Carbohydrate groups of alpha(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(1)m) 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-alpha(1)m were compared to nonmutated alpha(1)m, which carries two shor t non-sialylated N-linked oligosaccharides when expressed in the same syste m. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge, Circular dichroism spectra and antibody binding indicated a simi lar overall structure. However, the secretion of N17,95Q-alpha(1)m was sign ificantly reduced and similar to 75% of the protein were found accumulated intracellularly, The in vitro immunological effects of recombinant nonmutat ed aim and N17,96Q-alpha(1)m were compared to the effects of alpha(1)m isol ated from plasma, which is sialylated and carries an additional O-linked ol igosaccharide. All three alpha(1)m variants bound to human peripheral lymph ocytes 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(1)m was faster than that of plasma alpha(1)m, Nonmutated alpha(1)m was located primarily to the liver, most likely via b inding to asialoglycoprotein receptors, and N17,96Q-alpha(1)m was located m ainly to the kidneys. It is concluded that the carbohydrates of alpha(1)m a re important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties.