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PubMed2002

Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus.

Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases.

ADP Ribose Transferases↗
PubMed1997

Adhesion of tumour-infiltrating lymphocytes to endothelium: a phenotypic and functional analysis.

Efficacy of cancer immunotherapy with cultured tumour-infiltrating lymphocytes (TILs) depends upon infused TILs migrating into tumour-bearing tissue, in which they mediate an anti-tumour response. For TILs to enter a tumour, they must first bind to tumour endothelium, and this process depends on TILs expressing and regulating the function of relevant cell-surface receptors. We analysed the cell-surface phenotype and endothelial binding of TILs cultured from human melanoma and compared them with peripheral blood T cells and with allostimulated T cells cultured under similar conditions. Compared with peripheral blood T cells, TILs expressed high levels of five integrins, two other adhesion molecules, including the skin homing molecule CLA, and several activation markers and showed markedly enhanced integrin-mediated adhesion to a dermal microvascular endothelial cell line in vitro. Compared with the allostimulated T cells, TILs expressed higher levels of the cutaneous lymphocyte antigen (CLA), the adhesion molecule CD31 and the activation markers CD30 and CD69, but lower levels of several other adhesion and activation molecules. These phenotypic and functional properties of TILs should have complex effects on their migration in vivo. Expression of CLA, the skin homing receptor, may increase migration to melanoma (a skin cancer), whereas integrin activation may cause non-specific binding of TILs to other endothelium. Manipulation of the culture conditions in which TILs are expanded might result in a phenotype that is more conducive to selective tumour homing in vivo.

Cell Adhesion↗
PubMed2005

Functional analysis of Slac2-c/MyRIP as a linker protein between melanosomes and myosin VIIa.

Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c x myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A.Slac2-c x myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.

Animals↗
PubMed2006

Identification and functional analysis of novel inactivating thyrotropin receptor mutations in patients with thyrotropin resistance.

OBJECTIVE: We identified and analyzed novel thyrotropin (TSH) receptor mutations in three Japanese families with resistance to TSH. DESIGN: The TSH receptor gene was sequenced and the mutations were determined. The mutant TSH receptors were transfected into COS-7 cells, and their functions were analyzed. PATIENTS: The patients were compound-heterozygotes for the R450H mutation and novel mutations in the TSH receptor gene. The first patient was a compound-heterozygote for R450H and V473I. The second sibling possessed R450H and R519C. The third sibling had R450H and R519G. RESULTS: The R450H mutant exhibited moderately impaired receptor functions and a moderately decreased cell surface expression in agreement with previous results. The V473I mutant exhibited an almost normal TSH binding, a slightly decreased cyclic adenosine monophosphate (cAMP) response, a moderately decreased inositolphosphate (IP) response, and an almost normal cell surface expression. TSH binding and TSH stimulation of cAMP and IPs were markedly decreased in the R519C and R519G mutants. Cell surface expression was decreased in the R519C mutant and negligible in the R519G mutant. All of these mutants showed normal intracellular synthesis of TSH receptors. CONCLUSIONS: These novel inactivating mutations contribute to understanding of the structure-function relationship of the TSH receptor. To date, all of the patients with TSH resistance resulting from TSH receptor mutations identified in Japan possessed the R450H mutation at least in one allele. These observations suggest that the R450H mutation is a commonly observed TSH receptor mutation in patients with TSH resistance in Japan.

Animals↗
PubMed2005

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway.

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Bacterial Proteins↗
PubMed1999

Functional analysis of dynamin isoforms in Drosophila melanogaster.

Dynamin and dynamin-like proteins are required for endocytosis, synaptic vesicle recycling and membrane trafficking. From the shibire locus in Drosophila melanogaster, six different isoforms of dynamin are generated by alternative splicing. However, the roles of the individual isoforms in cellular processes are unknown. To investigate functional differences among the dynamin isoforms, transgenic lines were generated that individually expressed each of 3 different isoforms under UASGAL4 control. The expression of the isoforms was controlled by neural promoter (elav)-driven GAL4, or by a shibire-promoter driven GAL4 transgene. Reporter gene expression indicated that the shi promoter is active during embryogenesis, and in larvae, pupae, and adults in a pattern consistent with normal dynamin expression. To assay for the ability of dynamin isoforms to function in vivo, the isoforms expressed via these GAL4 drivers were tested for the ability to rescue shibire phenotypes. When expressed at very high levels all individual isoforms tested rescued the temperature-sensitive paralytic phenotype of shi(ts2) flies; however, this rescue was partial, suggesting that no single tested isoform is sufficient for synaptic vesicle recycling in vivo. When tested for ability to rescue lethality induced by heat-pulsing larvae during development, shi- promoter driven expression of individual isoforms conferred significant resistance to heat treatment during larval development. However, all 3 isoforms were unable to rescue the lethality of shi12-12B mutants which are severely hypomorphic (or null) for shibire function. Taken together, these observations suggest that individual shibire isoforms have specific molecular activities in vivo.

