Differential Modulation of SERCA2 Isoforms by Calreticulin

In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca²⁺ oscillations in a manner consistent with inhibition of Ca²⁺ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA)2 gene display differential Ca²⁺ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.     

Layilin,A Novel Talin-binding Transmembrane Protein Homologous with C-type Lectins,is Localized in Membrane Ruffles

Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten–amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. We have identified a short region within talin''s amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase.     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

Thermodynamic parameters of the interaction of Urtica dioica agglutinin with N-acetylglucosamine and its oligomers

The interaction between Urtica dioica agglutinin (UDA) and N-acetylglucosamine (GlcNAc) and its (1-4)-linked oligomers was studied by fluorescence titration and isothermal titration microcalorimetry. UDA possesses one significant binding site that can be measured calorimetrically. This site is composed of three subsites, each subsite accommodating one GlcNAc residue. The interaction is enthalpically driven, and the binding area of UDA is characterized by a H of interaction for a given oligosaccharide considerably smaller than that of wheat germ agglutinin (WGA), despite the fact that they both belong to a family of proteins composed entirely of hevein domains. Relatively high Cp values of the UDA-carbohydrate interactions and more favorable entropy term compared to WGA suggest that binding of the carbohydrate ligands by UDA has a higher hydrophobic component than that of WGA.     

草鱼胶原凝集素基因的克隆及其功能分析

从草鱼Ctenopharyngodon idella肝肾cDNA文库中克隆得到胶原凝集素基因。草鱼胶原凝集素全长cDNA为1128bp,其中5′非编码区229bp,3′非翻译区104bp,最大开放阅读框为795bp,编码264个氨基酸。系统进化分析表明草鱼胶原凝集素与斑马鱼的亲缘关系最近。根据草鱼胶原凝集素序列特征,克隆了包含糖基识别域(CRD)的cDNA,并进行原核表达、纯化获得其重组蛋白PCRD。进行PCRD与6种细菌的凝集和糖抑制实验,结果表明半乳糖、葡萄糖、甘露糖和麦芽糖4种糖都会使PCRD与嗜水气单胞菌的凝集明显下降甚至极大地干扰凝集;麦芽糖使金黄色葡萄球菌的凝集明显下降,而肽聚糖和甘露糖会使凝集受到抑制;此外,PCRD的凝集反应不依赖Ca2+。     

Production and characterization of the B chains of mistletoe toxic lectins

Mistletoe toxic lectins consist of two polypeptide chains: an enzymatically active A chain, which is a toxic component, and a disulfide-bonded B chain, which confers the lectin properties on the total molecule. Mistletoe leaves contain three toxic lectins encoded by three genes. The B chains of these lectins were overproduced in Escherichia coli in a soluble form. The recombinant proteins bound with asialofetuin, but had substantially lower affinity for simple sugars D-galactose and N-acetyl-D-galactosamine as compared with the natural proteins. The functional properties of the B chains strongly depended on the storage conditions (salt concentration and the presence of galactose); the dependence was explained by structural instability of nonglycosylated recombinant proteins. The lectin activity of one of the recombinant B chains was close to that of the native protein, which was attributed to the lack of N-glycosylation sites in the latter.     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

α-mannosidase from Erythrina indica seeds is a Zn²⁺ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol^− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

抗肿瘤蛋白AAL与靶蛋白MRG15的共定位研究

一种从食用真菌Agrocybe aegerita中提取的凝集素AAL具有显著的抗肿瘤效果。该蛋白的基因序列已经通过RT-PCR得到。T7噬菌体展示库筛选发现该蛋白可以在体外和细胞核内的细胞周期调控蛋白mortality factor related gene on chro-mosome 15(MRG15)发生相互作用。本实验目的是证实AAL和MRG15在细胞核内的共定位,为AAL与MRG15的体内的互作提供证据。构建质粒,将aal与mrg15分别连接到pEGFP-C1和pDsRed-C1上,共转染Hela细胞。将pEGFP-C1和pD-sRed-C1空载体共转染Hela细胞作为对照。利用激光共聚焦显微镜研究AAL和MRG15在细胞内的共定位。AAL和MRG15均定位在细胞核内,荧光图片的重叠表明AAL和MRG15在核内共定位。而对照组中的EGFP和DS-RED在细胞内呈弥散分布。这些为AAL和MRG15在细胞核内的相互作用提供了证据。