Browsing by Subject "Amino Acid Sequence"

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    Open Access
    A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase
    (2001) Alfalah, Marwan; Parkin, Edward T; Jacob, Ralf; Sturrock, Edward D; Mentele, Reinhard; Turner, Anthony J; HOOPER, Nigel M; Naim, Hassan Y
    Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.
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    Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans
    (2009) Lindemans, M; Liu, F; Janssen, T; Husson, S J; Mertens, I; Gäde, G; Schoofs, L
    In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC50 = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Gαq protein with Ca2+ as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.
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    Asn 102 of the Gonadotropin-releasing Hormone Receptor Is a Critical Determinant of Potency for Agonists Containing C-terminal Glycinamide
    (1996) Davidson, James S; McArdle, Craig A; Davies, Peter; Elario, Ricardo; Flanagan, Colleen A; Millar, Robert P
    We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand.
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    Development of plant-produced protein body vaccine candidates for bluetongue virus
    (2017) van Zyl, Albertha R; Meyers, Ann E; Rybicki, Edward P
    BACKGROUND: Bluetongue is a disease of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), which have caused serious outbreaks worldwide. Commercially available vaccines are live-attenuated or inactivated virus strains: these are effective, but there is the risk of reversion to virulence or reassortment with circulating strains for live virus, and residual live virus for the inactivated vaccines. The live-attenuated virus vaccines are not able to distinguish naturally infected animals from vaccinated animals (DIVA compliant). Recombinant vaccines are preferable to minimize the risks associated with these vaccines, and would also enable the development of candidate vaccines that are DIVA-compliant. RESULTS: In this study, two novel protein body (PB) plant-produced vaccines were developed, Zera®-VP2ep and Zera®-VP2. Zera®-VP2ep contained B-cell epitope sequences of multiple BTV serotypes and Zera®-VP2 contained the full-length BTV-8 VP2 codon-optimised sequence. In addition to fulfilling the DIVA requirement, Zera®-VP2ep was aimed at being multivalent with the ability to stimulate an immune response to several BTV serotypes. Both these candidate vaccines were successfully made in N. benthamiana via transient Agrobacterium-mediated expression, and in situ TEM analysis showed that the expressed proteins accumulated within the cytoplasm of plant cells in dense membrane-defined PBs. The peptide sequences included in Zera®-VP2ep contained epitopes that bound antibodies produced against native VP2. Preliminary murine immunogenicity studies showed that the PB vaccine candidates elicited anti-VP2 immune responses in mice without the use of adjuvant. CONCLUSIONS: These proof of concept results demonstrate that Zera®-VP2ep and Zera®-VP2 have potential as BTV vaccines and their development should be further investigated.
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    Functional microdomains in G-protein-coupled receptors: the conserved arginine-cage motif in the gonadotropin-releasing hormone receptor
    (1998) Ballesteros, Juan; Kitanovic, Smiljka; Guarnieri, Frank; Davies, Peter; Fromme, Bernard J; Konvicka, Karel; Chi, Ling; Millar, Robert P; Davidson, James S; Weinstein, Harel; Sealfon, Stuart C
    An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp3.49(138). Experimental results for the Asp3. 49(138) --> Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp3. 49(138)-Arg3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp3. 49(138)-Arg3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile3.54(143). The mutation Ile3.54(143) --> Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp3.49(138) and Ile3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.
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    Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone
    (1994) Flanagan, C A; Becker, I I; Davidson, J S; Wakefield, I K; Zhou, W; Sealfon, S C; Millar, R P
    The Arg residue at position 8 of mammalian GnRH is necessary for high affinity binding to mammalian GnRH receptors. This requirement has been postulated to derive from an electrostatic interaction of Arg8 with a negatively charged receptor residue. In order to identify such a residue, 8 conserved acidic residues of the mouse GnRH receptor were mutated to isosteric Asn or Gln. Mutant receptors were tested for decreased preference for Arg8-containing ligands by ligand binding and inositol phosphate production. One of the mutants, in which the Glu301 residue was mutated to Gln, exhibited a 56-fold decrease in apparent affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys8]GnRH, but its affinity for [Gln8]GnRH was unchanged compared with the wild type receptor. The apparent affinity of the mutant receptor for the acidic analogue, [Glu8]GnRH, was increased more than 10-fold. The mutant receptor did not, therefore, distinguish mammalian GnRH from analogues with amino acid substitutions at position 8 as effectively as the wild type receptor. This loss of discrimination was specific for the residue at position 8, because the mutant receptor did distinguish mammalian GnRH from analogues with favorable substitutions at positions 5, 6, and 7. These findings show that Glu301 of the GnRH receptor plays a role in receptor recognition of Arg8 in the ligand and are consistent with an electrostatic interaction between these 2 residues.
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    Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE
    (2003) Clausen, Johannes D; McIntosh, David B; Vilsen, Bente; Woolley, David G; Andersen, Jens Peter
    Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.
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    Molecular Characterization of trans -Golgi p230: a human peripheral membrane protein encoded by a gene on chromosome 6p12-22 contains extensive coiled-coil α-helical domains and a Granin Motif
    (1996) Erlich, Rebecca; Gleeson, Paul A; Campbell, Paul; Dietzsch, Erin; Toh, Ban-Hock
    Using autoantibodies from a Sjögren's syndrome patient, we have previously identified a 230-kDa peripheral membrane protein associated with the cytosolic face of the trans-Golgi (Kooy, J., Toh, B. H., Pettitt, J. M., Erlich, R. and Gleeson, P. A. (1992) J. Biol. Chem. 267, 20255-20263). Here we report the molecular cloning and sequence analysis of human p230 and the localization of its gene to chromosome 6p12 22. Partial cDNA clones, isolated from a HeLa cell cDNA library using autoantibodies, were used to obtain additional cDNAs, which together span 7695 base pairs (bp). The p230 mRNA is approximately 7.7 kilobases. Two alternatively spliced mRNAs for p230 were detected. These differed by 21- and 63-bp insertions in the 3'-sequence, resulting in differences in amino acid sequence at the carboxyl terminus. The predicted 261-kDa protein is highly hydrophilic with 17-20% homology with many proteins containing coiled-coil domains. Apart from two proline-rich regions (amino acids 1-117 and 239-270), p230 contains a very high frequency of heptad repeats, characteristic of alpha-helices that form dimeric coiled-coil structures. p230 also includes the sequence ESLALEELEL (amino acids 538-546), a motif found in the granin family of acidic proteins present in secretory granules of neuroendocrine cells. This is the first report of a cytosolic Golgi protein containing a granin motif. The structural characteristics of p230 indicate that it may play a role in vesicular transport from the trans-Golgi.