Alternative Splicing↗
PubMed2003

Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer.

The antitumor activity of IFN-alpha is well established. However, the role of the plasmacytoid dendritic cell (PDC), the major producer of IFN-alpha upon viral infection, in tumor biology is unknown. We sought to study the presence and function of PDC in a human solid tumor. Here, we demonstrate that PDCs infiltrate tumor tissue of patients with head and neck squamous cell carcinoma (HNSCC). Functional activity of PDC was examined by using CpG motif containing oligonucleotides, a defined microbial stimulus for PDCs (recognized via toll-like receptor 9). We found that HNSCC diminished the ability of PDC to produce IFN-alpha in response to CpG motif containing oligonucleotide. Tumor-induced down-regulation of toll-like receptor 9 was identified as one mechanism likely contributing to impaired PDC function within the tumor environment. In tumor-draining lymph nodes, suppression of CpG-induced IFN-alpha production was less pronounced than in single-cell suspensions of primary tumor tissue. In these lymph nodes, CpG-induced IFN-alpha production was associated with increased levels of interferon-induced protein 10 and IFN-gamma and activation of CD4 and CD8 T cells. These results show for the first time the presence of PDCs in human solid tumor tissue and that tumors suppress the capacity of PDCs to produce IFN-alpha. PDCs, which in the absence of appropriate stimulation are reported to promote regulatory CD8 T cells, may contribute to an impaired T-cell-mediated immune response in HNSCC.

Adolescent↗
PubMed1988

Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties.

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.

Cell Line↗
PubMed1993

Functional analysis of phosphorylation sites in human lamin A controlling lamin disassembly, nuclear transport and assembly.

We have constructed point mutations in human lamin A cDNA at conserved serine and threonine residues, some of which were shown to be phosphorylated in vitro by cdc2-kinase and protein kinase C and in vivo. Using a functional in vivo assay system, we identified three categories of mutant phenotypes. (i) Dominant negative phenotypes in mitosis result from mutation of Thr-19 and Ser-22 within the amino-terminal cdc2-kinase motif of lamin A. An increase of aberrant mitotic phenotypes in the double mutants Thr-19/Ser-392 and Ser-22/Ser-392 suggests that concomitant phosphorylation of the three residues regulates mitotic lamin A disassembly. (ii) Mutation of both Ser-403/Ser-404 within a PKC motif flanking the nuclear localization signal inhibits transport of mutant lamin A to the nucleus in 64% of the cells. It is proposed that phosphorylation of the motif in vivo positively regulates nuclear localization together with the nuclear localization sequence. (iii) The assembly of lamin A into the perinuclear lamina is disturbed by mutation of the carboxy-terminal Ser-525, previously shown to be interphase-specifically phosphorylated (Eggert et al., Eur. J. Biochem. 213, 659-671 (1993)). The phenotype shows discontinuous and patch-like aggregates of the mutant protein in the nucleus. We suggest that phosphorylation of the site either regulates lamina assembly or lamina-chromatin interaction in interphase.

Amino Acid Sequence↗
PubMed2002

(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

Alkyl and Aryl Transferases↗
PubMed1998

Expression and functional analysis of glycosyl-phosphatidyl inositol-linked CD46 in transgenic mice.

