Lysine Crosslinks Research Articles

Characterization of a Blue Fluorophore Isolated from in Vitro Reaction of N-α-Acetyllysine and 3-Deoxyglucosone

With the objective to investigate 3-deoxyglucosone (3-DG) mediated lysine crosslinks in vivo, we have isolated a lysine-3-DG-lysine crosslink from in vitro reaction of 3-DG and N-α-acetyllysine (NAL). This crosslink, named as furopyrrolopyridine crosslink (FPPC), has intense blue fluorescence with absorption maxima at 235, 270 and 370 nm and emission maximum at 470 nm. The absorption and fluorescence spectra of FPPC were not altered in pHs ranging from 2–12, but the characteristic spectrum of FPPC (at pH 7.0) disappeared when it was reduced with sodium borohydride. FAB-MS showed that FPPC has a molecular mass of 611, equivalent to the reaction of two molecules each of NAL and 3-DG with the concomitant loss of 5 molecules of water. NMR data showed that FPPC has a pyridinium ring and four free hydroxy groups. Since acid hydrolyzed FPPC can be detected by amino acid analysis, we have determined its levels in the acid hydrolyzates of proteins glycated by 3-DG or in the acid hydrolyzates of normal aged, cataractous, diabetic and brunescent human lens proteins as well as in the acid hydrolyzed glycated hemoglobin, A0.

Biochemical characterization and localization of transglutaminase in wild-type and cell-death mutants of the nematode Caenorhabditis elegans.

Transglutaminase activity was characterized in extracts of the nematode Caenorhabditis elegans using a microtiter plate method, and found to be Ca2+-dependent, optimal at pH 8.0, and to be inhibited by EGTA, ammonia, iodoacetamide and GTP. Monoclonal and polyclonal antibodies raised against human tissue transglutaminase also inhibited the activity and detected a 61-kDa protein from the worm lysate. Constitutive expression of the enzyme in the wild-type intestinal cells was revealed by immunohistochemistry. Potential protein substrates for the enzyme were found in worm lysates using a biotin-labelled amine substrate. There is a basal level of protein-bound epsilon(gamma-glutamyl)lysine cross-links, characteristic of transglutaminase activity, formed in situ in adult wild-type animals. Developmental studies have revealed that the enzyme activity is highest in adult animals, and relatively higher in L1 larvae than in other larval stages. As compared to wild types, lower transglutaminase activity has been measured in lysates of ced-3, ced-4 and ced-9 mutants. Cross-link levels were also low in ced-4 and ced-9 mutants. By contrast, the crosslink content was high in several phagocytosis mutants. The highest concentration was found in the ced-5; ced-7 double phagocytosis mutants which carry an extra number of dead cells during their lifespan. In accordance with this finding, several transglutaminase-immunopositive cells were found in both the embryos and in the head of these double phagocytosis mutants. The results suggest that a transglutaminase is involved in, or related to, the death program of cells in C. elegans and the expression and crosslinking activity of the enzyme may be perturbed in some ced mutants.

Novel Inhibitors Against the Transglutaminase-catalysed Crosslinking of Lens Proteins

Post-translational modifications by transglutaminase may contribute to the remodeling of cellular architecture in the development of lens fiber cells, and there is evidence that the enzyme may also play a role in cataract formation. It catalyses hydrolytic deamidations as well as amide exchanges on select glutamine side chains at endo positions in a small subset of proteins of the lens. Nϵ(γ-glutamyl)lysine crosslinks, the characteristic hallmarks of transglutaminase activity, were identified in polymers isolated from human cataract.Following up on our earlier studies relating to the inhibition of protein crosslinking by the Ca2+-activated transglutaminase in the lens, we have now examined the effects of 2-[(2-oxopropyl)thio]imidazolium derivatives, recently described as active site-directed inhibitors for this family of enzymes. First, we have shown that the compounds at concentrations of 1–2 μmwere effective in blocking the transamidating activities of partially purified lens transglutaminase. Then we focused on their efficacy in preventing the formation of the ca. 55 kDa β crystallin dimers in the whole lens tissue. The production of these dimers, crosslinked by Nϵ(γ-glutamyl)lysine isopeptide bridges, is an early sign of transglutaminase action in rabbit lens, and it can be readily documented by the SDS-PAGE analysis of proteins remaining in the soluble phase after brief exposure of the homogenate to Ca2+. The new compounds proved to be potent inhibitors of transglutaminase also in this preparation, preventing the crosslinking event at ca. 1 μmconcentration. Moreover, even when applied at a 1,000-fold greater concentration (2 mm),they did not interfere with the action of calpain which, similarly to the activation of the transglutaminase system, is triggered by the addition of Ca2+. The high selectivity of the new compounds for differentially blocking only the transglutaminase and not the calpain of the lens, is all the more remarkable because these two enzymes share several mechanistic and structural similarities.

