PMID: 17525160

 

    Legend: Gene, Sites

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
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycosylation, , -, -, threonine 27
  • 12. glycosylation, , -, -, Thr-27
  • 3. glycosylation, , -, -, Asn-15
  • 5. contains, , -, O-linked polysaccharides, terminus
  • 7. glycosylation, , -, -, site
Output(Part-Of) (sent_index, protein, site):
  • 11. hCTR1, polypeptide
  • 12. hCTR1, terminus
  • 5. hCTR1, terminus
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 0. hCTR1, -, threonine 27
  • 12. hCTR1, -, Thr-27
  • 3. hCTR1, -, Asn-15

 

 

Protein NCBI ID SENTENCE INDEX
hCTR1 1317 0,1,2,4,5,6,7,8,9,10,11,12