A 10-kDa class-CI sHsp protects<Emphasis Type="Italic"> E. coli</Emphasis> from oxidative and high-temperature stress

We report on a new cDNA clone (Qshsp10.4-CI) of a Quercus suber L. class-CI small heat-shock protein (sHsp) obtained from cork (phellem), a highly oxidatively stressed plant tissue. The deduced gene product lacks the C-terminal extension and the consensus I region of the alpha-crystallin domain, being the most C-terminally truncated sHsp reported to date. In an attempt to prove that a protective function is possible for such a truncated sHsp, we overexpressed in Escherichia coli three recombinant sHsp-CIs, one (rQsHsp10.4-CI) showing the same truncation as Qshsp10.4-CI, a second (rN49) lacking the whole alpha-crystallin domain, and a third (rN153) consisting of a full-length sHsp-CI. The overexpression of rN153 and, remarkably, rQsHsp10.4-CI but not rN49 enhanced cell viability under high temperature and, interestingly, under oxidative stress. These results show that the C-terminal extension and the consensus I region of the alpha-crystallin domain are dispensable, but amino acids 1-41 of the alpha-crystallin domain (including the consensus II region) are essential for the protective activity of sHsp-CIs. On the other hand, two-dimensional immunodetection patterns showed accumulation of ca. 10-kDa sHsp-CI immunorelated polypeptides in cork and other oxidatively stressed tissues but not in control and heat-stressed tissues. We discuss the possible role of highly truncated sHsps in relation to oxidative stress.     

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.     

Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23

The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.     

Role of N- and C-terminal domains and non-homologous region in co-refolding of Thermotoga maritima β-glucosidase

Thermotoga maritima β-glucosidase consists of three structural regions with 721 amino acids: the N-terminal domain, middle non-homologous region and a C-terminal domain. To investigate the role of these domains in the co-refolding of two fragments into catalytically active form, five sites coding the amino acid residue at 244, 331 in the N-terminal domain, 403 in the non-homologous region, 476 and 521 in the C-terminal domain were selected to split the gene. All the 10 resultant individual fragments were obtained as insoluble inclusion bodies and found to be catalytically inactive. However, the catalytic activity was recovered when the two fragments derived from N-terminal and C-terminal peptides were co-refolded together. It is quite interesting to find that not only the complement polypeptides such as N476/477C but also the truncated combination (N476/522C, amino acid residues from 477 to 521 is truncated) and overlapped combination (N476/245C and N476/404C, amino acid residues from 245 to 476 and from 404 to 476 are overlapped) also gave catalytically active enzymes. Our results showed that folding motifs consisted of the complete N-terminal domain play an important role in the co-refolding of the polypeptides into the catalytically active form.     

Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase

Glucoamylase (GA) from Aspergillus awamori (EC 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1-440), a starch-binding domain (aa 513-616), and a highly O-glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains. We have genetically engineered a series of truncated forms of GA to determine how much of the highly O-glycosylated region is necessary for the activity or stability of GAII, a fully active form of the enzyme that lacks the starch-binding domain. Mutations were made by inserting stop-codon linkers into restriction sites within the coding region of the GA gene, and mutated genes were expressed in Saccharomyces cerevisiae for analysis of the truncated enzymes. Our results show that up to 30 aa from the C-terminal end of GAII can be deleted with little effect on the activity, thermal stability, or secretion of the enzyme. Further deletions resulted in diminution or loss of enzyme activity on starch plates, and loss of detectable enzyme in culture supernatants, indicating that these residues are essential for GAII function.     

Characterization of telomere-binding activity of replication factor C large subunit p140.

The large subunit of RFC (RFC p140) has been suggested to be associated with the 3'-end of elongating DNA primer and to recruit proliferating cell nuclear antigen (PCNA) onto DNA polymerase delta. Previously, we isolated a cDNA clone encoding a DNA-binding domain of RFC p140 as a telomeric repeat (TTAGGG)n binding protein. This domain was shown to have a specific affinity for the 5'-phosphate ends of a telomere repeat sequence. In order to investigate the structure and function of RFC p140, we constructed the full-length recombinant RFC p140 as well as N- and/or C-terminal deleted mutants and analyzed their telomere-binding activities. South-Western blot and gel mobility shift analyses revealed that deletion of the N- but not the C-terminal region enhances recognition of the telomeric repeat sequence and 5'-phosphate ends, suggesting the negative effect of the N-terminal region of the RFC p140 binding to the telomeric repeat. On the other hand, the C-terminal truncated RFC inhibits the telomerase activity more than the N-terminal-deleted and full-length RFC p140. The inhibitory effect of RFC p140 on telomerase activity is completely diminished by both terminal deletions. Thus, a certain interaction of the N- and C-terminal regions is considered to be required for RFC p140 to suppress telomerase activity. Taken together, these results suggest that both telomeric repeat-binding and telomerase inhibitory activities of RFC p140 are finely regulated by the intrinsic N- and C-terminal regions.     

Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB

LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal α-helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp²⁵) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.     

An unusual class of PITX2 mutations in Axenfeld-Rieger syndrome

BACKGROUND: Mutations in the PITX2 homeobox gene are known to contribute to Axenfeld-Rieger syndrome (ARS), an autosomal-dominant developmental disorder. Although most mutations are in the homeodomain and result in a loss of function, there is a growing subset in the C-terminal domain that has not yet been characterized. These mutations are of particular interest because the C-terminus has both inhibitory and stimulatory activities. METHODS: In this study we used a combination of in vitro DNA binding and transfection reporter assays to investigate the fundamental issue of whether C-terminal mutations result in gain or loss of function at a cellular level. RESULTS: We report a new frameshift mutation in the PITX2 allele that predicts a truncated protein lacking most of the C-terminal domain (D122FS). This newly reported mutant and another ARS C-terminal mutant (W133Stop) both have greater binding than wild-type to the bicoid element. Of interest, the mutants yielded approximately 5-fold greater activation of the prolactin promoter in CHO cells, even though the truncated proteins were expressed at lower levels than the wild-type protein. The truncated proteins also had greater than wild-type activity in 2 other cell lines, including the LS8 oral epithelial line that expresses the endogenous Pitx2 gene. CONCLUSIONS: The results indicate that the PITX2 C-terminal domain has inhibitory activity and support the notion that ARS may also be caused by gain-of-function mutations.     

The N-terminal ATPase AT-hook-containing region of the Arabidopsis chromatin-remodeling protein SPLAYED is sufficient for biological activity

The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants.     

Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G.

We constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middle-size tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.     

Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis

Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20,000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.     

Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species

Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.     

Molecular cloning and characterization of cDNA coding for beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum.

In plants as well as in animals beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from oligomannosidic N-glycans to complex or hybrid type N-linked oligosaccharides. Employing degenerated primers deduced from known GlcNAc-TI genes from animals, we were able to identify the cDNA coding for GlcNAc-TI from a Nicotiana tabacum cDNA library. The complete nucleotide sequence revealed a 1338 base pair open reading frame that codes for a polypeptide of 446 amino acids. Comparison of the deduced amino acid sequence with that of already known GlcNAc-TI polypeptides revealed no similarity of the tobacco clone within the putative cytoplasmatic, transmembrane, and stem regions. However, 40% sequence similarity was found within the putative C-terminal catalytic domain containing conserved single amino acids and peptide motifs. The predicted domain structure of the tobacco polypeptide is typical for type II transmembrane proteins and comparable to known GlcNAc-TI from animal species. In order to confirm enzyme activity a truncated form of the protein containing the putative catalytic domain was expressed using a baculovirus/insect cell system. Using pyridylaminated Man(5)- or Man(3)GlcNAc(2)as acceptor substrates and HPLC analysis of the products GlcNAc-TI activity was shown. This demonstrates that the C-terminal region of the protein comprises the catalytic domain. Expression of GlcNAc-TI mRNA in tobacco leaves was detected using RT-PCR. Southern blot analysis gave two hybridization signals of the gene in the amphidiploid genomes of the two investigated species N. tabacum and N.benthamiana.     

Adjuvant activity of GP96 C-terminal domain towards Her2/neu DNA vaccine is fusion direction-dependent

