猪鼻支原体膜蛋白p37酶切修饰的质谱分析

p37是猪鼻支原体的膜脂蛋白,有潜在的致癌活性.在对真核表达的p37蛋白分析时发现,p37表现为不同大小的分子片段,提示其存在蛋白质修饰.为了对p37的确切修饰进行分析,进而为其功能研究奠定基础,在构建不同融合蛋白表达质粒并证明其存在修饰蛋白的基础上,通过飞行质谱及N端氨基酸序列测定,分析了p37蛋白的精确分子量和酶切修饰的大致部位,发现3种形式的p37蛋白的精确分子量分别为47 644、46 105和43 984.p37蛋白在其N端被信号肽酶切去1个21肽,在其C端被蛋白酶切去1个18～26小肽后,成为分子量为43 984的成熟p37蛋白. 对p37不同酶切修饰片段的分析将有助于对p37蛋白生物学功能及意义的研究.     

羊毛硫细菌素bovicin HJ50修饰酶BovM双功能域单独催化活性鉴定

【目的】体外重建羊毛硫细菌素bovicin HJ50修饰酶Bov M双功能域(脱水酶与环化酶功能域)各自的催化活性,为深入了解Bov M催化机制奠定基础。【方法】在大肠杆菌(Escherichia coli)中分别异源表达纯化Bov M含脱水功能域的N端与含环化功能域的C端重组蛋白,构建体外反应体系,分别对其前肽底物Bov A进行修饰,通过产物抑菌活性及MALDI-TOF MS分子量检测来鉴定两者的修饰活性;并通过体内体外两种方法检测双功能域蛋白之间的协同作用。【结果】Bov M的N端脱水功能域及C端环化功能域分别具有脱水与环化活性,但双功能域重组蛋白之间没有协同作用。【结论】Bov M双功能域均可独立行使各自的催化功能,但Bov M的完整结构对其正常的催化活性非常重要。     

多种S1断裂内含肽的体内交叉反应

断裂内含肽含有两个独立分离的多肽片段(N端内含肽和C端内含肽),它催化蛋白质反式剪接反应,在蛋白质研究与蛋白质工程中已得到诸多实际应用.在蛋白质反式剪接过程中,内含肽的N端内含肽和C端内含肽通过结构互补特异性地非共价组合.然而,Ssp DnaX S1型断裂内含肽的较大C端内含肽片段近来被发现能够与源自其它内含肽的N端内含肽片段交叉反应,表明蛋白质内含子Ssp DnaX具有结构杂交特征.本研究对另外2种S1型内含肽Rma DnaB和Ssp GyrB的较大C端内含肽与不同S1型断裂内含肽的N 端内含肽交叉反应活性进行分析检测.目的是探讨S1型断裂内含肽的结构杂交特征是否具有普遍性.结果发现,Rma DnaB的S1 C端内含肽能够与Ssp GyrB的S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应;与此相似,Ssp GyrB的S1 C端内含肽能够与Rma DnaB的 S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应.此外,某些交叉反应表现出温度依赖性.这些结果对于内含肽的结构 功能关系以及S1型断裂内含肽的应用研究具有重要的意义.     

镇痛多肽——内吗啡肽-1的人工合成及活性研究

 用液相合成方法合成了具有镇痛作用的μ阿片受体的内源性配体——内吗啡肽 - 1(endomorphin- 1 ) ,该四肽为 Tyr- Pro- Trp- Phe NH2 .液相合成法是在氨基酸的 N端用 Boc(叔丁氧羰酰基 )作保护基 ,C端用 HOSu(N-羟基琥珀酰亚胺 )活化 ,与未加保护基的氨基酸在碱性条件下接肽 .先分别合成 C端二肽和 N端二肽 ,再缩合为四肽 ,产物的保护基用盐酸脱帽去除 .中间产物用薄层层析和熔点鉴定其纯度 ,最终得到了高纯度的四肽 .小白鼠脑室注射 (i.c.v)测定表明 ,8.2 5nmol剂量给药 ,其镇痛活性为 87% ,明显高于吗啡 (morphine) .     

