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Membrane expression and interactions of human transcobalamin II receptor
S Bose1, S Seetharam, B Seetharam
1Department of Biochemistry, Medical College of Wisconsin, Milwaukee, USA.
Insights
The human transcobalamin II receptor (TC II-R) is synthesized as a 45-kDa protein, matures to 62 kDa via glycosylation, and forms a 124-kDa dimer in plasma membranes, interacting with lipids.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Transcobalamin II receptor (TC II-R) plays a crucial role in vitamin B12 transport.
- Understanding TC II-R synthesis and membrane expression is vital for cellular nutrient uptake.
Purpose of the Study:
- To investigate the synthesis, maturation, and membrane assembly of the human transcobalamin II receptor (TC II-R).
- To elucidate the molecular form and ligand-binding activity of TC II-R in native and reconstituted membrane environments.
Main Methods:
- Immunoprecipitation using antiserum against purified 62-kDa human placental TC II-R.
- Cell-free translation of human kidney mRNA.
- Immunoblotting of tissue membranes and Triton X-100 soluble fractions.
- Lipid extraction and liposome reconstitution experiments.
- Chemical cross-linking with 125I-TC II-cobalamin.
Main Results:
- A 45-kDa protein was immunoprecipitated from cell-free translation products.
- Immunoblotting revealed a 124-kDa band in tissue membranes, with highest expression in kidney.
- Triton X-100 and lipid extraction yielded a 62-kDa form, which dimerized to 124 kDa upon liposome insertion.
- Both 62-kDa and 124-kDa forms demonstrated ligand-binding activity.
Conclusions:
- TC II-R is synthesized as a 45-kDa polypeptide, matures to a 62-kDa form through glycosylation.
- The mature 62-kDa TC II-R exists as a noncovalent 124-kDa dimer in plasma membranes, stabilized by lipid interactions.
- Dimerization is essential for the receptor's function in ligand binding.
Abstract:
Antiserum raised to purified 62-kDa human placental transcobalamin II receptor (TC II-R) has been used to study its synthesis and membrane expression. The antiserum immunoprecipitated a 45-kDa protein from the cell-free translation using human kidney mRNA and recognized a single 124-kDa band on immunoblotting of placental and other human tissue membranes, and quantitation of the blots revealed high levels of TC II-R expression in the human kidney followed by placenta, intestine, and liver. Triton X-100 extraction of placental membranes resulted in the complete (100%) solubilization of the receptor, and immunoblotting of the Triton X-100-soluble fraction revealed a single band of 62 kDa. Lipid extraction of placental membranes with a mixture of chloroformmethanol (2:1) followed by immunoblotting revealed a single band of molecular mass 62 kDa. The molecular mass of the pure Triton X-100-bound receptor increased on SDS-polyacrylamide gel electrophoresis from 62 to 124 kDa upon its insertion in liposomes prepared using egg phosphatidylcholine and cholesterol. Chemical cross-linking of native membrane-or lipid vesicle-bound TC II-R or detergent-soluble extracts of the membrane with 125I-TC II-cobalamin revealed that both the 124- and 62-kDa forms of the receptor were active in ligand binding. Based on these results we suggest that TC II-R is synthesized as a single polypeptide of 45 kDa, and following its maturation (involving N- and O-glycosylation) the 62-kDa mature receptor is expressed in plasma membranes as a noncovalent dimer of 124 kDa. The dimerization of TC II-R in the plasma membranes is due to its interactions with annular lipids.