ABSTRACT
BACKGROUND: Gastric cancer (GC) is one of the most significant health problems worldwide. Some studies have reported associations between Phospholipase C epsilon 1 (PLCE1) single-nucleotide polymorphisms (SNPs) and GC susceptibility, but its relationship with GC prognosis lacked exploration, and the specific mechanisms were not elaborated fully yet. This study aimed to further explore the possible mechanism of the association between PLCE1 polymorphisms and GC. MATERIALS AND METHODS: A case-control study, including 588 GC patients and 703 healthy controls among the Chinese Han population, was performed to investigate the association between SNPs of PLCE1 and GC risk by logistic regression in multiple genetic models. The prognostic value of PLCE1 in GC was evaluated by the Kaplan-Meier plotter. To explored the potential functions of PLCE1, various bioinformatics analyses were conducted. Furthermore, we also constructed the spatial structure of PLCE1 protein using the homology modeling method to analyze its mutations. RESULTS: Rs3765524 C > T, rs2274223 A > G and rs3781264 T > C in PLCE1 were associated with the increased risk of GC. The overall survival and progression-free survival of patients with high expression of PLCE1 were significantly lower than those with low expression [HR (95% CI) = 1.38 (1.1-1.63), P < 0.01; HR (95% CI) = 1.4 (1.07-1.84), P = 0.01]. Bioinformatic analysis revealed that PLCE1 was associated with protein phosphorylation and played a crucial role in the calcium signal pathway. Two important functional domains, catalytic binding pocket and calcium ion binding pocket, were found by homology modeling of PLCE1 protein; rs3765524 polymorphism could change the efficiency of the former, and rs2274223 polymorphism affected the activity of the latter, which may together play a potentially significant role in the tumorigenesis and prognosis of GC. CONCLUSION: Patients with high expression of PLCE1 had a poor prognosis in GC, and SNPs in PLCE1 were associated with GC risk, which might be related to the changes in spatial structure of the protein, especially the variation of the efficiency of PLCE1 in the calcium signal pathway.
ABSTRACT
Glycyrrhetinic acid 3-O-mono-ß-d-glucuronide (GAMG), which possesses a higher sweetness and stronger pharmacological activity than those of glycyrrhizin (GL), can be obtained by removal of the distal glucuronic acid (GlcA) from GL. In this study, we isolated a ß-glucuronidase (TpGUS79A) from the filamentous fungus Talaromyces pinophilus Li-93 that can specifically and precisely convert GL to GAMG without the formation of the by-product glycyrrhetinic acid (GA) from the further hydrolysis of GAMG. First, TpGUS79A was purified and identified through matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) and deglycosylation, indicating that TpGUS79A is a highly N-glycosylated monomeric protein with a molecular mass of around 85 kDa, including around 25 kDa of glycan moiety. The gene for TpGUS79A was then cloned and verified by heterologous expression in Pichia pastoris TpGUS79A belonged to glycoside hydrolase family 79 (GH79) but shared low amino acid sequence identity (<35%) with the available GH79 GUS enzymes. TpGUS79A had strict specificity toward the glycan moiety but poor specificity toward the aglycone moiety. Interestingly, TpGUS79A recognized and hydrolyzed the distal glucuronic bond of GL but could not cleave the glucuronic bond in GAMG. TpGUS79A showed a much higher catalytic efficiency on GL (kcat/Km of 11.14 mM-1 s-1) than on the artificial substrate pNP ß-glucopyranosiduronic acid (kcat/Km of 0.01 mM-1 s-1), which is different from the case for most GUSs. Homology modeling, substrate docking, and sequence alignment were employed to identify the key residues for substrate recognition. Finally, a fed-batch fermentation in a 150-liter fermentor was established to prepare GAMG through GL hydrolysis by T. pinophilus Li-93. Therefore, TpGUS79A is potentially a powerful biocatalyst for environmentally friendly and cost-effective production of GAMG.IMPORTANCE Compared to chemical methods, the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-ß-d-glucuronide (GAMG), which has a higher sweetness and stronger pharmacological activity than those of GL, via catalysis by ß-glucuronidase is an environmentally friendly approach due to the mild reaction conditions and the high yield of GAMG. However, currently available GUSs show low substrate specificity toward GL and further hydrolyze GAMG to glycyrrhetinic acid (GA) as a by-product, increasing the difficulty of subsequent separation and purification. In the present study, we succeeded in isolating a novel ß-glucuronidase (named TpGUS79A) from Talaromyces pinophilus Li-93 that specifically hydrolyzes GL to GAMG without the formation of GA. TpGUS79A also shows higher activity on GL than those of the previously characterized GUSs. Moreover, the gene for TpGUS79A was cloned and its function verified by heterologous expression in P. pastoris Therefore, TpGUS79A can serve as a powerful biocatalyst for the cost-effective production of GAMG through GL transformation.
