棉花两个β-甘露糖苷酶cDNA的克隆及其特征

从陆地棉纤维cDNA文库中分离出两个B-甘露糖苷酶的cDNA克隆,GhManAl和GhManA2.它们的开放读码框编码长度分别为834个氨基酸和976个氨基酸的多肽序列,这两个多肽C-末端的747个氨基酸残基是完全一致的,而N-末端的序列差异较大.GhManAl和GhManA2均属于糖基水解酶家族2的成员,它们与其它植物来源的该家族中β-甘露糖苷酶之间具有较高的同源性,而与非植物来源的β-甘露糖苷酶之间的同源性较低,但不同蛋白序列中均存在糖基水解酶家族2的酶催化活性所需的两个Glu保守残基.这两个多肽的N-末端均没有信号肽序列,因此可能为胞内酶.从表达特征来看,GhManA1属于组成型表达基因,而GhManA2则为纤维细胞优势表达基因.     

SKW6细胞在转导6A8 α-甘露糖苷酶反义cDNA后对抗Fas抗体诱导的凋亡

鉴于蛋白质糖基化的重要生物学意义,以腺相关病毒为载体,把编码6A8 α-甘露糖苷酶的cDNA 3′端1358 bp的反向片段转导入EB病毒转化的B细胞株SKW6,探讨6A8 α-甘露糖苷酶表达抑制对抗Fas抗体诱导凋亡的影响.反义6A8转导成功及表达用Northern杂交及RT-PCR检测,6A8 α-甘露糖苷酶表达用Con A结合试验检测.Giemsa染色,AnnexinⅤ染色及梯形DNA电泳显示,Fas抗体能诱导SKW6细胞凋亡,但6A8 α-甘露糖苷酶表达抑制的细胞对Fas抗体诱导的凋亡发生抵抗,而转导正义6A8或空载载体则无影响.6A8 α-甘露糖苷酶表达状况对SKW6细胞表面Fas分子表达没有影响.     

外显子拼接法克隆紫杉烷2α-羟基化酶基因与生物信息学分析

紫杉烷2α-羟基化酶是形成紫杉醇核心骨架的羟基化反应关键酶之一,以taxusin作为底物进行氧化生成2α,7β-dihydroxytaxusin.利用蔓地亚红豆杉的总DNA为模板,采用PCR技术克隆出紫杉烷2α-羟基化酶的DNA序列,利用在线比对和生物学软件分析其内含子,采用外显子拼接法克隆出紫杉烷2α-羟基化酶基因的cDNA序列.测序结果表明该基因含有1个1 488 bp的开放阅读框,编码495个氨基酸的多肽;同源性比较分析结果表明,其碱基序列及氨基酸序列与已经报道的加拿大红豆杉的紫杉烷2α-羟基化酶基因的一致性为分别为98%和89%.利用SWISS-PROT、DNAMAN等生物信息学工具对其列进行了序列分析,为利用代谢工程的方法生产紫杉醇或其前体物质提供了分子基础.     

兔防御素(MCP-1)cDNA的克隆与序列分析

从兔脾脏细胞中分离提取总RNA,经反转录PCP(RT-PCR)扩增出兔巨嗜细胞阳离子多肽(MCP-1)cDNA,插入经EcoRI和XbaI双酶切的pUC19中,构建了克隆质粒pUCDEF.进行了限制性酶切鉴定和序列分析,结果在扩增出的cDNA288个碱基中,在前片段中有一个碱基与发表的兔MCP-1cDNA序列不同,即第157位碱基由G变为A,导致编码的氨基酸由丙氨酸变为苏氨酸.该cDNA全长288bp,编码94个氨基酸,由编码信号肽,前片段及成熟肽片段的序列组成.     

SKW6细胞在转导6A8 a-甘露糖苷酶反义cDNA后对抗Fas抗体诱导的凋亡

鉴于蛋白质糖基化的重要生物学意义, 以腺相关病毒为载体, 把编码6A8 a-甘露糖苷酶的cDNA 3′端1358 bp的反向片段转导入EB病毒转化的B细胞株SKW6, 探讨6A8 a-甘露糖苷酶表达抑制对抗Fas抗体诱导凋亡的影响. 反义6A8转导成功及表达用Northern杂交及RT-PCR检测, 6A8 a-甘露糖苷酶表达用Con A结合试验检测. Giemsa染色, AnnexinⅤ染色及梯形DNA电泳显示, Fas抗体能诱导SKW6细胞凋亡, 但6A8 a-甘露糖苷酶表达抑制的细胞对Fas抗体诱导的凋亡发生抵抗, 而转导正义6A8或空载载体则无影响. 6A8 a-甘露糖苷酶表达状况对SKW6细胞表面Fas分子表达没有影响.     

番茄GDP-D-甘露糖焦磷酸化酶cDNA的克隆、表达及定位

GDP-D-甘露糖焦磷酸化酶催化GDP-D-甘露糖的合成,是植物抗坏血酸生物合成途径中上游的关键酶。以马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列为信息探针,在GenBank dbEST数据库中找到65条高度同源的番茄EST序列,通过序列拼接及RACE-PCR得到了番茄该基因的全长cDNA序列,命名为LeGMP。LeGMP与马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列一致率为96％,推导的氨基酸序列与马铃薯、烟草、紫苜蓿、拟南芥的GDP-D-甘露糖焦磷酸化酶基因的一致率分别为99％、97％、91％、89％。经Northern杂交分析,LeGMP在番茄根、茎、叶、花、果实中都有表达,但表达水平有差异。利用75个番茄远缘杂交重组系（IL系）将LeGMP定位在番茄第3染色体上的D区段（3-D）。     

天麻中一种抗真菌蛋白基因的克隆

依据天麻(GastrodiaelataBl.f.flavidaS.Chow)抗真菌蛋白GAFP_1的N端部分氨基酸序列设计简并引物,通过RACE(快速分离cDNA末端)的方法扩增得到GAFP_1全长cDNA。该cDNA包含一个编码171个氨基酸的ORF,推导的多肽序列与测得的蛋白质部分序列相同;在5′端有一个长为55bp的5′非编码区;终止密码子下游有一个141bp长的3′非编码区,其中含有两个加poly(A)信号及长度为26个腺苷酸的poly(A)。经检索发现该推导蛋白序列与火烧兰(Epipactishelloborine)和二叶兰(Listeraovata)的甘露糖结合蛋白以及雪花莲(Galanthusnivalis)中的甘露糖结合凝集素具有很高同源性。     

甜瓜α-甘露糖苷酶亚家族基因的克隆及分析

在植物体中,α-甘露糖苷酶(α-man,α-mannosidase)是N-聚糖加工、修饰的关键酶,而N聚糖的加工对于植物生长发育是必不可少的,同时在果实成熟过程中起着重要作用。对甜瓜Ⅱ类α-甘露糖苷酶家族基因的结构特点和表达模式进行分析,为探讨α-甘露糖苷酶基因家族各成员在甜瓜不同组织器官发育中可能存在的作用奠定基础。本研究以甜瓜品种河套蜜瓜为材料,采用RT-PCR方法克隆了该家族的3个成员:Cm MAN1、Cm MAN2和Cm MAN3的全长c DNA,应用生物信息学方法对其相应蛋白质的理化性质、系统发育、保守基序进行了分析。分析研究表明,Cm MAN1、Cm MAN2和Cm MAN3的开放阅读框分别为3 063 bp、3 483 bp和3 024 bp,分别编码1 020、1 160和1 007个氨基酸,均为酸性蛋白质,且在不同物种间具有高度保守性。利用RT-q PCR方法检测了这些基因的表达模式,发现Cm MAN1在成熟甜瓜果实中表达量最高,Cm MAN2在叶、花和子房中表达量相对较高,Cm MAN3在子房中表达量较高,表明α-甘露糖苷酶各基因在甜瓜发育中可能具有不同的作用。     

