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1.
本文应用明胶、肝素亲和层析二步法首先纯化了人胚肺成纤维细胞培养液的纤连蛋白（Ｆｉｂｒｏｎｅｏｔｈｅ,Ｆｎ）,经ＳＤＳ－ＰＡＧＥ鉴定为一条带,然后用胰糜蛋白酶消化纯化的Ｆｎ所获得的酶解波,经分离分别得到明胶结合片段和肝素结合片段,再应用凝集素－ＨＲＰ染色的Ｗｅｓｔｅｎ转移电泳法研究糖链结构,结果证实：１．Ｆｎ中明胶结合片段（４４ｋｄ）中含有二天线和多天线复杂型糖链,并接有平分型ｇｌｃＮＡｃ糖基、核心力Ｆｕｃ。２肝素结合片段（３０ｋｄ）只含有二天线复杂型糖链,不含平分型ＧｌｃＮＡｃ糖基及核心Ｆｕｃ．  相似文献   

2.
    
The potent cellulose‐binding modules of cellulosomal scaffoldin subunits belong to the greater family of carbohydrate‐binding modules (CBMs). They have generally been classified as belonging to family 3a on the basis of sequence similarity. They form nine‐stranded β‐sandwich structures with jelly‐roll topology. The members of this family possess on their surface a planar array of aromatic amino‐acid residues (known as the linear strip) that form stacking interactions with the glucose rings of cellulose chains and have a conserved Ca2+‐binding site. Intriguingly, the CBM3 from scaffoldin A (ScaA) of Bacteroides cellulosolvens exhibits alterations in sequence that make it more similar to the CBMs of free cellulolytic enzymes, which are classified into CBM family 3b. X‐ray structural analysis was undertaken in order to examine the structural consequences of the sequence changes and the consequent family affiliation. The CBM3 crystallized in space group I4122 with one molecule in the asymmetric unit, yielding diffraction to a resolution of 1.83 Å using X‐ray synchrotron radiation. Compared with the known structures of other scaffoldin‐borne CBMs, a sequence insertion and deletion appear to compensate for each other as both contained an aromatic residue that is capable of contributing to cellulose binding; hence, even though there are alterations in the composition and localization of the aromatic residues in the linear strip its binding ability was not compromised. Interestingly, no Ca2+ ions were detected in the conserved calcium‐binding site, although the module was properly folded; this suggests that the structural role of Ca2+ is less important than originally supposed. These observations indicate that despite their conserved function the scaffoldin‐borne CBMs are more diverse in their sequences and structures than previously assumed.  相似文献   

3.
分离纯化了人胎盘纤连蛋白（Ｆｎ）,经ＳＤＳ－ＰＡＧＥ鉴定为一条带,纯化Ｆｎ仍保持其搞原性,得率为３８．７％。根据植物凝集素识别专一糖链结构的原理,应用斑点印迹法,亲和层析法和Ｗｅｓｔｅｒｎ转移电泳研究糖链结构,结果证实：１．人胎盘Ｆｎ分子中含有复杂型Ｎ糖链（包括二天线和大于二天线的结构）以及高甘露糖型和／或杂合型Ｎ糖链;复杂型Ｎ糖链中含有平分型ＧｌｃＮＡｃ,糖链末端也可连有唾液酸;２．胰糜蛋白酶水解而获得的明胶结合片段（４４ｋＤ）含有二天线和多天线复杂型糖链,也可接有平分型ＧｌｃＮＡｃ;３．肝素结合片段（３０ｋＤ）以及明胶、肝素均不结合的Ｆｎ片段不含有多天线复杂型Ｎ糖链。  相似文献   

4.
    
In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost‐effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate‐binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM‐encoding module named StX was expressed, purified and crystallized, and X‐ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging‐drop vapour‐diffusion method, belonged to space group P3121, with unit‐cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single‐wavelength anomalous diffraction method.  相似文献   

5.
    
Family 3 carbohydrate‐binding modules (CBM3s) are associated with the scaffoldin subunit of the multi‐enzyme cellulosome complex and with the family 9 glycoside hydrolases, which are multimodular enzymes that act on plant cell‐wall polysaccharides, notably cellulose. Here, the crystallization of CBM3b from cellobiohydrolase 9A is reported. The crystals are tetragonal and belong to space group P41 or P43. X‐ray diffraction data for CBM3b have been collected to 2.68 Å resolution on beamline ID14‐4 at the ESRF.  相似文献   

6.
    
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7.
    
