Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104

Elizabeth A. Rutledge, Caroline Enns

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

A soluble form of the human transferrin receptor (TfR) resulting from proteolytic cleavage at Arg 100 has been measured in human blood. In tissue culture cells elimination of the O-linked carbohydrate at Thr 104, four amino acids from the cleavage site, results in enhanced cleavage of the TfR (Rutledge et al., 1994, Blood, 83:580-586). In the present set of studies, the influence of amino acid substitution and the composition of the oligosaccharide at amino acid 104 on the cleavage of the TfR was examined. Site-directed mutagenesis was used to generate six different amino acids at position 104 which varied in size and charge. Measurement of the soluble TfR in the conditioned medium of the transfected cells of each mutant TfR showed that the large and charged side chains inhibited TfR cleavage the most. Otherwise the properties of the mutant TfRs were indistinguishable from the wild-type TfR in that the affinity of transferrin for these receptors, the extent of disulfide bond formation of the TfRs, and the proportion of TfRs at the cell surface were similar to that of the wild-type TfR. Removal of the sialic acid component of the carbohydrate from wild-type TfR by treatment of live cells with neuraminidase enhances TfR cleavage. Expression of wild-type TfR in CHO IdID cells (a glycosylation defective cell line) also shows enhanced cleavage under conditions that produce truncated or no O-linked carbohydrates. Treatment of IdID cells with neuraminidase reveals that the sialic acid of the O-linked carbohydrate protects against TfR cleavage, whereas the core sugars GalNAc and Gal do not protect as much. These results show that the terminal charged sialic acid residues are important for protection from proteolytic cleavage and suggest that cleavage could be regulated in the cell by removal of all or part of the carbohydrate.

Original languageEnglish (US)
Pages (from-to)284-293
Number of pages10
JournalJournal of Cellular Physiology
Volume168
Issue number2
DOIs
StatePublished - Aug 1996

Fingerprint

Transferrin Receptors
Carbohydrates
Chemical analysis
N-Acetylneuraminic Acid
Amino Acids
Neuraminidase
Blood
Glycosylation
Tissue culture
Mutagenesis
CHO Cells
Amino Acid Substitution
Conditioned Culture Medium
Site-Directed Mutagenesis
Oligosaccharides
Sugars
Disulfides
Substitution reactions

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104. / Rutledge, Elizabeth A.; Enns, Caroline.

In: Journal of Cellular Physiology, Vol. 168, No. 2, 08.1996, p. 284-293.

Research output: Contribution to journalArticle

@article{584e4366869344e098d7d695b83faea3,
title = "Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104",
abstract = "A soluble form of the human transferrin receptor (TfR) resulting from proteolytic cleavage at Arg 100 has been measured in human blood. In tissue culture cells elimination of the O-linked carbohydrate at Thr 104, four amino acids from the cleavage site, results in enhanced cleavage of the TfR (Rutledge et al., 1994, Blood, 83:580-586). In the present set of studies, the influence of amino acid substitution and the composition of the oligosaccharide at amino acid 104 on the cleavage of the TfR was examined. Site-directed mutagenesis was used to generate six different amino acids at position 104 which varied in size and charge. Measurement of the soluble TfR in the conditioned medium of the transfected cells of each mutant TfR showed that the large and charged side chains inhibited TfR cleavage the most. Otherwise the properties of the mutant TfRs were indistinguishable from the wild-type TfR in that the affinity of transferrin for these receptors, the extent of disulfide bond formation of the TfRs, and the proportion of TfRs at the cell surface were similar to that of the wild-type TfR. Removal of the sialic acid component of the carbohydrate from wild-type TfR by treatment of live cells with neuraminidase enhances TfR cleavage. Expression of wild-type TfR in CHO IdID cells (a glycosylation defective cell line) also shows enhanced cleavage under conditions that produce truncated or no O-linked carbohydrates. Treatment of IdID cells with neuraminidase reveals that the sialic acid of the O-linked carbohydrate protects against TfR cleavage, whereas the core sugars GalNAc and Gal do not protect as much. These results show that the terminal charged sialic acid residues are important for protection from proteolytic cleavage and suggest that cleavage could be regulated in the cell by removal of all or part of the carbohydrate.",
author = "Rutledge, {Elizabeth A.} and Caroline Enns",
year = "1996",
month = "8",
doi = "10.1002/(SICI)1097-4652(199608)168:2<284::AID-JCP7>3.3.CO;2-#",
language = "English (US)",
volume = "168",
pages = "284--293",
journal = "Journal of Cellular Physiology",
issn = "0021-9541",
publisher = "Wiley-Liss Inc.",
number = "2",

}

TY - JOUR

T1 - Cleavage of the transferrin receptor is influenced by the composition of the O-linked carbohydrate at position 104

AU - Rutledge, Elizabeth A.