The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.

Regulated Expression of Tissue Transglutaminase in Swiss 3T3 Fibroblasts: Effects on the Processing of Fibronectin, Cell Attachment, and Cell Death

Tissue transglutaminase (tTgase) catalyzes the posttranslational modification of proteins by forming Ca2+-dependent intermolecular ϵ(γ-glutamyl) lysine crosslinks; however, its physiological function is unclear despite increasing evidence for its involvement in the extracellular environment. To define where the enzyme is active and characterizes targets of crosslinking we have modulated tTgase expression in stably transformed Swiss 3T3 cell lines, generated by transfecting tTgase cDNA under the control of a tetracycline-regulated inducible promoter. Induced expression of tTgase enabled the detection of two pools of transglutaminase antigen, one intracellular and the other extracellular, which has a cellular distribution comparable to fibronectin. Incubation of cells with the fluorescent tTgase substrate fluorescein cadaverine indicated incorporation only in the extracellular matrix of healthy cells even though the amine was freely permeable to cells. Incorporation paralleled the deposition of fibronectin during fibril assembly when monitored by immunofluorescence. Fibronectin polymerization was confirmed by Western blotting. Cell surface-related tTgase was further demonstrated by preincubation of cells with tTgase antibody which led to inhibition of activity and cell attachment. Activation of the intracellular tTgase by increasing cytosolic Ca2+using ionomycin resulted in cell death accompanied by extensive crosslinking in the cytoplasm, nucleus, and cell substratum contacts of induced cells. These dead cells were not typical of those undergoing apoptosis or necrosis since they remained adherent, preserved their microtubule network, and showed little DNA fragmentation. Modulation of expression of tTgase has indicated a possible physiological function for the enzyme in cell attachment, the crosslinking of fibronectin during fibril assembly, and the maintenance of cellular integrity in a novel form of cell death.

Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia.

Transglutaminase 1 (TGase 1) is a tissue-specific enzyme which is expressed in the keratinized stratified squamous epithelia and which catalyzes straightepsilon-(gamma-glutamyl) lysine cross-links of proteins to form the cell envelope at the periphery of cornified cells. A transient expression assay using a luciferase reporter gene linked to the 2.5 kb 5' upstream region of the human TGase 1 gene (TGM1) showed phorbol ester-responsive promoter activity in cultured normal human keratinocytes. To assess its promoter activity in vivo, we generated transgenic mice expressing the Escherichia coli beta-galactosidase gene (lacZ) directed by the 5' upstream region. beta-Galactosidase histochemistry revealed that the TGM1-lacZ transgene was expressed in terminally differentiating keratinocytes in upper layers of stratified squamous epithelia in embryonic, neonatal and adult transgenic mice. The expression pattern was similar to that of endogenous TGase 1 mRNA detected by in situ hybridization. Furthermore, topical application of a phorbol ester to adult tail skin enhanced expression of the transgene as well as TGase 1 mRNA in the epidermis. Thus, the 2.5 kb 5' upstream sequence of TGM1 includes elements regulating tissue- and terminal differentiation-specific gene expression in stratified squamous epithelia.

Structure of a covalently cross-linked form of core histones present in the starfish sperm.

The post-translational modification of core histones plays an essential role in chromatin remodeling processes. We recently reported the occurrence of a novel histone modification, involving a epsilon-(gamma-glutamyl)lysine cross-link between a glutamine residue of histone H2B and a lysine residue of histone H4 in the testis of the starfish, Asterina pectinifera[Shimizu, T., Hozumi, K., Horiike, S., Nunomura, K., Ikegami, S., Takao, T., and Shimonishi, Y. (1996) Nature 380, 32]. In order to determine the complete structure of the modified histone heterodimer, p28 from both testis and sperm was purified. p28 was digested with Achromobacter lyticus protease I or Staphylococcus aureus V8 protease to give proteolytic fragments that were separated by HPLC. Amino acid analysis, sequencing, and mass spectrometric analysis of the fragments showed that the amino acid sequences of these fragments are identical to those of both histones H2B and H4, except for two NH2-terminal peptides obtained by digestion with A. lyticus protease I. One of the peptides, K8, was identical to that reported previously, and the other was a here-to-fore unidentified peptide, which was designated K10. Amino acid and positive-ion FAB-MS/MS analyses of K10 showed that it to be a fragment, derived from Gly8-Lys10 of histone H2B and Gly9-Lys16 of histone H4. The yields of K8 and K10 were calculated to be 47 and 42%, respectively, expressed as the percent of the total amount of p28 used in the experiment. Based on these data, the structure of p28 was determined to be a heterodimer, composed of histones H2B and H4, formed through a transglutaminase-catalyzed acyl transfer reaction between Gln9 of histone H2B and Lys5 or Lys12 of histone H4.

