Cell biology. ER-to-Golgi traffic--this bud's for you

How do protein-transporting vesicles, which bud from the endoplasmic reticulum (ER), specifically dock to, and fuse with, the Golgi apparatus? In their Perspective, Brittle and Waters discuss new work (Allan et al.) suggesting that some vesicle-associated docking and fusion proteins are "programmed" during vesicle budding from the ER and direct downstream events that occur during fusion of these transport vesicles with the membranes of the Golgi.     

Signal-mediated dynamic retention of glycosyltransferases in the Golgi

Golgi-resident glycosyltransferases are a family of enzymes that sequentially modify glycoproteins in a subcompartment-specific manner. These type II integral membrane proteins are characterized by a short cytoplasmically exposed amino-terminal tail and a luminal enzymatic domain. The cytoplasmic tails play a role in the localization of glycosyltransferases, and coat protein complex I (COPI) vesicle-mediated retrograde transport is also involved in their Golgi localization. However, the tails of these enzymes lack known COPI-binding motifs. Here, we found that Vps74p bound to a pentameric motif present in the cytoplasmic tails of the majority of yeast Golgi-localized glycosyltransferases, as well as to COPI. We propose that Vps74p maintains the steady-state localization of Golgi glycosyltransferases dynamically, by promoting their incorporation into COPI-coated vesicles.     

Bakers' yeast, a model for fungal biofilm formation

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Here we show that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation. When grown in low-glucose medium, the yeast cells adhered avidly to a number of plastic surfaces. On semi-solid (0.3% agar) medium they formed "mats": complex multicellular structures composed of yeast-form cells. Both attachment to plastic and mat formation require Flo11p, a member of a large family of fungal cell surface glycoproteins involved in adherence. The ability to study biofilm formation in a tractable genetic system may facilitate the identification of new targets for antifungal therapy.     

Chemical glycobiology

Chemical tools have proven indispensable for studies in glycobiology. Synthetic oligosaccharides and glycoconjugates provide materials for correlating structure with function. Synthetic mimics of the complex assemblies found on cell surfaces can modulate cellular interactions and are under development as therapeutic agents. Small molecule inhibitors of carbohydrate biosynthetic and processing enzymes can block the assembly of specific oligosaccharide structures. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. Carbohydrate biosynthetic pathways are often amenable to interception with synthetic unnatural substrates. Such metabolic interference can block the expression of oligosaccharides or alter the structures of the sugars presented on cells. Collectively, these chemical approaches are contributing great insight into the myriad biological functions of oligosaccharides.     

Automated solid-phase synthesis of oligosaccharides

Traditionally, access to structurally defined complex carbohydrates has been very laborious. Although recent advancements in solid-phase synthesis have made the construction of complex oligosaccharides less tedious, a high level of technical expertise is still necessary to obtain the desired structures. We describe the automated chemical synthesis of several oligosaccharides on a solid-phase synthesizer. A branched dodecasaccharide was synthesized through the use of glycosyl phosphate building blocks and an octenediol functionalized resin. The target oligosaccharide was readily obtained after cleavage from the solid support. Access to certain complex oligosaccharides now has become feasible in a fashion much like the construction of oligopeptides and oligonucleotides.     

