Lysine Bonds Research Articles

Biochemistry of Transglutaminases and Cross-Linking in the Skin

Transglutaminase is a calcium-dependent enzyme found widely in nature. It catalyzes the formation of epsilon-(gamma-glutamyl)lysine bonds that participate in processes varying from fibrin clot formation to epidermal cell envelope formation. Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the covalent cross-linking of a soluble cytoplasmic substrate into large polymers to form the cornified envelope that lines the inner membrane of keratinocytes in the stratum corneum. The soluble precursor from epidermis has been named keratolinin, and from keratinocyte culture, it has been named involucrin. Hair follicle transglutaminase is biochemically and immunochemically distinct from its epidermal counterpart. It has been localized to the inner root sheath and medulla of the hair follicle. The substrate of hair follicle transglutaminase has been poorly defined but appears to be rich in the amino acid citrulline. Transglutaminase has been shown to be an important marker of normal differentiation. There is a rise in its activity at the time of keratinization, and transglutaminase activity has been shown to be greatly decreased in basal cell epithelioma and in psoriasis. Keratinocyte cell culture has proven most helpful in delineating the processes of normal differentiation and keratinization, since the formation of the cell envelope in culture appears to parallel the formation in vivo.

Mechanism and basis for specificity of transglutaminase-catalyzed epsilon-(gamma-glutamyl) lysine bond formation.

Introduction Molecular Features and Catalytically Active Forms of the Transglutaminases Reactions Catalyzed by the Transglutaminases The Mechanism of Action of the Transglutaminases Substrate-Active Site Interactions and the Relationship of Conformations to Substrate Specificity and Catalysis Concluding Remarks

Calcium induction of transglutaminase and the formation of ɛ(γ-glutamyl)lysine cross-links in cultured mouse epidermal cells

Summary Terminal differentiation can be induced in cultured mouse epidermal cells by increasing the extracellular calcium concentration from 0.07 mM to 1.2 mM. An ultrastructural characteristic of this process is the formation of a cornified envelope in which membrane proteins are cross-linked by ɛ(γ-glutamyl) lysine bonds. This process is catalyzed by the calcium-requiring enzyme transglutaminase. Elevation of medium calcium levels increases both the activity of transglutaminase and the fraction of lysine residues involved in ɛ(γ-glutamyl)lysine cross-links.

Studies on the development of the definitive cell type of embryonic epidermis using the cross-linked envelope as a differentiation marker

During their late stages of differentiation, keratinocytes of epidermis form beneath the plasma membrane a protein envelope cross-linked by ϵ-(γ-glutamyl)lysine bonds. This property is a reliable and specific marker for the keratinocyte and has been used to study the development of this cell type in the skin during the embryonic life of the rabbit. Up to halfway through gestation, virtually no cells of the epithelium have the ability to make such an envelope. Between the 16 and 20th days of gestation, some cells acquire this ability, and by birth most can make envelopes. It may be concluded that prior to 16 days' gestation, the external epithelium consists of a cell type that is a precursor of the keratinocyte. In culture, colonies of typical keratinocytes develop from single epithelial cells of embryos 23 days or older, but not from the cells of epithelia of 14-day or younger embryos. Colonies of cells resembling keratinocytes can be grown from epithelia of 16- to 20-day embryos; the cells of these colonies develop envelopes with a somewhat higher frequency than the cells in the epithelia from which they were derived but not nearly as well as the cells grown from older epithelia. The epithelium during this period contains cells at an intermediate stage of development. The epithelial cells of 14-day embryos will not differentiate into keratinocytes in surface culture. In contrast, cultured explants of the 14-day epithelium do support the differentiation of the precursor cells into keratinocytes that are able to form cross-linked envelopes with a high frequency, like those of advanced epithelium. When primitive epithelium and underlying connective tissue of early embryos are disaggregated with trypsin and the cells reassembled in the form of aggregates, a significant though small number of keratinocytes develops from epithelial cells of 14-day embryos, and a considerably larger number from those of 16-day embryos. The failure of the differentiation in surface culture could be due to the absence of proper mesenchymal influence.