一种来源于青霉的新的α-半乳糖苷酶的分离纯化及其酶学性质

从丝状真菌中筛选到一株产α-半乳糖苷酶的菌株F63,对该菌株进行了形态观察和18SrDNA序列分析,该菌株属于青霉属。采用硫酸铵沉淀、阴离子交换层析和分子筛层析等方法分离纯化了该菌株的一种α-半乳糖苷酶。经过聚丙烯酰胺凝胶电泳,此酶蛋白的分子量约为82kDa。该α-半乳糖苷酶反应的最适pH为5.0,最适温度为45℃。此α-半乳糖苷酶的热稳定性在40℃以下,pH稳定性为pH5.0-6.0。与已报道的α-半乳糖苷酶的活性都受到Ag 的强烈抑制不同的是,该α-半乳糖苷酶受Ag 的抑制作用不显著。以pNPG为底物的Km值为1.4mmol/L和Vmax=1.556mmol/L.min-1.mg-1。该酶可以有效降解蜜二糖、棉子糖和水苏糖,但不能降解末端含α-半乳糖苷键的多糖。通过利用质谱技术对纯化的α-半乳糖苷酶进行鉴定以及内肽的N端测序证明该蛋白为一种新的α-半乳糖苷酶。     

人IL-6和TNF突变体重组融合蛋白表达质粒的构建及在大肠杆菌中的表达

本文利用PCR技术,对人肿瘤坏死因子α(hTNFα)基因进行了改造,并将其与人白细胞介素-6(hIL-6)成熟肽编码区cDNA进行融合,构建了5′IL-6-TNF△融合蛋白的表达质粒pBVIL6-TNFA△。DNA序列分析证明,PCR扩增片段核苷酸序列与引物设计序列及相应的cDNA序列完全一致;重组子用限制性内切酶酶切鉴定,含有正确的IL6-TNF△融合cDNA片段;表达产物经SDS-聚丙烯酰胺凝胶电泳,分子量约为37kD,与预计的相符合;生物学活性分析初步表明,该融合蛋白具有抗肿瘤活性。     

Alpha-mannosidase in human red cells.

1. A search for lysosomal hydrolases and related enzymes has been made in hemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for alpha-mannosidase, neutral alpha-glucosidase and beta-hexosaminidase. 2. alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5--6.0. Electrophoresis on cellulose acetate showed three bands. Hemolysates from four patients with mannosidosis were not deficient in alpha-mannosidase. pH activity curves and elctrophoretic pattern were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell mannosidase differs from the lysosomal acid mannosidase.     

Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.     

The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate that the alkaloid is the principal toxin responsible for the induction of locoism

A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.     

Direct enzyme transfer from lymphocytes corrects a lysosomal storage disease

Fibroblasts from patients with mannosidosis, the lysosomal storage disease resulting from an inherited deficiency of lysosomal alpha-D-mannosidase (EC 3.2.1.24), accumulate specific mannose-containing oligosaccharides which are characteristic of the disease (1,2). The present study shows that these substances were extensively degraded following transfer of the missing enzyme from normal lymphocytes to mannosidosis fibroblasts on direct contact in tissue culture. Moreover, prolonged correction of the metabolic abnormality of the recipient cells was sustained if contact with fresh donor lymphocytes was periodically renewed. These findings may be highly relevant to lymphocyte function in enzyme replacement therapy by transplantation procedures currently being attempted.     

