Synthesis of glycosyl amino acids by light-induced coupling of photoreactive amino acids with glycosylamines and 1-C-aminomethyl glycosides

The glycosylamines of O-acetyl-protected GlcNAc and chitobiose, as well as two partially unprotected 1-C-aminomethyl glucosides, were photochemically coupled with orthogonally protected N-aspartyl-5-bromo-7-nitroindoline derivatives. The reactions proceeded under neutral conditions by irradiation with near-UV light. The glycosyl asparagines with N- or C-glycosyl linkages were afforded in 60-85% yield on a 10-70 mg scale. Moreover, the ability of a highly photoreactive N-glutamyl-4-methoxy-7-nitroindoline derivative to acylate amino saccharides was tested. Upon irradiation in the presence of a dimeric 1-C-aminomethyl glycoside, or a glycosylamine, the corresponding glycosyl glutamines were obtained in 50% and 30% yield, respectively. Preparations of the photoreactive aspartates and the 1-C-aminomethyl glycosides are also described.     

Characterization of Gtf1p, the connector subunit of yeast mitochondrial tRNA-dependent amidotransferase

The bacterial GatCAB operon for tRNA-dependent amidotransferase (AdT) catalyzes the transamidation of mischarged glutamyl-tRNA(Gln) to glutaminyl-tRNA(Gln). Here we describe the phenotype of temperature-sensitive (ts) mutants of GTF1, a gene proposed to code for subunit F of mitochondrial AdT in Saccharomyces cerevisiae. The ts gtf1 mutants accumulate an electrophoretic variant of the mitochondrially encoded Cox2p subunit of cytochrome oxidase and an unstable form of the Atp8p subunit of the F(1)-F(0) ATP synthase that is degraded, thereby preventing assembly of the F(0) sector. Allotopic expression of recoded ATP8 and COX2 did not significantly improve growth of gtf1 mutants on respiratory substrates. However, ts gft1 mutants are partially rescued by overexpression of PET112 and HER2 that code for the yeast homologues of the catalytic subunits of bacterial AdT. Additionally, B66, a her2 point mutant has a phenotype similar to that of gtf1 mutants. These results provide genetic support for the essentiality, in vivo, of the GatF subunit of the heterotrimeric AdT that catalyzes formation of glutaminyl-tRNA(Gln) (Frechin, M., Senger, B., Brayé, M., Kern, D., Martin, R. P., and Becker, H. D. (2009) Genes Dev. 23, 1119-1130).     

Immunoassay of in vitro activated human tissue transglutaminase

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that exerts a variety of physiological functions and is involved in various pathoprocesses. To characterize the role of tTG in disease, simple assays are necessary for detection. We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that combines a transamidation step with immunological detection to determine tTG. This assay is based on covalent binding of in vitro activated tTG to N,N′-dimethylated casein and subsequent detection of coupled tTG by specific immunoglobulin G (IgG) antibodies directed against tTG followed by binding of a secondary biotin-labeled antibody. The assay reaches a detection limit of 0.05 ng of tTG/ml. Type 1 and 3 transglutaminases and factor XIII are not detected. By use of this assay, tTG in several cell lines and the stimulatory effect of retinoic acid on tTG expression could be demonstrated. The new assay represents a promising tool for the study of tTG in normal cell physiology and under pathological conditions.     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.     

Continuous enzyme-coupled assay for microbial transglutaminase activity

Transglutaminases (protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a γ-glutamyl-ε-lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG’s promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K_M = 3 mM) than the commonly used Cbz-Gln-Gly (K_M = 58 mM), attesting to its potential for application in biocatalysis and bioconjugation.     

Tissue Transglutaminase Is an In Situ Substrate of Calpain: Regulation of Activity

Abstract: Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'- O -(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 n M maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an ∼50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.     

Co-evolution of the archaeal tRNA-dependent amidotransferase GatCAB with tRNA(Asn)

The important identity elements in tRNA(Gln) and tRNA(Asn) for bacterial GatCAB and in tRNA(Gln) for archaeal GatDE are the D-loop and the first base pair of the acceptor stem. Here we show that Methanothermobacter thermautotrophicus GatCAB, the archaeal enzyme, is different as it discriminates Asp-tRNA(Asp) and Asp-tRNA(Asn) by use of U49, the D-loop and to a lesser extent the variable loop. Since archaea possess the tRNA(Gln)-specific amidotransferase GatDE, the archaeal GatCAB enzyme evolved to recognize different elements in tRNA(Asn) than those recognized by GatDE or by the bacterial GatCAB enzyme in their tRNA substrates.     

Keratin transamidation

Low molecular weight keratin was self-crosslinked by microbial transglutaminase (mTGase) for application to wool fabric. From amino acid determination, keratin produced by the alkaline hydrolysis of wool showed requisite glutamine and lysine required for mTGase-mediated transamidation. Keratin showed less lysyl amine content after combination with mTGase as proof of self-crosslinking. Gel electrophoretic patterns provided evidence of self-crosslinking with the development of relatively higher molecular weight protein bands within 30 min after mTGase was combined with keratin at 30 degrees C. Increase in the deconvoluted amide II band from IR spectra of keratin after combination with mTGase provided further evidence of transamidation. By examining the ability of keratin to self-crosslink, past findings were elucidated whereby mTGase-mediated crosslinking imparted strength to wool and keratin controlled its dimensional stability. mTGase-catalyzed isopeptide bond formation of keratin to form monosubstituted gamma-amides of peptide-bound glutamine in epsilon-amino-(gamma-glutamyl)lysine crosslinks. This system was effective for binding wool to wool, keratin to wool, and keratin to keratin in self-crosslinking.     

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.