In contrast to the human serum protein which is approximately one-half erythro-β-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant Cls expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gin 159) yields a faster migrating recombinantCls still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with Clq and either human serum or recombinant Clr, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between Cls and Clq, Clr, C2, and C4 required for reassembly of the Cl complex, activation, and initiation of the classical complement pathway.The 4.0S recombinant Cls dimerizes to yield 5.6S Cls2 in the presence of Ca2+ and forms the 9. IS Cls-Clr-Clr-Cls tetramer upon the addition of human serum Clr and the 15.6S Cl complex upon the addition of Clq to the tetramer. The recombinant Cls and human serum Cls have identical N-terminalamino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant Cls is secreted and isolated as the unactivated zymogen, and it may be activated by human serum Clr which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between Cl-inhibitor and the light chain of recombinant Cls. To summarize, recombinant Cls expressed in insect cells is not β-hydroxylated, lacks sialic acid, and has been mutated to remove one-half of the carbohydrate, yet reassembles with the remaining subcomponents to form a functional Cl complex.
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