酸粒细胞增多的机制需进一步的研究。     

The Role of Glycosaminoglycan Binding of Staphylococci in Attachment to Eukaryotic Host Cells

Attachment of microorganisms to host cells is believed to be a critical early step in microbial pathogenesis. The aim of the study was to determine the role of the known glycosaminoglycan (GAG) binding activity of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in their attachment to six different eukaryotic cell lines. Three staphylococcal species expressing GAG binding capacity—S. aureus, S. epidermidis, and S. hemolyticus—were chosen for investigation. Six different eukaryotic cell lines, endothelial HUVEC and EA. hy 926 cells, epithelial A549 and HeLa S3 cells, fibroblasts HEL Sp 12 and macrophages J774.A1, were included. A modified ELISA with biotinylated bacteria was used for estimating the adhesion of staphylococci to each of the cell lines. Our results showed that staphylococci adhered to each of the cell lines studied, although the binding of CoNS strains to epithelial cells was lower than to the other cells. The attachment to all cell types could be partially decreased by pretreatment of the bacteria with various polysulfated agents (highest inhibition was 60%), as well as by chlorate and heparitinase treatment of the cells. These observations may suggest that at least one mode of staphylococcal attachment utilizes GAG chains present on the surface of virtually all adherent cells. Received: 6 September 2000 / Accepted: 29 December 2000     

Adherence of Lactobacillus to Intestinal 407 Cells in Culture Correlates with Fibronectin Binding

Lactobacilli are members of the normal mucosal microflora of most animals. Many isolates of Lactobacillus spp. are adherent to epithelial cells. In this study, using Lactobacillus acidophilus and L. agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins. Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected. In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers. Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin. Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces. Received: 19 January 2000 / Accepted: 2 March 2000     

纤维连接蛋白细胞结合区功能多肽在杆状病毒表达系统中的表达与纯化

目的：利用Bac-to-Bac杆状病毒表达系统表达纤维连接蛋白（FN）细胞结合区功能多肽（CBD）,并对其进行纯化和鉴定。方法：经PCR获得人血浆FN-CBD基因,酶切后定向克隆到T载体上,经测序正确后插入pFastBacHTB载体,转化大肠杆菌DH10Bac感受态细胞;用抗生素平板筛选重组杆粒,脂质体介导重组杆粒转染sf9昆虫细胞并进行蛋白表达;经Ni-NTA层析柱对重组多肽进行纯化,对纯化的多肽行SDS-PAGE和Western-blot分析。结果：得到融合6个组氨酸残基的FN-CBD,SDS-PAGE显示其相对分子质量约为36000,Western-blot表明该多肽能与FN的多克隆抗体结合。结论：利用Bac-to-Bac杆状病毒表达系统能成功表达出人血浆FN-CBD,且表达产物具有良好的免疫原性,为后续结构、功能研究奠定了基础。     

Periodate-Modified Gangliosides Enhance Surface Binding of Tetanus Toxin to PC 12 Pheochromocytoma Cells

The interaction of 125I-labeled tetanus toxin with PC12 pheochromocytoma cells in monolayer cultures has been examined. Under regular growth conditions, the PC12 cells bind 125I-tetanus toxin to a limited degree compared with dissociated cerebral neuron cultures. After exposure to nerve growth factor for 2 days in low serum-containing media with growth factor supplements, binding of toxin increases over twofold compared with untreated PC12 cells. Binding can also be enhanced (greater than 2.5-fold) after treatment of cells with 2 mM sodium metaperiodate for 20 min. Dissociated cerebral neurons but not fibroblasts in cell culture bind more toxin after periodate treatment. The effect of periodate can be abolished by 5 mM sodium borohydride. A ganglioside isolated from periodate-treated PC12 cells and tentatively identified as GT1b [(N-acetylneuraminyl)galactosyl-N-acetylgalactosaminyl(N- acetylneuraminyl-N-acetylneuraminyl)-galactosyl-glucosylceramide] binds 125I-tetanus toxin on silica gel chromatoplates and on nitrocellulose paper. There are no indications to suggest binding to a polypeptide from treated cells after polyacrylamide gel electrophoresis. Cells artificially supplemented with GT1b and subsequently treated with periodate effectively bind the toxin. The data suggest that modified sialyl groups linked to gangliosides, and not to proteins, are preferential targets for tetanus toxin.     

利用荧光共振能量转移技术研究活细胞TLR4与MD-2作用结构域

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用．研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义．运用基于强度的三通道荧光共振能量转移技术(FRET)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域．结果表明：N端Glu²⁴～Met⁴¹缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu²⁴～Met⁴¹的TLR4不能聚合．上述结果提示,TLR4的Glu²⁴～Met⁴¹不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用．     

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the ⁸F1⁹F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to ⁸F1⁹F1 and that UR binding to ⁸F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.     

Specific Lectin Binding to Beta1 Integrin and Fibronectin on the Apical Membrane of Madin-Darby Canine Kidney Cells

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.     

N－糖链在纤连蛋白分泌及纤连蛋白受体与配体结合中的作用

应用能阻断糖蛋白Ｎ－糖链合成的衣霉素（ＴＭ）,研究了Ｎ－糖链缺失对ＨＴ１０８０细胞分泌纤连蛋白（Ｆｎ）以及纤连蛋白受体（ＦｎＲ）与配体结合的影响。结果发现,１μｇ／ｍｌ的ＴＭ可抑制Ｎ－糖链的合成（此时,３Ｈ－甘露糖掺入下降６３％）,但细胞分泌Ｆｎ的量仅下降３３％,这主要是由于蛋白合成受ＴＭ抑制（２５％）而引起,因而,Ｎ－糖链缺失可能并不影响Ｆｎ的分泌。而在同样条件下,单个细胞结合１２５Ｉ－Ｆｎ的量显著下降,显示Ｎ－糖链的缺失可能导致了膜上ＦｎＲ总量或其与配体结合的亲和力的改变。ＴＭ处理组的ＦｎＲ的内吞率与对照组相比较无明显差异,提示受体分子中的Ｎ－糖链缺失不影响其内吞过程．     

Fusion of Carbohydrate Binding Modules to Bifunctional Cellulase to Enhance Binding Affinity and Cellulolytic Activity

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (K_M), for which K_M decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (k_cat/K_M) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9.     

Binding of CD157 Protein to Fibronectin Regulates Cell Adhesion and Spreading

CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.