Determination of amino- and carboxyl-terminal sequences of guinea pig liver transglutaminase: evidence for amino-terminal processing

171

169

Mammalian transglutaminase (TGase) catalyzes covalent crosslinking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPD(P), QxP, and QxxDP; Factor XIIIa, QxxxWP (where x and represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.Transglutaminase (TGase, 2 2.3.2.13) is an enzyme that catalyzes the formation of isopeptide cross-links between glutamine and lysine residues in a variety of proteins and also attaches other primary amines to peptide-bound glutamines (1-5). To date, eight human TGase isozymes (Factor XIII, TGases 1-7) have been identified, comprising a large protein family with unique tissue distributions and physiological roles. Among the isozymes, TGase 2 and Factor XIII are two major members, although their locations and regulation differ. TGase 2 is expressed ubiquitously and has been implicated in many biological processes, including apoptosis, stabilization of the extracellular matrix, and regulation of growth factors (6, 7). Plasma Factor XIII is synthesized as a zymogen that comprises two A and two B subunits (8). The A subunit contains the catalytic domain and is converted by thrombin-dependent proteolysis into its active form during clot formation. Activated Factor XIII (Factor XIIIa) is involved in fibrin stabilization and wound healing (9).Generally, TGase reactions involve a Ca 2ϩ -dependent acyl transfer via a double displacement mechanism. In the first step, a glutamine ␥-carboxyamide group in the substrate binds to a cysteine at the active site, resulting in formation of a ␥-glutamylthioester bond and release of ammonia. Formation of the covalent acyl enzyme intermediate is the rate-limiting step. The -amino group of a peptide-bound lysine, as a nucleophilic substrate, binds to the acyl enzyme intermediate and then attacks the thioester bond, thereby generating an intermolecular isopeptide -(␥-glutamyl)lysine cross-link. Primary amines can replace lysine in transamidation reactions and become incorp...

Section: Methodsmentioning

confidence: 99%

Screening for the Preferred Substrate Sequence of Transglutaminase Using a Phage-displayed Peptide Library

Sugimura

Hosono

Wada

et al. 2006

171

169

“…In the intracellular environment its binding to these nucleotides consequently prevents Ca 2ϩ activation of the enzyme (15), consistent with an extracellular function (16). Despite the growing evidence for an extracellular role for tTG, the enzyme presents the features of a cytosolic protein such as N-terminal acetylation, lack of disulfide bridges, and lack of glycosylation (17,18). A further feature of this protein and other members of this protein family with an extracellular function including factor XIIIa is the lack of a classical leader sequence necessary for the translocation of proteins into the endoplasmic reticulum for their secretion (19).…”

mentioning

confidence: 90%

“…Similar to Ala in wild-type tTG, a Val in the penultimate position is known to have a stabilizing effect on the translated polypeptide. tTG as well as factor XIII a-subunit are known to be N terminally processed by removal of the terminal Met and acetylation of the penultimate residue (18,39). Ala and Ser found in this position in wild-type tTG and factor XIII a-subunit, respectively, unlike Val in the generated fusion proteins, favor co-translational N-acetylation (40).…”

Section: Processing Of Pericellular Fibronectin By Transglutaminasementioning

confidence: 99%

Cell Surface Localization of Tissue Transglutaminase Is Dependent on a Fibronectin-binding Site in Its N-terminal β-Sandwich Domain

Gaudry¹,

Verderio²,

Aeschlimann³

et al. 1999

128

129

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme ␤-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal ␤-sandwich domain and that this interaction is crucial for cell surface association of tTG.

“…Although a key trigger for TG2 export is cell stress (2,21,22), TG2 is not unspecifically released, because extracellular trafficking occurs in the absence of leakage of intracellular components and cells remain viable (23). We know that TG2 requires the tertiary structure of its active site region to be secreted (9); moreover, TG2 is acetylated on the N terminus (24), a process reported to affect membrane targeting of nonconventional secreted proteins (25). Two main binding partners for TG2, FN and integrin-␤1, have both been attributed a possible role in the transport of TG2 to the cell surface (8,26).…”

mentioning

confidence: 99%

Heparan Sulfate Proteoglycans Are Receptors for the Cell-surface Trafficking and Biological Activity of Transglutaminase-2

Scarpellini

Germack

Lortat‐Jacob

et al. 2009

132

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.