BACKGROUND: Complement activation plays a pivotal role in hyperacute xenograft rejection. In humans, activation of complement is regulated by a number of cell surface regulatory proteins. Membrane cofactor protein (CD46) is one such regulator that protects cells by acting as a cofactor for the factor I-mediated cleavage of C3b and C4b. Transgenic animals expressing human CD46 may provide organs that are resistant to complement attack. However, attempts to generate mice expressing human CD46 using cDNA-based constructs have been largely unsuccessful. METHODS: Transgenic mice expressing a glycosylphosphatidyl inositol (GPI)-linked form of CD46 were generated by microinjection of a hybrid CD46/CD55 cDNA under the control of the human intercellular adhesion molecule-2 promoter. Expression of CD46-GPI on the vascular endothelium was determined by immunohistochemistry. The ability of CD46-GPI to protect mouse tissues from human complement attack was determined using an ex vivo isolated perfused heart model. RESULTS: Three founder animals expressing CD46-GPI were identified. Histological analysis showed strong and uniform expression of CD46-GPI on the vascular endothelium of all organs examined. Ex vivo perfusion of transgenic mouse hearts with human plasma showed a reduction in C3c deposition and a slightly prolonged function compared with controls. CONCLUSIONS: High-level expression of CD46-GPI was achieved in transgenic mice by using a modified cDNA-based construct. The CD46-GPI was functional, providing some protection from complement-mediated damage in the ex vivo model, and may be useful in xenotransplantation if expressed in combination with CD55 and CD59.

Animals↗
PubMed2005

Functional analysis of the p53 pathway in response to ionizing radiation in uveal melanoma.

PURPOSE: Uveal melanomas are notoriously radioresistant and thus necessitate treatment with extremely high radiation doses that often cause ocular complications. The p53 tumor suppressor pathway is a major mediator of the cellular response to radiation-induced DNA damage, suggesting that this pathway may be defective in uveal melanoma. The current study was conducted to analyze the functional integrity of the p53 pathway in primary uveal melanoma cells. METHODS: The p53 gene was sequenced in three primary uveal melanoma cells lines. Cultured primary uveal melanoma cells (MM28, MM50, Mel202, Mel270, and Mel290), MCF7 breast carcinoma cells, normal uveal melanocytes (UM47), and normal human diploid fibroblasts (NHDFs) were irradiated at 250 kVp and 12 mA at a dose rate of 1.08 Gy/min for a total dose of up to 20 Gy. Cell viability was analyzed with trypan blue exclusion. Western blot analysis was used to analyze the expression of p53, p53-phospho-Ser15, p21, Bax, PUMA, and Bcl-x(L). RESULTS: No p53 gene mutations were found in MM28, MM50, or Mel270 cells. Upstream signaling to p53 was intact, with normal induction of p53 and phosphorylation of p53-Ser15, in all five cell lines. Radiation-induced downstream activation of p21 was defective in MM28 and MM50 cells, and activation of Bax was defective in MM50 and Mel290 cells. MM28, MM50, and Mel202 cells failed to deamidate Bcl-x(L) in response to radiation-induced DNA damage. Overall, four of the five uveal melanoma cell lines exhibited at least one downstream defect in the p53 pathway. CONCLUSIONS: Expression of p53 and upstream signaling to p53 in response to radiation-induced DNA damage appear to be intact in most uveal melanomas. In contrast, functional defects in the p53 pathway downstream of p53 activation appear to be common. Further elucidation of p53 pathway abnormalities in uveal melanoma may allow therapeutic interventions to increase the radiosensitivity of the tumors.

Apoptosis Regulatory Proteins↗
PubMed1990

Functional analysis of the transcriptional promoter for the CYP1A1 gene.

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases the transcription rate of the CYP1A1 gene, which encodes a cytochrome P-450 enzyme. In this study, we analyzed the DNA region immediately upstream of the CYP1A1 gene. A domain that extends upstream to nucleotide--166 was found to function as a transcriptional promoter. The promoter was silent when uncoupled from the dioxin-responsive enhancer located farther upstream. DNase footprinting experiments indicated that nuclear proteins interact with distinct domains of the promoter in a TCDD-independent fashion. Mutational analyses indicated that the CYP1A1 promoter contains at least three functional domains, including a TATAAA sequence, a CCAAT box transcription factor/nuclear factor I-like recognition motif, and a guanine-rich G box.

Animals↗
PubMed2005

Comparative functional analysis of the hyolingual anatomy in lacertid lizards.