内毒素结合肽的原核表达、纯化及生物学活性鉴定

重组人内毒素结合肽 (endotoxinbindingpeptide ,EBP)融合蛋白在大肠杆菌中表达 ,分离和纯化后对其进行生物学活性观察 .将构建好的PinpointⅩa3 EBP生物素融合表达载体转化大肠杆菌DH5α ,IPTG诱导表达菌株 ,亲和层析法纯化表达产物 ,因子Ⅹa(factorⅩa)切割分离内毒素结合肽 ,采用凝胶过滤和反相液相高效色谱法两步纯化 ,从相对分子质量、N端 1 0个氨基酸的序列分析等方面进行鉴定 ;利用人单核细胞U937对重组内毒素结合肽进行了生物学活性的检测 .结果发现 ,内毒素结合肽以包涵体形式存在 ,因子Ⅹa酶切融合蛋白后得到 3 5kD的内毒素结合肽 ,纯化后内毒素结合肽纯度达 99%以上 ,N端 1 0个氨基酸的分析结果与预期相符 ;初步证实内毒素结合肽具有较好的LPS结合活性 ,能够抑制LPS的作用 .经原核表达及纯化复性 ,获得了具有较好生物学活性的内毒素结合肽 ,为进一步研究其功能奠定了良好的基础     

马尔堡病毒GP蛋白C肽的抑制活性及作用机理研究

目的:研究马尔堡病毒(MARV)包膜糖蛋白(GP)C肽的抗该病毒进入活性及初步作用机制。方法:选取MARV的GP2蛋白上CHR区域序列(611～637残基),合成该序列多肽(C肽)及突变体,利用pVAX1-MGP与pNL-4.3-Luc-R-E-质粒包装假病毒体系,测定各肽对马尔堡假病毒融合的抑制活性,利用圆二色谱、凝胶色谱分析C肽及其突变体对MARV的N肽的相互作用。结果:C肽对马尔堡假病毒融合抑制的IC_(50)为32.67μmol/L,C肽突变体的IC_(50)为9.61～33.47μmol/L,抑制活性高于C肽或与其相当;C肽与N肽形成双螺旋结构,在C肽的7元螺旋体的b、f、c、g位引入离子相互作用氨基酸可提高多肽本身的α螺旋结构含量,一些突变可提高复合物的α螺旋结构含量,但C肽与N肽混合后形成的复合物含量不高。结论:MARV的GP蛋白C肽与N肽相互作用较弱,C肽的抗病毒活性较弱,但改造后可提高活性。     

人骨形成蛋白-2C端102肽的诱骨活性

为分析更短的Hbmp-2C端肽是否具有诱骨活性 ,寻求新型的有诱骨活性的基因工程Hbmp-2产品。利用温度诱导的大肠杆菌表达系统表达肽段长度为102个氨基酸的Hbmp-2C端肽及其Cys的突变体。表达产物经纯化复性后 ,植入小鼠肌袋模型中测试其诱骨活性。获得了能稳定表达Hbmp 2C端肽的工程菌 ,测序结果与预期的序列完全一致。表达产物以包涵体形式存在 ,表达量占细胞总蛋白的 3 0 %。产物经纯化复性后 ,小鼠肌袋模型测试结果表明 :Hbmp 2 10 2肽仍具有诱骨活性 ,而将C端第一位Cys突变的 10 2肽诱骨活性丧失。实验表明 :比Hbmp 2成熟肽 ( 114个氨基酸 )更短的C端 10 2肽仍具有良好诱骨活性 ,这 10 2肽N端第一个Cys对其诱骨活性可能是必需的。     

rhIL-12二硫键、N-糖基化位点及C端氨基酸序列分析

重组人白细胞介素12（rhIL-12）是一种已经用于治疗肿瘤,寄生虫、病毒性感染及造血障碍等疾病研究的异二聚体糖蛋白。结构确证是质量控制的重要内容,此研究对CHO细胞表达的rhIL-12二硫键配对方式、N-糖基化位点以及C端氨基酸序列进行了分析,使用Trypsin、Chymotrypsin和Glu-C三种酶分别对rhIL-12进行非还原酶解,尽可能地在其所有半胱氨酸残基之间断裂而形成二硫键相连的肽段,然后使用LC-MS/MS对酶解后的肽段样品进行分析,确定了rhIL-12样品中存在和理论配对方式相符的7对二硫键。将rhIL-12二硫键还原后并烷基化修饰保护,分别采用Trypsin,Chymotrypsin和GluC进行酶解,并用LC-MS/MS对酶解后肽段进行了质谱肽图及C端氨基酸序列分析,确定了rhIL-12 p35亚基C端氨基酸序列的8个氨基酸、p40亚基C端氨基酸序列的15个氨基酸。对rhIL-12样品还原及烷基化后用Trypsin变性酶解,所得肽段在H₂O及H₂¹⁸O水中分别用PNGase F糖苷酶处理酶切产物。并通过二级质谱分析脱糖后糖肽段分子量变化,从而确定了rhIL-12的3个N糖基化修饰位点,分别为p35亚基的71位和85位以及p40亚基的200位。通过建立酶解结合二级质谱鉴定的方法,证明了新药rhIL-12的二硫键位点、C端氨基酸序列和糖基化位点与理论一致。     