Mushroom tyrosinase‐associated lectin‐like protein (MtaL) binds to mature Agaricus bisporus tyrosinase in vivo, but the exact physiological function of MtaL is unknown. In this study, the crystal structure of recombinant MtaL is reported at 1.35 Å resolution. Comparison of its structure with that of the truncated and cleaved MtaL present in the complex with tyrosinase directly isolated from mushroom shows that the general β‐trefoil fold is conserved. However, differences are detected in the loop regions, particularly in the β2–β3 loop, which is intact and not cleaved in the recombinant MtaL. Furthermore, the N‐terminal tail is rotated inwards, covering the tyrosinase‐binding interface. Thus, MtaL must undergo conformational changes in order to bind mature mushroom tyrosinase. Very interestingly, the β‐trefoil fold has been identified to be essential for carbohydrate interaction in other lectin‐like proteins. Comparison of the structures of MtaL and a ricin‐B‐like lectin with a bound disaccharide shows that MtaL may have a similar carbohydrate‐binding site that might be involved in glycoreceptor activity.  相似文献   

8.
    
There is an urgent need to provide effective anti‐HIV microbicides to resource‐poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin‐N (rCV‐N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV‐N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV‐N matched the expected sequence of native CV‐N, as did the mass of rCV‐N (11 009 Da). Purified rCV‐N from soya is active in anti‐HIV assays with an EC50 of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli‐produced CV‐N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV‐N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti‐HIV microbicide development.  相似文献   

9.
    
Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome‐producing anaerobic bacteria that efficiently degrade plant cell‐wall polysaccharides. The assembly of carbohydrate‐active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate‐binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysis. The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD‐1 contains over 200 modular proteins containing the cellulosomal signature dockerin module. One of these is an endoglucanase Cel5A comprising two family 5 glycoside hydrolase catalytic modules (GH5) flanking an unclassified CBM (termed CBM‐Rf2) and a C‐terminal dockerin. This novel CBM‐Rf2 has been purified and crystallized, and data from cacodylate‐derivative crystals were processed to 1.02 and 1.29 Å resolution. The crystals belonged to the orthorhombic space group P212121. The CBM‐Rf2 structure was solved by a single‐wavelength anomalous dispersion experiment at the As edge.  相似文献   

10.
    
The anaerobic, thermophilic, cellulosome‐producing bacterium Clostridium thermocellum relies on a variety of carbohydrate‐active enzymes in order to efficiently break down complex carbohydrates into utilizable simple sugars. The regulation mechanism of the cellulosomal genes was unknown until recently, when genomic analysis revealed a set of putative operons in C. thermocellum that encode σI factors (i.e. alternative σ factors that control specialized regulon activation) and their cognate anti‐σI factor (RsgI). These putative anti‐σI‐factor proteins have modules that are believed to be carbohydrate sensors. Three of these modules were crystallized and their three‐dimensional structures were solved. The structures show a high overall degree of sequence and structural similarity to the cellulosomal family 3 carbohydrate‐binding modules (CBM3s). The structures of the three carbohydrate sensors (RsgI‐CBM3s) and a reference CBM3 are compared in the context of the structural determinants for the specificity of cellulose and complex carbohydrate binding. Fine structural variations among the RsgI‐CBM3s appear to result in alternative substrate preferences for each of the sensors.  相似文献   

11.
To accurately characterize the carbohydrate moieties of oligosaccharide chains in glycosylated proteins, it is necessary to distinguish exactly which types of oligosaccharides are present at which site. We describe lectin overlay assays, which take advantage of the ability of lectins to distinguish between different types of glycoproteins via recognition of terminal sugars, thus allowing the chain type and peripheral antigenic components to be determined. Three microassays involving lectins are reported in this paper: non-proteasetreated intact glycoproteins; glycopeptides released by prior digestion of the glycoprotein and then separated by HPLC; and release of sugars from glycoproteins by hydrazinolysis and then coupling them to a multivalent support.  相似文献   

12.
    
The starch-synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII-specific domain (SSIII-SD) with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch-binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved.  相似文献   

13.
    
The range of genome‐editing tools has recently been expanded. In particular, an RNA‐guided genome‐editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)‐associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV‐1 was designed and used to generate gRNA‐expressing lentiviral vectors. An HIV‐1‐specific gRNA and Cas9 were stably dually transduced into a highly HIV‐1‐susceptible human T‐cell line and the inhibitory ability of the anti‐HIV‐1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV‐1 infection was observed, as evaluated by a VSV‐G‐pseudotyped HIV‐1 reporter system, the anti‐HIV‐1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti‐HIV‐1 treatment with the CRISPR/Cas9 system alone.  相似文献   

14.
    
