PlumX Metrics
Embed PlumX Metrics

O -Linked Glycosylation at Threonine 27 Protects the Copper Transporter hCTR1 from Proteolytic Cleavage in Mammalian Cells *

Journal of Biological Chemistry, ISSN: 0021-9258, Vol: 282, Issue: 28, Page: 20376-20387
2007
  • 72
    Citations
  • 0
    Usage
  • 62
    Captures
  • 1
    Mentions
  • 0
    Social Media
Metric Options:   Counts1 Year3 Year

Metrics Details

Article Description

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.

Provide Feedback

Have ideas for a new metric? Would you like to see something else here?Let us know