Further enhancement of analgesic activity: enkephalin analogs with terminal guanidino group

There is a great body of evidence that a basic amino-terminal is essential for the opioid activity of enkephalins, Tyr-Gly-Gly-Phe-Met and -Leu [ 1 I, and their analogs. Removal of the amino-group of Tyr results in a practically inactive peptide [2], as does its acetylation [3]. Introduction of two methylgroups into the terminal amino-moiety brings about a substantial loss in the biological activity [4], while mono-methylation decreases the potency in the mouse vas deferens preparation but enhances activity in the guinea-pig ileum [S]. It was tempting to speculate that interaction between the peptide N-terminus and its counterpart on the receptor (presumably a carboxyl-group) might be favourably influenced by replacing the terminal amino-group with a guanidino function. The resonance stabilized guanidinium cation has the capacity to interact.with some bidentate structure in the receptor site establishing hydrogen bridges in addition to the electrostatic interaction. One could anticipate that the improved binding properties of the peptide N-terminus would result in enhanced biological activity. It has also to be taken into account, however, that this replacement leads to a distal shift of the positive charge (~1.2 A). Such modification at the e-amino group of the lysine side chain in certain peptides, i.e., conversion of lysine into homoarginine residue, makes the lysine bond resistant to trypsin [6]. Thus trypsin seems to be unable to combine simultaneously with both the carbonyl-group and the positive charge of the basic residue when the distance between these is greater than occurs in a lysine or arginine residue. In this context one may recall that papain, which is less specific than trypsin, can hydrolyse homoarginine

New findings on epidermal transglutaminase substrates.

Recent studies suggest the presence of natural substrates for epidermal transglutaminase in buffer extracts of epidermis. We have investigated a soluble substrate of 36,000 molecular weight present in neutral buffered extracts of cow snout epidermis. When purified to homogeneity, this protein was cross-linked by transglutaminase with 70-80% efficiency into a series of high molecular weight soluble polymers and a highly insoluble protein aggregate. The epsilon-(gamma-glutamyl) lysine bond was present in both high molecular weight soluble polymers and insoluble aggregate, but was absent from the initial 36,000 molecular weight protein. Substrate cross-linking was completely inhibited by EDTA, iodoacetamide, and 25 mM putrescine. When subjected to electrophoresis in gels containing sodium dodecyl sulfate (SDS), 90% of the 36,000 molecular weight protein was reduced to subunits of 8,000-10,000 molecular weight. We postulate intracellular synthesis of the 36,000 molecular weight initial substrate molecule, followed by insolubilization and cross-linking at the cell membrane.

Digestibility of acetylated and succinylated proteins by pepsin-pancreatin and some intracellular peptidases.

The molecular sizes of hydrolysates of acetylated and succinylated caseins by pepsin-pancreatin were examined by gel filtration on Sephadex G-15. There were more large peptides (average residual number > 15) in the hydrolysates of the acylated caseins than there were in the hydrolysate of unmodified casein. In these large peptides from the acylated caseins the contents of Ne-acyl-lysines were high. The digestibility of Ne-acetyl and Ne-succinyl lysine bonds in peptides by aminopeptidases [(EC 3.4.11.1) and (EC 3.4.11.2)] and watermelon carboxypeptidase [model enzyme of cathepsin A (EC 3.4.16.1)] was examined using digest of acylated caseins by pepsin, trypsin and α-chymotrypsin and some synthetic peptides. All peptidases released either Ne-acetyl or Ne-succinyl-lysine from peptides.The hydrolytic processes of acetylated and succinylated proteins before and after intestinal absorption are discussed.

Coupling of amines to and cross-linking of endogenous cytosol or membrane proteins by hepatic transglutaminase

Endogenous protein acceptors for rabbit transglutaminase were demonstrated in the cytosol fraction of liver homogenates by the coupling of labeled compounds containing primary amino groups, such as putrescine, and by the apparent cross-linking of proteins, as indicated by a low mobility band observed in gel electrophoresis in 0.1% sodium dodecyl sulfate. When Ca 2+ was omitted or when iodoacetamide was added to either of these reactions, no coupling or cross-linking occurred. Cross-linking of proteins of isolated rabbit liver plasma membranes could be demonstrated in the presence of purified rabbit liver transglutaminase, Ca 2+, and a reducing agent. A marked diminution of at least two peaks corresponding to proteins of high molecular weight (> 150,000 daltons) was observed in densitometric scans of gels of proteins from membranes or cytosol cross-linked by the enzyme. It is proposed that these proteins participated in the crosslinking reaction. Since cross-linking was inhibited only weakly by relatively high concentrations of various amines, it is probable that a physiological role of hepatic transglutaminase is the catalysis of ϵ(γ-glutamyl) lysine bonds between cellular proteins, a process that might be controlled by the amount of intracellular Ca 2+ available to the enzyme.

Presence of the ϵ-(γ-glutamic)lysine crosslink in cellular proteins

The ϵ-(γ-glutamic)lysine bond is a covalent interaction which has been found to crosslink polypeptide chains of a number of extracellular proteins. Among known covalent bonds crosslinking protein chains, it is unique in that it is formed directly by enzymatic catalysis, a property which may also endow Glu-Lys crosslink formation with important intracellular functions. We found glutamic-lysine bonds to be present in the procaryote, Escherichia coli, in primitive eucaryotes such as the slime mold, Physarum polycephalum, and the ciliate, Paramecium aurelia, and in muscle cells of a bird and a mammal. Our data show that, although Glu-Lys bonds occur in low concentrations in cellular proteins, they are nevertheless widely distributed. Evidence is also presented indicating that the low levels of the Glu-Lys bonds we measure in the proteins of various cells types are not artifacts of our analytical procedures.

