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

Abstract

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 wildtype 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.

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

“…In experiments measuring the kinetics of copper uptake, no correction was made for initial nonspecific binding. Copper uptake was stopped by removing 64 Cu and adding ice-cold buffer (28), after which the cells were washed twice with the cold buffer and then dissolved in 0.4% Triton X-100, 10 mM DTT, 2 mM EDTA, and 20 mM Tris, pH 8.0, and incubated for Ͼ10 min at 37°C. Lysed cells were collected, and half the volume was counted in a Beckman LS 6500 scintillation counter.…”

“…Normalizing Expression Levels of hCTR1-expressing LinesFor experiments involving 64 Cu uptake assays in which several different lines expressing FLAG-tagged hCTR1 WT and mutants were compared, hCTR1 expression levels were normalized as described previously (28). Western blots from total membranes from each line were probed with FLAG or Na,KATPase antibodies and quantified as described (28), and copper uptake levels were adjusted for expression when expressed as the percentage of control uptake.…”

“…As described previously (28), an N-terminal FLAG tagged hCTR1 clone was constructed using this cDNA. The H139R and C-terminal mutants were derived from the FLAGtagged hCTR1 clone pEM94 (Ref.…”

Posttranslational regulation of copper transporters

“…Specific targeting of ATP7B towards the apical membrane is dependent on an ATP7B-specific sequence, F 37 AFDNVGYE 45 , and disruption of this motif results in basolateral targeting [71] (Fig. 1).…”

“…In contrast, CTR2 lacks these Mets-motifs with the exception of the penultimate methionine, providing an explanation for the reduced affinity compared with CTR1. The amino terminus of hCTR1 is substituted with N-and O-linked oligosaccharides [37,45]. The function of the N-linked glycosylation is not completely clear, whereas the O-linked glycans are possibly involved in the stability of the protein [45].…”