title: Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase αβ and γ subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology.
abstract: Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and ß subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg(-/-) embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg(-/-) embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg(-/-) embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal.
authors.affiliation: Complex Carbohydrate Research Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602, United States.
authors.forename: Heather
authors.initials: H
authors.lastname: Flanagan-Steet
authors.affiliation: Complex Carbohydrate Research Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602, United States.
authors.forename: Courtney
authors.initials: C
authors.lastname: Matheny
authors.affiliation: Complex Carbohydrate Research Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602, United States.
authors.forename: Aaron
authors.initials: A
authors.lastname: Petrey
authors.affiliation: Complex Carbohydrate Research Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602, United States.
authors.forename: Joshua
authors.initials: J
authors.lastname: Parker
authors.affiliation: Complex Carbohydrate Research Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602, United States. Electronic address: rsteet@ccrc.uga.edu.
authors.forename: Richard
authors.initials: R
authors.lastname: Steet
mesh_terms: D000818:Animals; D002403:Cathepsins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D008358:Mannose / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D009081:Mucolipidoses / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation / Q000502:physiology*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*
chemical_list: D008360:Mannosephosphates; C027693:mannose-6-phosphate; D017855:Transferases (Other Substituted Phosphate Groups); D006867:Hydrolases; D006026:Glycoside Hydrolases; D002403:Cathepsins; D010447:Peptide Hydrolases; D008358:Mannose
keywords: Cathepsin; Lysosomal hydrolase; Mannose phosphorylation; Zebrafish