Cloning and characterization of glycogen branching and debranching enzymes from the parasitic protist Trichomonas vaginalis

Abstract

The protist Trichomonas vaginalis is an obligate parasite of humans and the causative agent of trichomoniasis, a common sexually transmitted infection. The organism has long been known to accumulate glycogen, a branched polymer of glucose, and to mobilize this reserve in response to carbohydrate limitation. However, the enzymes required for the synthesis and degradation of glycogen by T. vaginalis have been little studied. Previously, we characterized T. vaginalis glycogen synthase and glycogen phosphorylase, the key enzymes of glycogen synthesis and degradation, respectively. We determined that their regulatory properties differed from those of well-characterized animal and fungal enzymes. Here, we turn our attention to how glycogen attains its branched structure. We first determined that the glycogen from T. vaginalis resembled that from a related organism, T. gallinae. To determine how the branched structure of T. vaginalis glycogen arose, we identified open reading frames encoding putative T. vaginalis branching and debranching enzymes. When the open reading frames TVAG_276310 and TVAG_330630 were expressed recombinantly in bacteria, the resulting proteins exhibited branching and debranching activity, respectively. Specifically, recombinant TVAG_276310 had affinity for polysaccharides with long outer branches and could add branches to both amylose and amylopectin. TVAG_330630 displayed both 4-α-glucanotransferase and α1,6-glucosidase activity and could efficiently debranch phosphorylase limit dextrin. Furthermore, expression of TVAG_276310 and TVAG_330630 in yeast cells lacking endogenous glycogen branching or debranching enzyme activity, restored normal glycogen accumulation and branched structure. We now have access to the suite of enzymes required for glycogen synthesis and degradation in T. vaginalis.