Functional characterization of glutamate carboxypeptidase II: a multifunctional Zn‐metalloprotease

Abstract

Glutamate Carboxypeptidase II (GCPII) is a dimeric, transmembrane, zinc metallopeptidase produced in many organisms including humans. Human GCPII (hGCPII) is expressed in the brain, prostate, intestine, and kidney, and is therefor predicted to exhibit varying tissue‐specific hydrolase activities. The only documented GCPII hydrolase activities are the breakdown of N‐acetyl‐aspartyl‐glutamate (NAAG) – GCPII is involved in the development of neurotoxicity due to excess glutamate – and folyl‐poly‐γ‐glutamate – GCPII is involved in folate uptake across intestinal mucosal membrane. However, functions in other tissues have not been reported. A comprehensive understanding of potentially diverse tissue‐specific proteolytic and other biochemical functions of GCPII requires detailed molecular level in vivo and in vitro studies using the full‐length GCPII and suitable model organism respectively.Homology modeling and protein structure analyses identified a hGCPII ortholog in the roundworm Caenorhabditis elegans, (hereafter referred as cGCPII), thus C. elegans has been chosen for the in vivo studies. The hGCPII and the cGCPII share all essential protein structural features. The cGCPII is encoded as three paralogs (gcp‐2.1, gcp‐2.2, gcp‐2.3) compared to five paralogs in humans. Whole animal phenotypic studies carried out using the wild‐type (N2) and the mutant RB1055, which lacks the gcp‐2.1, showed a larger brood size for RB1005 than N2 and longevity assays showed similar lifespans for both strains. The N2 and RB1055 strains raised in the presence of the folate hydrolase inhibitor sulfasalazine (SSZ) and GCPII specific inhibitors 2‐(Phosphonomethyl)pentane‐1,5‐dioicacid (PMPA) or 2‐(3‐Mercaptopropyl)pentanedioic acid (2‐MPPA) and the showed significantly reduced brood size for RB1055 compared to N2. The functional roles of individual cGCPII paralogs were investigated by RNAi mediated knockout experiments. Collective results indicate that the all three cGCPII paralogs may be involved in functional complementation in C. elegans. For the in vitro studies, the full‐length hGCPII was cloned and overexpression was carried out in Pichia pastoris. Several hGCPII expressing transformants have been identified and expression optimization is underway. Parallel cloning in insect cells in process to compare the functionally important glycosylation levels in Pichia versus the insect cells in order to choose an optimal expression system. Together, the in vivo and in vitro findings will aid in further understanding of the physiological functions of GCPII.Support or Funding InformationEastern Illinois UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.