F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Homophilic adhesion mechanism of neurofascin, a member of the L1 family of neural cell adhesion molecules

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.     

Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae

Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive. The nonpathogenic protozoa Leishmania tarentolae is an easy-to-handle alternative expression system for production of proteins requiring the eukaryotic protein folding machinery and post-translational modifications. We used and evaluated the system for the secretory expression of extracellular domains from human glycoprotein VI and the receptor for advanced glycation end products from rat. Both proteins were well expressed and homogeneously glycosylated. Analysis of the glycosylation pattern identified the structure as the conserved core pentasaccharide Man₃GlcNac₂.     

由反转录病毒载体转移的马立克病毒糖蛋白D基因在感染细胞中的表达

用ＥｃｏＲＩ、ＰｓｔＩ将已分离和克隆的马立克氏病病毒糖蛋白Ｄ基因从重组ｐＭｇＤ１８质粒中切出,克隆进反转录病毒质粒载体（ＲＣＡＳ）的连接质粒载全ＰＵＣＣｌａ１１２Ｎ的相同位点。用ＣｌａＩ再次ｇＤ基因切出,构建于ＲＣＡＳ的ＣｌａＩ位点。通过原位杂交筛选重组ＲＣＡＳ,并结合酶切分析鉴定出ｇＤ插入方向正确的重组ＲＣＡＳ。用磷酸钙沉淀法将ｇＤ重组ＲＣＡＳ转染鸡胚成纤维细胞（ＣＥＦ）,转染后第９天收集细胞上     

Nutrient–secretion coupling in the pancreatic islet β-cell: recent advances

Insulin secretion from pancreatic islet β-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total β-cell mass is essentially unaltered, but β-cells become progressively “glucose blind” and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K⁺(K_ATP) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca²⁺, then prompt the “first phase” on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K_ATP channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.     

Production of functional IgM Fab fragments by Saccharomyces cerevisiae

The aim of this study was to express and secrete functional mouse IgM fragments in yeast. The heavy chain cDNA was truncated at two different sites, yielding genes coding for the complete VH domain. In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3′ terminus of the CH1 region. Both shortened genes were coexpressed in Saccharomyces cerevisiae with a cDNA gene encoding a full length mouse Ig light chain. We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments.     

稻瘟病菌含信号肽小蛋白的计算机分析

结合计算机技术和生物信息学的方法,采用组合的信号肽分析软件SignalPv3.0、TargetPv1.1、Big-PIpredictor、TMHMMv2.0和SecretomeP对已公布的1486个稻瘟菌(magnaporthegrisea)小蛋白基因的N-端氨基酸序列进行信号肽分析,同时系统分析了信号肽的类型及结构。分析结果表明,在1486个稻瘟病菌小蛋白中,119个具有N-端信号肽的典型分泌蛋白。其中116个具有分泌型信号肽,1个具RR-motif型信号肽,2个具信号肽酶II型信号肽。在稻瘟病菌基因组中,分泌型小蛋白的序列是高度趋异的,仅出现少数氨基酸组成完全一致的信号肽,为进一步确认具有相同信号肽的分泌蛋白是否具有同源性,分别用BLAST2SEQUENCES对具有相同信号肽的分泌蛋白进行了序列对比。结果表明,具有相同信号肽的分泌蛋白同源性非常高。同时还采用Sublocv1.0对1486个小蛋白的亚细胞位置进行了预测,结果显示小蛋白的可能功能场所包括细胞质、细胞外、线立体和细胞核,功能场所位于细胞核的小蛋白是最多的。     

特超强毒648株马立克氏病病毒的囊膜糖蛋白gE基因的克隆和序列比较

鸡马立克氏病毒特超强毒 (vv+MDV) 648株的囊膜糖蛋白 gE基因经PCR扩增并克隆入pUC1 8载体。 648株gE基因经核酸序列分析测定 ,全长为 1 494碱基。所编码的蛋白具有跨膜糖蛋白的一些特征。它含有 8个潜在的糖基化位点、N端有一段疏水区 (1～ 1 9aa)所构成的信号肽、C端有一段疏水区 (391～ 41 9aa)所构成的膜锚着序列。经 648株 (vv+MDV)和马立克氏病毒强毒 (vMDV)GA株的 gE相比较 ,在MDV血清 1型中 gE是较为保守的 ,二者仅有 2个核苷酸的差异 (第 51 2位、第 1 472位 ) ,并导致了有二个相应的氨基酸的改变 (第1 71位Leu/Pro、第 491位Arg/Lys)。     

西瓜柱头乳突细胞分泌活动期间ATP酶活性超微结构定位

研究了西瓜柱头乳突细胞ＡＴＰ酶活性的超微结构定位。分泌活动旺盛的细胞中,质膜、内质网、质体的内部片层、胞间连丝以及多数大液泡的膜上面都有大量ＡＴＰ酶活性反应产物,线粒体和小泡上只有少量酶活性反应产物。分泌活动停止后处于解体状态的细胞内,反应产物主要定位于液泡膜上。分泌旺盛的乳突细胞质膜具有高的ＡＴＰ酶活性表明分泌物运出需要大量能量,内质网ＡＴＰ酶活性强可能意味着该细胞参与分泌物合成。