昆虫糖基水解酶20家族β-N-乙酰己糖胺酶研究进展

糖基水解酶20家族β-N-乙酰己糖胺酶是N-乙酰己糖胺代谢过程中一个重要的酶,其可将位于非还原端以β糖苷键连接的N-乙酰己糖胺从寡糖及糖复合物中水解下来。昆虫糖基水解酶20家族β-N-乙酰己糖胺酶参与昆虫多个生理过程,包括表皮几丁质水解、蛋白质N糖基化修饰、糖复合物水解及精卵识别,因此可能成为生态农药设计的潜在靶标,受到国内外学者的普遍关注。根据昆虫20家族β-N-乙酰己糖胺酶的进化关系和生理功能,可以将其分为4个亚家族,分别为Hex1、Hex2、Hex3和Hex4。Hex1主要在昆虫蜕皮时期的表皮中表达,RNA干扰该基因能够导致昆虫在发育过程中不能正常蜕皮而死亡。通过酶学性质研究发现,Hex1能够专一性水解以β 1-4糖苷键连接的几丁质寡糖底物,而不能水解以其他形式连接的底物,是专一性的参与几丁质水解的β-N-乙酰己糖胺酶。目前获得的唯一一个昆虫β-N-乙酰己糖胺酶的晶体结构来自于亚洲玉米螟的OfHex1,其参与底物结合的“+1”位点存在一个特殊的三明治结构,使得Hex1对几丁质寡糖具有高度的选择性和水解活性。Hex2在双翅目昆虫中并不存在,与人类的β-N-乙酰己糖胺酶具有高度的相似性,RNA干扰该基因的表达能够导致幼虫化蛹时异常,成虫翅、附肢、触角等异常。通过酶学性质研究表明,Hex2能够水解释放几丁质寡糖,糖复合物等底物中的β-N-乙酰己糖胺,具有广泛的底物谱。Hex2的功能可能与人类Hex酶的功能类似,参与糖复合物的水解。Hex3是目前研究较少的一类β-N-乙酰己糖胺酶,RNA干扰该基因的表达能够导致幼虫蜕皮过程异常。Hex3存在于昆虫蜕皮液中,与Hex1存在相互作用,可能形成二聚体。通过酶学性质研究表明,Hex3能够水解几丁质寡糖底物,但活性较弱,说明其可能参与几丁质的水解。此外,Hex3、Hex1和Hex4被发现存在于双翅目昆虫精子的表面,可能参与精卵识别过程。Hex4又被称为FDL,是因为该基因的缺失能够导致果蝇左右大脑发生融合（fused lobe）。它是另外一种具有严格底物选择性的β-N-乙酰己糖胺酶,能够专一性地水解糖复合物GnGn中α1-3分支上以β 1-2糖苷键连接的GlcNAc β 1-2Man,通过细胞定位研究表明Hex4主要存在于高尔基体当中。以上表明Hex4的主要功能是参与细胞内的糖基化修饰过程。尽管目前对于昆虫β-N-乙酰己糖胺酶的研究取得了很大的进展。对Hex1的功能、酶学性质和晶体结构研究得较为透彻,对于设计环境友好的杀虫剂具有较强的指导意义。但其他3种β-N-乙酰己糖胺酶在生理条件下如何发挥功能,以及4种β-N-乙酰己糖胺酶活性差异的结构基础并不是十分清楚。作者从进化关系、晶体结构、酶学性质和生理功能等方面对昆虫糖基水解酶20家族β-N-乙酰己糖胺酶的研究进展进行综述。     

Chaperone selection during glycoprotein translocation into the endoplasmic reticulum

A variety of molecular chaperones and folding enzymes assist the folding of newly synthesized proteins in the endoplasmic reticulum. Here we investigated why some glycoproteins interact with the molecular chaperone BiP, and others with the calnexin/calreticulin pathway. The folding of Semliki forest virus glycoproteins and influenza hemagglutinin was studied in living cells. The initial choice of chaperone depended on the location of N-linked glycans in the growing nascent chain. Direct interaction with calnexin and calreticulin without prior interaction with BiP occurred if glycans were present within about 50 residues of the protein's NH2-terminus.     

Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.     

Secreted kinase phosphorylates extracellular proteins that regulate biomineralization

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.     

疯草研究进展

疯草是指含有苦马豆素的豆科黄芪属和棘豆属有毒植物,动物疯草中毒病是危害草原畜牧业最严重的问题之一.苦马豆素是疯草的主要毒性成分,它能抑制动物细胞α-甘露糖苷酶和甘露糖苷酶Ⅱ的活性,影响低聚糖的代谢和糖蛋白的加工,导致低聚糖蓄积和糖蛋白合成障碍.中毒动物表现为行为异常、瘫痪、不育、流产、体质量下降,严重的发生死亡,造成巨大的经济损失.本文主要对疯草的毒性成分、中毒机理、化学防除和脱毒利用等方面的研究进行了综述,旨在为疯草中毒病的防治和疯草的利用提供思路和认识.     

固醇调节元件结合蛋白裂解激活蛋白(SCAP)的研究进展

对SCAP的结构、功能、单核苷酸多态性等方面作一综述,并就固醇对SCAP的反馈性调节机制进行讨论。     

Toward automated synthesis of oligosaccharides and glycoproteins

The discovery of previously unknown functions associated with carbohydrates and the study of their structure-function relations are of current interest in carbohydrate chemistry and biology. Progress in this area is, however, hampered by the lack of convenient and effective tools for the synthesis and analysis of oligosaccharides and glycoconjugates. Development of automated synthesis of such materials is necessary to facilitate research in this field. This review describes recent advances in carbohydrate synthesis, with particular focus on developments that have potential application to the automated synthesis of oligosaccharides, glycopeptides, and glycoproteins.     

