O-linked glycosylation at threonine 27 protects the copper transporter hCTR1 from proteolytic cleavage in mammalian cells.
The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wild-type hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.
Maryon
Edward B
EB
Department of Biochemistry and Molecular Genetics, University of Illinois, 900 S. Ashland Avenue, Chicago, IL 60607, USA.
Molloy
Shannon A
SA
Kaplan
Jack H
JH
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D018938:Caco-2 Cells; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079923:Copper Transporter 1; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D020125:Mutation, Missense; D046188:Protein Modification, Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; C490079:SLC31A1 protein, human; D013912:Threonine; D003300:Copper