Partial amino acid sequence of human factor D:homology with serine proteases.

Abstract

Human factor D purified to homogeneity by a modified procedure was subjected to NH2-terminal amino acid sequence analysis by using a modified automated Beckman sequencer. We identified 48 of the first 57 NHrterminal amino acids in a single sequencer run, using microgram quantities of factor D. The deduced amino acid sequence represents approximately 25% of the primary structure of factor D. This extended NH2-terminal amino acid sequence of factor D was compared to that of other trypsin-related serine proteases. By visual inspection, strong homologies (33-50% identity) were observed with all the serine proteases included in the comparison. Interestingly, factor D showed a higher degree of homology to serine proteases of pancreatic origin than to those of serum origin.Human factor D (D) is a trace serum protein playing an essential role in the activation sequence of the alternative complement pathway (1, 2). It is an endopeptidase catalyzing the cleavage of a single peptide bond of factor B, which results in the generation of the C3-cleaving enzyme of the alternative pathway, C3bBb. Cleavage of factor B by D occurs only when factor B is in the form of a Mg2+-dependent, reversiblc complex with C3b, the major fragment of C3 activation (1) or with the cobra equivalent of C3b (3), cobra venom factor (4). The enzymatic activity of D is irreversibly inhibited by di-isopropyl fluorophosphate (5), which led to its characterization as a serine protease. Structurally, D consists of a single, Mr 22,900 polypeptide chain with isoleucine at the NH2 terminus (6). A nine-amino acid NH2-terminal sequence recently reported by Davis et al. (7) indicated that D is homologous with other serine proteases. We report here an extended NH2-terminal amino acid sequence of D, which allows for a more detailed comparison with other trypsin-like pancreatic and serum serine proteases of known primary structure.MATERIALS AND METHODS Purification of Factor D. Factor D was isolated from outdated human plasma by a combination of ion-exchange chromatography on Bio-Rex 70 and gel filtration on Bio-Gel P-60 as described (6) except that, after the batch elution of D from the first Bio-Rex 70 column, serum lipoproteins were precipitated with 50 mM MnCl2 (8). This resulted in removal of approximately 45% of the total absorbance at 280 nm without appreciable loss of D activity, as measured by the hemolytic diffusion plate assay of Martin et al. (9). Approximately 2 mg of D were isolated from 7 liters of serum. D purified by this procedure gave a single protein band on electrophoresis on NaDodSO4-containing polyacrylamide gels (10). An aliquotThe 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. 1116 of this D preparation was reduced with 0.7 M 2-mercaptoethanol for 2 min in a boiling water bath in the presence of 1% NaDodSO4 and then subjected to electrophoresis on polyacrylamide gradient (5-...

Active site amino acid sequence of human factor D.

“…The data confirm the sequence of the nine NH2-terminal amino acids previously reported (15). The combined NH2-terminal sequence and the sequence of CNBr peptide I agree, for the most part, with the extended NH2-terminal sequence reported by Volanakis et al (16). The major differences occur at residues 10 and 15, which were reported by them as phenylalanine and valine, respectively.…”

“…This histidine is analogous to histidine-57 of chymotrypsin and is required for the charge-relay system of serine proteases. However, neither I nor Volanakis and colleagues (16) has been able to identify the amino acid at this position definitively.…”

“…Its activity is inhibited by diisopropyl phosphorofluoridate (iPr2P-F) (7)(8)(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.…”

Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella.

“…Functionally active human C3 was prepared by the procedure of Hammer et al (33) with minor modifications, and was radiolabeled with '25I-Na using Iodobeads (Pierce Chemical Co.) according to manufacturer's instructions. Human factor D (25 Mg/ml in isotonic VBS, pH 7.4) was purified by a modification of the method of Volanakis et al (34). Factor B (4.3 mg/ml) was prepared using a minor modification of the method of Hammer et al (33).…”

Alpha, Beta, Gamma, Omega—The Structure of the Plasma Proteins

“…Partial structural data have been reported including the sequence at the amino terminus, some of the CNBr fragments, and the region around the serine at the active site (Volanakis et al, 1980;Johnson et al, 1980). Partial structural data have been reported including the sequence at the amino terminus, some of the CNBr fragments, and the region around the serine at the active site (Volanakis et al, 1980;Johnson et al, 1980).…”