拟南芥细胞分裂素糖基转移酶UGT76C2的N端亮氨酸替换对酶活性的影响

UGT76C2是负责细胞分裂素N-糖基化修饰的糖基转移酶,该基因对于维持植物体内细胞分裂素动态平衡有重要作用。为了进一步研究UGT76C2酶蛋白结构与催化活性的关系,本文采用定点突变方法,将UGT76C2的N端第31位的保守亮氨酸替换为组氨酸。结果发现,突变型UGT76C2在离体实验中完全丧失了对细胞分裂素的糖基化修饰活性,该突变基因的过表达转基因植物出现与UGT76C2突变体类似的表型,转基因植物体内的两类主要细胞分裂素的N-糖苷含量显著降低。实验结果证明了UGT76C2 N端亮氨酸残基对于糖基化修饰活性的重要性。     

人胸腺素α1在大肠杆菌中的融合表达

利用基因工程表达的方法,在大肠杆菌中通过与GST蛋白融合的方式高效表达了胸腺素α1前体基因,随后经亲和层析和SP强阳离子树脂纯化相结合的方式,得到了胸腺素前体肽段31肽和N端未经乙酰化修饰的28肽。融合蛋白表达量达到菌体总蛋白的35%～40%,样品肽的产量也达到了约200mg/L（肽/发酵液）的产量。经质谱测定,分子量分别为3366和3066。BalB/C小鼠脾脏淋巴细胞体外测活表明,所构建的GSTTα1融合蛋白和纯化后的产物对于淋巴细胞具有比较明显的增殖作用,其中N端未经乙酰化的28肽产物与31肽产物活性相近,均对淋巴细胞具有明显的刺激增殖作用。     

Role of hydroxyprolines in the in vitro oxidative folding and biological activity of conotoxins

Hydroxylation of proline residue occurs in specific peptides and proteins derived from plants and animals, but the functional role of this modification has been characterized primarily in collagen. Marine cone snails produce disulfide-rich peptides that have undergone a plethora of posttranslational modifications, including proline hydroxylation. Although Conus snails extensively utilize proline hydroxylation, the consequences of this modification remain largely unexplored. In this work, we investigated the function of 4-hydroxyproline (Hyp) in conotoxins from three distinct gene families: mu-, omega-, and alpha-conotoxins. Analogues of mu-GIIIA, omega-MVIIC, alpha-GI, and alpha-ImI were synthesized with either Pro or Hyp, and their in vitro oxidative folding and biological activity were characterized. For GIIIA, which naturally contains three Hyp residues, the modifications improved the ability to block NaV1.4 sodium channels but did not affect folding. In contrast, the presence of Hyp in MVIIC had a significant impact on the oxidative folding but not on the biological activity. The folding yields for the MVIIC[Pro7Hyp] analogue were approximately 2-fold higher than for MVIIC under a variety of optimized oxidation conditions. For alpha-conotoxins ImI and GI, the hydroxylation of the conserved Pro residue improved their folding but impaired their activities against target receptors. Since prolyl-4-hydroxylase and protein disulfide isomerase coexist as a heterotetramer in the ER, we discuss the effects of Hyp on the folding of conotoxins in the context of cis-trans isomerization of Pro and Hyp. Taken together, our data suggest that proline hydroxylation is important for both in vitro oxidative folding and the bioactivity of conotoxins.     

A novel conotoxin from Conus delessertii with posttranslationally modified lysine residues

A major peptide, de13a from the crude venom of Conus delessertii collected in the Yucatan Channel, Mexico, was purified. The peptide had a high content of posttranslationally modified amino acids, including 6-bromotryptophan and a nonstandard amino acid that proved to be 5-hydroxylysine. This is the first report of 5-hydroxylysine residues in conotoxins. The sequence analysis, together with cDNA cloning and a mass determination (monoisotopic mass of 3486.76 Da), established that the mature toxin has the sequence DCOTSCOTTCANGWECCKGYOCVNKACSGCTH, where O is 4-hydroxyproline, W 6-bromotryptophan, and K 5-hydroxylysine, the asterisk represents the amidated C-terminus, and the calculated monoisotopic mass is 3487.09 Da. The eight Cys residues are arranged in a pattern (C-C-C-CC-C-C-C) not described previously in conotoxins. This arrangement, for which we propose the designation of framework #13 or XIII, differs from the ones (C-C-CC-CC-C-C and C-C-C-C-CC-C-C) present in other conotoxins which also contain eight Cys residues. This peptide thus defines a novel class of conotoxins, with a new posttranslational modification not previously found in other Conus peptide families.     

