‘Sulfo‐click’ for ligation as well as for site‐specific conjugation with peptides,fluorophores, and metal chelators

The ‘sulfo‐click’ reaction, which is a chemoselective amidation reaction involving the reaction of an aminoethane sulfonyl azide with a thio acid, encompasses a new approach for ligation and conjugation. Detailed protocols are provided for decorating biologically active peptides or dendrimers with biophysical tags, fluorescent probes, metal chelators, and small peptides by using this reaction as a novel, metal‐free ‘sulfo‐click’ approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.     

Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C‐terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C‐terminal lysine (‐K) or lysine and glycine (‐GK). Interestingly, clones that express antibodies lacking HC C‐terminal lysine (‐K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (‐GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C‐terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:786–794, 2017     

Cloning,co-expression with an amidating enzyme,and activity of the scorpion toxin BmK ITa1 cDNA in insect cells

BmK ITa1 is an insect-specific neurotoxin from the Chinese scorpion Buthus martensi Karsch (Bmk). We succeeded in obtaining biologically active recombinant BmK ITa1 protein by simultaneous expression in insect cells of BmK ITa1 cDNA with an amidating enzyme expressed by the rat peptidylglycine α-amidating monooxygenase (PAM) gene. We investigated the insecticidal efficacy of recombinant BmK ITa1/W (without coexpression of PAM), and of BmK ITa1/A (with coexpression of PAM) in 5th instar Bombyx mori, by injecting these recombinant toxins into larvae. The lethal time for 50% of larvae (LT₅₀) was 9 h for BmK ITa1/A and 17 h for BmK ITa1/W. At 19 h after injection all of the larvae exposed to BmK ITa1/A had been killed, whereas only half of the larvae exposed to BmK ITa1/W had been killed. These results show that the simultaneous expression of an amidating enzyme can result in apparently higher insecticidal activity of BmK ITa1.     

Economically pertinent continuous amide formation by direct lipase-catalyzed amidation with ammonia

An economically pertinent process for the lipase-catalyzed synthesis of amides was developed. A continuous plug flow reactor was used. The model reaction was the production of oleamide, a lubricant and anti-slip agent, via direct Candida antarctica lipase B-catalyzed amidation of oleic acid with ammonia. Of all solvents tested, 2-methyl-2-butanol was found to respond optimally to the demands formulated in our specifications. A continuous conversion of oleic acid into oleamide of 85% was obtained. A productivity of 4.5 tons oleamide per kg of enzyme per year was calculated, indicating a contribution of enzyme to the product price of only 4%. The volumetric productivity, 100 g. L(-1). h(-1), is 4 to 100 times higher than in literature procedures. A simple crystallization procedure leads to 99% purity.     

Peptide backbone cleavage by α‐amidation is enhanced at methionine residues

Cleavage reactions at backbone loci are one of the consequences of oxidation of proteins and peptides. During α‐amidation, the C_α–N bond in the backbone is cleaved under formation of an N‐terminal peptide amide and a C‐terminal keto acyl peptide. On the basis of earlier works, a facilitation of α‐amidation by the thioether group of adjacent methionine side chains was proposed. This reaction was characterized by using benzoyl methionine and benzoyl alanyl methionine as peptide models. The decomposition of benzoylated amino acids (benzoyl‐methionine, benzoyl‐alanine, and benzoyl‐methionine sulfoxide) to benzamide in the presence of different carbohydrate compounds (reducing sugars, Amadori products, and reductones) was studied during incubation for up to 48 h at 80 °C in acetate‐buffered solution (pH 6.0). Small amounts of benzamide (0.3–1.5 mol%) were formed in the presence of all sugars and from all benzoylated species. However, benzamide formation was strongly enhanced, when benzoyl methionine was incubated in the presence of reductones and Amadori compounds (3.5–4.2 mol%). The reaction was found to be intramolecular, because α‐amidation of a similar 4‐methylbenzoylated amino acid was not enhanced in the presence of benzoyl‐methionine and carbohydrate compounds. In the peptide benzoyl‐alanyl‐methionine, α‐amidation at the methionine residue is preferred over α‐amidation at the benzoyl peptide bond. We propose here a mechanism for the enhancement of α‐amidation at methionine residues. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Hydrophobic benzyl amines as supports for liquid‐phase C‐terminal amidated peptide synthesis: application to the preparation of ABT‐510

C‐terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C‐terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C‐terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work‐up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag‐assisted liquid‐phase peptide synthesis as supports, leading to the total synthesis of ABT‐510 ( 2 ). Although an ethyl amide‐forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C‐terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION*

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsA_Cg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsA_Cg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsA_Cg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH₄⁺ could serve as the donor substrate for LtsA_Cg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.     

Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2

Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.