Epsilon-(gamma-Glutamyl)lysine cross-links in human stratum corneum.

epsilon-(gamma-Glutamyl)lysine has been found in human stratum corneum in the fraction containing the alpha helical fibrous proteins (keratins) and other high molecular weight proteins. The S-carboxymethylated fractions were enzymatically digested with pronase, carboxypeptidase A and B, leucine aminopeptidase and prolidase, and epsilon-(gamma-glutamyl)lysine isolated from digests by gel filtration and cation ion exchange chromatography. Acid hydrolysis of the purified epsilon-(gamma-glutamyl)lysine yielded equimolar amounts of lysine and glutamic acid, and end group analysis of the peptide by dansylation (application of 5-dimethylaminonaphthalene-1-sulfonyl) confirmed the isomer assignment to be epsilon-(gamma-glutamyl)lysine. About 9 nmol of the peptide per mg of protein were found in the fraction by isotope dilution after the enzymatic digestion. These results suggest that proteins in stratum corneum may be covalently cross-linked through epsilon-(gamma-glutamyl)lysine bonds.

Keratin Cross-Linking And Epidermal Transglutaminase. A Review with Observations on the Histochemical and Immunochemical Localization of the Enzyme

Enzymes known as transglutaminases mediate cross-linking of polypeptide chains by epsilon-(gamma-glutamyl) lysine bonds. Such bonds stabilize structural proteins of many tissues; transglutaminases specific for these tissues have been identified. A calcium- and sulfhydryl-dependent transglutaminase with a molecular weight of 55,000 has been purified from bovine snout epidermis and used to elicit a specific antiserum to the enzyme. Sites of epidermal transglutaminase activity have been localized in the cytoplasm of upper malpighian and granular cells by two complementary methods. When thin-tissue sections were incubated with a fluorescent lysine analog(dansyl cadaverine) and calcium, tissue acceptor sites became fluorescent. Localization was confirmed by fluorescein-conjugated antibody labeling of the enzyme in situ. These observations indicate that epidermal transglutaminase cross-links epidermal proteins during the final stages of keratinization.

Increase in fibrinogen and fibrin-related antigen in human serum due to in vitro lysis of fibrin by thrombin.

In vitro lysis of fibrin, as indicated by increased fibrinogen-fibrin-related antigen (FR-antigen) in serum is usually seen when whole blood, or plasma, or highly purified fibrinogen prepared by several different procedures is clotted and kept at temperatures above 0 degrees C. This increase is both time and temperature dependent, occurs despite the addition of various plasmin and cathepsin inhibitors, and is probably caused by thrombin evolved during clotting and/or added in vitro. In these experiments, the FR-antigen was measured by a sensitive, reproducible hemagglutination inhibition immunoassay adapted to the AutoAnalyzer. Serum from whole blood contained more than serum from plasma, and fibrin rather than fibrinogen proved to be essential for the in vitro lysis. The phenomenon was also caused by Arvin or Reptilase, suggesting that splitting of one or more arginine or lysine bonds in fibrin may be at least partially responsible. To obtain minimal levels of FR-antigen (< 0.5 mug/ml), plasma is clotted for 4 hr at 0 degrees C with 1.0-5.0 U/ml thrombin, CaCl(2) (0.0125 mole/liter), and epsilon aminocaproic acid (0.05 mole/liter). Slightly higher levels, probably adequate for clinical diagnosis, are obtained by 10-30 min clotting at room temperature. Since endogenous and/or exogenous thrombin is essential for the collection of serum FR-antigen, all the FR-antigen found in normal serum probably results from an irreducible amount of in vitro lysis rather than from continuous intravascular clotting and fibrinolysis.

Transglutaminase from hair follicle of guinea pig (crosslinking-fibrin-glutamyllysine-isoenzymes-purified enzyme).

Two transglutaminases are found in homogenates of the inner root sheaths of guinea pig hair-follicles. One is indistinguishable from the well-characterized liver transglutaminase [J. Biol. Chem., 246, 1093 (1971)]. The other, which is present in far greater quantity, has not been detected in other organs or tissues. Gel filtration and polyacrylamide gel electrophoresis studies indicate that the native hair-follicle enzyme, of molecular weight 54,000, is composed of two subunits of identical molecular weight. Specificity studies suggest that the intermolecular cross-linking of fibrin and fibrinogen that is catalyzed by this enzyme is a result of the formation of epsilon(gamma-glutamyl)lysine bonds. The probable participation of hair-follicle transglutaminase in the formation of these cross-links in the proteins of hair is discussed.