维生素K的羧化反应与钙化作用

自从阐明维生素K有促进血浆中一些凝血因子组成结构中N末端谷氨酸残基的γ碳发生羧化作用以来,又发现某些组织结构蛋白中也有γ羧化谷氨酸残基,也与维生素K有关。由此推论维生素K可能有较广泛的调节钙代谢的作用。此谷氨酸残基的γ碳羧化,同时也是体内CO_2固定的一种方式。     

Ydj1p C端二聚体中螺旋α3与domain Ⅲ分离的拉伸分子动力学模拟

许多遗传性疾病或基因突变疾病实际上与蛋白质错误折叠相关,由于分子伴侣在调控蛋白折叠、聚集、降解方面发挥功能,研究分子伴侣的结构、功能、作用机制等对于人类某些疾病的阐释意义重大。作为分子伴侣,Hsp40以二聚体的形式调控多肽的折叠。文章通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中α3与domainⅢ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。由研究结果可知,α2、α3及α3之前loop结构中的残基L274、I278、F335、P336、F340、L346、L349、L352、L353之间形成疏水盒子,使螺旋α2、α3之间作用紧密,稳固二聚体结构。其中,F335与domainⅢ内的残基V302、I278、I303和P305,P336与domainⅢ内的残基V302形成疏水作用力来维持二聚体的稳定。     

组氨酸在代谢中的作用

组氨酸是含有异吡唑环的氨基酸,是机体蛋白质的构成氨基酸,也是一些功能蛋白质(如组蛋白、血红蛋白)的主要组成氨基酸。组氨酸残基及异吡唑环是一些酶蛋白(如二氢叶酸还原酶、过氧化物歧化酶)和功能蛋白质(如血红蛋白)的功能部位或功能基团。组氨酸是天然螯合剂,许多含锌的金属酶(如羧基肽酶等)其功能性锌原子与活性中心的组氨酸残基相结合;自由组氨酸、由组氨酸构成的小肽以及组氨酸脱去羧基生成的组胺等都具有特殊的生理功能,所以组氨酸在代谢中起重要作用。食物中组氨酸含量影响体内组氨酸水平;药用组氨酸(组氨酸作为药物治疗某些疾病及氨基酸输液等)用量不当也影响体内组氨酸水平,能影响某些代谢,甚至发生疾病。     

Kunitz 型丝氨酸蛋白酶抑制剂结构与功能研究

蛋白酶抑制剂在酶学及蛋白质的结构与功能关系研究中有重要意义,Kunitz型丝氨酸蛋白酶抑制剂是其中最重要的,也是研究最广泛的蛋白酶抑制剂之一．该类蛋白酶抑制剂三维结构高度保守：由一个明显的疏水核心、三对高度保守的二硫键桥、三链β-折叠和一个N端3 10螺旋及一个C端α-螺旋组成．3对二硫键对分子空间结构的稳定起着非常重要的作用．这一类型抑制剂有5个主要的活性位点：P1、P1’、P3、P3’、P4,它们都位于一个溶剂暴露的环上．P1位点是抑制作用的关键活性位点,抑制剂的专一性由P1位点氨基酸残基的性质决定;P1’位点氨基酸残基的侧链大小对抑制剂．酶的结合常数有很大影响,用大的侧链残基取代会导致结合常数降低;P4位点残基被取代经常产生负效应,会导致活性区域环的构象发生很大改变,从而影响酶与抑制剂的结合．     

PR-39,一种多功能的抗菌肽

PR－39（proline-arginine-rich)是哺乳动物体内的一种含有39个氨基酸残基的小分子抗菌肽,因其富含脯氨酸而得名。分子量小（4719．7）,结构稳定,具有广谱抗菌活性。近年来许多实验研究表明,PR－39在抗肿瘤,组织修复等方面同样具有生物学作用。     

组蛋白去甲基化酶JMJD 及其抑制剂的研究进展

组蛋白去甲基化酶JMJD 家族可通过催化去除组蛋白N 末端赖氨酸残基上甲基,参与表观遗传调控,包括基因表达调控,并与某些疾病,特别是癌症密切相关。因此,该酶已成为令人关注的癌症治疗新靶点,其抑制剂也成为药物研究与开发的热点。综述JMJD 与肿瘤的关联、JMJD 的结构和催化机制及其抑制剂的研究进展。     

Val55点突变对鸡胱抑素I66Q结构稳定性影响的分子动力学研究

Val55是鸡胱抑素(Chicken cystatin,cC)铰链环状区的重要位点.本文采用分子动力学模拟的方法研究了V55位点突变对cC典型的淀粉样突变体I66Q结构稳定性的影响情况,并深入探讨了其分子机制.研究表明V55N和V55D对I66Q突变体都有稳定其结构的作用,但V55N的稳定作用更显著.进一步研究发现V55N和V55D对I66Q的这种稳定作用是由于突变后的55位残基与邻近残基形成了较多稳定的氢键,从而增加了自身位点及Loop1、β2 - β3的稳定性,并进一步稳定了I66Q的α-螺旋和疏水核心结构.这可能最终阻碍胱抑素淀粉样突变体I66Q结构域交换的发生.     

香菇多糖硫酸化衍生物的制备及其结构分析

采用改良的Ｗｏｌｆｒｏｍ方法制备了一系列的硫酸化香菇多糖衍生物。硫酸基含量测定结果表明,硫酸基的取代程序受反应时间和酯化试剂中氯磺酸与吡啶的比例的控制;证明甲基化分析方法不适合硫酸化香菇多糖衍生物的结构分析,＾１３Ｃ－ＮＭＲ数据表明,硫酸基取供在香菇多糖中Ｃ－６上,表明Ｃ－６位羟基的反应活性高于其他位置的羟基。     

Kazal型蛋白酶抑制剂结构与功能研究进展

蛋白酶抑制剂广泛存在于生物体内,在许多生命活动过程中发挥必不可少的作用,特别是对蛋白酶活性进行精确调控。其中Kazal型蛋白酶抑制剂是最重要的、研究最为广泛的酶抑制剂之一,该类抑制剂一般由一个或几个结构域组成,每一个结构域具有保守的序列和分子构象,同时发现该类抑制剂与蛋白酶作用的结合部位高度易变,它们大多数暴露于与溶剂接触的环上,其中P1部位是抑制作用的关键部位,抑制剂的专一性由P1部位氨基酸残基的性质决定,其它残基取代结合部位残基对抑制剂-酶的结合常数有显著的影响。Laskowski算法可直接从Kazal型丝氨酸蛋白酶抑制剂的序列推测其与6种丝氨酸蛋白酶之间的抑制常数(Ki)。目前在生物体内发现大量的Kazal型蛋白酶抑制剂,并证实其有重要的生物学功能。     

组蛋白修饰与基因调控

基因表达是一个受多因素调控的复杂过程,组蛋白是染色体基本结构－核小体中的重要组成部分,其N－末端氨基酸残基可发生乙酰化、甲基化、磷酸化、泛素化、多聚ADP糖基化等多种共价修饰作用,组蛋白的修饰可通过影响组蛋白与DNA双链的亲性,从而改变染色质的疏松或凝集状态,或通过影响其它转录因子与结构基因启动子的亲和性来发挥基因调控作用,组蛋白修饰对基因表达的调控有类似DNA遗传密码的调控作用。     

Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation

Cell lines, selected from two independent clones of an established mouse embryo cell line by their ability to grow as solid tumors in immunocompetent syngeneic hosts, were found to have the same alteration in anion exchange properties as was previously reported for simian virus 40 (SV40)-transformed subclones. One tumor cell line (219CT) and one SV40-transformed subclone (215CSC) were selected for further detailed comparison with their common parent clone (210C). Cellulose acetate electrophoresis at pH 1.0 showed that 215CSC heparan sulfate had a slight overall decrease in sulfation compared with heparan sulfate from 210C; however, no gross difference in sulfation could be detected between heparan sulfate from 219CT and 210C. Analysis of the products of deaminative cleavage of heparan sulfate by nitrous acid under conditions where cleavage occurs quantitatively at N-sulfated glucosamine residues showed that, although heparan sulfate from the three cell lines gave similar yields of O-sulfated disaccharides, both 215CSC and 219CT had only about half as many O-sulfate residues in higher molecular weight oligosaccharides compared to heparan sulfate from 210C. Enzymatic degradation of heparan sulfate with a mixture of enzymes from Flavobacterium heparinum showed that this common alteration in heparan sulfate from both 215CSC and 219CT resulted from a 30% decrease in glucosamine residues bearing 6-O-sulfate groups. As this decrease in 6-O-sulfate glucosamine residues occurs in regions of the chain containing relatively few sulfate groups, it is clear that certain sequences of charged groups present in heparan sulfate frm 210C will be found only rarely in heparan sulfate from 215CSC and 219CT. It is suggested that this will result in alterations of the interaction of heparan sulfate with other molecules in the microenvironment at the cell surface which may be important in the control of such phenomena as cell growth and adhesion.     

