rhsBLyS及其突变体对小鼠血清免疫球蛋白分泌及B细胞增殖分化的影响

为了进一步研究所构建的人可溶性B淋巴细胞刺激因子 (hsBLyS)及其突变体hsBY A(Cys14 6→Ala14 6 )和hsBY V(Cys14 6→Val14 6 )在体内的生物活性 ,将 3种重组蛋白注射入BALb C小鼠体内 ,进而分析重组蛋白对小鼠血清免疫球蛋白分泌和脾脏B细胞增殖分化的影响 ,并对 3种蛋白的体内生物活性进行了比较 .结果表明 ,3种蛋白均能促进小鼠血清总IgG和IgM的分泌明显增加 ,统计学检验显示突变体rhsBY V诱导这 2种免疫球蛋白分泌的能力最强 ;3种蛋白还诱导小鼠脾脏增大、脾脏成熟B细胞的相对数量增加 .该高活性突变体hsBY V的获得提示 ,Cys14 6并非BLyS发挥正常功能所必需 ,将Cys14 6突变成Val14 6有可能提高BLyS的生物活性 .     

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity.

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.     

Construction and function of two Cys146-mutants with high activity, derived from recombinant human soluble B lymphocyte stimulator

B lymphocyte stimulator (BLyS) is a novel member of tumor necrosis factor (TNF) ligand family that is important in B cell maturation and survival. Previous studies were almost related to the function or mechanism of its wild type. Here, we constructed two site-directed mutants of the recombinant human soluble BLyS, the BY-A and BY-V, and found that BY-V ranked the highest whenever in the process of promoting proliferation of B lymphocytes in vitro or stimulating total serum IgG and IgM secretion in vivo. Besides, assays for the biological responses of human leukemic cell lines to BLyS, BY-A and BY-V demonstrated that they could suppress the proliferation of Raji cells but promote the growth of THP-1. The discovery of BY-V with high activity will help come to a conclusion that the mutation of Cys146 to Val146 might improve the biological activity of BLyS.     

Nucleotide excision repair by mutant xeroderma pigmentosum group A (XPA) proteins with deficiency in interaction with RPA

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.     

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope

The DNA encoding soluble B lymphocyte stimulator (134–285 amino acids, sBLyS) mutant with residues 217–224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5′-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5α after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.     

GDNF缺失突变体的设计及其结构与功能关系(英文)

研究了GDNF结构与功能的关系 .基于鼠源GDNF的晶体结构 ,利用计算机SGIIndigo2(R4 4 0 0 )工作站和InsightⅡ (95.5)蛋白质分析软件模拟了人GDNF三维结构 ,设计了GDNF分子的两个缺失突变体ΔN1 2 8和ΔN78 90 .以野生型GDNFcDNA作为模板 ,用PCR法得到编码缺失突变体的DNA片段 .将大肠杆菌作为表达系统 ,使缺失突变体GDNF在大肠杆菌中表达 ,对表达产物纯化和复性后进行生物活性测定 .两株突变体在大肠肝菌中获得了高效表达 ,纯化后的GDNF突变体ΔN1 2 8可以与存在于KG 1a细胞表面的受体结合 ,但不能促进 8日龄鸡胚背根节突起的生长 .突变体ΔN78 90既不能与受体结合 ,同时也失去了促背根节突起生长的功能 .说明GDNF分子的N端氨基酸对分子的生物学活性很重要 ,但对分子与受体GDNFR α的结合并不是必需的 ,而分子中的螺旋区对分子与受体的结合以及生物学活性都必不可少 .     

Deletion mutants of human granulocyte-macrophage colony-stimulating factor

To study the structure-function relationship of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), genes were constructed that encode its three deletion mutants: D1, a mutant with the deletion of six amino acid residues (37-42) some of which are a part of a beta-structural region; D2, a mutant with the deletion of the unstructured six-aa sequence of a loop (45-50); and D3, a mutant with the deletion of 14 aa residues (37-50) corresponding to the A-B loop and encoded by the second exon of the gmcsf gene. The expression products of these genes in E. coli were accumulated in a fraction of insoluble proteins. The secondary structures of the mutant proteins were similar to that of the full-size GM-CSF, but the biological activity of the deletion mutants was 130 times lower than that of the GM-CSF: they stimulated the proliferation of the TF-1 cell line at 3 ng/ml concentration. The resulting proteins displayed antagonistic properties toward the full-size GM-CSF, with the inhibition degree of its colony-stimulating activity being 27%. A decrease in the mutant activity in the row D2 > D1 > D3 implies the importance of the conserved hydrophobic residues involved in the formation of the beta-structure for the formation of the GM-CSF functional conformation.     

Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin.

Structural requirements for the activation of transducin by rhodopsin have been studied by site-specific mutagenesis of bovine rhodopsin. A variety of single amino acid replacements and amino acid insertions and deletions of varying sizes were carried out in the two cytoplasmic loops CD (amino acids 134-151) and EF (amino acids 231-252). Except for deletion mutant delta 137-150, all the mutants bound 11-cis-retinal and displayed normal spectral characteristics. Deletion mutant delta 236-239 in loop EF caused a 50% reduction of transducin activation, whereas deletion mutant delta 244-249 and the larger deletions in loop EF abolished transducin activation. An 8-amino acid deletion in the cytoplasmic loop CD as well as a replacement of 13 amino acids with an unrelated sequence showed no transducin activation. Several single amino acid substitutions also caused significant reduction in transducin activation. The conserved charged pair Glu-134/Arg-135 in the cytoplasmic loop CD was required for transducin activation; its reversal or neutralization abolished transducin activation. Three amino acid replacements in loop EF (S240A, T243V, and K248L) resulted in significant reduction in transducin activation. We conclude that 1) both the cytoplasmic loops CD and EF are required for transducin activation, and 2) effective functional interaction between rhodopsin and transducin involves relatively large peptide sequences in the cytoplasmic loops.     

Effects of site-specific mutations on biologic activities of recombinant human IL-6

To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.     

Remarkably simple sequence requirement of the M-factor pheromone of Schizosaccharomyces pombe

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMC(Far)-OCH(3)) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.     

Influence of acylation sites of influenza B virus hemagglutinin on fusion pore formation and dilation

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.     

Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading

The human maintenance DNA (cytosine-5) methyltransferase (hDNMT1) consists of a large N-terminal regulatory domain fused to a catalytic C-terminal domain by randomly repeated Gly-Lys dipeptides. Several N-terminal deletion mutants of hDNMT1 were made, purified, and tested for substrate specificity. Deletion mutants lacking 121, 501, 540, or 580 amino acids from the N-terminus still functioned as DNA methyltransferases, methylated CG sequences, and preferred hemimethylated to unmethylated DNA, as did the full-length hDNMT1. Methylated DNA stimulated methylation spreading on unmethylated CpG sequences for the full-length and the 121 amino acid deletion hDNMT1 equally well but not for the mutants lacking 501, 540, or 580 amino acids, indicating the presence of an allosteric activation determinant between amino acids 121 and 501. Peptides from the N- and C-termini bound methylated DNA independently. Point mutation analysis within the allosteric region revealed that amino acids 284-287 (KKHR) were involved in methylated DNA-mediated allosteric activation. Allosteric activation was reduced in the double point mutant enzymes D25 (K284A and K285A) and D12 (H286A and R287A). Retinoblastoma gene product (Rb), a negative regulator of DNA methylation, bound to the allosteric site of hDNMT1 and inhibited methylation, suggesting Rb may regulate methylation spreading.     

Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants.

To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.     

Substituted cysteine accessibility of the third transmembrane domain of the creatine transporter: defining a transport pathway

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining approximately 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an alpha-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321-28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl- -dependent neurotransmitter transporters.     

Inhibition of cytosolic phospholipase A2 by annexin I. Specific interaction model and mapping of the interaction site

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA(2). "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA(2) inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism of cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activity was determined. The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA(2) and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA(2). ANX-I-(1-274) did not interact with cPLA(2). Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2). These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.     

Differential determinants for peptide and non-peptidyl ligand binding to the motilin receptor. Critical role of second extracellular loop for peptide binding and action.

The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518-35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs DeltaCys(235), Delta179-182, and Delta241-246. Cys(235) is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val(179) and Leu(245) and Arg(246), were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand.     

Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis

Anthrax toxin is a complex of protective antigen (PA, 735 aa), lethal factor (LF, 776 aa), and edema factor (EF, 767 aa). PA binds to cell surface receptors and is cleaved by cell surface proteases into PA63, while LF and EF compete for binding to PA63. The PA63-LF/EF complex is internalized into the cytosol and causes different pathogenic responses in animals and cultured cells. 1-300 amino acid residues of LF have been viewed as the region responsible for the high affinity binding of LF to PA. Amino acid analysis of LF and EF revealed a common stretch of 7 amino acids (147VYYEIGK153). In the present study, each amino acid of this stretch was replaced by alanine at a time. Y148A, Y149A, I151A, and K153A mutants were found to be deficient in their ability to lyse J774A.1 cells and their binding ability to PA63 was drastically reduced. We propose that these four amino acids play a crucial role in the process of binding of LF to PA63.