The role of transglutaminase in the rat subtotal nephrectomy model of renal fibrosis.

Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P </= 0.01. Levels of epsilon-(gamma-glutamyl) lysine cross-link correlated well with the renal fibrosis score throughout the 120 observation days (r = 0.78, P </= 0.01). Tissue homogenates showed no significant change in overall transglutaminase activity (14C putrescine incorporation assay) unless adjusted for the loss of viable tubule cells, when an increase from 5.77+/-0.35 to 13.93+/-4.21 U/mg DNA in cytosolic tissue transglutaminase activity was seen. This increase was supported by Western blot analysis, showing a parallel increase in renal tissue transglutaminase content. Immunohistochemistry demonstrated that this large increase in epsilon-(gamma-glutamyl) lysine cross-link and tissue transglutaminase took place predominantly in the cytoplasm of tubular cells, while immunofluorescence also showed low levels of the epsilon-(gamma-glutamyl) lysine cross-link in the extracellular renal interstitial space. The number of cells showing increases in tissue transglutaminase and its cross-link product, epsilon-(gamma-glutamyl) lysine appeared greater than those showing signs of typical apoptosis as determined by in situ end-labeling. This observed association between tissue transglutaminase, epsilon-(gamma-glutamyl) lysine cross-link, and renal tubulointerstitial scarring in rats submitted to SNx suggests that tissue transglutaminase may play an important role in the development of experimental renal fibrosis and the associated loss of tubule integrity.

Hydrolysis of γ:ϵ Isopeptides by Cytosolic Transglutaminases and by Coagulation Factor XIIIa

Nepsilon-(gamma-glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the gamma:epsilon linkage. Branched compounds were synthesized, in which the backbone on the gamma-side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an epsilon-aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the Nalpha position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca2+-activated human coagulation Factor XIIIa, cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with Km values of 10(-4) to 10(-5) M. Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of Nepsilon-(gamma-glutamyl)lysine isopeptides.

Epsilon(gamma-Glutamyl)lysine crosslinks are concentrated in a non-collagenous microfibrillar fraction of cartilage.

Fractions of rib cartilage were obtained by homogenization and extracted with 4 M guanidinium chloride, and the washed residue was digested with purified collagenase. Differential centrifugation of the insoluble residue from this digestion yielded a non-collagenous fraction that earlier work had shown to contain microfibrils. This material contains a much higher concentration of epsilon(gamma-glutamyl)lysine than the ribs or any of the other cartilage fractions. This transglutaminase-derived crosslink may be a common component of extracellular matrix microfibrils.

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A Novel Modification of the Lysine Residue at Position 12 of Histone H4 in Starfish Sperm

Post-translational modification of core histones is essential in processes requiring chromatin remodeling. We report here a novel modification in histones of the sperm of the starfish, Asterina pectinifera, which involves an ε-(γ-glutamyl)lysine cross-link between the glutamine residue at position 9 of histone H2B and the lysine residue at position 12 of histone H4.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: relevance to diseases of the nervous system.

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl-lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado-Joseph disease (SCA3), or dentato-rubral pallidoluysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl-lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.

ϵ-(γ-Glutamyl)lysine cross-links of spore coat proteins and transglutaminase activity in Bacillus subtilis

Spores of Bacillus subtilis are resistant to proteolytic enzymes. Some of the spore coat proteins cannot be solubilized even by treatment with strong chemical agents like dithiothreitol. The spore coat has been suggested to possess cross-links between proteins in addition to disulfide bonds. We analyzed B. subtilis spore coat proteins to find specific cross-link structures. We detected ϵ-(γ-glutamyl)lysine in the spore coat proteins after sequential protease digestion followed by HPLC. We also detected transglutaminase activity in sporulating cells, which incorporated putrescine into N,N′-dimethylcasein. This enzyme was suggested to create ϵ-(γ-glutamyl)lysine bonds between spore coat proteins.

Crosslinking of Gelatin in the Reaction with Formaldehyde: An FT-IR Spectroscopic Study

The reaction of an aqueous solution of formaldehyde with gelatin dispersed in a potassium bromide pellet is monitored in real time by FT-IR spectroscopy. Principal component regression analysis of the spectra recorded at different times is carried out. On the whole, the latter results and the observed spectral changes are in agreement with a previously reported interpretation for the kinetics of the crosslinking reaction of gelatin with formaldehyde, according to which the reaction is initialized by the lysine–methylol formation and is subsequently followed by arginine–methylol, which, in turn, reacts with lysine–methylol to originate arginine–lysine crosslinks.

