RECOMBINANT HUMAN-COMPLEMENT SUBCOMPONENT CLS LACKING BETA-HYDROXYASPARAGINE, SIALIC-ACID, AND ONE OF ITS 2 CARBOHYDRATE CHAINS STILL REASSEMBLES WITH CLQ AND CLR TO FORM A FUNCTIONAL CL COMPLEX

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxy-asparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s 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 Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1rBAR which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1sBAR. To summarize, recombinant C1s expressed in insect cells is not beta-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 C1 complex.