Identification of the 1H-NMR spectra of complex oligosaccharides with artificial neural networks

Artificial networks can be used to identify hydrogen nuclear magnetic resonance (1H-NMR) spectra of complex oligosaccharides. Feed-forward neural networks with back-propagation of errors can distinguish between spectra of oligosaccharides that differ by only one glycosyl residue in twenty. The artificial neural networks use features of the strongly overlapping region of the spectra (hump region) as well as features of the resolved regions of the spectra (structural reporter groups) to recognize spectra and efficiently recognized 1H-NMR spectra even when the spectra were perturbed by minor variations in their chemical shifts. Identification of spectra by neural network-based pattern recognition techniques required less than 0.1 second. It is anticipated that artificial neural networks can be used to identify the structures of any complex carbohydrate that has been previously characterized and for which a 1H-NMR spectrum is available.     

海藻寡糖在农业领域的应用研究进展

海藻寡糖是海藻多糖经过降解得到的一系列寡糖片段,分子量低、水溶性好,具有广泛的生物活性优势,在农业生产中具有广阔的应用前景。文章对近年来海藻寡糖在土壤修复和促进植物生长两方面的研究进展进行综述,同时指出海藻寡糖对土壤重金属的不同作用效果限制了其在土壤修复中的进一步应用、土壤微生物对海藻寡糖—重金属络合物的影响机理不明及海藻寡糖作为植物诱导剂在实际应用中存在局限性等问题。因此,今后在海藻寡糖的利用中应注意以下问题:进行土壤修复时,应针对土壤的酸碱度及重金属污染情况,合理选择海藻寡糖的施用量,确定适宜的修复方式;可将海藻寡糖与其他诱导剂或杀菌剂联合使用以解决其作为诱导剂的局限性;此外,海藻寡糖可作为新型肥料添加剂进行深入开发,以促进其在农业领域的进一步应用。     

Production of complex human glycoproteins in yeast

We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.     

胆固醇平衡调控中的SREBP途径

胆固醇在生物体内发挥着极其重要的作用。细胞主要通过调节胆固醇的合成、吸收、酯化及外流等途径之间的平衡以维持正常的胆固醇浓度。本文介绍甾醇调控元件结合蛋白(SREBP,Sterolregulatoryelement-bindingproteins)转录因子对胆固醇平衡的调控。SREBP主要调节胆固醇的合成、LDL受体介导的胆固醇的吸收和脂肪酸的合成。SREBP前体是一内质网膜蛋白,SREBP经两次蛋白酶水解后释放出其N端结构域,进入细胞核,激活靶基因的转录。随着对SREBP领域研究的深入,人们对胆固醇平衡调节将认识更多,并有可能开发出更为有效的控制胆固醇的药物。     

Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle

The mechanisms that determine how folding attempts are interrupted to target folding-incompetent proteins for endoplasmic reticulum-associated degradation (ERAD) are poorly defined. Here the alpha-mannosidase I-like protein EDEM was shown to extract misfolded glycoproteins, but not glycoproteins undergoing productive folding, from the calnexin cycle. EDEM overexpression resulted in faster release of folding-incompetent proteins from the calnexin cycle and earlier onset of degradation, whereas EDEM down-regulation prolonged folding attempts and delayed ERAD. Up-regulation of EDEM during ER stress may promote cell recovery by clearing the calnexin cycle and by accelerating ERAD of terminally misfolded polypeptides.     

A vital stain for the Golgi apparatus

The Golgi complex, a membranous organelle with important functions in membrane traffic and macromolecular synthesis, has been stained in living cells with a fluorescent sphingolipid. Cells were first incubated with liposomes containing N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), or with a bovine serum albumin complex of the fluorescent lipid, and then examined by fluorescence microscopy. An intensely fluorescent perinuclear structure was identified as the Golgi apparatus by its colocalization with known Golgi markers in fixed cells. C6-NBD-ceramide was used to observe the morphology of the Golgi apparatus in living cells in the presence or absence of monensin or Colcemid, and during mitosis. In all cases, C6-NBD-ceramide revealed a Golgi apparatus in the living cell that was identical to that obtained with conventional procedures that require fixation.     

BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis

BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.     

牛卵母细胞发育的超微结构研究

本实验根据卵细胞的数量及分布、透明带的变化,卵泡大小等把牛卵母细胞的整个发育过程分为六个阶段,在超微结构的水平上进行了研究。随着卵母细胞的发育,高尔基体、线粒体、滑面内质网、皮质颗粒等细胞器数量不断增加,且逐渐迁移到皮质区。卵母细胞成熟时。高尔基体及粗面内质网消失,皮质颗粒开始在质膜下呈线状排列,而线粒体又向胞质中央区扩散。直径2毫米以上卵泡卵母细胞中,少嵴圆形线粒体一律转变为带帽线粒体,核仁发生致密化。此外,牛卵母细胞中有带有界膜的、均匀分布的囊泡。皮质颗粒成团发生和分布也是牛卵母细胞的特点之一。实验事实证明,皮质颗粒的形成与滑面内质网关系更为密切。