Structural requirements for heparin/heparan sulfate binding to type V collagen

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.     

Characterization of heparanase from a rat parathyroid cell line

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-beta-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.     

Multi-faceted substrate specificity of heparanase

Heparan sulfate is a highly sulfated polysaccharide abundantly present in the extracellular matrix. Heparan sulfate consists of a disaccharide repeating unit of glucosamine and glucuronic and iduronic acid residues. The functions of heparan sulfate are largely dictated by its size as well as the sulfation patterns. Heparanase is an enzyme that cleaves heparan sulfate polysaccharide into smaller fragments, regulating the functions of heparan sulfate. Understanding the substrate specificity plays a critical role in dissecting the biological functions of heparanase and heparan sulfate. The prevailing view is that heparanase recognizes specific sulfation patterns in heparan sulfate. However, emerging evidence suggests that heparanase is capable of varying its substrate specificities depending on the saccharide structures around the cleavage site. The plastic substrate specificity suggests a complex role of heparanase in regulating the structures of heparan sulfate in matrix biology.     

Activin A Binds to Perlecan through Its Pro-region That Has Heparin/Heparan Sulfate Binding Activity

Activin A, a member of the transforming growth factor-β family, plays important roles in hormonal homeostasis and embryogenesis. In this study, we produced recombinant human activin A and examined its abilities to bind to extracellular matrix proteins. Recombinant activin A expressed in 293-F cells was purified as complexes of mature dimeric activin A with its pro-region. Among a panel of extracellular matrix proteins tested, recombinant activin A bound to perlecan and agrin, but not to laminins, nidogens, collagens I and IV, fibronectin, and nephronectin. The binding of recombinant activin A to perlecan was inhibited by heparin and high concentrations of NaCl and abolished by heparitinase treatment of perlecan, suggesting that activin A binds to the heparan sulfate chains of perlecan. In support of this possibility, recombinant activin A was capable of directly binding to heparin and heparan sulfate chains. Site-directed mutagenesis of recombinant activin A revealed that clusters of basic amino acid residues, Lys²⁵⁹-Lys²⁶³ and Lys²⁷⁰-Lys²⁷², in the pro-region were required for binding to perlecan. Interestingly, deletion of the peptide segment Lys²⁵⁹-Gly²⁷⁷ containing both basic amino acid clusters from the pro-region did not impair the activity of activin A to stimulate Smad-dependent gene expressions, although it completely ablated the perlecan-binding activity. The binding of activin A to basement membrane heparan sulfate proteoglycans through the basic residues in the pro-region was further confirmed by in situ activin A overlay assays using frozen tissue sections. Taken together, the present results indicate that activin A binds to heparan sulfate proteoglycans through its pro-region and thereby regulates its localization within tissues.     

乙酰肝素酶与胃癌发展的关系及其检测

乙酰肝素酶是目前发现的哺乳动物细胞中惟一能切割细胞外基质中硫酸乙酰肝素蛋白多糖侧链——硫酸乙酰肝素的一种葡萄糖醛酸内切酶,在胃癌侵袭转移中起重要作用。我们就乙酰肝素酶的分子结构特点、在胃癌侵袭转移中的作用机制及其检测等方面的研究进展进行综述。     

Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase

Heparan sulfate N-sulfotransferase catalyzes the transfer of sulfate groups from adenosine 3'-phosphate, 5'-phosphosulfate to the free amino groups of glucosamine residues in heparan sulfate. We have identified a Chinese hamster ovary cell mutant, designated pgsE-606, which is 3-5-fold defective in N-sulfotransferase activity. The residual enzyme activity is indistinguishable from the wild-type enzyme with respect to Km values for adenosine 3'-phosphate,5'-phosphosulfate and N-desulfoheparin, pH dependence, Arrhenius activation energy, and thermal lability. The mutation is recessive, and mixing experiments indicate that the mutant does not produce soluble antagonists of N-sulfotransferase. Inspection of the heparan sulfate chains from the mutant showed that the extent of N-sulfation is reduced about 2-3-fold. The addition of sulfate to hydroxyl groups on the chain is reduced to a similar extent, suggesting that N-sulfation and O-sulfation are normally coupled. Nitrous acid fragmentation of the chains showed that N-sulfated glucosamine residues are spaced much less frequently than in heparan sulfate from wild-type cells. The close correlation of enzyme activity to the number and position of N-sulfate groups indicates that N-sulfotransferase plays a pivotal role in determining the extent of sulfation of heparan sulfate.     

Characterization of a heparan sulfate octasaccharide that binds to herpes simplex virus type 1 glycoprotein D

Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate d-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gD-binding site is an octasaccharide, and has a binding affinity to gD around 18 microm, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is DeltaUA-GlcNS-IdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH(2)3S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry.     

A peptide found by phage display discriminates a specific structure of a trisaccharide in heparin

A number of recent studies have shown that heparan sulfate can control several important biological events on the cell surface through changes in sulfation pattern. The in vivo modification of sugar chains with sulfates, however, is complicated, and the discrimination of different sulfation patterns is difficult. Heparin, which is primarily produced by mast cells, is closely approximated by the structural analog heparan sulfate. Screening of heparin-associating peptides using phage display and antithrombin-bound affinity chromatography identified a peptide, heparin-associating peptide Y (HappY), that acts as a target of immobilized heparin. The peptide consists of 12 amino acid residues with characteristic three arginines and exclusively binds to heparin and heparan sulfate but does not associate with other glycosaminoglycans. HappY recognizes three consecutive monosaccharide residues in heparin through its three arginine residues. HappY should be a useful probe to detect heparin and heparan sulfate in studies of glycobiology.     

Transport of heparan sulfate into the nuclei of hepatocytes

Monolayer cultures of a rat hepatocyte cell line shown previously to accumulate a nuclear pool of free heparan sulfate chains that are enriched in sulfated glucuronic acid (GlcA) residues (Fedarko, N.S., and Conrad, H.E., (1986) J. Cell Biol. 587-599) were incubated with 35SO4(2-), and the rate of appearance of heparan [35S]sulfate in the nuclei was measured. Heparan [35S]sulfate began to accumulate in the nuclei 2 h after the administration of 35SO4(2-) to the cells and reached a steady state level after 20 h. Heparan [35S]sulfate was lost from the nuclei of prelabeled cells with a t1/2 of 8 h. Chloroquine did not inhibit the transport of heparan sulfate into the nucleus, but increased the t1/2 for the exit of heparan sulfate from the nucleus to 20 h and led to a doubling of the steady state level of nuclear heparan sulfate. Heparan [35S]sulfate which was obtained from the medium or from the cell matrix of a labeled culture and which contained only low levels of GlcA-2-SO4 residues was incubated with cultures of unlabeled cells, and the uptake of the exogenous heparan [35S]sulfate was studied. At 37 degrees C the cells took up proteoheparan [35S]sulfate and transported about 10% of the internalized heparan [35S]sulfate into the nucleus, where it appeared as free chains. The heparan [35S]sulfate isolated from the nucleus was enriched in GlcA-2-SO4 residues, whereas the heparan [35S]sulfate remaining in the rest of the intracellular pool showed a corresponding depletion in GlcA-2-SO4 residues. At 16 degrees C, where endocytosed materials do not enter the lysosomes, the cells also transported exogenous proteoheparan [35S]sulfate to the nucleus with similar processing. Thus, the metabolism of exogenous heparan sulfate by hepatocytes follows the same pathway observed in continuously labeled cells and does not involve lysosomal processing of the internalized heparan sulfate.