▽5-人粒系集落刺激因子(▽5-hG-CSF)的cDNA克隆、序列测定与表达

从LPS刺激的正常中国人外周血单核细胞中提取mRNA,经反转录(RT)-PCR扩增出不含信号肽和成熟型N端5氨基酸(V5-hG-CSF)的cDNA片段,酶切后组入大肠杆菌表达载体pJLA602中。序列测定表明,克隆片段与国外报道的高活性hG-CSF cDNA序列一致。重组子经诱导表达、小鼠骨髓细胞体外CFU-G测试表明,表达产物具明显的粒细胞集落刺激活性。     

Tryptophan-216 is essential for the transglycosylation activity of endo-beta-N-acetylglucosaminidase A

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has a high level of transglycosylation activity. To determine which amino acids are involved in this activity, we employed deletion analysis, as well as random and site-directed mutagenesis. Using PCR random mutagenesis, 11 mutants with greatly decreased levels of enzyme activity were isolated. Six catalytically essential amino acids were identified by site-directed mutagenesis. Mutants E173G, E175Q, D206G, and D270N had markedly reduced hydrolysis activity, while mutants V109D, E173D, and E173Q lost all enzymatic activity, indicating that Val-109 and Glu-173 are important for the catalytic function. Moreover, we isolated a random mutation that abolished the transglycosylation activity without affecting the hydrolysis activity. The Trp-216 to Arg mutation was identified, by site-directed mutagenesis, as that responsible for the loss of transglycosylation activity. While other mutants of Trp-216 showed reduced activity, mutation to another positively charged residue (Lys) also abolished the transglycosylation activity. Sequence comparison with two other endo-beta-N-acetylglucosaminidases, that possess transglycosylation activity and that have been cloned recently, reveals a high degree of identity in the N-terminal regions of the three enzymes. These results indicate that the tryptophan residue at position 216 of Endo-A has a key role in the transglycosylation.     

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.     

Site-specific mutagenesis of the human interleukin-1 beta gene: the role of arginine residue at the N-terminal region

Using the expression system for site-specific mutagenesis in Escherichia coli, we have made deletion mutants at the N-terminal or C-terminal region of human interleukin-1 beta (IL-1 beta) consisting of 153 amino acids. The truncated mutants showed that at least 147 amino acids (numbers 4-150) in IL-1 beta are necessary for the exertion of biological activity. When we changed the arginine at the 4th position (Arg4) in IL-1 beta to other specific amino acids, there was a marked difference in the relative extent of biological and receptor binding activities among the mutants. The order of the mutants was Arg4 = Lys4 greater than Gln4 greater than Gly4 = des-Arg4 greater than Asp4. Our results demonstrate that the arginine residue at the 4th position in IL-1 beta is important, but not essential, for IL-1 beta to exhibit its biological and receptor binding activities, and the positive charge at this site plays a key role for IL-1 beta to exert the activities.     

Mutational analysis of conserved residues in HhaI DNA methyltransferase

HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase.     

Secretory production of human granulocyte colony-stimulating factor in Escherichia coli.

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein, consisting of 174 amino acids, which plays an important role in hematopoietic cell proliferation, differentiation of hemopoietic precursor cells, and activation of mature neutrophilic granulocytes. In this study, secretory production of hG-CSF in the periplasmic space of Escherichia coli using the Bacillus sp. endoxylanase signal peptide was examined. For the efficient expression of hG-CSF gene, the first five codons at the N-terminal were altered based on the E. coli high-frequency codon database. The hG-CSF gene fused to the endoxylanase signal sequence was expressed using an inducible trc promoter. However, recombinant E. coli cells were completely lysed after induction with 1 mM isopropyl-beta-D-thiogalactopyranoside. Insertion of a small oligopeptide (13 amino acids) containing the histidine hexamer and factor Xa cleavage site between the signal peptide and the mature hG-CSF protein allowed successful secretion of hG-CSF into the periplasm without cell lysis. Among the several E. coli strains examined, E. coli BL21(DE3) and E. coli MC4100 allowed production of hG-CSF to the highest levels (20-22% of total proteins) with the secretion efficiencies greater than 98%. The circular dichroism spectra showed that the conformation of purified hG-CSF is almost identical to native hG-CSF.     

Identification of an N-Terminal Region of Sigma 54 Required for Enhancer Responsiveness

Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.     

An oligomeric form of simian virus 40 large T-antigen is immunologically related to the cellular tumor antigen p53.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.     

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.     

Mutational analysis of the herpes simplex virus triplex protein VP19C

Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal approximately 100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.     

Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants.

We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity. Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity. These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity. We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.     

Essential residues in the chromate transporter ChrA of Pseudomonas aeruginosa

The chrA gene of Pseudomonas aeruginosa plasmid pUM505 encodes the hydrophobic protein ChrA, which confers resistance to chromate by the energy-dependent efflux of chromate ions. Chromate-sensitive mutants were isolated by in vivo random mutagenesis. Transport experiments with cell suspensions of selected mutants showed that 51CrO4(2-) extrusion was drastically lowered as compared to suspensions of the strain with the wild-type plasmid, confirming that the mutations affected a chromate efflux system. DNA sequence analysis showed that most point mutations affected amino acids clustered in the N-terminal half of ChrA, altering either cytoplasmic regions or transmembrane segments, and replaced residues moderately to highly conserved in ChrA homologs. PhoA and LacZ translational fusions were used to confirm the membrane topology at the N-terminal half of the ChrA protein.     

Molybdopterin dinucleotide biosynthesis in Escherichia coli: identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides

The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.     

Characterization of the DNA binding properties of polyomavirus capsid protein.

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.     

Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis

The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD_W-135 and SiaD_Y, differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD_W-135 and SiaD_Y were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.     

Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues

Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1-, VirD2-dependent border-nicking activity in Escherichia coli harbouring a border-containing substrate plasmid. Analysis of the mutants led to the identification of a region from amino acids 45–60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA-binding domain. Deletion mutagenesis indicated that amino acids 2–16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5–27, completely inactivated VirD1.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.     

Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus.

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.