Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate alpha-galactosidase and alpha-N-acetylgalactosaminidase.
BACKGROUND
Human alpha-galactosidase A (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD)--Fabry (alpha-GAL deficiency) and Schindler (alpha-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins alpha-GAL and alpha-NAGA.


RESULTS
BlastP searches for orthologs of human alpha-GAL and alpha-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (alpha-galactosidase and alpha-N-acetylgalactosaminidase)--gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization.Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken alpha-NAGA, rice alpha-GAL and human alpha-GAL suggest a close evolutionary relationship of GANA-1 to both human alpha-GAL and alpha-NAGA. Both alpha-GAL and alpha-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, alpha-GAL activity on an artificial substrate was completely inhibited by the alpha-NAGA inhibitor, N-acetyl-D-galactosamine.A GANA-1::GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH4Cl, agents that increase the pH of the cellular acidic compartment. Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes.Inhibition of gana-1 by RNA interference resulted in a decrease of both alpha-GAL and alpha-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype.


CONCLUSIONS
GANA-1 is a single C. elegans ortholog of both human alpha-GAL and alpha-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment.
Hujová
Jana
J
Institute of Inherited Metabolic Disorders, Charles University, 1st Medical Faculty, Prague, Czech Republic. jhujo@lf1.cuni.cz
Sikora
Jakub
J
Dobrovolný
Robert
R
Poupetová
Helena
H
Ledvinová
Jana
J
Kostrouchová
Marta
M
Hrebícek
Martin
M
D000818:Animals; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D006801:Humans; D008247:Lysosomes; D008969:Molecular Sequence Data; D010802:Phylogeny; D011506:Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D040681:Structural Homology, Protein; D000519:alpha-Galactosidase / Q000235:genetics* / Q000378:metabolism; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D011506:Proteins; C018589:lysosomal proteins; D000519:alpha-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase; C509345:gana-1 protein, C elegans