Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Abstract

Transglutaminases (TGs) are a family of enzymes that catalyze Ca 2þ -dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40-and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.

Transglutaminase 2 is involved in autophagosome maturation

“…38 In this connection it is relevant to note that five TG2 candidate protein substrates (i.e.,: arginase-1, fructose-1,6-bisphosphatase, betaina-homocysteine S-methyltransferase BHMT, glyceraldehyde-3-phosphate dehydrogenase and glutathione S-transferase) have been shown to be enriched on the autophagosomal membrane. 39,40 In particular, a BHMT fragment has been shown to accumulate in the autolysosome fraction obtained from rat hepatocytes treated with inhibitors of intralysosomal protein degradation, similar to what we observed in this study for ANT-1 (Fig. 8A).…”

Global identification and analysis of isozyme-specific possible substrates crosslinked by transglutaminases using substrate peptides in mouse liver fibrosis

“…In many articles in research on the enzymes, lower molecular weight substrate molecules such as labeled primary amine (biotinylated pentylamine as glutamine-acceptor substrate) and Gln-containing peptide (as lysine-acceptor substrate) have been commonly used for the identification of possible protein substrates as well as in vitro detection of enzymatic activity15163536. Because the identified substrate candidates by this procedure appeared mostly crosslinked components in vivo , we believe the identified molecules in this study are highly possible as substrates by our identified substrate peptide.…”

Identification of hepatic protein-protein interaction targets for betaine homocysteine S-methyltransferase

“…BHMT interaction targets listed in the IntAct and BioGRID databases, as well as in the literature include approximately 22 interaction partners ( Table 9 ) [ 12 , 33 – 36 , 50 – 57 ]. However, only seven of these targets have been also identified in our study.…”

“…Datasets derived from several high-throughput studies focused on the identification of protein targets for certain PTMs or on the elucidation of the human interactome have rendered data concerning BHMT. For example, phosphorylation [ 26 , 27 ], ubiquitination [ 28 ], acetylation [ 29 , 30 ], succinylation [ 30 ], carbonylation [ 31 ], and nitrosylation [ 32 ] are some of the PTMs detected on BHMT in these analyses, whereas other studies also identify BHMT as a target for modification by transglutaminase [ 33 ]. Nevertheless, in many cases the existence of these PTMs has not been verified by additional methods, neither the enzymes catalyzing these modifications have been identified.…”