The Her2 is one of tumor-associated antigens (TAA), regarded as an ideal target of immunotherapy. DNA encoding full-length or truncated rat Her2/neu have shown protective and therapeutics potentials against Her2/neu-expressing mammary tumors. However, the efficacy of active vaccination is limited since Her2 is a self-tolerated antigen. Hence, new strategies are required to enhance both the quality and quantity of the immune response against Her2-expressing tumors. Many studies have used Her2/neu gene with cytokine or other molecules involved in regulation of immune response to enhance the potency of Her2/neu DNA vaccines. Some studies fused adjuvant gene to C-terminal domain of Her2/neu gene, while others fused the adjuvant gene N-terminally to Her2/neu gene, but no comparison on how direction of fusion could affect efficiency of DNA vaccine has ever been made. Based on previous reports demonstrating potent adjuvant activity of gp96 C-terminal domain, we chose it as adjuvant. The aim of this study was to investigate if direction of fusion could affect adjuvant activity of gp96 C-terminal domain or potency of Her2/neu DNA vaccination. To do so, we fused C-terminal domain of gp96 to downstream or C-terminal end of transmembrane and extracellular domain (TM+ECD) of rat Her2/neu and resultant immune response to DNA vaccination was evaluated. The results were compared with that of N-terminally fusion of gp96 C-terminal domain to TM+ECD of rat Her2/neu. Our results revealed that adjuvant activity of gp96 C-terminal domain is enhanced when fused N-terminally to TM+ECD of rat Her2/neu. It suggests that adjuvant activity of gp96 C-terminal domain towards Her2/neu is fusion direction-dependent.     

Functional analysis of the C-terminal region of γ-glutamyl kinase of Saccharomyces cerevisiae

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.     

Analysis of a dextran-binding domain of the dextranase of Streptococcus mutans

AIMS: To examine the dextran-binding domain of the dextranase (Dex) of Streptococcus mutans. METHODS AND RESULTS: Deletion mutants of the Dex gene of Strep. mutans were prepared by polymerase chain reaction and expressed in Escherichia coli cells. Binding of the truncated Dexs to dextran was measured with a Sephadex G-150 gel. Although the Dexs which lacked the N-terminal variable region lost enzyme activity, they still retained dextran-binding ability. In addition, further deletion into the conserved region from the N-terminal did not influence the dextran-binding ability. However, the Dex which carried a deletion in the C-terminus still possessed both enzyme activity and dextran-binding ability. Further deletion into the conserved region from the C-terminal resulted in complete disappearance of both enzyme and dextran-binding activities. CONCLUSIONS: Deletion analysis of the Dex gene of Strep. mutans showed that the C-terminal side (about 120 amino acid residues) of the conserved region of the Dex was essential for dextran-binding ability. SIGNIFICANCE AND IMPACT OF THE STUDY: The dextran-binding domain was present in a different area from the catalytic site in the conserved region of the Dex molecule. The amino acid sequence of the dextran-binding domain of the Dex differed from those of glucan-binding regions of other glucan-binding proteins reported.     

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.     

Deletion mutagenesis using an 'M13 splint': the N-terminal structural domain of tyrosyl-tRNA synthetase (B. stearothermophilus) catalyses the formation of tyrosyl adenylate.

The X-ray crystallographic structure of tyrosyl-tRNA synthetase (TyrTS) comprises only the N-terminal 320 amino acids of the molecule as the C-terminal 99 amino acids are poorly ordered in the crystal. A new technique, employing a single-stranded M13 splint, has been used to direct a deletion in the cloned gene of TyrTS so as to remove the disordered C-terminal region. We find that the truncated enzyme catalyses the formation of tyrosyl adenylate with unchanged Kcat and Km values and the crystallographic model must therefore include all the binding and catalytic residues involved in tyrosine activation. However, the truncated enzyme no longer binds tRNATyr or transfers tyrosine to tRNATyr. This indicates that the structural division of TyrTS is equally a functional one: the N-terminal structural domain catalyses tyrosine activation while the disordered C-terminal domain carries major determinants in tRNA binding.     

Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains

ClpB is a member of a protein-disaggregating multi-chaperone system in Escherichia coli. The mechanism of protein-folding reactions mediated by ClpB is currently unknown, and the functional role of different sequence regions in ClpB is under discussion. We have expressed and purified the full-length ClpB and three truncated variants with the N-terminal, C-terminal, and a double N- and C-terminal deletion. We studied the protein concentration-dependent and ATP-induced oligomerization of ClpB, casein-induced activation of ClpB ATPase, and ClpB-assisted reactivation of denatured firefly luciferase. We found that both the N- and C-terminal truncation of ClpB strongly inhibited its chaperone activity. The reasons for such inhibition were different, however, for the N- and C-terminal truncation. Deletion of the C-terminal domain inhibited the self-association of ClpB, which led to decreased affinity for ATP and to decreased ATPase and chaperone activity of the C-terminally truncated variants. In contrast, deletion of the N-terminal domain did not inhibit the self-association of ClpB and its basal ATPase activity but decreased the ability of casein to activate ClpB ATPase. These results indicate that the N-terminal region of ClpB may contain a functionally significant protein-binding site, whereas the main role of the C-terminal region is to support oligomerization of ClpB.