The present study investigates the GGT enzyme in parasitic helminth, Gigantocotyle explanatum (GeGGT), which may shed light on a previously unexplored area of tegumental biology pertaining to the GGT enzyme. The GGT enzyme from adult G. explanatum worms was successfully isolated, purified, and some of its biochemical properties, particularly the kinetic parameters, have been studied. Adult G. explanatum worms were subjected to a saponin-containing membrane disrupting solution, which resulted in the tegumental layer being delicately stripped off. Scanning electron micrographs revealed the purity of the plasma membrane separation, which was free of any lesions or physical damage. The GGT enzyme is evidently membrane-bound in this amphistome parasite as indicated by the enrichment of GGT activity by several folds observed following isolation of the surface plasma membrane. The enriched enzyme activity in the vicinity of the tegumental areas of these amphistomes was further supported by the histo-enzymological localization of GeGGT.
GeGGT needs to be membrane solubilized in order to be evaluated for activity because it is a membrane-anchored enzyme. To achieve the necessary solubilization procedure, a variety of detergents and salts are being used. 1% Triton X-100 produced the most solubilized protein with the highest enzyme activity when the isolated membrane pellets were solubilized in different detergents like CHAPS, Tween-20, and Triton X-100 for various times (results not shown). It was also noticed that the rate of solubilization also affected the rate at which the enzyme penetrated the polyacrylamide gels. The Triton X-100 solubilized membrane samples readily penetrated the 6 percent Native gels, resulting in a sharp band of about 120 kDa, while the insolubilized membrane samples did not. This may be because of the high hydrophobicity and aggregative properties of the insolubilized membrane samples, which are a typical characteristic of membrane proteins.
The amphistome enzyme GeGGT was effectively purified to an apparent homogeneous form and bound to the Con-A Sepharose. As determined by SDS-PAGE, the purified enzyme appeared to be dimeric and produced two subunits of 65.5 kDa and 55 kDa. This finding explains the typical property of the GGT enzyme, which is the dissociation of enzyme in its different subunits in the presence of SDS; heavy subunit with higher molecular weight and light subunit with lower molecular weight. The molecular weights of the two subunits supported the 120 kDa molecular weight of the native form. The molecular weights of the two subunits were found to be different in comparison to mammalian GGT subunits (bovine liver 68 kDa and 27 kDa, rat kidney 46 kDa and 22 kDa), but fairly similar to those of A. suum GGT subunits, which were reported to be of 43 and 30 kDa (Goldberg, 1980; Furukawa et al., 1983; Hussein and Walter, 1996). It has been noted that the size heterogeneity of the GeGGT subunits closely resembles that of the GGT of the worm parasite A. suum. The GeGGT described in this study exhibited a difference of just 11.5 kDa, which was found to be close to A. suum GGT where a difference of 13 kDa was identified between the two subunits. Normally most GGTs published show a size heterogeneity of a minimum of 20 kDa between their two subunits. In this regard, some bacterial GGTs, such as those from E. coli and B. pumilus, only show 15 kDa and 16–17 kDa variances between their subunits, respectively (Suzuki et al., 1986; Moallic et al., 2006). The large component (65.5 kDa) of GeGGT shown 58.3% identity with B. malayi GGT (NCBI: txid6279), while the light subunit of GeGGT (55 kDa) displayed 26.33% identity with that previously reported GGT. The homology of GeGGT also revealed similarities with other helminth parasites, including Schistosoma mansoni (NCBI: txid6183), Schistosoma haematobium (NCBI: txid6185), Loa loa (NCBI: txid7209), and A. suum (NCBI: txid6253), with light and heavy chain similarities of 50 and 21.47%, 27 and 21%, 35.5 and 31%, 33 and 23%, respectively. Due to its higher percentage of similarity with other helminth GGTs than the heavier subunit, it is concluded that the lighter subunit of GeGGT may have been evolutionary conserved.
According to biochemical analyses, GeGGT that had been affinity purified had the highest specific activity (62.48 U/mg protein) in comparison to somatic extract (0.33 U/mg protein), showing 189.33-fold increase in enzyme enrichment after con-A Sepharose affinity purification. GGT has also been purified from a variety of organisms, including certain helminth species like A. suum (Hussein and Walter, 1996), B. malayi (Lobos et al., 1996)d cervi (Gupta et al., 2005), while the bacterial and human GGT has been described up to its crystalline state (Suzuki et al., 1986; Okada et al., 2006; West et al., 2013). The con-A purified GGT from B. subtilis (Ogawa et al., 1991)d suum (Hussein and Walter, 1996) similarly shown somewhat comparable specific activity, 65 U/mg protein and 62.9 U/mg protein, respectively. It was shown that the specific enzyme activity dramatically increased when the concentration of the substrate, α-methyl glucopyranoside, increased up to a maximum concentration of 0.2 M in the elution buffer, during the affinity purification process of GeGGT. Shaw et al. (1978) used a substrate concentration of 0.2 M to successfully elute human liver GGT, while Furukawa et al. (1983) used a substrate concentration of 0.1 M to successfully elute bovine liver GGT.
The Michaelis constant, Km, of purified GeGGT is calculated to 2.43 ± 0.059 mM. The Km value of GeGGT is consistent with the GGTs of A. suum GGT (1.28 mM), bovine liver (0.8 mM), human kidney (0.8 mM), and B. subtilis SK 11.004 (1.73 mM) (Hussein and Walter, 1996; Miller et al., 1976; Furukawa et al., 1983; Shuai et al., 2011). The pH is one of the most important parameters influencing the GGT activity. The GeGGT is pH-optimized at 8, but there is still significant enzyme activity (73%) at pH 9; nevertheless, additional increases in pH considerably (p < 0.001) reduced the enzyme activity. The enzyme activity was strongly impacted by both the acidic range and the alkaline range above optimal pH. The pH optimal of GeGGT observed in the current work is equivalent to the pH optimum of GGTs observed in various human tissues, rabbit liver, E. coli, B. pumilus, B. subtilis TAM-4, and A. suum, which were observed between the pH 8 and pH 9.(Miller et al., 1976; Shaw et al., 1978; Bagrel et al., 1981; Suzuki et al., 1986b; Hussein and Walter, 1996; Sener and Yardimci, 2005; Moallic et al., 2006; Abe et al., 2009).
Other biochemical characterizations revealed the optimal GeGGT activity at temperature 37°C and incubation time of 15 min. Given that 78% and 25%, respectively, of the enzyme activity were observed after incubation at 50°C and 60°C, the GeGGT seems to be a remarkably thermo stable enzyme. GeGGT's thermostability is also found to be time-dependent, as the enzyme loses up to 80–90% of its initial activity after 20 minutes at a temperature of 37°C. An earlier study on A. suum GGT indicated that the enzyme performed best at a temperature of 37°C and that its activity was linear for the first 30 minutes of incubation (Hussein and Walter, 1996). The most popular inhibitor, DON, permanently binds at the acceptor site of the enzyme and completely suppresses the GeGGT activity after about 40 min of incubation at ambient temperature. These outcomes are consistent with earlier studies on GGT inhibition, where Moallic et al. (2006) used a variety of inhibitors, including 1 mM DON for 1 hour, which completely inhibited the GGT activity of B. pumilus. 1 mM DON exhibited a 67% enzyme inhibition for the suppression of human kidney GGT after 15 minutes of incubation (Tate and Ross, 1977). The molecular docking studies also confirmed the high binding affinity of DON towards the GGT molecule.