Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component C1s.
A 159 residue, N-terminal fragment of the human C1s complement component, C1s alpha(159), was expressed in the baculovirus, insect cell system. The protein was abundantly produced 3 days after infection, reaching levels as high as 40 microg/ml in cell culture media. It had a molecular weight of 18,100 (+/-4.9) Da by laser desorption mass spectrometry, close to the theoretical value of 18,111 Da, confirmed by sequencing. Sedimentation equilibrium and gel filtration column chromatography showed that C1s alpha(159) was a monomer in the presence of EDTA, and a dimer in the presence of Ca2+. The C1s alpha(159)2 dimer had a sedimentation coefficient of 3.1 S. When the C1s alpha(159)2 was mixed with Clq, there was little or no interaction. Likewise, unactivated C1r2 dimer had a sedimentation coefficient of 6.8 S, and when mixed with C1q little or no interaction was observed. When C1s alpha(159)2 was mixed with the 6.8 S C1r2 in Ca2+, a 7.5 S complex was formed, presumably the C1s alpha(159) x C1r x C1r x C1s alpha(159) tetramer. When C1q, which migrated at 10.1 S was mixed with C1s alpha(159)2 and C1r2 in the presence of Ca2+, a C1-like complex, but containing C1s alpha(159) instead of C1s, was formed which migrated at 14.0 S. This C1-like molecule remained unactivated unless challenged with an ovalbumin-antiovalbumin immune complex. In the presence of immune complex, the C1r became activated. This suggested that the presence of the 159 amino acid C1s alpha domain, which held the C1r to the C1q, was sufficient to permit activation by an immune complex, even though the catalytic domains of C1s were not present.
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles 90095, USA.
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Tsai
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Poon
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Schumaker
D000595:Amino Acid Sequence; D001483:Base Sequence; D003167:Complement Activation; D003172:Complement C1 / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology
D003172:Complement C1; D010446:Peptide Fragments