Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome
Tamara Manuelian, Jens Hellwage, Seppo Meri, Jessica Caprioli, Marina Noris, Stefan Heinen, Mihaly Jozsi, Hartmut P.H. Neumann, Giuseppe Remuzzi, Peter F. Zipfel
Tamara Manuelian, Jens Hellwage, Seppo Meri, Jessica Caprioli, Marina Noris, Stefan Heinen, Mihaly Jozsi, Hartmut P.H. Neumann, Giuseppe Remuzzi, Peter F. Zipfel
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Article Nephrology

Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome

Abstract

Hemolytic uremic syndrome (HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Recent studies have identified a factor H–associated form of HUS, caused by gene mutations that cluster in the C-terminal region of the complement regulator factor H. Here we report how three mutations (E1172Stop, R1210C, and R1215G; each of the latter two identified in three independent cases from different, unrelated families) affect protein function. All three mutations cause reduced binding to the central complement component C3b/C3d to heparin, as well as to endothelial cells. These defective features of the mutant factor H proteins explain progression of endothelial cell and microvascular damage in factor H–associated genetic HUS and indicate a protective role of factor H for tissue integrity during thrombus formation.

Authors

Tamara Manuelian, Jens Hellwage, Seppo Meri, Jessica Caprioli, Marina Noris, Stefan Heinen, Mihaly Jozsi, Hartmut P.H. Neumann, Giuseppe Remuzzi, Peter F. Zipfel

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Heparin affinity chromatography binding of recombinant wild-type deletio...
Heparin affinity chromatography binding of recombinant wild-type deletion mutant factor H (SCRs 8-20). (a) Culture supernatant of insect cells infected with recombinant baculovirus coding for the recombinant wild-type protein (FH 8-20) and the two mutant forms, R1210C mutant (i.e., FH 8-20/R1210C) or the R1215G mutant (i.e., FH 8-20/R1215G) was applied to heparin affinity chromatography. After loading, the column was thoroughly washed, and bound proteins were eluted by an NaCl gradient. Absorbencies are indicated for recombinant wild-type protein by the dashed line, the R1210C mutant by the solid line, and the R1215 mutant by the dotted line. The identical conductivity graphs show that elution was performed under identical conditions. mAu, milliampere units. (b) SDS-PAGE and Western blot analysis of fractions 34–41 of the individual proteins. (c) The separation yields pure protein as shown for wild-type protein fractions 34–41 after SDS-PAGE separation in combination with silver staining. The mobility of the marker proteins is indicated on the left.