Inhibition of tissue transglutaminase and ɛ (γ‐glutamyl) lysine cross‐linking in human hypertrophic scar

Immunohistochemical staining with monoclonal antibody to tissue transglutaminase was used to study cryostat sections of human skin wounds. The enzyme was found in acute wounds and chronic hypertrophic scars but not in normal mature scars. Because tissue transglutaminase is responsible for the formation of isopeptide cross-links, a two-stage high-performance liquid chromatographic analysis was used to quantitate the epsilon (gamma-glutamyl) lysine cross-link produced by various types of wound tissues. Eighteen patients with hypertrophic scars between 6 months' and 10 years' duration after injury underwent a double-blind trial with putrescine 50 mmol/L in a eutectic vehicle for 2 months under nonocclusive dressings (Biofill). For the control portion of the same or different scar, sham vehicle and non-occlusive dressing were simultaneously applied. Both scars were harvested at biopsy or elective revision surgery 2 months later. After homogenization and exhaustive proteolysis, digests were studied with the use of high-performance liquid chromatography analysis. The results of treatment were a significant decrease in the levels of isopeptide cross-link formation from 0.018 +/- .006 nmol/micromol amino acids in untreated scars to 0.008 +/- .001 nmol/micromol amino acid in the treated group (p < 0.05). The isopeptide cross-link content in treated scars was nearly as low as that in normal mature scars (0.003 +/- 0.001 nmol/micromol amino acid). These results show that cross-link formation by tissue transglutaminase activity is inhibited during treatment of hypertrophic scar by putrescine. These results support the possible therapeutic use of topical putrescine in the treatment of hypertrophic scar formation.

Influence of gelatin matrices cross-linked with transglutaminase on the properties of an enclosed bioactive material using β-galactosidase as model system

Transglutaminase (protein-glutamine: amine γ-glutamyltransferase, EC 2.3.2.13) from Streptoverticillium mobaraense has been used to stabilize immobilisates produced with β-galactosidase (β- d-galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae and acid-processed gelatins of different qualities as support. The isopeptide level of N ε-(γ- l-glutamyl)- l-lysine bonds formed by transglutaminase was determined to estimate their influence on the kinetic properties of the enclosed β-galactosidase. An HPLC procedure using precolumn derivatization of the gelatin hydrolysates with FMOC-chloride was chosen which permits the analysis of cross-linked lysine with satisfactory precision. Depending on the gelatin quality, the degree of cross-links necessary for the transformation of gelatin into an insoluble protein was in the range 0.3–32.3% of the available lysine residues. β-Galactosidase was entrapped in the gelatin matrices with a yield of 8–46% of the initial activity. Long reaction times for cross-linking were due to low yields rather than to the number of isopeptide bonds. Repeated use of the immobilisates did not lead to an appreciable loss of activity. The V max of β-galactosidase were diminished by immobilization caused by a tighter package of the protein chains rather than by the extent of cross-links, while the obtained K m values of the free enzyme and the immobilisates were quite similar. Also, the pH and temperature optima of the free enzyme and the gelatin immobilisates differ only slightly. The data suggest that the immobilization procedure only moderately affects the activity of enzymes catalysing the reaction of a small compound if gelatin with high jelly strength is cross-linked in a 10% solution with transglutaminase.

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

Site-Directed Mutation in Conserved Anionic Regions of Guinea Pig Liver Transglutaminase

Transglutaminases (EC 2.3.2.13) catalyze the formation of ϵ-(γ-glutamyl) lysine cross-links and the substitution of primary amines for the γ-carboxamide groups of protein-bound glutamine residues. There are conserved anionic regions in transglutaminases, some of which are thought to be possible calcium-binding sites. By site-directed mutagenesis, three mutant forms of recombinant guinea-pig liver transglutaminase, in which some acidic amino acid residues in two conserved regions became nonionic, were expressed in Escherichia coli: TGM1, with Asp-231 and -232 changed to Asn; TGMS, with Glu-445, -448, -449, -450, and -452 changed to Gin; and TGM3, with the mutations of both TGM1 and TGM2. The size and level of synthesis of the mutant proteins were unchanged when monitored by immunoblotting. All mutants retained enzyme activity, and their apparent K m values for substrates during histamine incorporation into acetyl α s1-casein were similar to those of the wild-type enzyme, but their V max values were smaller. The deamidation rate of glutamine residues in the acetyl α s1-casein was unaffected, but the rate of protein cross-linking catalyzed by these mutants was very low. All mutations caused with the enzyme a decrease in the sensitivity to activation by calcium and an increase in the sensitivity to inhibition by GTP. These results indicated that the negative charges of some acidic amino acid residues in the two conserved anionic regions of transglutaminase are not essential for its activity but the loss of their negative charges affects some catalytic properties.