PMID: 17525160

 

    Legend: Sugar

Title : O-linked glycosylation at threonine 27 protects the copper transporter hCTR1 from proteolytic cleavage in mammalian cells

Abstract :

1. The major human copper uptake protein , hCTR1 , has 190 amino acids and a predicted mass of 21 kDa
2. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa
3. Part of this increased mass is due to N-linked glycosylation at Asn-15
4. 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
5. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides
6. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa
7. 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
8. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations
9. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1
10. 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
11. 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
12. 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