Effects of mutations in the helix G region of horseradish peroxidase

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F′ (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state Vm/E values occurred with reducing substrate ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site

Structural and biochemical characterization of a new type of lectin isolated from carp eggs.

A previously unidentified glycoprotein present in the eggs of the carp (Cyprinus carpio) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N-acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N-acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp (Carassius auratus gibelio) and 78% identity with a gene in the cDNA library of the zebrafish (Danio rerio)

The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region.

A family of chimeric immunoglobulins (Igs) bearing the murine variable region directed against the hapten dansyl linked to human IgG1, -2, -3, and -4 has been characterized with respect to binding to the human high affinity Fc gamma receptor, Fc gamma RI. Chimeric IgG1 and -3 have the highest affinity association (Ka = 10(9) M-1), IgG4 is 10-fold reduced from this level, and IgG2 displays no detectable binding. A series of genetic manipulations was undertaken in which domains from the strongly binding subclass IgG3 were exchanged with domains from the nonbinding subclass IgG2. The subclass of the CH2 domain was found to be critical for determining IgG receptor affinity. In addition, the hinge region was found to modulate the affinity of the IgG for Fc gamma RI, possibly by determining accessibility of Fc gamma RI to the binding site on Fc. A series of amino acid substitutions were engineered into the CH2 domain of IgG3 and IgG4 at sites considered potentially important to Fc receptor binding based on homology comparisons of binding and nonbinding IgG subclasses. Characterization of these mutants has revealed the importance for Fc gamma RI association of two regions of the genetic CH2 domain separated in primary structure by nearly 100 residues. The first of these is the hinge-link or lower hinge regions, in which two residues, Leu (234) and Leu(235) in IgG1 and -3, are critical to high affinity binding. Substitution at either of these sites reduces the IgG association constant by 10-100-fold. The second region that appears to contribute to receptor binding is in a hinge-proximal bend between two beta strands within the CH2 domain, specifically, Pro(331) in IgG1 and -3. As a result of beta sheet formation within this domain, this residue lies within 11 A of the hinge-link region. Substitution at this site reduces the Fc receptor association constant by 10-fold

The role of cysteine 230 and lysine 238 of biotin carboxylase in the deprotonation of biotin and synthesis of bisubstrate analogy inhibitor of carboxyltransferase

Acetyl-CoA carboxylase catalyzes the first step in the synthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. This enzyme uses the cofactor biotin as a carboxyl carrier. In order for the carboxylation of biotin to occur, biotin must be deprotonated at its N-1 position. It has been proposed that the active site residues cysteine 230 and lysine 238 act as an acid-base pair to deprotonate biotin. To test this hypothesis, site-directed mutagenesis was used to mutate cysteine 230 to alanine (C230A) and lysine 238 to glutamine (K238Q). Mutations at either residue resulted in a 50-fold increase in the Km for ATP. The C230A mutation had no effect on the formation of carboxybiotin, indicating that cysteine 230 does not play a role in the deprotonation of biotin. However, the K238Q mutation resulted in no formation of carboxybiotin, which showed that lysine 238 has a role in the carboxylation reaction. However, the pK value for lysine 238 was 9.4 or higher, suggesting lysine 238 is not a catalytic base. Thus, the results suggest that cysteine 230 and lysine 238 do not act as an acid-base pair in the deprotonation of biotin. A bisubstrate analog inhibitor of carboxyltransferase was synthesized by covalently linking biotin to Coenzyme A via an acyl bridge between the sulfur of Coenzyme A and the N-1 of biotin. The inhibitor was found to have an inhibition constant of 23 ± 2 ìM, which means it binds the enzyme 350-times tighter than biotin. The bisubstrate analog demonstrated competitive inhibition versus malonyl-CoA and noncompetitive inhibition versus biocytin. This is consistent with an ordered kinetic mechanism with malonyl-CoA binding first. A precursor to the inhibitor, chloroacylated biotin, was capable of inhibiting the differentiation of 3T3-L1 cells in a dose-dependent manner. Treatment with chloroacylated biotin resulted in a decrease in acetyl-CoA carboxylase activity and inhibited lipid accumulation. Our results support recent studies that indicate acetyl-CoA carboxylase may be a suitable target as an anti-obesity therapeutic

Functional characterisation of constitutive expresser of pathogenesis-related genes 5 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand

As reported previously, CPR5 negatively regulates the onset of leaf death, hypersensitive response, disease resistance and early leaf senescence. cpr5 plants contain aberrant trichomes and higher levels of ROS, SA and JA. Cell-cycle, JA/ET, ABA and sugar signalling are also affected in cpr5 plants. These results suggest that CPR5 is a master regulator of multiple processes. However, how CPR5 manages to exert pleiotropic effects is still poorly understood. The first objective of the current study was the purification of the CPR5 protein to solve its crystal structure. Extensive in silico analyses were carried out and the results showed that CPR5 is predicted to be a membrane protein with 4 or 5 transmembrane (TM) domains. Additionally, CPR5 contains intrinsically disordered regions (IDRs) at its N-terminus. Proteins containing IDRs and TM domains are often difficult to purify for crystallization studies. Therefore, the undesirable regions of CPR5 such as, IDR and TM domains were deleted and a set of 24 constructs were developed. Despite several efforts, none of the CPR5 recombinant proteins were isolated. In addition to predicting IDR and TM domains, in silico results also predicted three NLS-encoding clusters, casein kinase phosphorylation sites, multiple start codons, coiled-coil domains and glycine motifs. To find out the roles of these putative structural elements on CPR5 functions, firstly a CPR5 cDNA was synthesised and termed as SynCPR5. Subsequently, predicted sites or motifs were mutated in SynCPR5 through sitedirected mutagenesis and a set of 25 mutated CPR5 transgenes (cDNA constructs) were developed. Using a complementation strategy, all the constructs were transformed into cpr5- 2 plants. The results show that the complementation of cpr5-2 plants with SynCPR5, fully restored HR-like lesions, wildtype-like trichomes and leaves on SynCPR5 plants. Further physiological characterization such as, transcript abundance of SynCPR5, PR1, PR5 and PDF1.2, leaf area measurements and ploidy levels showed that CPR5 regulates some of its functions and phenotypes quantitatively as well as qualitatively. When compared with the wildtype, better growth (larger leaves) but enhanced disease susceptibility was found in metCPR5 transgenic lines (in which putative start codons were mutated), indicating that CPR5 regulates a balance between growth and resistance. Functional characterization of NLS mutants (nlsCPR5) showed that NLS-encoding clusters are important for CPR5 proper functions. However, current evidence is insufficient to relate their role in CPR5 localization. Moreover, in silico results show that putative NLS clusters are present in the region of CPR5 which were annotated as intrinsically disordered region (IDR). Similar phenotypes shown by both nlsCPR5 and Del63CPR5 (in which the first 63 amino acids of CPR5 including putative NLS were deleted), indicate that the putative NLS clusters could be part of IDR and may have dual functions. Loss-of-function phenotypes shown by coiled-coil domain mutants (ccdCPR5) reinforce the role of coiled-coil domains in CPR5 homo-dimerization. Moreover, in contrast to previous reports, the downregulation of PDF1.2 in the majority of CPR5 complementation lines proposes CPR5 to be a positive regulator of PDF1.2. Based on the results presented in the current study, putative CPR5 IDRs and coiled-coil domains are proposed to facilitate CPR5 dimerization in order to restrict the entry of deregulated cargos into the nucleus. Moreover, these results uncover a novel role of CPR5 in the regulation of balance between plant growth and resistance. Furthermore, this study, for the first time, reports evidence of the requirement of NLS clusters for CPR5 functions

Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4.

The integrin alpha 6 beta 4 is a heterodimer predominantly expressed by epithelia. While no definite receptor function has yet been assigned to it, this integrin may mediate adhesive and/or migratory functions of epithelial cells. We have determined the complete primary structure of both the alpha 6 and beta 4 subunits from cDNA clones isolated from pancreatic carcinoma cell line libraries. The deduced amino acid sequence of alpha 6 is homologous to other integrin alpha chains (18-26% identity). Antibodies to an alpha 6 carboxy terminus peptide immunoprecipitated alpha 6 beta 4 complexes from carcinoma cells and alpha 6 beta 1 complexes from platelets, providing further evidence for the association of alpha 6 with more than one beta subunit. The deduced amino acid sequence of beta 4 predicts an extracellular portion homologous to other integrin beta chains, and a unique cytoplasmic domain comprised of greater than 1,000 residues. This agrees with the structures of the beta 4 cDNAs from normal epithelial cells (Suzuki, S., and Y. Naitoh. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:757-763; Hogervost, F., I. Kuikman, A. E. G. Kr. von dem Borne, and A. Sonnenberg. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:765-770). Compared to these structures, however, the beta 4 cDNAs that we have cloned from carcinoma cells contain extra sequences. One of these is located in the 5'-untranslated region, and may encode regulatory sequences. Another specifies a segment of 70 amino acids in the cytoplasmic tail. Amplification by reverse transcription-polymerase chain reaction of mRNA indicated that multiple forms of beta 4 may exist, possibly due to cell-type specific alternative splicing. The unique structure of beta 4 suggests its involvement in novel cytoskeletal interactions. Consistent with this possibility, alpha 6 beta 4 is mostly concentrated on the basal surface of epithelial cells, but does not colocalize with components of adhesion plaques

Glycosylation in the thyroid gland : vital aspects of glycoprotein function in thyrocyte physiology and thyroid disorders

The key proteins responsible for hormone synthesis in the thyroid are glycosylated. Oligosaccharides strongly affect the function of glycosylated proteins. Both thyroid-stimulating hormone (TSH) secreted by the pituitary gland and TSH receptors on the surface of thyrocytes contain N-glycans, which are crucial to their proper activity. Thyroglobulin (Tg), the protein backbone for synthesis of thyroid hormones, is a heavily N-glycosylated protein, containing 20 putative N-glycosylated sites. N-oligosaccharides play a role in Tg transport into the follicular lumen, where thyroid hormones are produced, and into thyrocytes, where hyposialylated Tg is degraded. N-glycans of the cell membrane transporters sodium/iodide symporter and pendrin are necessary for iodide transport. Some changes in glycosylation result in abnormal activity of the thyroid and alteration of the metabolic clearance rate of hormones. Alteration of glycan structures is a pathological process related to the progression of chronic diseases such as thyroid cancers and autoimmunity. Thyroid carcinogenesis is accompanied by changes in sialylation and fucosylation, β1,6-branching of glycans, the content and structure of poly-LacNAc chains, as well as O-GlcNAcylation, while in thyroid autoimmunity the main processes affected are sialylation and fucosylation. The glycobiology of the thyroid gland is an intensively studied field of research, providing new data helpful in understanding the role of the sugar component in thyroid protein biology and disorders

The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components

The Protein Precursors of Peptides That Affect the Mechanics of Connective Tissue and/or Muscle in the Echinoderm Apostichopus japonicus

PMCID: PMC3432112This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited