Active site amino acid sequence of human factor D.

Abstract

C3 and C5 (3-6). Factor B has recently been characterized as a serine protease (11). Factor D is thus required for alternative pathway activation (4,(12)(13)(14). Its activity is inhibited by diisopropyl phosphorofluoridate (iPr2P-F) (7-9), and its NH2-terminal amino acid sequence is homologous with the NH2-terminal sequences of the serine proteases (15, 16). However, this enzyme differs from other plasma proteases in several characteristics. Factor D probably circulates in its active proteolytic form. The data of Fearon et al. (7) suggest that a zymogen exists, but others suggest that it does not and that only the active proteolytic form is present in plasma (8). Factor.D has restricted substrate specificity. It probably does not cleave peptide bonds other than the susceptible bond in factor B (9). Among the synthetic substrates hydrolyzed by most serine proteases, factor D inefficiently hydrolyzes only the chromogenic substrate N-benzoylisoleucylglutamylglycylarginine-p-nitroanilide hydrochloride (9, 15). Although high concentrations of iPr2P-F inhibit factor D functional activity (7, 9), stoichiometric incorporation of iPr2P-F into the active site of factor D has not been demonstrated. In addition, factor D is not inhibited by the plasma protease inhibitors (9). Determination of the primary structure of factor D should clarify some of these unusual characteristics.The present study characterized the cyanogen bromide peptides of factor D and their NH2-terminal amino acid sequences, which includes the active site serine residue. MATERIALS AND METHODSFresh frozen human plasma was obtained from the American Red Cross Blood Services, Northeast Region. CM-Sephadex C-50 and Sephadex G-75 were purchased from Pharmacia. Hydroxylapatite (Bio-Gel HT) and NaDodSO4 were purchased from Bio-Rad. Amicon positive-pressure ultrafiltration cells and PM10 ultrafiltration membranes werepurchased from Amicon (Lexington, MA). Acrylamide, N,N'-rnethylenebisacrylamide, N,N,NlNl-tetramethylethylenediamine, iodoacetic acid, and CNBr were purchased from Eastman. Molecular weight markers for NaDodSO4/polyacrylamide gel electrophoresis were insulin, lysozyme, bovine trypsinogen, carbonic anhydrase, and ovalbumin, all obtained from Sigma. All sequencing reagents (5% phenylisothiocyanate in heptane, 0.1 M Quadrol buffer, and heptafluorobutyric acid) and solvents (benzene, ethyl acetate, and butyl chloride) were obtained from Pierce. Polybrene was also from Pierce.Protein Purification. Factor D was isolated from 4-8 liters of fresh frozen human plasma essentially as described (15) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Amino acid sequence of human D of the alternative complement pathway

“…Residue 24 was identified as histidine, 48 as aspartic acid, and 50 as lysine. This is in agreement with the work of Davis et al (1979Davis et al ( , 1980 but differs from the glutamic acid, glycine, and tyrosine determinations, respectively, published by Reid et al (1981) at these positions. In addition, we have identified residue 38 as serine, residue 58 as threonine, residue 112 as glutamine, residue 154 as threonine, and residue 213 as threonine, whereas Reid et al (1981) have identified threonine, isoleucine, glycine, lysine, and serine, respectively, at these positions.…”

“…The study of D has been hampered by its low serum concentration and by the difficulty in removing contaminants of similar apparent molecular weight and behavior on ion-exchange chromatography (Johnson et al, 1980). Recently, however, several reports have appeared in the literature detailing the isolation of purified D in sufficient quantities to carry out partial amino acid sequence analysis (Davis et al, 1979;Volanakis et al, 1980;Davis, 1980;Johnson et al, 1980;Reid et al, 1981). We now report the determination of the essentially complete amino acid sequence of this key complement component.…”