title: Inefficient glycosylation leads to high steady-state levels of actively degrading cardiac triadin-1.
abstract: In junctional sarcoplasmic reticulum, binding to cardiac triadin-1 provides a mechanism by which the Ca(2+)-release channel/ryanodine receptor may link with calsequestrin to regulate Ca(2+) release. Calsequestrin and triadin-1 both contain N-linked glycans, but about half of triadin-1 in the heart remains unglycosylated. To investigate mechanisms for this incomplete glycosylation, we overexpressed triadin-1 as a series of glycoform variants in non-muscle cell lines and neonatal heart cells using plasmid and adenoviral vectors. We showed that the characteristic incomplete glycosylation stemmed from properties of the glycosylation sequence that are conserved among triadin splice variants, including the close proximity of Asn(75) to the sarcoplasmic reticulum inner membrane. Although triadin-1 appeared by SDS-PAGE analysis as a 35/40-kDa doublet in all cells, variations occurred in the relative levels of the two glycoforms depending on the cell type and whether overexpression involved a plasmid or adenoviral vector. Treatment of triadin-1 with the proteasome inhibitor MG-132 led to striking changes in the relative levels of triadin-1 that indicated active breakdown of unglycosylated, but not glycosylated, triadin-1. Besides substantial increases in the relative levels of unglycosylated triadin-1, proteasome inhibition led to an accumulation of two new modified forms of triadin-1 that were seen with triadin-1 only when it is not glycosylated on Asn(75). Effects of tunicamycin and endoglycosidase H confirmed that these novel isoforms represent two alternative N-linked glycosylation sites, indicating that an alternative topology occurs infrequently leading to yet other glycoforms with short half-lives.
authors.affiliation: Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Milstein
authors.forename: Timothy P
authors.initials: TP
authors.lastname: McFarland
authors.forename: James D
authors.initials: JD
authors.lastname: Marsh
authors.forename: Steven E
authors.initials: SE
authors.lastname: Cala
mesh_terms: D000256:Adenoviridae; D000818:Animals; D000831:Animals, Newborn; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019556:COS Cells; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004285:Dogs; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins / Q000494:pharmacology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046188:Protein Modification, Translational / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D002155:Calsequestrin; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D009124:Muscle Proteins; D061988:Proteasome Inhibitors; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; C498269:TRDN protein, human; C498271:Trdn protein, rat; D014415:Tunicamycin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde