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Carbohydrate groups of 1-microglobulin are important for secretion and tissue localization but not for immunological properties
The role of the carbohydrates for the structure and functions of the plasma and tissue protein [alpha]1-microglobulin ([alpha]1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96[Right arrow]Q). The mutated cDNA was expressed in a baculovirus-insect...
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Published in: | Glycobiology (Oxford) 2000-09, Vol.10 (9), p.891-900 |
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Main Authors: | , , , , , , |
Format: | Article |
Language: | English |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The role of the carbohydrates for the structure and functions of the plasma and tissue protein [alpha]1-microglobulin ([alpha]1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96[Right arrow]Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-[alpha]1m were compared to nonmutated [alpha]1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-[alpha]1m was significantly reduced and ~75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated [alpha]1m and N17,96Q-[alpha]1m were compared to the effects of [alpha]1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three [alpha]1m variants bound to human peripheral lymphocytes and mouse T cell hybridomas to the same extent. They also inhibited the antigen-stimulated proliferation of peripheral lymphocytes and antigen-stimulated interleukin 2-secretion of T cell hybridomas in a similar manner. After injection of rats intravenously, the blood clearance of recombinant nonmutated and N17,96Q- [alpha]1m was faster than that of plasma [alpha]1m. Non-mutated [alpha]1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-[alpha]1m was located mainly to the kidneys. It is concluded that the carbohydrates of [alpha]1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties. |
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ISSN: | 0959-6658 1460-2423 |
DOI: | 10.1093/glycob/10.9.891 |