Human immunodeficiency virus type-1 (HIV-1) infection generally provokes antibody responses to the viral envelope glycoprotein. Two major regions of gp120, the third variable (V3) domain and the CD4-binding site, have been identified as neutralization targets. The precise mechanism of HIV-1 neutralization by antibodies against the V3 domain is still unknown. It is shown that by kinetic neutralization studies, one molecule of V3-targeted monoclonal antibody (0.5beta) is enough to neutralize one virion. This antibody, which neutralized more than 99% of the virus, inhibited the binding of the virus to cells by 42%. HIV-1 pseudotyped with G glycoprotein from vesicular stomatitis virus was also neutralized by 0.5beta, suggesting that the antibody did not inhibit the viral attachment but caused some alteration in the envelope. These results indicate that the antibody plays an additional role on steric change of the envelope involved in inhibition of viral entry.  相似文献   

15.
    
Galectin‐4, a member of the tandem‐repeat subfamily of galectins, participates in cell‐membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin‐4 carbohydrate‐recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N‐terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 Å resolution. The lactose‐binding affinity was characterized by fluorescence measurements and two lactose‐binding sites were identified: a high‐affinity site with a Kd value in the micromolar range (Kd1 = 600 ± 70 µM) and a low‐affinity site with Kd2 = 28 ± 10 mM.  相似文献   

16.
Sialic acid-specific lectins have been detected in the serum of the “whip scorpion,” Mastigoproctus giganteus. When compared to Limulus lectins, Mastigoproctus agglutination profiles for a panel of untreated and enzyme-treated vertebrate erythrocytes were almost identical except for the agglutination of nonhuman primate erythrocytes. However, both chelicerate species exhibited heterogeneous serum lectins which showed some differences in their serological reactivity. At least three distinct specific fractions could be demonstrated in Mastigoproctus serum by crossed absorption and hemagglutination-inhibition experiments. These fractions are specific for sialic acids and/or sialoconjugates but also bind substances such as N-acetylglutamic acid, N-acetylmuramic acid, chitobiose, and chitotriose. These adjunct specificities are important clues in the interpretation of the possible biological role of chelicerate lectins.  相似文献   

17.
    
Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate‐binding module connected through a serine/threonine‐rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 Å resolution and the crystal system is primitive orthorhombic, with unit‐cell parameters a = 90.9, b = 123.4, c = 135.4 Å. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 Å3 Da−1 and a solvent content of 50.88%(v/v).  相似文献   

18.
    
Carbohydrate‐binding modules (CBMs) are discrete parts of carbohydrate‐hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high‐resolution X‐ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate–protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate‐binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X‐2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β‐glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A of Rhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm3) were grown at room temperature and subjected to H/D exchange. Both neutron and X‐ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X‐ray refinement using phenix.refine showed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen‐bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein–carbohydrate binding interactions.  相似文献   

19.
    
Galectin‐3 is a multifunctional carbohydrate‐binding protein that has roles in cancer progression. In addition to carbohydrate‐dependent extracellular functions, galectin‐3 participates in carbohydrate‐independent intracellular signalling pathways, including apoptosis, via protein–protein interactions, some of which engage the carbohydrate‐binding groove. When ligands bind within this site, conformational rearrangements are induced and information on unliganded galectin‐3 is therefore valuable for structure‐based drug design. Removal of cocrystallized lactose from the human galectin‐3 carbohydrate‐recognition domain was achieved via crystal soaking, but took weeks despite low affinity. Pre‐soaking to remove lactose enabled the subsequent binding of cryoprotectant glycerol, whereas when the lactose was not removed a priori the glycerol could not displace it in the short cryosoaking time frame. This slow diffusion of lactose out of the crystals contrasts with the entrance of glycerol, which takes place within minutes. The importance of the removal of incumbent ligands prior to attempts to introduce alternative ligands is indicated, even for proteins exhibiting low affinity for ligands, and has significance for ligand exchange in structure‐based drug design.  相似文献   

20.
    
A construct containing the CBM22‐1–CBM22‐2 tandem forming the N‐terminal domain of Paenibacillus barcinonensis xylanase 10C (Xyn10C) has been purified and crystallized. A xylan‐binding function and an affinity for mixed β‐1,3/β‐1,4 glucans have previously been demonstrated for some members of the CBM22 family. The sequence of the tandem is homologous to the N‐terminal domains found in several thermophilic enzymes. Crystals of this tandem were grown by the streak‐seeding method after a long optimization strategy. The structure has been determined by molecular replacement to a resolution of 2.43 Å and refinement is under way. This study represents the first structure containing two contiguous CBM22 modules, which will contribute to a better understanding of the role that this multiplicity plays in fine‐tuning substrate affinity.  相似文献   

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