Glutamine, Proline, Ornithine, Citrulline
5.3 Glutamine
5.3.3 Transglutaminases
As discussed in section 2.1.2 , a number of proteins contain isopeptide bonds between theε - amino group of a lysine residue and the γ - carboxyl group of a glutamate or aspartate residue, which reduce the digestibility of the protein and the availability of lysine. The availability of lysine in these isopeptide links is higher than might be expected, because some of the isopeptides are absorbed from the small intestine and are substrates forγ - glutamyl cyclotrans-ferase (Yasumoto & Suzuki, 1990 ; see section 5.4.5 ).
The ε amino lysine – γ carboxyl glutamate bonds are formed by post synthetic modifi cation of the protein, catalyzed by transglutaminases. The carboxyl group of the isopeptide bond is derived from a glutamine residue in the precursor protein, as shown in Figure 5.4 . The various mammalian transglutaminases show signifi cant sequence homology; they are members of the papain - like superfamily of cysteine proteases and have a conserved Table 5.1 Glutamine - dependent amidotransferases.
anthranilate synthase EC 4.1.3.27 Figure 9.3
asparagine synthase EC 6.3.5.4 Section 1.3.2.5
carbamoyl phosphate synthase EC 6.3.5.5 Figures 1.11 and 1.16
CTP synthase EC 6.3.4.2 Figure 1.11
formylglycinamide amidotransferase EC 6.3.5.3 Figure 1.6
glutamate synthase EC 1.4.1.13 Figure 1.4
GMP synthase EC 6.3.5.2 Figure 1.7
imidazole glycerol phosphate synthase no EC number Figure 8.1
iminodeoxychorismate synthase no EC number in folic acid biosynthesis
PRPP synthetase EC 2.7.6.1 Figure 1.6
Figure 5.4 Formation of isopeptide bonds by transglutaminase.
Transglutaminase EC 2.3.2.13, also known as clotting factor XIIIa or fi brin stabilizing factor.
H C C O
CH2
NH
CH2
CH2
CH2
NH3+
C H C O
CH2 NH CH2 C NH2
O
NH4+
H C C O
CH2
NH
CH2
CH2
CH2
C H C O
CH2
NH CH2
C O transglutaminase NH
Tyr - Gly - Gln - Cys - Trp sequence at the catalytic site. The cysteine residue forms a thioester with the glutamine residue in the substrate, displacing ammonium (Beninati & Piacentini, 2004 ).
Transglutaminases (either from animal blood or from bacterial sources) are used in food manufacture to bind proteins in products such as surimi, fi sh balls and mechanically recovered meat products, where they are commonly known as ‘ protein glue ’ . They are also used to cross - link pectin and chitosan to proteins in order to produce edible fi lms for food packaging and coating, and they are important in tissue engineering and textile and leather process-ing (Porta et al ., 2011 ).
Transglutaminase 1 is a membrane - bound enzyme in the plasma membrane of epidermal cells. It catalyzes the formation of a highly cross - linked protein layer, the cornifi ed or cross - linked cell envelope, inside the cell. It also cata-lyzes the formation of ester bonds between glutamine residues in the precur-sor protein to ω - hydroxyceramides to form the lipid envelope of surface epidermal keratinocytes. Keratinocytes migrate to the surface of the skin as an interlocking layer of dead stratum corneum cells.
Genetic lack of transglutaminase 1 leads to the condition of lamellar ich-thyosis, which is characterized by defective epidermal barrier function. There is reduced activity of transglutaminase 1 in psoriasis and basal cell epitheli-oma (Peterson et al ., 1983 ).
Recessive X - linked lamellar ichthyosis is due to the accumulation of cholesterol 3 - sulphate in the epidermis, which inhibits the cross - linking and esterifi cation actions of transglutaminase 1. The enzyme now catalyzes deamidation of glutamine residues in the precursor protein instead of trans-glutamination, because cholesterol 3 - sulphate distorts the active site of the enzyme, permitting the access of water and hydrolysis (Nemes et al ., 2000 ).
Transglutaminase 2 is the so - called tissue transglutaminase, and it has a dual action; in addition to its transglutaminase activity, the carboxy - terminal functions as a G - protein. The activity of transglutaminase 2 increases mark-edly in apoptosis, when it has a role in forming an intracellular scaffold of insoluble protein, stabilizing intracellular membranes and preventing the release of lysosomal enzymes. Over - expression of transglutaminase 2 in cells in culture leads to increased susceptibility to apoptosis, and knocking out the enzyme with antisense cDNA leads to a decreases in both spontaneous and induced apoptosis.
Transglutaminase 2 is involved in the pathogenesis of neurodegenerative, auto - immune and infl ammatory diseases, including coeliac disease. Inhibitors or antibody targeting have therapeutic potential in these conditions. Trans-glutaminase 2 is a member of the α h family of G - proteins, involved in the activity of theα 1 adrenoceptor, leading to the activation of phospholipase C.
Adrenergic stimulation leads to inhibition of transglutaminase activity and an increase in G - protein signalling. It is also involved in receptor - mediated
endocytosis (Autuori et al ., 1998 ; Caccamo et al ., 2010b ; Levitzki et al ., 1980 ; Nakaoka et al ., 1994 ; Park et al ., 2010 ; Piacentini et al ., 1991 ).
Transglutaminase 3 is a pro - enzyme in terminally differentiating epidermal and hair keratinocytes that requires activation by partial proteolysis. Like transglutaminase 1, it acts in the formation of the cornifi ed cell envelope of epidermis and hair follicles. It is the major auto - antigen in coeliac disease and dermatitis herpetiformis, both of which are due to sensitivity to the gliadin fraction of gluten, the major protein of wheat and other cereals.
Transglutaminase is involved in the deamidation of glutamine residues in gliadin, but then catalyzes the formation of highly immunogenic aggregates (Beninati & Piacentini, 2004 ; Sardy et al ., 2002 ).
Transglutaminase 4 is the prostate - specifi c transglutaminase, although it is also expressed in a number of other tumour cell lines. In rodents, it is respon-sible for formation of the copulatory plug in the female genital tract after coitus, and it may mask the immunogenicity of the male gametes. The mRNA for the human enzyme undergoes differential splicing in the development of benign prostate hyperplasia and prostate cancer, and it has a role in the adhe-sion of prostate cells to epithelial cells (Cho et al ., 2010 ; Davies et al ., 2007 ; Jiang et al ., 2009 ; Jiang & Ablin, 2011 ).
Blood clotting Factor XIII (section 5.6.1 ) is the zymogen of a transglutami-nase (fi broligase) that catalyzes stabilization of the fi brin clot by forming inter - chain isopeptide bonds. It also protects the newly formed fi brin against fi brinolysis by binding plasmin inhibitor to the fi brin network. It consists of two A - chains (that have the active site) and two B - chains (with no catalytic activity). The zymogen is activated by partial proteolysis, releasing a 36 - amino acid peptide, followed by a conformational change to expose the catalytic site cysteine. This partial proteolysis is catalyzed by thrombin, but only occurs if uncross- linked fi brin is also present. The A chain of Factor XIII is also expressed in platelets, monocytes, macrophages and the placenta, and it is activated non - proteolytically in response to an increase in the intracellular concentration of calcium ions, which leads to a conformational change, expos-ing the catalytic site (Bereczky & Muszbek, 2011 ; Board et al ., 1993 ; Lewis et al ., 1985 ; Muszbek et al ., 1996, 2011 ).
Transglutaminases also catalyze the incorporation of amines into proteins, formingγ - glutamyl amides by displacement of the amide group of glutamine residues. Putrescine, spermine and spermidine (see section 5.8 ) are good substrates for incorporation, and either or both of the primary amino groups can be linked to glutamate. Reaction of the free amino group of a γ - glutamyl amide with a second glutamine residue leads to formation of inter and intra chain cross - links in the target protein. There is a great deal of specifi city for the environment of the glutamine residues that are amidated, but the trans-glutaminases generally have a broad specifi city for the polyamines that they incorporate (Lentini et al ., 2004 ).
Excessive stimulation of glutamate receptors in the central nervous system leads to over - stimulation of calcium - dependent kinases and other down-stream effectors, as a result of a massive infl ux of calcium ions via the iono-tropic glutamate receptors, leading to neurodegeneration. At least part of the pathogenesis of this excitotoxicity is due to the activation of calcium dependent transglutaminases, leading to cross - linking of proteins.
There is some evidence that transglutaminases are also involved in cross linking neuronal proteins in Alzheimer ’ s and Huntington ’ s diseases, so meas-urement ofε - ( γ - glutamyl)lysine isopeptides in plasma may be a useful marker of tissue damage in these and other conditions. Alzheimer ’ s, Huntington ’ s and Parkinson’ s diseases are all associated with proteolytic stress due to incorrectly folded and isopeptide cross linked proteins that overload the ubiquitin proteasome system (section 2.1.5.2 ). Tau protein, amyloid - beta, α - synuclein and huntingtin are all substrates for transglutaminase (Caccamo et al ., 2004, 2010a ).