Human C.lovin.1 inhibitor: improved isolation and preliminary structural characterization

Abstract

An improved procedure for the isolation of the C.hivin.1 [activated complement component 1] inhibitor (C.hivin.1-INH) component of human complement is reported. Following preliminary steps to remove plasminogen, fibrinogen and aggregated material, 3 conventional chromatographic steps are used to isolate C.hivin.1-INH in high (70%) overall yield. An extinction coefficient of 3.60 was determined. The isolated protein exhibits a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a mobility corresponding to an apparent MW of 105,000. After removal of carbohydrate, the protein shows an increased mobility, corresponding to an apparent MW of 78,000. A total carbohydrate content of 33% was calculated, and from this and the size of the deglycosylated polypeptide, a true MW of 116,000 was estimated. Further analysis of the carbohydrate indicated a galactose:mannose ratio of 2:1 and approximately equimolar amounts of N-acetylglucosamine and N-acetylgalactosamine. This composition is unusual for a plasma protein and suggests that much of the carbohydrate is contained in linkages other than the typical N-glycosidic structures. Values found for the amino acid composition are compared with those reported previously. The amino-terminal sequence (40 residues) of C.hivin.1-INH is also reported. Asparagine lies at the amino terminus. Neither high-performance liquid chromatography of the released phenylthiohydantoin derivative nor back-hydrolysis of the thiazolinone permitted identification of the residue contained at position 3. The sequence around this position is compatible with an N-glycosidic linkage to residue 3. The 1st 10 residues also contain an unusual run of 5 hydroxyl-containing amino acids (-Thr-Ser-Ser-Ser-Ser-) at positions 5-9. Visual comparison of the amino-terminal sequences with those reported for other protease inhibitors does not indicate any sequence homology.