The tongue is often considered a key innovation in the evolution of a terrestrial lifestyle as it allows animals to transport food items through the oral cavity in air, a medium with low density and viscosity. The tongue has been secondarily coopted for a wide diversity of functions, including prey capture, drinking, breathing, and defensive behaviors. Within basal lizard groups, the tongue is used primarily for the purpose of prey capture and transport. In more derived groups, however, the tongue appears specialized for chemoreceptive purposes. Here we examine the tongue structure and morphology in lacertid lizards, a group of lizards where the tongue is critical to both food transport and chemoreception. Because of the different mechanical demands imposed by these different functions, regional morphological specializations of the tongue are expected. All species of lacertid lizards examined here have relatively light tongue muscles, but a well developed hyobranchial musculature that may assist during food transport. The intrinsic musculature, including verticalis, transversalis, and longitudinalis groups, is well developed and may cause the tongue elongation and retraction observed during chemoreception and drinking. The papillary morphology is complex and shows clear differences between the tongue tips and anterior fore-tongue, and the more posterior parts of the tongue. Our data show a subdivision between the fore- and hind-tongue in both papillary structure and muscular anatomy likely allowing these animals to use their tongues effectively during both chemoreception and prey transport. Moreover, our data suggest the importance of hyobranchium movements during prey transport in lacertid lizards.

Animals↗
PubMed2006

Functional analysis of PKHD1 splicing in autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene on chromosome 6p12. The longest continuous open reading frame comprises 66 exons encoding a novel 4,074 aa multidomain integral membrane protein (polyductin/fibrocystin) of unknown function. Various alternatively spliced transcripts may additionally result in different isoproteins. Overall, the large size of PKHD1, its complex pattern of splicing, multiple allelism and lack of knowledge of the encoded protein's/proteins' functions pose significant challenges to DNA-based diagnostic testing. Nucleotide substitutions, particularly if residing in regulatory elements or introns outside the splice consensus sites, are often difficult to assess without further functional analyses and cannot be unambiguously classified as disease-associated. Investigations on the transcript level, however, are hampered as PKHD1 is not widely expressed in blood lymphocytes. We thus determined the functional significance of the novel splice site mutation c.53-3C>A in intron 2 by RNA analyses by minigene-construction. The mutant allele was shown to cause skipping of exon 3. Thus, given the minigene results together with 400 control chromosomes negative for this change, segregation of the mutation with the phenotype, and a significant lowering of the strength of the splice site by bioinformatics, the mutant allele is most likely pathogenic. To the best of our knowledge, this is the first study that defines the consequences of a PKHD1 splice mutation and underlines the relevance of functional analyses in determining the pathogenicity of changes of unknown significance.

Base Sequence↗
PubMed1991

Chemical mutagenesis and fine-structure functional analysis of the mouse genome.

Heritable mutations constitute important raw materials for mammalian developmental genetics and general genome studies. Mutations induced by high-efficiency chemical mutagenesis of germ cells in mice can be used in genetic and molecular studies to complement physical-mapping strategies and to examine the nature and extent of the functional complexities hidden within the mammalian genome.

Animals↗
PubMed2003

Functional analysis of human memory B-cell subpopulations: IgD+CD27+ B cells are crucial in secondary immune response by producing high affinity IgM.

The number of memory B cells in peripheral blood has been assayed in various diseases by using CD27 as a memory B-cell marker. However, the defining differences of characteristic and function between the two memory B-cell subpopulations separated by immunoglobulin (Ig)D expression remain to be clearly elucidated. We analyzed here IgD(+)CD27(+) B cells (circulating B cells 2, cB2) and IgD(-)CD27(+) memory B cells (cB3) in comparison with IgD(+)CD27(-) naive B cells (cB1). cB2 were found to be morphologically similar to cB3 with abundant cytoplasm, whereas cB3 expressed CD80, CD86, and CD95 on their surface more predominantly than cB2. A majority of cB2 expressed both IgD and IgM, and cB3 expressed IgA or IgG. Mature gamma1 and gamma2 transcripts were found in cB3, but at very low levels in cB2, and activation-induced cytidine deaminase (AID) mRNA expression was recognized only in cB3. The frequencies of somatic hypermutation in cB2 and cB3 were comparable levels studied by VH5. cB2 did not shift to cB3 in vitro by the stimuli such as via B-cell receptor or CD40. cB2 produced large amounts of IgM predominantly and promptly, which is in accordance with the known characteristics of memory B cells. Taken together, although cB2 are unclass-switched, cB2 have the functions of memory B cells and are not in the process of transition from naive to switched memory B cells, playing a crucial role in secondary immune response by producing high-affinity IgM in the early phase of infections.

Adolescent↗