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Related Experiment Videos

The molecular basis for therapeutic concepts utilizing CD14

F Stelter1, M Bernheiden, R Menzel

  • 1Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany.

Progress in Clinical and Biological Research
|May 12, 1998
PubMed
Summary

Soluble CD14 (sCD14) shows therapeutic potential for septic shock. Alanine scanning identified key regions (9-13 and 91-101) crucial for lipopolysaccharide (LPS) binding and sCD14-mediated cell signaling.

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Area of Science:

  • Immunology
  • Molecular Biology

Background:

  • CD14 is a myeloid cell receptor recognizing lipopolysaccharide (LPS) and Gram-negative bacteria.
  • Soluble CD14 (sCD14) offers potential therapeutic benefits for LPS-induced septic shock.

Purpose of the Study:

  • To investigate the functional domains of human CD14 involved in LPS binding and signaling.
  • To identify specific amino acid residues critical for CD14-LPS interaction and downstream cellular responses.

Main Methods:

  • Alanine scanning mutagenesis of human CD14 (amino acids 1-152).
  • Generation and characterization of 23 substitution mutants in CHO cells.
  • Assays for anti-CD14 antibody recognition, LPS/E. coli binding, and LPS-induced IL-6 release.

Main Results:

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  • Twenty-one mutants expressed on cell surfaces, 18 secreted as soluble forms.
  • CD14(39-41,43-44)A mutant lost all LPS and E. coli binding ability.
  • A combined mutant CD14(9-13/57,59,61-63)A showed significantly reduced LPS interaction, suggesting a conformational LPS-binding site.
  • Regions 9-13 and 91-101 were identified as critical for sCD14-mediated LPS signaling.

Conclusions:

  • The LPS-binding site on human CD14 is a conformational epitope.
  • Specific N-terminal and internal regions of CD14 are essential for LPS recognition and downstream signaling pathways relevant to septic shock treatment.