Golgi alpha-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals

The maturation of N-glycans to complex type structures on cellular and secreted proteins is essential for the roles that these structures play in cell adhesion and recognition events in metazoan organisms. Critical steps in the biosynthetic pathway leading from high mannose to complex structures include the trimming of mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and Golgi complex. These exo-mannosidases comprise two separate families of enzymes that are distinguished by enzymatic characteristics and sequence similarity. Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include enzymes involved in trimming reactions in N-glycan maturation in the Golgi complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and cytosol. Studies on the biological roles of complex type N-glycans have employed a variety of strategies including the treatment of cells with glycosidase inhibitors, characterization of human patients with enzymatic defects in processing enzymes, and generation of mouse models for the enzyme deficiency by selective gene disruption approaches. Corresponding studies on Golgi mannosidase II have employed swainsonine, an alkaloid natural plant product that causes "locoism", a phenocopy of the lysosomal storage disease, alpha-mannosidosis, as a result of the additional targeting of the broad-specificity lysosomal mannosidase by this compound. The human deficiency in Golgi mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Mouse models for Golgi mannosidase II deficiency recapitulate many of the pathological features of the human disease and confirm that the unexpectedly mild effects of the enzyme deficiency result from a tissue-specific and glycoprotein substrate-specific alternate pathway for synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of a systemic autoimmune disorder as a consequence of the altered glycosylation. This review will discuss the biochemical features of Golgi mannosidase II and the consequences of its deficiency in mammalian systems as a model for the effects of alterations in vertebrate N-glycan maturation during development.     

Human lysosomal α‐mannosidases exhibit different inhibition and metal binding properties

Two structurally‐related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal α‐mannosidase (hLM, MAN2B1) and the human core α‐1, 6‐specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II α‐mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine‐based compounds designed for selective inhibition of Golgi α‐mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal α‐mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.     

Deficiency of α-mannosidase in Angus cattle. An inherited lysosomal storage disease

A disease of Angus cattle previously known as pseudolipidosis has been shown to be an inherited lysosomal storage disease, in which an oligosaccharide containing mannose and glucosamine is the storage substance. Diseased animals have a near-absolute deficiency of the lysosomal enzyme, alpha-mannosidase, whereas heterozygotes have a partial deficiency of this enzyme. The condition is analogous to the human disease known as mannosidosis.     

The storage products in genetic and swainsonine-induced human mannosidosis.

Swainsonine induces the accumulation of mannose-rich oligosaccharides in human fibroblasts. The composition of the storage products shows that swainsonine completely inhibits lysosomal alpha-D-mannosidase and alters processing of glycoproteins by inhibiting Golgi alpha-D-mannosidase II. Comparison of the storage products in genetic and swainsonine-induced mannosidosis suggests that human fibroblasts contain a lysosomal alpha-D-mannosidase that is unaffected in genetic mannosidosis.     

Golgi α-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals

The maturation of N-glycans to complex type structures on cellular and secreted proteins is essential for the roles that these structures play in cell adhesion and recognition events in metazoan organisms. Critical steps in the biosynthetic pathway leading from high mannose to complex structures include the trimming of mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and Golgi complex. These exo-mannosidases comprise two separate families of enzymes that are distinguished by enzymatic characteristics and sequence similarity. Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include enzymes involved in trimming reactions in N-glycan maturation in the Golgi complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and cytosol. Studies on the biological roles of complex type N-glycans have employed a variety of strategies including the treatment of cells with glycosidase inhibitors, characterization of human patients with enzymatic defects in processing enzymes, and generation of mouse models for the enzyme deficiency by selective gene disruption approaches. Corresponding studies on Golgi mannosidase II have employed swainsonine, an alkaloid natural plant product that causes “locoism”, a phenocopy of the lysosomal storage disease, α-mannosidosis, as a result of the additional targeting of the broad-specificity lysosomal mannosidase by this compound. The human deficiency in Golgi mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Mouse models for Golgi mannosidase II deficiency recapitulate many of the pathological features of the human disease and confirm that the unexpectedly mild effects of the enzyme deficiency result from a tissue-specific and glycoprotein substrate-specific alternate pathway for synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of a systemic autoimmune disorder as a consequence of the altered glycosylation. This review will discuss the biochemical features of Golgi mannosidase II and the consequences of its deficiency in mammalian systems as a model for the effects of alterations in vertebrate N-glycan maturation during development.