Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.
The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D003180:Complement C3b Inactivator Proteins; D017242:Complement Factor H; D004591:Electrophoresis, Polyacrylamide Gel; D005361:Ficain; D008954:Models, Biological; D010196:Pancreatic Elastase; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D003180:Complement C3b Inactivator Proteins; D010446:Peptide Fragments; D017242:Complement Factor H; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D005361:Ficain; D010434:Pepsin A