Glycoside hydrolase family 31 proteins in Caenorhabditis elegans

Filip Majer¹, Jana Uřinovská¹, Jakub Sikora¹, Helena Poupětová¹, Jitka Hlavatá¹, Karel Jelínek², Marta Kostrouchová¹, Jana Ledvinová¹, Martin Hřebíček¹

¹Charles University in Prague, 1st Faculty of Medicine, Institute of Inherited Metabolic Disorders, Charles University in Prague

²Charles University in Prague, Faculty of Science, Department of Physical and Macromolecular Chemistry, Charles University in Prague

filip.majer@lf1.cuni.cz

Human acid alpha-glucosidase (GAA, EC 3.2.1.20) catalyses lysosomal glycogen degradation and belongs to glycoside hydrolase family 31 (GH31). The objective of our study was to assess GH31 proteins in Caenorhabditis elegans (C. elegans), to identify the acid active GAA ortholog and to evaluate C. elegans as a model organism for glycogen storage disease type II (acid alpha-glucosidase deficiency).

C. elegans genome contains four acid alpha-glucosidase related (aagr-1-4) genes. These predicted ORFs were amplified from mixed stage N2 culture’s cDNA and sequenced. Multiple protein sequence alignment demonstrated common evolutionary origin of AAGR-1-4 and other selected GH31 members. We further performed protein structure homology modeling of all four AAGR proteins on the basis of available GH31 templates (YicI, MalA and Nt-MGA) as well as molecular docking of the specific inhibitor of non-alpha-glucosidase GH31 enzymes - acarbose. All these bioinformatic analyses suggested clustering of AAGR-1 and 2 with acid-active, and AAGR-3 and 4 with neutral pH optimum GH31 enzymes. The expression of AAGR-1 and 2, evaluated by transcriptional GFP tagging, was in both cases limited to intestinal cells and six coelomocytes. RNA interference (RNAi) of each of the four nematode’s genes did not reveal any changes in morphological phenotype. Additionaly, RNAi efficacy was assessed by glucosidase activity measurements at two pH values (4.0 and 6.5) with and without addition of acarbose (pH 4.0), a clinically used alpha-glucosidase inhibitor. We observed predominant neutral or acidic glucosidase activities associated with individual AAGRs. Nevertheless, AAGR-1 was found to possess acidic glucosidase activity with relatively pronounced acarbose resistance, a result further replicated in aagr-1 deletion mutant.

To conclude, four C. elegans (AAGR-1-4) orthologs of human GAA were evaluated by combination of bioinformatic, cellular and biochemical approaches. It was determined that AAGR-2 has predominating acid and AAGR-3 neutral glucosidase activity, though AAGR-1 was the least acarbose sensitive acidic AAGR and therefore could represent the most probable ortholog of human GAA.

Acknowledgements

This work was supported from research project 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.