AU - Enns, Caroline

PY - 1996/8

Y1 - 1996/8

N2 - A soluble form of the human transferrin receptor (TfR) resulting from proteolytic cleavage at Arg 100 has been measured in human blood. In tissue culture cells elimination of the O-linked carbohydrate at Thr 104, four amino acids from the cleavage site, results in enhanced cleavage of the TfR (Rutledge et al., 1994, Blood, 83:580-586). In the present set of studies, the influence of amino acid substitution and the composition of the oligosaccharide at amino acid 104 on the cleavage of the TfR was examined. Site-directed mutagenesis was used to generate six different amino acids at position 104 which varied in size and charge. Measurement of the soluble TfR in the conditioned medium of the transfected cells of each mutant TfR showed that the large and charged side chains inhibited TfR cleavage the most. Otherwise the properties of the mutant TfRs were indistinguishable from the wild-type TfR in that the affinity of transferrin for these receptors, the extent of disulfide bond formation of the TfRs, and the proportion of TfRs at the cell surface were similar to that of the wild-type TfR. Removal of the sialic acid component of the carbohydrate from wild-type TfR by treatment of live cells with neuraminidase enhances TfR cleavage. Expression of wild-type TfR in CHO IdID cells (a glycosylation defective cell line) also shows enhanced cleavage under conditions that produce truncated or no O-linked carbohydrates. Treatment of IdID cells with neuraminidase reveals that the sialic acid of the O-linked carbohydrate protects against TfR cleavage, whereas the core sugars GalNAc and Gal do not protect as much. These results show that the terminal charged sialic acid residues are important for protection from proteolytic cleavage and suggest that cleavage could be regulated in the cell by removal of all or part of the carbohydrate.

AB - A soluble form of the human transferrin receptor (TfR) resulting from proteolytic cleavage at Arg 100 has been measured in human blood. In tissue culture cells elimination of the O-linked carbohydrate at Thr 104, four amino acids from the cleavage site, results in enhanced cleavage of the TfR (Rutledge et al., 1994, Blood, 83:580-586). In the present set of studies, the influence of amino acid substitution and the composition of the oligosaccharide at amino acid 104 on the cleavage of the TfR was examined. Site-directed mutagenesis was used to generate six different amino acids at position 104 which varied in size and charge. Measurement of the soluble TfR in the conditioned medium of the transfected cells of each mutant TfR showed that the large and charged side chains inhibited TfR cleavage the most. Otherwise the properties of the mutant TfRs were indistinguishable from the wild-type TfR in that the affinity of transferrin for these receptors, the extent of disulfide bond formation of the TfRs, and the proportion of TfRs at the cell surface were similar to that of the wild-type TfR. Removal of the sialic acid component of the carbohydrate from wild-type TfR by treatment of live cells with neuraminidase enhances TfR cleavage. Expression of wild-type TfR in CHO IdID cells (a glycosylation defective cell line) also shows enhanced cleavage under conditions that produce truncated or no O-linked carbohydrates. Treatment of IdID cells with neuraminidase reveals that the sialic acid of the O-linked carbohydrate protects against TfR cleavage, whereas the core sugars GalNAc and Gal do not protect as much. These results show that the terminal charged sialic acid residues are important for protection from proteolytic cleavage and suggest that cleavage could be regulated in the cell by removal of all or part of the carbohydrate.

UR - http://www.scopus.com/inward/record.url?scp=0029780466&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029780466&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-4652(199608)168:2<284::AID-JCP7>3.3.CO;2-#

DO - 10.1002/(SICI)1097-4652(199608)168:2<284::AID-JCP7>3.3.CO;2-#

M3 - Article

C2 - 8707864

AN - SCOPUS:0029780466

VL - 168

SP - 284

EP - 293

JO - Journal of Cellular Physiology

JF - Journal of Cellular Physiology

SN - 0021-9541

IS - 2

ER -