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Related Experiment Videos

Structure-function relationship of a recombinant human galactoside-binding protein

J Ochieng1, D Platt, L Tait

  • 1Metastasis Research Program, Michigan Cancer Foundation, Detroit 48201.

Biochemistry
|April 27, 1993
PubMed
Summary

Human tumor cells contain a galactoside-binding lectin (hL-31) homologous to known macrophage and murine lectins. Recombinant hL-31 (rhL-31) expression and analysis confirm its Y-shaped structure and membrane protein function.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • A galactoside-binding lectin, human lectin-31 (hL-31), identified in human tumor cells, shares homology with IgE-binding protein, Mac-2 antigen, and murine lectins (CBP35, RL-29, mL-34).
  • This lectin contains a collagen-like sequence, suggesting potential roles in cellular interactions and signaling.

Purpose of the Study:

  • To express and functionally characterize recombinant human lectin-31 (rhL-31) in Escherichia coli.
  • To elucidate the structural and functional properties of rhL-31, including its oligomerization state, cellular localization, and membrane orientation.

Main Methods:

  • Recombinant hL-31 (rhL-31) was expressed in E. coli and purified using an asialofetuin affinity column.
  • Functional assays included testing reactivity with anti-lectin antibodies and lactose-dependent hemagglutination.

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  • Electron microscopy was used to determine the lectin's structure, and cell surface iodination followed by collagenase treatment assessed membrane association.

    • Main Results:

    • rhL-31 was successfully purified and demonstrated reactivity with specific antibodies.
    • The recombinant lectin retained its ability to agglutinate erythrocytes in a lactose-dependent manner.
    • Electron microscopy revealed a Y-shaped structure for rhL-31, which exists as a monomer but dimerizes at higher concentrations relevant for hemagglutination.
    • rhL-31 was identified as a peripheral membrane protein with both N- and C-termini exposed on the cell surface.

    Conclusions:

    • The functional analysis of rhL-31 confirms its biological activity and structural characteristics.
    • The findings suggest a mechanism for the lectin's structure-function relationship, highlighting its role as a membrane-associated protein involved in cellular recognition.