Oxidative folding of conotoxins sharing an identical disulfide bridging framework

Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.     

Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.     

Isolation and characterization of the bovine hypothalamic corticotropin-releasing factor

A 41 amino acid peptide with high intrinsic corticotropin-releasing activity was isolated from 1000 bovine hypothalami by means of immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The primary structure of the amino terminal 39 amino acids was characterized by gas phase sequence analysis. The sequence of the amidated carboxyl terminal dipeptide was established by digestion of the intact natural product with Staphylococcus aureus V8 protease, dansylation of the digest and comparative reverse phase liquid chromatography studies with the synthetic dansylated dipeptides Ile-Ala-NH2, Ile-Ala-OH, Ala-Ile-NH2 and Ala-Ile-OH. The complete structure of the bovine corticotropin-releasing factor was established as: Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu- Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Asn-Asn-Arg-Lys-Leu- Leu- Asp-Ile-Ala-NH2 using approximately 650 pmol of material.     

Phospholamban: dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms

Structural characterization of a 40 amino acid peptide with high intrinsic growth hormone releasing activity isolated from a human pancreatic tumor which had caused acromegaly was accomplished by gas phase sequence analyses of the intact peptide and its carboxy terminal cyanogen bromide digestion fragment. High pressure liquid chromatography of the native peptide and synthetic replicates showed that the molecule possessed a free acid rather than an amidated carboxy terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly- Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-OH using 1.8 nmoles of material. The structural identity of this material with a previously characterized fragment of a larger growth hormone releasing peptide isolated from a different human tumor is discussed.     

Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus

The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.     

从织锦芋螺中克隆α芋螺毒素序列

为了从我国南海产织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中分离新的毒素序列并研究其应用价值,进行了织锦芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍＲＮＡ,以Ａ族芋螺毒素的信号肽编码部分和３′端非翻译部分的保守序列为引物,通过ＲＴ－ＰＣＲ扩增和序列分析方法获得新的芋螺毒素序列．结果得到两种不同的α芋螺毒素序列,两者都属于α４／７亚型芋螺毒素,预测其成熟肽序列分别为Ｐｒｏ－Ｇｌｕ－Ｃｙｓ－Ｃｙｓ－Ｓｅｒ－Ａｓｐ－Ｐｒｏ－Ａｒｇ－Ｃｙｓ－Ａｓｎ－Ｓｅｒ－Ｓｅｒ－Ｈｉｓ－Ｐｒｏ－Ｇｌｕ－Ｌｅｕ－Ｃｙｓ－Ｇｌｙ（Ｃ端Ｇｌｙ可能被酰胺化）和Ｐｒｏ－Ｇｌｕ－Ｃｙｓ－Ｃｙｓ－Ｓｅｒ－Ｈｉｓ－Ｐｒｏ－Ａｌａ－Ｃｙｓ－Ａｓｎ－Ｖａｌ－Ａｓｐ－Ｈｉｓ－Ｐｒｏ－Ｇｌｕ－Ｉｌｅ－Ｃｙｓ－Ａｒｇ．采用传统的生化分离手段尚未从织锦芋螺中获得过α芋螺毒素序列,这两种α芋螺毒素作用的种属特异性、受体类型特异性和在小细胞肺癌的诊断和治疗中的应用价值有待进一步研究     

The activity of prolactin releasing peptide correlates with its helicity

The prolactin releasing peptide (PrRP) is involved in regulating food intake and body weight homeostasis, but molecular details on the activation of the PrRP receptor remain unclear. C‐terminal segments of PrRP with 20 (PrRP20) and 13 (PrRP8‐20) amino acids, respectively, have been suggested to be fully active. The data presented herein indicate this is true for the wildtype receptor only; a 5‐10‐fold loss of activity was found for PrRP8‐20 compared to PrRP20 at two extracellular loop mutants of the receptor. To gain insight into the secondary structure of PrRP, we used CD spectroscopy performed in TFE and SDS. Additionally, previously reported NMR data, combined with ROSETTA NMR, were employed to determine the structure of amidated PrRP20. The structural ensemble agrees with the spectroscopic data for the full‐length peptide, which exists in an equilibrium between α‐ and 3₁₀‐helix. We demonstrate that PrRP8‐20's reduced propensity to form an α‐helix correlates with its reduced biological activity on mutant receptors. Further, distinct amino acid replacements in PrRP significantly decrease affinity and activity but have no influence on the secondary structure of the peptide. We conclude that formation of a primarily α‐helical C‐terminal region of PrRP is critical for receptor activation. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 273–281, 2013.     

A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.