Carbohydrate groups of alpha1-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 alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, 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-alpha1m was significantly reduced and approximately 75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated alpha1m and N17,96Q-alpha1m were compared to the effects of alpha1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three alpha1m 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-alpha1m was faster than that of plasma alpha1m. Nonmutated alpha1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-alpha1m was located mainly to the kidneys. It is concluded that the carbohydrates of alpha1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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