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Related Experiment Videos

Molecular and functional analysis of mouse decay accelerating factor (CD55).

C L Harris1, N K Rushmere, B P Morgan

  • 1Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.

The Biochemical Journal
|July 27, 1999
PubMed
Summary

Researchers discovered novel mouse decay accelerating factor (DAF; CD55) isoforms generated by alternative splicing. These DAF forms, including transmembrane and GPI-anchored variants, regulate complement activity and protect cells from damage.

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Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • The mouse decay accelerating factor (DAF; CD55) exists in transmembrane (TM) and glycosylphosphatidylinositol (GPI)-anchored forms.
  • These forms were initially thought to be encoded by separate genes, Daf-GPI and Daf-TM.

Purpose of the Study:

  • To identify and characterize novel isoforms of mouse DAF generated through alternative splicing.
  • To investigate the functional consequences of DAF expression on complement regulation.

Main Methods:

  • Northern-blot analysis and reverse transcriptase-PCR to detect DAF mRNA expression in various tissues.
  • Molecular cloning and sequence analysis of DAF cDNA from spleen and testis.
  • Stable expression of DAF isoforms in Chinese hamster ovary (CHO) cells.

Related Experiment Videos

  • Complement-mediated C3b deposition assays and cell-lysis assays.
  • Hemolytic assays using soluble recombinant DAF.

    • Main Results:

    • Several novel DAF isoforms were identified, arising from alternative splicing of Daf-GPI and Daf-TM genes.
    • Novel TM isoforms with altered GPI-anchor addition sequences or deleted STP regions were found.
    • A novel secreted form of DAF derived from the Daf-GPI gene was identified.
    • DAF-transfected CHO cells exhibited significantly reduced C3b deposition and resistance to complement-mediated lysis.
    • Soluble recombinant DAF inhibited complement activity in a hemolytic assay.

    Conclusions:

    • Alternative splicing generates a diverse repertoire of mouse DAF isoforms with distinct structural features.
    • These DAF isoforms play a crucial role in regulating the complement system.
    • DAF confers protection against complement-mediated cell damage, highlighting its importance in immune regulation.