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Structure/function studies of human decay-accelerating factor.

W G Brodbeck1, L Kuttner-Kondo, C Mold

  • 1Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.

Immunology
|September 30, 2000
PubMed
Summary
This summary is machine-generated.

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The decay-accelerating factor (DAF) uses specific amino acids, not its N-linked glycan, for complement regulation. Residues L147F148 are crucial for both classical and alternative pathways, while KKK125-127 are vital for the alternative pathway.

Area of Science:

  • Immunology
  • Complement System Biology

Background:

  • The decay-accelerating factor (DAF) regulates the complement system via its four complement control protein repeats (CCPs).
  • DAF's classical pathway activity is linked to CCPs 2 and 3, while alternative pathway activity involves CCPs 2, 3, and 4.
  • Molecular modeling suggested potential ligand-binding sites involving charged and hydrophobic residues within CCPs 2 and 3.

Purpose of the Study:

  • To investigate the functional roles of DAF's N-linked glycan and specific amino acid residues (KKK125-127 and L147F148) in complement regulation.
  • To determine the contribution of these elements to DAF's activity on both classical and alternative C3 and C5 convertases.

Main Methods:

  • Site-directed mutagenesis was used to create DAF variants (N61Q, KKK125-127TTT, L147F148SS).

Related Experiment Videos

  • Mutant DAF proteins were expressed, purified, and incorporated into erythrocytes.
  • Complement regulatory activity was assessed using C3b deposition assays, hemolytic assays, and fluid-phase C3a generation assays.

    • Main Results:

    • The N-linked glycan at N61 is not essential for DAF's regulatory functions.
    • The hydrophobic residues L147F148 on CCP3 are critical for regulating both classical and alternative pathway C3 convertases.
    • The charged residues KKK125-127 are essential for alternative pathway regulation and play a minor role in classical pathway regulation.

    Conclusions:

    • DAF's complement regulatory activity is primarily mediated by specific amino acid residues, not its N-linked glycan.
    • The L147F148 motif is a key functional site for DAF in both complement pathways.
    • The KKK125-127 motif is specifically important for DAF's alternative pathway function.