title: N-glycosylation of human sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is essential for stability, secretion and activity.
abstract: Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secretedN-linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its functionin vivo, remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of itsN-glycans and secretion. Glycosylation of Asn356 in SMPDL3A, anN-linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specific N-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A.
authors.affiliation: Atherosclerosis Laboratory, ANZAC Research Institute, University of Sydney, NSW, Australia mathew.traini@sydney.edu.au.
authors.forename: Mathew
authors.initials: M
authors.lastname: Traini
authors.affiliation: Atherosclerosis Laboratory, ANZAC Research Institute, University of Sydney, NSW, Australia.
authors.forename: Raani
authors.initials: R
authors.lastname: Kumaran
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW, Australia.
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.affiliation: Atherosclerosis Laboratory, ANZAC Research Institute, University of Sydney, NSW, Australia.
authors.forename: Maaike
authors.initials: M
authors.lastname: Kockx
authors.affiliation: Atherosclerosis Laboratory, ANZAC Research Institute, University of Sydney, NSW, Australia.
authors.forename: Wendy
authors.initials: W
authors.lastname: Jessup
authors.affiliation: Atherosclerosis Laboratory, ANZAC Research Institute, University of Sydney, NSW, Australia.|Department of Cardiology, Concord Repatriation General Hospital, Concord, NSW, Australia.
authors.forename: Leonard
authors.initials: L
authors.lastname: Kritharides
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D021382:Protein Sorting Signals; D055550:Protein Stability / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017026:Swainsonine / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
chemical_list: D007212:Indolizines; D007976:Leupeptins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011480:Protease Inhibitors; D046988:Proteasome Endopeptidase Complex; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013108:Sphingomyelin Phosphodiesterase; D017026:Swainsonine; D014415:Tunicamycin; C469297:SMPDL3A protein, human; C037806:castanospermine; D054836:Indolizidines; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: SMPDL3A; glycoproteins; glycosylation; phosphodiesterase; sphingomyelinase